Research organism

elife

eLife

eLife

2050-084X

eLife Sciences Publications, Ltd

79648

10.7554/eLife.79648

Research Article

Neuroscience

α₁-Adrenergic receptor–PKC–Pyk2–Src signaling boosts L-type Ca²⁺ channel Ca_V1.2 activity and long-term potentiation in rodents

Man

Kwun Nok Mimi

https://orcid.org/0000-0002-0132-9129

1Bartels

Peter

https://orcid.org/0000-0001-5852-1835

1†Henderson

Peter B

1†Kim

Karam

1Shi

Mei

2Zhang

Mingxu

12Ho

Sheng-Yang

1Nieves-Cintron

Madeline

https://orcid.org/0000-0003-1935-8400

1Navedo

Manuel F

https://orcid.org/0000-0001-6864-6594

1Horne

Mary C

mhorne@ucdavis.edu12Hell

Johannes W

https://orcid.org/0000-0001-7960-7531

jwhell@ucdavis.edu121

https://ror.org/05rrcem69

Department of Pharmacology, University of California

Davis

United States2

https://ror.org/036jqmy94

Department of Pharmacology, University of Iowa

Iowa City

United States

Goda

Yukiko

Reviewing Editor

https://ror.org/02qg15b79

Okinawa Institute of Science and Technology

JapanSwartz

Kenton J

Senior Editor

https://ror.org/01cwqze88

National Institute of Neurological Disorders and Stroke, National Institutes of Health

United States

†

These authors contributed equally to this work

20062023

2023

12

e79648

2104202219062023

This manuscript was published as a preprint at .

03072022

© 2023, Man et al

2023

Man et al

http://creativecommons.org/licenses/by/4.0/

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

The cellular mechanisms mediating norepinephrine (NE) functions in brain to result in behaviors are unknown. We identified the L-type Ca²⁺ channel (LTCC) Ca_V1.2 as a principal target for G_q-coupled α₁-adrenergic receptors (ARs). α₁AR signaling increased LTCC activity in hippocampal neurons. This regulation required protein kinase C (PKC)-mediated activation of the tyrosine kinases Pyk2 and, downstream, Src. Pyk2 and Src were associated with Ca_V1.2. In model neuroendocrine PC12 cells, stimulation of PKC induced tyrosine phosphorylation of Ca_V1.2, a modification abrogated by inhibition of Pyk2 and Src. Upregulation of LTCC activity by α₁AR and formation of a signaling complex with PKC, Pyk2, and Src suggests that Ca_V1.2 is a central conduit for signaling by NE. Indeed, a form of hippocampal long-term potentiation (LTP) in young mice requires both the LTCC and α₁AR stimulation. Inhibition of Pyk2 and Src blocked this LTP, indicating that enhancement of Ca_V1.2 activity via α₁AR–Pyk2–Src signaling regulates synaptic strength.

L-type calcium channelsα1-adrenergic receptorprotein kinase Ctyrosine phosphorylationlong-term potentiation

Research organismMouse

http://dx.doi.org/10.13039/100000002

National Institutes of Health

R01 MH097887

Hell

Johannes W

http://dx.doi.org/10.13039/100000002

National Institutes of Health

RF1 AG055357

Hell

Johannes W

http://dx.doi.org/10.13039/100000002

National Institutes of Health

R01 HL098200

Navedo

Manuel F

http://dx.doi.org/10.13039/100000002

National Institutes of Health

R01 HL121059

Navedo

Manuel F

http://dx.doi.org/10.13039/100000002

National Institutes of Health

T32 GM099608

Henderson

Peter B

http://dx.doi.org/10.13039/100000002

National Institutes of Health

R01 NS123050

Nieves-Cintron

Madeline

The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.

Author impact statement

The α₁-adrenergic receptor augments the activity of the L-type Ca²⁺ channel Ca_V1.2 through PKC and the tyrosine kinases Pyk2 and Src and thereby synaptic plasticity.

Introduction

Norepinephrine (NE) causes arousal and augments behavioral acuity and learning (Berman and Dudai, 2001; Cahill et al., 1994; Carter et al., 2010; Hu et al., 2007; Minzenberg et al., 2008). NE signals via the G_q-coupled α₁-adrenergic receptor (AR), G_i-coupled α₂AR, and G_s-coupled β₁, β₂, and β₃ ARs. βARs act through adenylyl cyclase (AC), cAMP, and PKA (Sanderson and Dell’Acqua, 2011). The β₂AR, G_s, AC, and PKA are all associated with the L-type Ca²⁺ channel (LTCC) Ca_V1.2 for efficient signaling in neurons (Davare et al., 2001; Dittmer et al., 2014; Murphy et al., 2014; Oliveria et al., 2007; Patriarchi et al., 2016; Qian et al., 2017) and heart (Balijepalli et al., 2006). The formation of this signaling complex identifies Ca_V1.2 as a major effector of signaling by NE. We now find that Ca_V1.2 is also a major effector for signaling via the α₁AR, which has a higher affinity for NE than βARs (Giustino and Maren, 2018; Ramos and Arnsten, 2007). Importantly, a large body of evidence implicates the α₁AR in NE’s role in attention and vigilance (Bari and Robbins, 2013; Berridge et al., 2012; Hahn and Stolerman, 2005; Hvoslef-Eide et al., 2015; Liu et al., 2009; Puumala et al., 1997; Robbins, 2002).

Ca_V1.2 fulfills a remarkably broad spectrum of functions. Dysfunctions due to mutations in Ca_V1.2 span from impaired cardiac contractility to the autistic-like behaviors seen in Timothy syndrome (Splawski et al., 2004). Furthermore, Ca_V1.2 has been linked to filopodia formation in invasive cancer cells (Jacquemet et al., 2016). Ca_V1.2 is by far the most abundant LTCC in heart and accounts for ~80% of all LTCCs in brain (Hell et al., 1993a; Sinnegger-Brauns et al., 2004). It governs the heartbeat, vascular tone, and neuronal functions including long-term potentiation (LTP) (Ghosh et al., 2017; Grover and Teyler, 1990; Moosmang et al., 2005; Patriarchi et al., 2016; Qian et al., 2017), long-term depression (Bolshakov and Siegelbaum, 1994), neuronal excitability (Berkefeld et al., 2006; Marrion and Tavalin, 1998), and gene expression (Dolmetsch et al., 2001; Li et al., 2016; Li et al., 2012; Ma et al., 2014; Marshall et al., 2011; Murphy et al., 2014; Wheeler et al., 2012). Studies on Ca_V1.2 mutant mice suggest that this channel plays a central role in anxiety disorders, depression, and self-injurious behavior (Sinnegger-Brauns et al., 2004). Congruently, LTCC blockers elicit antidepressant effects while agonists induce depression-like behavior (Mogilnicka et al., 1987; Mogilnicka et al., 1988) and self-biting in mice, a symptom associated with autism (Jinnah et al., 1999).

Ca_V1.2 consists of the pore-forming subunit α₁1.2, a β subunit and the α₂δ subunit (Catterall, 2000; Dai et al., 2009; Zamponi et al., 2015). The β and α₂δ subunits facilitate release of α₁1.2 subunits from the endoplasmic reticulum, inhibit ubiquitin-mediated degradation of voltage-gated calcium channels, influence electrophysiological properties of Ca²⁺ channels, such as activation and inactivation, and play diverse roles in the regulation of these channels (Catterall, 2000; Dai et al., 2009; Zamponi et al., 2015).

In the cardiovascular system, the α₁AR, the endothelin receptor ET1, and the angiotensin receptor AT₁ are important regulators of LTCC currents via G_q signaling (Catterall, 2000; Kamp and Hell, 2000; Voelker et al., 2023). G_q stimulates phospholipase C-β to induce production of diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP₃), which triggers Ca²⁺ release from intracellular stores. DAG and Ca²⁺ act in concert with phosphatidyl-serine to activate different PKC isoforms. Stimulation of PKC mostly leads to an increase in Ca_V1.2 activity (Bkaily et al., 1995; Dai et al., 2009; Döşemeci et al., 1988; He et al., 2000; Kamp and Hell, 2000; Lacerda et al., 1988; Navedo et al., 2005). However, an inhibitory effect of PKC on Ca_V1.2 currents has been reported in cardiomyocytes (Cheng et al., 1995; Voelker et al., 2023). This inhibition is mediated by phosphorylation of residues T27 and T31 by PKC in an isoform of α₁1.2 that is expressed in heart (McHugh et al., 2000). T27/T31 are not present in the most prevalent brain isoform due to alternative splicing (Snutch et al., 1991); thus, the inhibitory effect of PKC on LTCC currents is typically absent in neurons and neural crest-derived PC12 cells, or in vascular smooth muscle (Navedo et al., 2005; Taylor et al., 2000). Here, we show that stimulation of the α₁AR and of PKC consistently augments LTCC in hippocampal neurons.

Despite the prominent role of PKC in augmentation of Ca_V1.2 activity, how PKC mediates this effect has been unknown. PKC activates the nonreceptor tyrosine kinase Pyk2, a signaling process first shown in PC12 cells (Dikic et al., 1996; Lev et al., 1995) and later primary neurons (Bartos et al., 2010; Huang et al., 2001), and cardiomyocytes (Sabri et al., 1998). Activation of PKC triggers autophosphorylation of residue Y402 on Pyk2 to create a binding site for the SH2 domain of Src, which upon binding to Pyk2 becomes activated (Dikic et al., 1996). Src increases LTCC activity in smooth muscle cells (Gui et al., 2006; Hu et al., 1998; Wu et al., 2001), retinal pigment epithelium (Strauss et al., 1997), and neurons (Bence-Hanulec et al., 2000; Endoh, 2005; Gui et al., 2006). Furthermore, PKC (Navedo et al., 2008; Yang et al., 2005) and Src (Bence-Hanulec et al., 2000; Chao et al., 2011; Hu et al., 1998) are physically and functionally associated with Ca_V1.2. These findings underscore the physiological relevance of Src in regulating Ca_V1.2. Importantly, the pathway by which Src is activated in the context of Ca_V1.2 regulation has not been determined.

Once we established that stimulation of PKC or the G_q/PKC-coupled α₁AR strongly augments LTCC activity in neurons, we tested whether Pyk2 mediates this upregulation of channel activity. We link the α₁AR–PKC signaling to Src, which thus emerges as an important mediator of tyrosine phosphorylation on Ca_V1.2 downstream of G_q-coupled receptors. In neurons, the nearly twofold increase in LTCC currents upon stimulation of PKC with phorbol-12-myristate-13-acetate (PMA) or via the α₁AR was blocked by inhibitors of Pyk2 and Src, consistent with earlier data showing that Src elevates Ca_V1.2 activity to a comparable degree (Bence-Hanulec et al., 2000; Gui et al., 2006). Furthermore, we found that Pyk2 co-immunoprecipitated with Ca_V1.2 in parallel to Src. We identified the loop between domains two and three of α₁1.2 as the Pyk2-binding site. Stimulation of PKC either directly with PMA or through the G_q-coupled bradykinin (BK) receptor leads to tyrosine phosphorylation of α₁1.2 in PC12 cells. Abrogation of Pyk2 or Src activity ablated the phosphorylation. Finally, we discovered that the LTP in young mice mediated by LTCC-dependent Ca²⁺ influx during 200 Hz tetani (termed LTP_LTCC) that is not NMDAR dependent, required α₁AR stimulation and both Pyk2 and Src activity. These findings implicate upregulation of Ca_V1.2 activity by α₁AR–Pyk2–Src signaling as a critical process for control of synaptic strength. Our findings indicate that Ca_V1.2 forms a supramolecular signaling complex (signalosome) with PKC, Pyk2, and Src and that α₁AR–PKC–Src–Ca_V1.2 signaling constitutes a central regulatory mechanism of neuronal activity and synaptic plasticity by NE.

Resultsα<sub>1</sub>AR signaling augments LTCC activity in hippocampal neurons via PKC, Pyk2, and Src

We performed cell-attached recordings from cultured hippocampal neurons for single-channel analysis, which allows pharmacological isolation of LTCCs by application of ω-conotoxins GVIA and MVIIC (Hall et al., 2013; Oliveria et al., 2007; Patriarchi et al., 2016; Qian et al., 2017). LTCC channel activity was measured by cell-attached recordings, which yielded the product of the number of channels (N) and the open probability (Po) of each single channel. Application of phenylephrine (PHE), a selective agonist for all three α₁ARs, augmented N × Po of LTCCs from 0.18 ± 0.0433 (H₂O vehicle Control, n = 11) to 0.6156 ± 0.1386 (PHE; n = 13, p ≤ 0.01; Figure 1A–C). This increase was blocked by the selective α₁AR antagonist prazosin (0.2954 ± 0.0607; n = 10, p ≤ 0.05), indicating that PHE acted through α₁ARs and not other G-protein-coupled receptors. Prazosin by itself had no effect, vs. vehicle control (0.1846 ± 0.04624; n = 10) suggesting that there is little if any regulation of LTCCs under basal conditions in neurons by α₁ARs. PHE also increased the peak current of the ensemble average current in a prazosin-sensitive manner (Figure 1D, E).

Figure 1.The α<sub>1</sub>AR agonist phenylephrine (PHE) augments NPo of L-type Ca<sup>2+</sup> channels (LTCCs) in hippocampal neurons.

(A) Neurons were preincubated with vehicle, PHE and prazosin (PRAZ) before seal formation. (B) Ten consecutive traces from representative cell-attached single-channel recordings of LTCCs from cultured hippocampal neurons with vehicle (water; black), 10 µM PHE (red), PHE plus 20 nM prazosin (bright green), and prazosin alone (dark green). (C) The increase in NPo by PHE was blocked by prazosin. F_3,40 = 5.474. Control vs. PHE, p = 0.0036; PHE vs. Prazosin + PHE, p = 0.0334; Control vs. Prazosin only, p = 0.9723. (D) Ensemble averages during depolarization. (E) The increase in ensemble average peak currents by PHE was blocked by prazosin. F_3,40 = 4.506. Control vs. PHE, p = 0.0101; PHE vs. Prazosin + PHE, p = 0.0316; Control vs. Prazosin only, p = 0.9722. (C, E) Data are presented as means ± standard error of the mean (SEM). n represents the number of cells (*p ≤ 0.05, **p ≤ 0.01; analysis of variance [ANOVA] with post hoc Holm–Sidak’s multiple comparisons test). Panel A was created using Biorender.com.

Because direct phosphorylation of α₁1.2 by PKC inhibits Ca_V1.2 activity in heart (McHugh et al., 2000), we explored whether PKC might upregulate Ca_V1.2 activity indirectly via other kinases. PKC can activate Pyk2 (Bartos et al., 2010; Dikic et al., 1996; Huang et al., 2001; Lev et al., 1995) and thereby Src (Dikic et al., 1996; Huang et al., 2001). Src, in turn, augments LTCC activity (Bence-Hanulec et al., 2000; Endoh, 2005; Gui et al., 2006; Hu et al., 1998; Strauss et al., 1997; Wu et al., 2001). Therefore, we tested whether block of Pyk2 and Src affects upregulation of LTCC activity by PHE. In a new set of recordings augmentation of LTCC activity by PHE from NPo of 0.2008 ± 0.03348 (dimethyl sulfoxide (DMSO) vehicle control, n = 33) to 0.3272 ± 0.04412 (PHE, n = 33; p ≤ 0.01; Figure 2A–C) was completely blocked by two different PKC inhibitors, bisindolylmaleimide I (GF109203X; Bis I; 0.1412 ± 0.03305; n = 11, p ≤ 0.01) and chelerythrine (Chel; 0.05801 ± 0.01508; n = 12, p ≤ 0.001), the Pyk2-selective inhibitor PF-719 (0.09118 ± 0.02828; n = 10, p ≤ 0.01) and two structurally different Src family kinase inhibitors, PP2 (0.01487 ± 0.006808; n = 7, p ≤ 0.001) and SU6656 (0.09149 ± 0.02866; n = 9, p ≤ 0.01). Peak currents of ensemble averages showed respective changes (Figure 2D, E). Accordingly, α₁AR signaling increases LTCC activity via a PKC–Pyk2–Src signaling cascade. Notably, stimulation of two other major G_q-protein-coupled receptors in neurons, that is, the metabotropic mGluR1/5 receptors with dihydroxyphenylglycine (DHPG) and muscarinic receptors with muscarine, did not significantly increase LTCC activity, although there was a tendency for DHPG to do so (Figure 2—figure supplement 1).

Figure 2.The phenylephrine (PHE)-induced increase in NPo of L-type Ca<sup>2+</sup> channels (LTCCs) in hippocampal neurons requires PKC, Pyk2, and Src.

(A) Neurons were preincubated with vehicle, PHE, and the indicated kinase inhibitors before seal formation. (B) Ten consecutive traces from representative cell-attached single-channel recordings of LTCCs with vehicle (0.1% DMSO; black) and PHE either alone (red) or with the PKC inhibitors chelerythrine (Chel; 10 µM; bright brown) and bisindolylmaleimide I (Bis I; 100 nM; dark brown), the Pyk2 inhibitor PF-719 (1 µM; green), or the Src inhibitors PP2 (10 µM; blue) and SU6656 (10 µM; purple). (C) The increase in NPo by PHE was blocked by all inhibitors. F_6,108 = 6.434. Control vs. PHE, p = 0.0076; PHE vs. Chel + PHE, p = 0.0001; PHE vs. Bis I + PHE, p = 0.0076; PHE vs. PF-719 + PHE, p = 0.0018; PHE vs. PP2 + PHE, p = 0.0003; PHE vs. SU6656 + PHE, p = 0.0022. (D) Ensemble averages during depolarization. (E) The increase in ensemble average peak currents by PHE was blocked by PKC inhibitors chelerythrine, bisinolylmaleimide I, and Src inhibitor PP2. F_6,108 = 4.839. Control vs. PHE, p = 0.0242; PHE vs. Chel + PHE, p = 0.0004; PHE vs. Bis I + PHE, p = 0.0242; PHE vs. PF-719 + PHE, p = 0.0723; PHE vs. PP2 + PHE, p = 0.0006; PHE vs. SU6656 + PHE, p = 0.0723. (C, E) Data are presented as means ± standard error of the mean (SEM). n represents the number of cells (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; analysis of variance [ANOVA] with post hoc Holm–Sidak’s multiple comparisons test). Panel A was created using Biorender.com.

Figure 2—figure supplement 1.Group I mGluR and muscarinic receptor agonists did not change NPo of L-type Ca<sup>2+</sup> channels (LTCCs) in hippocampal neurons.

(A) Ten consecutive traces from representative cell-attached single-channel recordings of LTCCs from cultured hippocampal neurons with vehicle (H₂O; black), 100 µM dihydroxyphenylglycine (DHPG, pink), and 10 µM muscarine (cyan). (B) DHPG and muscarine did not alter NPo. F_2,35 = 0.6559. Control vs. DHPG, p = 0.648; Control vs. Muscarine, p = 0.6843. (C) Ensemble averages during depolarization. (D) DHPG and muscarine did not alter the amplitudes of the peak ensemble average current. F_2,35 = 0.007689. Control vs. DHPG, p = 0.9904; Control vs. Muscarine, p = 0.9904. (B, D) Data are presented as means ± standard error of the mean (SEM). n represent the number of cells (n.s., not significant; analysis of variance [ANOVA] with post hoc Holm–Sidak’s multiple comparisons test).

PKC augments LTCC activity in hippocampal neurons via Pyk2 and Src

To further establish a role of Pyk2 and Src, we directly stimulated PKC by including PMA in the bath solution during single-channel recording of LTCCs in hippocampal neurons. PMA increased NPo of LTCCs by around twofold from 0.1099 ± 0.0173 (DMSO control, n = 45) to 0.232 ± 0.03269 (PMA, n = 38; p ≤ 0.001, Figure 3A–C). This increase was blocked by Pyk2 inhibitors PF-719 (NPo = 0.1407 ± 0.02705, n = 13, p ≤ 0.05) and PF-431396 (NPo = 0.09282 ± 0.01765, n = 18, p ≤ 0.001) and by Src inhibitors PP2 (NPo = 0.05614 ± 0.01815, n = 14, p ≤ 0.001) and SU6656 (NPo = 0.02951 ± 0.00555, n = 8, p ≤ 0.001). Peak currents of ensemble averages showed respective changes (Figure 3D, E). The L-type calcium channel blocker isradipine completely blocked L-type currents in the presence of PMA, indicating successful isolation of L-type single-channel currents (Figure 3—figure supplement 1). These results show that in hippocampal neurons, PKC activation stimulates LTCC activity and this augmentation requires Pyk2 and Src activity.

Figure 3.The increase in NPo of L-type Ca<sup>2+</sup> channels (LTCCs) in hippocampal neurons by PKC requires Pyk2 and Src.

(A) Neurons were preincubated with vehicle, the phorbol ester phorbol-12-myristate-13-acetate (PMA), and the indicated kinase inhibitors before seal formation. (B) Ten consecutive traces from representative cell-attached single-channel recordings of LTCCs with vehicle (0.06% DMSO; black) and 2 µM PMA either alone (red) or with the Pyk2 inhibitors PF-719 (1 µM; green) and PF-431396 (3 µM; orange), or the Src inhibitors PP2 (10 µM; blue) and SU6656 (10 µM; purple). (C) The increase in NPo by PMA was blocked by all inhibitors. F_5,130 = 6.530. DMSO vs. PMA, p = 0.0003; PMA vs. PF-719 + PMA, p = 0.0372, PMA vs. PF-431396 + PMA, p = 0.0009; PMA vs. PP2 + PMA, p = 0.0003; PMA vs. SU6656 + PMA, p = 0.0005. (D) Ensemble averages during depolarization. (E) The increase in ensemble average peak currents by PMA was blocked by all inhibitors. F_5,130 = 5.665. DMSO vs. PMA, p = 0.0003; PMA vs. PF-719 + PMA, p = 0.0303, PMA vs. PF-431396 + PMA, p = 0.0051; PMA vs. PP2 + PMA, p = 0.0003; PMA vs. SU6656 + PMA, p = 0.0051. (C, E) Data are presented as means ± standard error of the mean (SEM). n represents the number of cells (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; analysis of variance [ANOVA] with post hoc Holm–Sidak’s multiple comparisons test). Panel A was created using Biorender.com.

Figure 3—figure supplement 1.L-type channel blocker isradipine completely blocks L-type single-channel currents in the presence of phorbol-12-myristate-13-acetate (PMA).

PMA (2 μM) in the bath solution potentiated the open probability of L-type channels during single-channel recording. Isradipine (10 μM) in the pipette solution completely blocked L-type currents in PMA-containing bath solution. Data are presented as as means ± standard error of the mean (SEM). n represents the number of cells (*p ≤ 0.05; analysis of variance [ANOVA] with post hoc Holm–Sidak’s multiple comparisons test). F_2,26 = 6.004. DMSO vs. PMA, p = 0.0139; PMA vs. Isradipine + PMA, p = 0.0129.

α<sub>1</sub>AR signaling augments single-channel open probability Po of LTCCs in neurons

Preincubation of neurons with PHE could promote either surface insertion or Po of LTCCs. To test whether PHE augmented specifically Po, we used pipettes with smaller diameters to minimize patch size and channel number in the patch, as reflected by pipette resistences of 7–12 vs. 3.5–5.5 MΩ in the preceding experiments. This approach typically resulted in <4 channels per patch, allowing exact determination of channel number and thereby calculation of single-channel Po. PHE was acutely washed on after establishing baseline activity to avoid delays as occurring when recording the effect of preincubation of neurons with PHE during which new channels could have been inserted (Figure 4A). PHE consistently increased within 2–3 min Po and peak currents as determined by ensemble averages (Figure 4B–D).

Figure 4.α<sub>1</sub>AR signaling augments Po of L-type Ca<sup>2+</sup> channels (LTCCs) in hippocampal neurons.

(A, E) Seals were formed by the recording pipettes before application of phenylephrine (PHE) or norepinephrine (NE) and ultimately of either isradipine or nimodipine to ensure channel activity was mediated by LTCCs. (B) Sample diary shows time course of Po before and after application of 10 µM PHE and then 10 µM isradipine. The number of channels under the patch was estimated based on the maximal number of observed stagged openings in each patch (k; upper left). (C) Ten consecutive traces from representative cell-attached single-channel LTTC recordings before and after application of PHE and then isradipine. Bottom panels show ensemble averages. (D) PHE increases Po (left) and peak currents of ensemble averages (n = 12 cells; right). (F) Sample diary shows time course of Po before and after application of 10 µM NE and then 10 µM isradipine. (G) Ten consecutive traces from representative cell-attached single-channel recordings of LTCCs before and after application of NE and then nimodipine. Bottom panels show ensemble averages. (H) NE increases Po (left) and peak currents of ensemble averages (n = 8 cells; right). (D, H) Data are presented as means ± standard error of the mean (SEM). Statistical significance was tested by a paired, two-tailed Student’s t-test, *p ≤ 0.05. Panels A and E were created using Biorender.com.

Application of the endogenous agonist NE to the outside of the cell-attached pipette was equally able to augment single-channel Po and peak currents of ensemble averages (Figure 4E–G). Of note, the β₂AR-selective adrenergic agonist albuterol can also augment Po of Ca_V1.2 by stimulating the Ca_V1.2-associated β₂AR, adenylyl cyclase and PKA (Davare et al., 2001; Dittmer et al., 2014; Murphy et al., 2014; Oliveria et al., 2007; Patriarchi et al., 2016; Qian et al., 2017). However, it does so only when applied inside the patch pipette and not when applied after seal formation to the outside, reflective of highly localized, spatially restricted signaling events (Davare et al., 2001; Dittmer et al., 2014; Murphy et al., 2014; Oliveria et al., 2007; Patriarchi et al., 2016; Qian et al., 2017). Accordingly, NE applied to the outside of the pipette augments Po not via β₂AR but rather via α₁AR signaling. Consistently, the increases in single-channel Po and peak currents of ensemble averages seen with NE were remarkably similar to the respective PHE effects.

To further test the role of α₁AR vs. β₂AR signaling in this recording configuration, we applied NE either alone or together with the α₁AR antagonist prazosin to the neurons before seal formation and recording of channel activity (Figure 5A). NE increased Po and peak current of ensemble averages more strongly in this approach than when applied only to the outside of the pipettes (Figure 5B–D). This effect was inhibited but not fully blocked when prazosin was co-applied with NE. These two effects are consistent with upregulation of Ca_V1.2 activity by NE via both α₁AR and β₂AR signaling.

Figure 5.Norepinephrine (NE) can augment Po of L-type Ca<sup>2+</sup> channels (LTCCs) via α<sub>1</sub>AR signaling in hippocampal neurons.

(A) Neurons were preincubated with NE ± prazosin (PRAZ) before seal formation. (B, C) Sample diaries show time courses of Po recordings obtained after preincubation with either NE alone or NE + PRAZ and seal formation. The number of channels under the patch was estimated based on the maximal number of observed stagged openings in each patch (k; upper left). (D) Ten consecutive traces from representative cell-attached single-channel recordings of LTCCs under control conditions or upon pre-incubation with either NE alone or NE plus PRAZ. Bottom panels show ensemble averages. (E) NE strongly increases Po (left) and peak currents of ensemble averages (right), which was strongly but not fully inhibited by PRAZ. Data are presented as means ± standard error of the mean (SEM; Control, n = 8 cells; NE, n = 12 cells; NE/PRAZ, n = 11 cells). Statistical significance was tested by a one-way analysis of variance (ANOVA) with Bonferroni correction, *p ≤ 0.05. Panel A was created using Biorender.com.

BK augments Po of LTCCs in neurons

The above results indicate that signaling by the G_q-coupled α₁AR promotes LTCC activity in a manner that is spatially much less localized if at all as opposed to signaling by the G_s-coupled β₂AR. Stimulation of other prominent G_q-coupled receptors, mGluR and muscarinic receptors, yielded little or no effects, respectively, on LTCC activity (Figure 2—figure supplement 1), possibly because those might be to far removed from the LTCCs in soma where the recordings were performed. Another G_q-coupled receptor that is prominent in the hippocampus is the BK receptor 2 (BK2). Application of BK to the outside of the patch pipette after establishing baseline activity of LTCCs (Figure 6A) significantly augmented Po (Figure 6B–D). In this set of experiments, the identity of the Ca²⁺ channels in the patch was confirmed by applying the LTCC activity promoting Bay K8644, which, consistently, augmented the current under the patch.

Figure 6.Bradykinin (BK) signaling augments Po of L-type Ca<sup>2+</sup> channels (LTCCs) in hippocampal neurons.

(A) Seals were formed by the recording pipettes before application of BK and ultimately of BayK8644 (BayK) to ensure channel activity was mediated by LTCCs. (B) Sample diary shows time course of Po before and after application of 5 µM BK and then 5 µM BayK to not only provide further evidence that the channels in the patch were LTCC but also aid in determining channel number k (upper left), which is the number of channels under the patch as estimated based on the maximal number of observed stagged openings in each patch. (C) Ten consecutive traces from representative cell-attached single-channel recordings of LTCCs before and after application of BK and then BayK. Bottom panels show ensemble averages. (D) BK increases Po (left) and peak currents of ensemble averages (right). Data are presented as means ± standard error of the mean (SEM; n = 7 cells). Statistical significance was tested by a paired, two-tailed Students t-test, *p ≤ 0.05. Panel A was created using Biorender.com.

Pyk2 co-immunoprecipitates with Ca<sub>V</sub>1.2 from brain and heart

Kinases and proteins that regulate kinase activity are often found in complexes with their ultimate target proteins (i.e., their ultimate substrates) including different ion channels for efficient and specific signaling (Dai et al., 2009; Dodge-Kafka et al., 2006). Both, PKC (Navedo et al., 2008; Yang et al., 2005) and Src (Bence-Hanulec et al., 2000; Chao et al., 2011; Hu et al., 1998), are associated with Ca_V1.2. We tested in brain and heart (where Ca_V1.2 is most abundant) whether the same is true for Pyk2. The Pyk2 antibody detected a single immunoreactive band with an apparent M_R of ~120 kDa in brain lysate (Figure 7A) and two bands in the same range in heart (Figure 7A, B), as reported earlier (Dikic et al., 1998). The shorter form is missing 42 residues in the proline-rich region of Pyk2, which affects its binding selectivity to proteins with SH3 domains. The single size form of Pyk2 present in brain and its two size forms expressed in heart co-immunoprecipitated with Ca_V1.2 (Figure 7A, B). No Pyk2 immunoreactive band was detectable when the immunoprecipitation (IP) was performed with control IgG, demonstrating that the co-IP of Pyk2 with Ca_V1.2 was specific. The detergent extracts from brain and heart were cleared of non-soluble material by ultracentrifugation prior to co-IP of Pyk2 with Ca_V1.2. Thus, our findings indicate that Pyk2 forms a bona fide protein complex with Ca_V1.2 rather than just co-residing in a detergent-resistant subcellular compartment. We also confirmed earlier work (Figure 7A, bottom panel) that indicated association of Src with Ca_V1.2 in vitro (Bence-Hanulec et al., 2000; Endoh, 2005; Gui et al., 2006; Hu et al., 1998; Strauss et al., 1997; Wu et al., 2001) and in intact cells (Bence-Hanulec et al., 2000; Chao et al., 2011; Hu et al., 1998).

Figure 7.Pyk2 binds to the loop between domains II and III of α<sub>1</sub>1.2. Co-immunoprecipitation of Pyk2 and Src with Ca<sub>V</sub>1.2 from brain (<bold>A</bold>) and heart (<bold>B</bold>).

Triton X-100 extracts were cleared from non-soluble material by ultracentrifugation before immunoprecipitation (IP) with antibodies against α₁1.2, Pyk2 itself, or non-immune control antibodies (rabbit IgG) and immunoblotting (IB) with anti-Pyk2 and anti-Src. Brain lysate (A, Input; 20 μl) and Pyk2 immunoprecipitates (B) served as positive control for detection of Pyk2 and Src by IB. Lanes for rabbit IgG control and α₁1.2 IP in B are from the same IB as the Pyk2 IP, which is depicted from a shorter exposure than the IgG and α₁1.2 IP lanes because IB signal was much stronger after Pyk2 IP than α₁1.2 IP. Comparable results were obtained in four independent experiments. (C) Schematic diagram of the intracellular α₁1.2 fragments used in the pulldown assay (Supplementary file 1). (D) Pulldown assay of Pyk2 binding to α₁1.2 fragments. GST fusion proteins of the N-terminus, the loops between domains I and II, II and III, III and IV, the whole C-terminus, and three different overlapping fragments covering the C-terminus were expressed in Escherichia coli, immobilized on glutathione Sepharose, washed and incubated with purified His-tagged Pyk2. Comparable amounts of fusion proteins were present (data not shown but see Hall et al., 2013; Hall et al., 2007; Patriarchi et al., 2016; Xu et al., 2010). Comparable results were obtained in five independent experiments.

Figure 7—source data 1.Original files of the full raw unedited blots with bands labeled in red boxes.

Pyk2 binds to the loop between domains II and III of α<sub>1</sub>1.2

To further confirm a direct interaction between Pyk2 and Ca_V1.2 we performed pulldown experiments using purified solubilized His-tagged Pyk2 and purified bead-bound GST-tagged α₁1.2 fragments covering all intracellular regions of α₁1.2 (Supplementary file 1, Snutch et al., 1990). As demonstrated earlier, all α₁1.2 fragments were present in comparable amounts (Hall et al., 2013; Patriarchi et al., 2016). The GST fusion protein covering the loop between domains II and III of α₁1.2 specifically pulled down Pyk2 (Figure 7C, D) indicating that Pyk2 directly binds to this region of the α₁ subunit.

Inhibitors of Pyk2 and Src block the increase in α<sub>1</sub>1.2 tyrosine phosphorylation upon stimulation of PKC

PC12 cells are of neural-endocrine crest origin and widely used as model cells for neuronal signaling and development. They express high levels of Ca_V1.2 (Eiki et al., 2009; Mustafa et al., 2010; Taylor et al., 2000; Walter et al., 2000), the BK receptor, and Pyk2 (Bartos et al., 2010; Dikic et al., 1996; Lev et al., 1995), making them an ideal model system for the difficult biochemical analysis of Ca_V1.2 phosphorylation. To characterize tyrosine phosphorylation of α₁1.2 we performed IP with the general anti-phosphotyrosine antibody 4G10 (Clifton et al., 2004; Ward et al., 1992). For this purpose, lysates were extracted with 1% sodium dodecyl sulfate (SDS) at 65°C followed by neutralization of SDS and ultracentrifugation before IP with the general anti-phosphotyrosine antibody 4G10 (Clifton et al., 2004; Ward et al., 1992). IP with 4G10 followed by immunoblotting (IB) with antibodies against the protein of interest is more reliable and more broadly applicable than the inverse. Because α₁1.2 does not re-associate with its binding partners after complex dissociation with SDS and the neutralization and dilution of SDS with Triton X-100 (Davare et al., 1999; Hell et al., 1995; Hell et al., 1993b; see also Leonard and Hell, 1997), detection of α₁1.2 by IB in the 4G10 IP would reflect specific tyrosine phosphorylation of the α₁1.2 subunit and not its artefactual re-association with an α₁1.2-associating tyrosine-phosphorylated protein that had been pulled down by the 4G10 antibody. This approach also allows analysis of tyrosine phosphorylation of Pyk2 within the same sample.

PC12 cells were pretreated with vehicle (0.02% DMSO), the Pyk2 inhibitor PF-431396, or the Src inhibitors SU6656 and PP2 or its inactive analogue PP3 for 5 min before application of BK or PMA for 10 min. BK strongly activates Pyk2 in PC12 cells via its G_q-coupled cognate receptor (Dikic et al., 1996; Lev et al., 1995). Using the 4G10 IP method, we found that both PMA and BK increased tyrosine phosphorylation of Pyk2 as previously described (Dikic et al., 1996; Lev et al., 1995; Figure 8A–C). This increase was prevented by PF-431396. In parallel, we determined phosphorylation of Pyk2 on Y402 and Y579 by direct IB of PC12 lysates with corresponding phosphospecific antibodies. Upon stimulation via PKC, Pyk2 phosphorylates itself in trans on Y402 (Bartos et al., 2010; Park et al., 2004) and then binds with phosphoY402 to the SH2 domain of Src (Dikic et al., 1996). This binding stimulates Src (Dikic et al., 1996), which in turn phosphorylates Pyk2 on Y579 in its activation loop for full activation (Avraham et al., 2000; Figure 8A). Src also phosphorylates itself in trans on Y416 in its activation loop for its own full activation (Roskoski, 2015), which was determined in parallel with a phosphospecific antibody. We found that BK and PMA increased phosphorylation of Pyk2 on Y402 (Figure 8B–D) and Y579 (Figure 8E, F) and of Src on Y416 (Figure 8G, H). PF-431396 blocked all of these phosphorylations indicating that Pyk2 acts downstream of PKC and upstream of Src. Furthermore, the Src inhibitor PP2, but not its inactive analog, PP3, also prevented PMA-induced Src autophosphorylation on Y416 (Figure 8I, J), as expected. Finally, PP2 inhibited PMA-induced phosphorylation of Pyk2 on Y402 and Y579 indicative of a self-maintaining positive feedback loop between Pyk2 and Src (Figure 8K–M). These results support the specific activation of both, Pyk2 and Src under our conditions and suggest that this activation occurs in a self-sustaining manner, which creates a quasi-molecular memory (Figure 8A).

Figure 8.PKC activates interdependent Pyk2 and Src.

PC12 cells were pretreated with vehicle (0.02% DMSO), the Pyk2 inhibitor PF-431396 (3 μM), and the Src inhibitor PP2 (10 μM) or its inactive analogue PP3 (10 μM) for 5 min before application of bradykinin (Brad., 2 μM) or phorbol-12-myristate-13-acetate (PMA, 2 μM) for 10 min, extraction with 1% sodium dodecyl sulfate (SDS) at 65°C to ensure dissociation of all proteins, neutralization of SDS with excess of Triton X-100, and ultracentrifugation. Supernatants were analysed by direct immunoblotting (IB) with the indicated Pyk2 and Src antibodies.

Some samples underwent immunoprecipitation (IP) with the anti-phosphotyrosine antibody 4G10 before IB with anti-Pyk2 antibody (top panel in A and quantification in B). IgG indicates control IP with non-immune mouse IgG. (A) Schematic diagram depicting the bradykinin receptor–PKC–Pyk2/Src signaling cascade and drugs used to target each molecular entity. (B) Upper panel: Total pY levels of Pyk2 determined by IP with 4G10 and IB with anti-Pyk2. Middle panel: pY402 levels of Pyk2 detected with anti-pY402 in corresponding lysates. Lower panel: Levels of total Pyk2 detected with anti-Pyk2 in same lysates. (C) Ratios of total pY of Pyk2 after 4G10 IP to total Pyk2 in lysates, normalized to control. F_5,63 = 12.73. DMSO vs. Brad., p = 0.012; DMSO vs. PMA, p < 0.0001; Brad. vs. PF-431396 + Brad., p < 0.0001; PMA vs. PF-431396 + PMA, p = 0.0001. (D) Ratios of pY402 to total Pyk2 signals in lysates, normalized to control. F_5,35 = 10.94. DMSO vs. Brad., p = 0.039; DMSO vs. PMA, p = 0.0052; Brad. vs. PF-431396 + Brad., p = 0.0005; PMA vs. PF-431396 + PMA, p < 0.0001. (E) Upper panel: pY579 levels of Pyk2 detected with anti-pY579. Lower panel: Levels of total Pyk2 detected with anti-Pyk2 in same lysates. (F) Ratios of pY579 to total Pyk2 signals in lysates, normalized to control. F_5,36 = 10.18. DMSO vs. Brad., p = 0.0072; DMSO vs. PMA, p = 0.021; Brad. vs. PF-431396 + Brad., p = 0.0008; PMA vs. PF-431396 + PMA, p = 0.0011. (G, I) Upper panels: pY416 levels of Src detected with anti-pY416. Lower panels: Levels of total Src detected with anti-Src in same lysates. (H, J) Ratios of pY416 to total Src signals in lysates, normalized to control. (H) F_5,72 = 4.464. DMSO vs. Brad., p = 0.0167; DMSO vs. PMA, p = 0.0226. (J) F_5,65 = 11.06. DMSO vs. PMA, p = 0.001; PMA vs. PP2 + PMA, p < 0.0001; DMSO vs. PP3 + PMA, p = 0.0042; PP3 vs. PP3 + PMA, p = 0.0086. (K) Upper panel: pY402 levels of Pyk2 detected with anti-pY402. Middle panel: pY579 levels of Pyk2 detected with anti-pY579. Lower panel: Levels of total Pyk2 detected with anti-Pyk2 in same lysates. (L, M) Ratios of pY402 and pY579 to total Pyk2 signals in lysates, normalized to control. (L) F_5,42 = 35.85. DMSO vs. PMA, p < 0.0001; PMA vs. PP2 + PMA, p < 0.0001; PP3 vs. PP3 + PMA, p = 0.0001; DMSO vs. PP3 + PMA, p = 0.0068; DMSO vs. PP2 + PMA, p = 0.0001; DMSO vs. PP2, p < 0.0001. (M) F_5,68 = 13.40. DMSO vs. PMA, p < 0.0001; PMA vs. PP2 + PMA, p < 0.0001; PP3 vs. PP3 + PMA, p = 0.0362; DMSO vs. PP3 + PMA, p = 0.0202. (C, D, F, H, J, L, M) Data are presented as mean ± standard error of the mean (SEM). Number (n) of independent experiments for each condition are indicated inside bars. Statistical analysis was by analysis of variance (ANOVA) with post hoc Bonferroni’s multiple comparisons test; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Bradykinin- and PMA-induced phosphorylation of Pyk2 on Y402 and Y579 and of Src on Y416, all of which were blocked by PF-431396 and PP2 but not the inactive PP3. Panel A was created using Biorender.com.

Figure 8—source data 1.Original files of the full raw unedited blots with bands labeled in red boxes.

Importantly, PMA and BK induced tyrosine phosphorylation of α₁1.2 (Figure 9). This effect was blocked by the Pyk2 inhibitor PF-431396 (Figure 9A–C) and the Src inhibitors SU6656 and PP2, whereas the inactive PP2 analogue PP3 was without effect (Figure 9D, E). These data show that activation of PKC translates into tyrosine phosphorylation of α₁1.2 and that this requires both Pyk2 and Src.

Figure 9.Increase in α<sub>1</sub>1.2 tyrosine phosphorylation by PKC is blocked by inhibitors or Pyk2 and Src. PC12 cells were treated as in <xref ref-type="fig" rid="fig8">Figure 8</xref> for analysis of tyrosine phosphorylation by immunoprecipitation (IP) with 4G10 and immunoblotting (IB) with anti-α<sub>1</sub>1.2.

IgG indicates control IP with non-immune mouse IgG. Vehicle (0.02% DMSO), PF-431396 (3 μM), PP2 (10 μM), PP3 (10 μM), or SU6656 (SU, 10 μM) were applied 5 min before phorbol-12-myristate-13-acetate (PMA) or bradykinin (Brad.) when indicated. (A) Schematic diagram depicting the bradykinin receptor–PKC–Pyk2/Src–Ca_V1.2 signaling cascade and drugs used to target each molecular entity. (B, D) Upper panels: pY of α₁1.2 determined by 4G10 IP and α₁1.2 IB. Lower panels: Levels of total α₁1.2 detected with anti-α₁1.2 in corresponding lysates. (C, E) Ratios of pY signals in 4G10 IPs by IB with anti-α₁1.2 to α₁1.2 signals in lysates, normalized to control. Data are presented as mean ± standard error of the mean (SEM). Number (n) of independent experiments for each condition are indicated inside bars. Statistical analysis was by analysis of variance (ANOVA) with post hoc Bonferroni’s multiple comparisons test. (C) F_5,50 = 10.65. DMSO vs. Brad., p = 0.0021; DMSO vs. PMA, p = 0.0036; Brad. vs. PF-431396 + Brad., p = 0.0003; PMA vs. PF-431396 + PMA, p < 0.0001. (E) F_7,31 = 23.67. DMSO vs. PMA, p < 0.0001; PMA vs. PP2 + PMA, p < 0.0001; PMA vs. SU + PMA, p < 0.0001 (**p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). Bradykinin- and PMA-induced α₁1.2 tyrosine phosphorylation was blocked by PF-431396, SU6656 and PP2 but not the inactive PP3. Panel A was created using Biorender.com.

Figure 9—source data 1.Original files of the full raw unedited blots with bands labeled in red boxes.

Knock down of Pyk2 and Src prevents the increase in α<sub>1</sub>1.2 tyrosine phosphorylation upon stimulation of PKC

To control for any potential side effects of PF-431396 and determine whether Pyk2 is required for PKC-induced tyrosine phosphorylation of α₁1.2 we employed FIV and HIV lentiviral expression vectors for shRNAs targeting Pyk2 in PC12 cells. We first designed and cloned an shRNA-targeting rat Pyk2 (Sh1) into the FIV-based plasmid pVETL-GFP (Bartos et al., 2010; Boudreau and Davidson, 2012; Harper et al., 2006) and tested the ability and specificity of this construct to knockdown ectopically expressed Pyk2 in HEK293T/17 cells. Cells were co-transfected with vectors for expression of GFP-tagged rat Pyk2 (rPyk2-GFP) and the pVETL-Sh1-GFP or no shRNA control pVETL-GFP (Figure 10A) and Pyk2 expression levels in the transfected cell lysates were examined via IB. Expression of rPyk2-GFP was virtually abolished by pVETL-Sh1-GFP whereas the control pVETL-GFP had no effect (Figure 10A). IB with both tubulin and GAPDH antibodies confirmed that total protein levels were not affected by transfection of these plasmids (Figure 10A).

Figure 10.Increase in α<sub>1</sub>1.2 tyrosine phosphorylation by PKC is blocked by knockdown of Pyk2 and Src.

(A) Lysates from HEK293T/17 cells transfected with vectors encoding rat Pyk2 (rPyk2-GFP) and either the Pyk2-targeting FIV lentivirus-derived, pVETL-Sh1-GFP (pFV-Pyk2-Sh1) or control (empty) pVETL-GFP (pFV-GFP) expression vectors, were immunoblotted (IB) with indicated antibodies. (B, C) IB analysis of indicated proteins in PC12 cultures incubated with viral particles containing pFV-Sh1-GFP (Sh1) FIV-based expression vector used in A or medium vehicle alone for 72 hr prior to treatment with either phorbol-12-myristate-13-acetate (PMA, B), bradykinin (Brad.; C), or vehicle alone (−; B, C). Upper blots in B and C show anti-α₁1.2 IBs of 4G10-anti-phosphotyrosine (pY) immunoprecipitation (IP) while middle and lower blots show direct IBs of indicated protein levels in input lysates. (D) Statistical analysis of the relative pY α₁1.2 levels. F_5,41 = 8.276. NT vs. PMA, p = 0.0031; NT vs. Brad., p = 0.0017; PMA vs. Sh1 + PMA, p = 0.001; Brad vs. Sh1 + Brad, p = 0.0433. (E, F) Direct IB analysis of indicated proteins in lysates of PC12 cultures transduced with HIV vector-derived lentiviral particles (e.g., pGFP-Pyk2-ShB-Lenti) containing expression cassettes for GFP and either the Pyk2-targeting (denoted pHV-Pyk-ShB and -ShC), Src-targeting (denoted pHV-Src-ShC and -ShD), or scrambled hairpin control (Cont.) shRNAs. In some cases (right blot in F) cultures were treated with PMA (+) or vehicle alone (−) before harvesting for IB. (G, H) IB analysis of indicated proteins from PC12 cultures infected with lentiviral particles containing HIV-GFP expression vectors as in E and F prior to treatment with either PMA (+) or vehicle (−). Upper panels show anti-α₁1.2 IBs of 4G10-anti-pY IP while lower blots show direct IBs of input lysates with indicated antibodies. (I) Statistical analysis of relative α₁1.2 pY levels. F_11,129 = 6.180. NT vs. PMA, p < 0.0001; PMA vs. Pyk2-ShB, p = 0.0005; PMA vs. Pyk2-ShB + PMA, p = 0.0029; PMA vs. Pyk2-ShC, p < 0.0001; PMA vs. Pyk2-ShC + PMA, p = 0.0002; PMA vs. Cont.-Sh, p = 0.0019; PMA vs. Cont.-Sh + PMA, p > 0.9999; PMA vs. Src-ShB, p = 0.0007; PMA vs. Src-ShB + PMA, p < 0.0001; PMA vs. Src-ShC, p < 0.0001; PMA vs. Src-ShC + PMA, p < 0.0001. The bar graphs in (D) and (I) show ratios of quantified anti-α₁1.2 IB signals in 4G10 IPs relative to α₁1.2 IB signals in total lysates, normalized to not treated (NT) control. Comparisons are made between samples treated with PMA (or bradykinin in D) and each of the other indicated conditions. Data are presented as mean ± standard error of the mean (SEM). Number (n) of independent experiments for each condition are indicated inside bars. Statistical analysis by analysis of variance (ANOVA) with post hoc Bonferroni’s multiple comparisons test (ns = not significant vs. PMA, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001).

Figure 10—source data 1.Original files of the full raw unedited blots with bands labeled in red boxes.

Next we tested whether pVETL-Sh1-GFP would inhibit PMA- and BK-induced α₁1.2 phosphorylation in PC12 cells. pVETL-Sh1-GFP lentiviral particles carrying the Sh1-shRNA and GFP expression cassettes were used to efficiently infect PC12 cells. Infected cells were monitored for GFP expression and then subjected to overnight serum starvation (to ensure low signaling levels) before treatment with PMA or BK. Fully SDS-dissociated tyrosine-phosphorylated proteins were immunoprecipitated with 4G10 before SDS–polyacrylamide gel electrophoresis (PAGE) and α₁1.2 IB (Figure 10B, C). As before, PMA and BK induced in average an about 2.5-fold increase in tyrosine phosphorylation of the α₁1.2 subunit, which was strongly repressed by Sh1 (Figure 10B–D). IB for total Pyk2 content confirmed Pyk2 knockdown by ~70–90% (Figure 10B, C, bottom panels). These findings indicate that depletion of Pyk2 potently blunts the PKC-mediated increase in α₁1.2 tyrosine phosphorylation. Total α₁1.2 content was not altered by Sh1. These findings indicate that Pyk2 knockdown does not affect α₁1.2 expression levels and that the reduction in tyrosine-phosphorylated α₁1.2 is likely not due to any potential off-target effects of the pVETL-Sh1-shRNA.

To further verify and extend these findings we obtained HIV-GFP lentiviral vectors for expression of validated unique 29mer shRNAs targeting Pyk2 (HIV-GFP-Pyk2ShA-D) and Src (HIV-GFP-SrcShA-D) as well as a scrambled, non-silencing control (HIV-GFP-Shscr). HIV-GFP-Pyk2ShB and C and HIV-GFP-SrcShB and C were most effective in knocking down endogenous Pyk2 and Src, respectively (Figure 10E, F and data not shown). PC12 cells were transduced with HIV-GFP-Pyk2ShB and C and HIV-GFP-Shscr, serum starved, stimulated with PMA, harvested, and lysed before 4G10 IP and IB for α₁1.2, Pyk2, Src, and tubulin. Total protein levels of α₁1.2, Pyk2, Src, and tubulin in lysate were monitored in parallel. The Pyk2-targeting HIV-GFP-Pyk2ShB and C but not the scrambled control shRNA abrogated the PMA-induced increase in α₁1.2 tyrosine phosphorylation (Figure 10G). Similarly, the Src-targeting HIV-GFP-SrcShB and C but not the scrambled control shRNA blocked the increase in α₁1.2 tyrosine phosphorylation upon PMA application (Figure 10G, H). For quantification, phosphotyrosine signals were normalized to total α₁1.2 in lysate (Figure 10I). None of the HIV viral constructs exhibited any detectable effects on protein expression of α₁1.2, Pyk2, Src, or α-tubulin, vinculin, and GAPDH as determined in lysates suggesting these constructs did not affect general protein expression. Collectively, the above findings indicate that knockdown effects were specific and not simply the consequence of viral infection or expression of non-specific stem-loop RNAs. Taken together, our findings strongly support the hypothesis that PKC signaling mediates its effects on Ca_V1.2 through Pyk2 and Src.

Inhibition of Pyk2 and Src blocks LTCC-dependent LTP

Ca_V1.2 is concentrated in dendritic spines (Hall et al., 2013; Hell et al., 1996; Leitch et al., 2009) where it mediates Ca²⁺ influx (Bloodgood and Sabatini, 2007; Hoogland and Saggau, 2004) and several forms of LTP (Grover and Teyler, 1990; Moosmang et al., 2005; Patriarchi et al., 2016; Qian et al., 2017; Tigaret et al., 2021). Notably, in older mice and rats, about half of the LTP (called LTP_LTCC) induced by four 200 Hz tetani, each 0.5 s long and 5 s apart, is insensitive to NMDAR blockade but abrogated by inhibition or elimination of Ca_V1.2 (Boric et al., 2008; Grover and Teyler, 1990; Moosmang et al., 2005; Shankar et al., 1998; Wang et al., 2016). Pharmacological inhibition and genetic disruption of Ca_V1.2 also abolish LTP induced by either pairing presynaptic stimulation with backpropagating action potentials (Magee and Johnston, 1997; Tigaret et al., 2021; Tigaret et al., 2016) or by 5 Hz/3 min tetani, the latter form of LTP requiring β₂AR signaling to upregulate Ca_V1.2 activity (Patriarchi et al., 2016; Qian et al., 2012; Qian et al., 2017). Thus, we hypothesized that upregulation of Ca_V1.2 activity by α₁AR signaling can augment LTCC-dependent forms of LTP.

LTP_LTCC is prominent in mice older than 1 year (30–40% above baseline) but small in mice younger than 3 months (10–15% above baseline) (Boric et al., 2008; Shankar et al., 1998). LTP_LTCC requires Ca_V1.2 activity (Moosmang et al., 2005) and stimulation of PKC signaling via type I metabotropic glutamate receptors (mGluR) (Wang et al., 2016). We tested whether increasing Ca_V1.2 activity through α₁AR–PKC–Pyk2–Src signaling can augment LTP_LTCC. Similar to previous reports (Boric et al., 2008; Shankar et al., 1998), LTP_LTCC was ~10% and was not statistically significant above baseline in our 13- to 20-week-old mice (Figure 11A). However, when Ca_V1.2 activity was upregulated by stimulation of α₁ARs with PHE, robust LTP_LTCC occurred (p ≤ 0.05, Figure 11A). This augmentation of LTP_LTCC was completely blocked by the LTCC inhibitor nimodipine and the α₁AR antagonist prazosin (both p ≤ 0.001, Figure 11A). Thus, this elevated potentiation strictly depends on both the activity of LTCCs and signaling through α₁ARs. Importantly, this LTP_LTCC is also blocked by the Pyk2 inhibitor PF-719 and the Src inhibitor PP2 (both p ≤ 0.001, Figure 11B). These data indicate that robust LTP_LTCC in 13- to 20-week-old mice requires Pyk2 and Src activity downstream of engaging α₁AR to boost LTCC activation to sufficient levels.

Figure 11.α<sub>1</sub>AR signaling augments LTP<sub>LTCC</sub> through L-type Ca<sup>2+</sup> channel (LTCC) activity, Pyk2, and Src.

LTP_LTCC was induced by four 200 Hz tetani, each 0.5 s long, in the CA3 Schaffer collateral projections to CA1 in acute hippocampal slices from 13- to 20-week-old mice. (A) LTP_LTCC required phenylephrine (PHE; 10 μM) and was prevented by the LTCC blocker nimodipine (10 μM; NIMO) and the α₁AR antagonist prazosin (1 μM; PRAZ). F_3,36 = 9.937. Control vs. PHE, p = 0.012; PHE vs. PHE/PRAZ, p = 0.0001; PHE vs. PHE/NIMO, p = 0.0003. (B) PHE-mediated long-term potentiation (LTP) is blocked by inhibitors of Pyk2 (1 μM PF-719) and Src (10 μM PP2). F_2,30 = 13.90. PHE vs. PHE/PF-719, p = 0.0002; PHE vs. PHE/PP2, p = 0.0003. Dot plots on the right show potentiation of field excitatory postsynaptic potentials (fEPSPs) determined as the averages of all responses between 45 and 50 min after high-frequency stimulation (HFS) as % of averages of all responses in the 5 min preceding HFS. Bars and whiskers represent means ± standard error of the mean (SEM; *p ≤ 0.05, ***p ≤ 0.001; one-way analysis of variance [ANOVA] with the Bonferroni correction). The number of slices and mice used is indicated.

Discussion

NE is arguably the most important neuromodulator for alertness and attention, augmenting multiple behavioral and cognitive functions (Berman and Dudai, 2001; Cahill et al., 1994; Carter et al., 2010; Hu et al., 2007; Minzenberg et al., 2008). The G_q-coupled α₁AR has a higher affinity for NE than βARs and has been implicated in many studies in attention and vigilance (Bari and Robbins, 2013; Berridge et al., 2012; Hahn and Stolerman, 2005; Hvoslef-Eide et al., 2015; Liu et al., 2009; Puumala et al., 1997; Robbins, 2002). Inspired by our earlier findings that Ca_V1.2 forms a unique signaling complex with β₂AR, G_s, AC, and PKA, making it a prominent effector of NE (Davare et al., 2001; Patriarchi et al., 2016; Qian et al., 2017), we tested and found that Ca_V1.2 is also a main target for NE signaling via the α₁AR. Given that Ca_V1.2 fulfills numerous functions in many cells this is a key and critical finding (Jacquemet et al., 2016; Splawski et al., 2004). In the following paragraphs, we discuss the four central and notable outcomes of our study.

Firstly, we found that stimulation of the α₁AR or the BK receptor strongly increased LTCC activity in neurons (Figures 1, 2,, 4—6). Stimulation of two other major classes of G_q-coupled receptors in neurons, mGluR1/5 and muscarinic M1/3/5 receptors affected LTCC activity at the cell soma only modestly or not at all, respectively (Figure 2—figure supplement 1). Accordingly, G_q-mediated signaling augments LTCC activity upon stimulation of defined but not all G_q-coupled receptors. Thus, activity of LTCCs is selectively regulated by α₁AR and BK receptor signaling. Perhaps G_q-coupled mGluR and muscarinic receptors are not as close to the LTCCs that were recorded in somata than the α₁AR or BK receptor, limiting their contribution to regulating Ca_V1.2. Defining what restricts the receptor type that can regulate LTCCs will be an interesting avenue of future investigation.

Remarkably, inhibitors of PKC, Pyk2, and Src reduce under nearly all conditions Ca_V1.2 baseline activity and also tyrosine phosphorylation of Ca_V1.2, Pyk2, and Src even when activators for α₁AR and PKC were present. Especially notable is the strong reduction of channel activity way below the control conditions by the Src inhibitor PP2 as well as the PKC inhibitor chelerythrine in Figure 2C. This effect is consistent with PP2 strongly reducing down below control conditions tyrosine phosphorylation of Src (Figure 8J), Pyk2 (Figure 8L), and Ca_V1.2 (Figure 9E) even with the PKC activator PMA present. These findings suggest that Pyk2 and Src experience significant although clearly by far not full activation under basal conditions as reflected by their own phosphorylation status, which translates into tyrosine phosphorylation of Ca_V1.2 under such basal conditions.

Secondly, we identified a complex PKC/Pyk2/Src cascade that mediates regulation of Ca_V1.2 by the α₁AR. Clear evidence for this signaling pathway is provided by the inhibition of PHE-induced upregulation of LTCC activity by inhibitors of PKC, Pyk2, and Src (Figure 2), which is further supported by the finding that direct stimulation of PKC also upregulates LTCC activity via Pyk2 and Src (Figure 3). The role of the PKC/Pyk2/Src pathway in regulating Ca_V1.2 is also substantiated by the association of Pyk2 in addition to Src and PKC with Ca_V1.2 (Figure 7) and inhibition of PKC-induced tyrosine phosphorylation of Ca_V1.2 by Pyk2 and Src inhibitors (Figure 9) and Pyk2 and Src knockdown (Figure 10). Multiple shRNAs specifically targeting both Pyk2 and Src efficiently prevented the PKC-mediated increase in α₁1.2 tyrosine phosphorylation. These observations not only confirm that Pyk2 mediates the Ca_V1.2 regulation downstream of PKC but also indicates that Src itself is in this context a relevant member of the Src kinase family.

Thirdly, we found that Pyk2 is firmly associated with Ca_V1.2 under basal conditions as reflected by their co-IP (Figure 7). This association places Pyk2 into a complex that also contains its immediate upstream activator and downstream effector, that is, PKC and Src. PKC can directly bind to the distal C-terminal region of α₁1.2, which also contain S1928 (Yang et al., 2005). Given that S1928 is a phosphorylation site for PKC (Yang et al., 2005), it is conceivable that PKC binding to this region reflects a temporary kinase–substrate interaction rather than a more permanent association of PKC with Ca_V1.2, although this consideration does not rule out that PKC can stably bind to another region in the C-terminus of α₁1.2. In addition, the A kinase anchor protein AKAP150, which is a major interaction partner for Ca_V1.2 (Davare et al., 1999; Hall et al., 2007; Oliveria et al., 2007), binds not only PKA but also PKC (Klauck et al., 1996) and constitutes another potentially constitutive link between PKC and Ca_V1.2 (Navedo et al., 2008). Furthermore, like Pyk2, Src co-precipitates with Ca_V1.2 (Figure 7) and binds directly to α₁1.2 (Bence-Hanulec et al., 2000; Chao et al., 2011; Hu et al., 1998). Our determination that Pyk2 co-precipitates with Ca_V1.2 from not only brain but also heart indicates that Ca_V1.2 forms a signaling complex with Pyk2 and Src and possibly also PKC in various tissues.

We identified the loop between domains II and III of α₁1.2 as the binding site for Pyk2. This observation lends further support to the association of Pyk2 with Ca_V1.2. Of note, Src binds to residues 1955–1973 in rat brain α₁1.2 (corresponding to residues 1982–2000 in the original rabbit cardiac α₁1.2 Mikami et al., 1989; Bence-Hanulec et al., 2000; Chao et al., 2011). This interaction with α₁1.2 might bring Src in close proximity to loop II/III-associated Pyk2 to augment their structural and functional interaction once Pyk2 has been activated by PKC.

Formation of supramolecular signaling complexes or ‘signalosomes’ consisting of kinases and their ‘customers’ ensures fast, efficient, and specific signaling (Dai et al., 2009; Dodge-Kafka et al., 2006). Our work establishes the PKC–Pyk2–Src–Ca_V1.2 complex as such a signalosome. Furthermore, it defines how various G_q-coupled receptors stimulate the activity of Ca_V1.2 in different cells. The remarkably strong upregulation of Ca_V1.2 channel activity by Src (Bence-Hanulec et al., 2000; Gui et al., 2006) and upon activation of the PKC–Pyk2–Src signaling cascade as shown here rivals the upregulation by β-adrenergic signaling, which is a central and thus widely studied mechanism of regulating Ca²⁺ influx into cardiomyocytes during the fight or flight response (Balijepalli et al., 2006; Bean et al., 1984; Fu et al., 2013; Fu et al., 2014; Fuller et al., 2010; Lemke et al., 2008; Liu et al., 2020; Reuter, 1983). Of note, Ca_V1.2 assembles all components required for β-adrenergic signaling including the β₂AR, G_s, adenylyl cyclase, and PKA in brain (Davare et al., 2001; Davare et al., 1999; Man et al., 2020) and heart (Balijepalli et al., 2006). Formation of this complex is important for upregulation of Ca_V1.2 activity (Balijepalli et al., 2006; Patriarchi et al., 2016) and LTP of glutamatergic synapses induced by a 5-Hz theta rhythm during β-adrenergic stimulation (Patriarchi et al., 2016; Qian et al., 2017). Analogously, assembly of the PKC–Pyk2–Src–Ca_V1.2 signalosome may be important for fast and specific regulation of Ca_V1.2 by the α₁AR. This hypothesis can now be tested by pursuing determination of the precise binding site of Pyk2 in the loop between domains II and III of α₁1.2 and then disrupting this interaction with peptides and point mutations. However, our initial attempts to narrow down the binding region by binding studies with six synthetic ~25 residue long overlapping peptides that spanned loop II/III failed (data not shown). Perhaps the Pyk2-binding site in loop II/III requires tertiary structural elements or sequences that were distributed between two neighboring peptides (which overlapped by five residues).

In rat brain neurons, LTCC activity can be increased by Src via phosphorylation of α₁1.2 on Y2122 (Bence-Hanulec et al., 2000; Gui et al., 2006). However, this phosphorylation site is not conserved even within rodents. It is equivalent to position 2150 in rabbit cardiac α₁1.2, which is a Cys and not Tyr residue (Mikami et al., 1989). Accordingly, other Tyr residues must serve as phosphorylation sites. It will be an interesting challenge for future work to identify the exact phosphorylation site and then test its functional relevance.

Remarkably, the Src inhibitor PP2 also completely blocked PKC-induced autophosphorylation of Pyk2 on Y402 (Figure 8K, L). This finding indicates a close interdependence between Pyk2 and Src activation by PKC in PC12 cells (depicted in Figure 8A). It is consistent with earlier results indicating that Pyk2 activation (assessed by Y402 phosphorylation) requires catalytically active Src (Cheng et al., 2002; Shi and Kehrl, 2004; Sorokin et al., 2001; Zhao et al., 2016), although in other systems Y402 phosphorylation was not dependent on Src (Corvol et al., 2005; Park et al., 2004; Yang et al., 2013). Accordingly, Pyk2 autophosphorylation on Y402 and the consequent binding of Src to phosphoY402 induces Src-mediated phosphorylation of Pyk2 on Y579 or Y580 in its activation loop, which further enhances Pyk2 activity beyond the level achieved by Pyk2 autophosphorylation on Y402 (Dikic et al., 1996; Lakkakorpi et al., 2003; Li et al., 1999; Park et al., 2004). Such interdependence was supported by the observation that Y579 phosphorylation upon PKC stimulation by either PMA or BK was also completely blocked by the Src inhibitor PP2 (Figure 8K, M).

Fourthly and finally, LTP_LTCC induced by 200 Hz tetani in 13- to 20-week-old mice required stimulation of α₁ARs and is completely blocked by inhibitors of LTCC, Pyk2, and Src (Figure 11). Incidentally, we were not able to induce any LTP_LTCC in mice younger than 13 weeks. Taken together, our findings suggest that LTP_LTCC requires stimulation of Ca_V1.2 activity by α₁AR–PKC–Pyk2–Src signaling. While we focus here on the importance of α₁AR signaling for LTP_LTCC, this is not the only form of LTP that requires upregulation of Ca²⁺ influx through Ca_V1.2. Prolonged theta tetanus LTP (PTT-LTP), which is induced by a 3-min-long 5 Hz tetanus, also depends on upregulation of Ca_V1.2 activity (Boric et al., 2008; Cavuş and Teyler, 1996; Grover and Teyler, 1990; Moosmang et al., 2005; Wang et al., 2016). In PTT-LTP, this upregulation is accomplished by β₂AR–G_s–adenylyl cyclase/cAMP–PKA signaling and the ensuing phosphorylation of the central pore-forming α₁1.2 subunit of Ca_V1.2 on S1928 by PKA (Patriarchi et al., 2016; Qian et al., 2012; Qian et al., 2017). Whether signaling by NE through α₁AR and β₂AR can act in parallel and is additive will be an interesting question for future studies. However, we already know that at least for classic PTT-LTP β₂AR signaling is sufficient and does not require engagement of α₁AR signaling (Qian et al., 2012). Because regulation of Ca_V1.2 by β₂AR signaling is highly localized (Davare et al., 2001; Patriarchi et al., 2016), it is conceivable that α₁AR signaling might engage a subpopulation of Ca_V1.2 channels whose spatial distribution differs from that of β₂AR-stimulated Ca_V1.2 in dendrites. Alternatively, parallel engagement of α₁AR and β₂AR signaling might ensure more robust and possibly additive or synergistic responses both at the Ca_V1.2 channel level and in the synaptic potentiation that results.

LTP is thought to underlie learning and memory (Choi et al., 2018; Whitlock et al., 2006). Conditional knock out of Ca_V1.2 in the hippocampus and forebrain impaired LTP_LTCC as well as initial learning (Moosmang et al., 2005) and long-term memory of spatial Morris water maze tasks (White et al., 2008). Moreover, decreased Ca_V1.2 expression or infusion of LTCC blockers into the hippocampus impaired both, LTP induced by pairing backpropagating action potentials in dendrites with synaptic stimulation and latent inhibition (LI) of contextual fear conditioning, the latter requiring learning to ignore non-relevant environmental stimuli (Tigaret et al., 2021). These Ca_V1.2-related learning deficits might be in part due to impaired attention the animals pay to their experimental environment during learning phases, processes requiring concerted attention (Panichello and Buschman, 2021). Attention, in turn, depends on the neurotransmitter NE, which might augment spatial learning through regulation of Ca_V1.2 via α₁AR–PKC–Pyk2–Src signaling.

Ca_V1.2 is increasingly implicated in not just the postsynaptic physiological functions discussed above. Multiple genome-wide association studies point to variants in the Ca_V1.2 gene, CACNA1C, as major risk factors for schizophrenia, bipolar disorder, and other mental diseases (Bhat et al., 2012; Ferreira et al., 2008; Green et al., 2010; Nyegaard et al., 2010; Smoller, 2013; Splawski et al., 2004). Other studies link chronic upregulation of Ca_V1.2 activity to etiologies behind senility and Alzheimer’s disease (e.g., Davare and Hell, 2003; Deyo et al., 1989; Disterhoft et al., 1994; Thibault and Landfield, 1996). Thus, it appears likely that dysfunctional regulation of Ca_V1.2 contributes to these diseases, making the detailed molecular analyses of the signaling paradigms that regulate its functionality especially important to advance our mechanistic understanding for development of future therapies.

Here, we establish for the first time that NE upregulates Ca_V1.2 activity via a complex α₁AR signaling cascade through PKC, Pyk2, and Src, the activity of each component being essential for LTP_LTCC and thus most likely relevant for learning. Our work forms the foundation for future studies to uncover the physiological context in which this action of NE is specifically engaged, what the precise role for each kinase is in this signaling cascade regulating Ca_V1.2 activity, and how the individual kinases could be coordinately regulated to further fine-tune Ca_V1.2 function. Given the central role of NE in attention and the many physiological and pathological aspects of Ca_V1.2, regulation of this channel via NE–α₁AR signaling predictably will elicit widespread and profound functional effects.

Materials and methodsMaterials availability statement

Further information and requests for resources and reagents should be directed to and will be fulfilled by the corresponding authors, Mary C. Horne (mhorne@ucdavis.edu) and Johannes W. Hell (jwhell@ucdavis.edu).

Experimental model and subject detailsAnimals

Pregnant Sprague-Dawley (SD) rats were ordered from Envigo (Order code 002) or Charles River (Strain code 001) and E18 embryos were used for preparation of dissociated hippocampal neuronal cultures. SD rats used for preparation of tissue extracts from heart and brain were of either sex and around 3 months old. For LTP experiments, mice of the strain B6129SF1/J aged between 13 and 18 weeks (both males and females) were used.

Animals were maintained with a 12/12 hr light/dark cycle and were allowed to access food and water ad libitum. All procedures followed NIH guidelines and had been approved by the Institutional Animal Care and Use Committee (IACUC) at UC Davis (Protocol # 20673 and 22403).

Primary hippocampal neuronal cultures

Primary hippocampal neurons were maintained at 37°C in humidified incubators under 5% CO₂ and 95% air. Both male and female rat embryos were used to prepare the cultures. Neurons were maintained in a medium containing 1× B-27 supplement (Gibco Cat#17504044), 1× Glutamax (Gibco Cat#35050061), 5% fetal bovine serum (FBS, Corning Cat#35-010-CV), and 1 µg/ml gentamicin (Gibco Cat#15710-064) in Neurobasal medium (Gibco Cat#21103-049). 10 µM each of 5-fluoro-2′-deoxyuridine (Sigma-Aldrich Cat#F0503) and uridine (Sigma-Aldrich Cat#U3003) were added around DIV7 to block the growth of glial cells.

Cell lines

All cells were grown at 37°C in humidified incubators under 5% CO₂ and 95% air. Rat pheochromocytoma cell line PC12 (ATCC Cat# CRL-1721; RRID:CVCL_0481, male) was grown in RPMI 1640 media (Gibco Cat#11875-101) containing 10% horse serum (HS, Gemini Bio Products Cat#100-508) and 5% FBS. For serum starvation, PC12 were incubated for 18 hr in RPMI 1640 media containing 1% HS and 0.5% FBS. HT-1080 cells (ATCC CCL-121; RRID:CVCL_0317, male) used for virus titration were grown and maintained in MEM (Gibco Cat#11095-080) supplemented with 10% FBS. HEK293T/17 (ATCC Cat# CRL-11268, RRID:CVCL_1926, female) cells were routinely cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS (Gibco Cat#11995-065). Cell lines used were obtained from ATCC, expanded and frozen at low passage number. Care was taken during the use of cell lines to ensure that only one cell line was processed in the culture hood at a time, and that they were used within 25–30 passages. Their morphology in culture and doubling time were routinely monitored as were other distinguishing properties such as high transfectability (HEK293T/17 cells) or high Ca_V1.2 expression (PC12 cells) before the time of experimental use.

Authentication of cell lines

All three cell lines HEK293T/17, HT1080,and PC12 cells were obtained from ATCC, a well-established and highly reliable source for cell lines. These are the only cell lines currently used in our lab, minimizing further any potential for confusion.

HEK293T/17 was only used for production of virus for knockdown of Pyk2 and Src and initial testing for efficacy of respective knockdown and not for data collection. It was mostly authenticated by inspection of shape and determination of viability as well as the absence of voltage-gated ion channels including Ca_V1.2 as tested electrophysiologically (all hallmarks of endothelial cells like HEK293 cells). All viruses produced with this cell line were of the expected titer and infectivity as tested. Further, the knockdown results for ectopically expressed Pyk2 and Src in these HEK293T cells were consistent with subsequent knockdown of endogenous Pyk2 and Src in our PC12 cells by several viruses with respective shRNA (Figure 9 and data not shown).

HT1080 was exclusively used for testing viral titer and mostly authenticated by inspection of shape and determination of viability.

PC12 cells are derived from a pheochromocytoma tumor and were verified in different ways. Firstly, they had the typical appearance described earlier, with some ‘rugged’ edges under basal conditions. Upon addition of nerve growth factor (NGF) they adopted a more neuron-like appearance with elongated protrusions reminiscent of short neurites. This response to NGF is a clear hallmark of PC12 cells and the reason why they are popular for use in biochemical experiments when neuron-like cultured cells are needed. Additional parameters were expression of L-type Ca channels as determined electrophysiologically (data not shown) and biochemically specifically for Ca_V1.2 as thoroughly analyzed in this study (Figures 8—10). In addition, expression of the BK receptor and its downstream effectors Pyk2 and Src is known to be very prominent in PC12 cells. (Pyk2 was first identified in PC12 cells) and again regularly observed in our thorough biochemical analysis.

Test for mycoplasma was performed per commercial PCR and was negative.

Methods detailsCulture of primary hippocampal neurons

Hippocampal neurons were cultured from wild-type E18 male and female embryos from SD rats. Hippocampus was excised from the brains of embryos in ice-cold Hank’s Buffer (Sigma-Aldrich Cat#H2387) with 10 mM 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Gibco Cat#15630-080), 0.35 g/l NaHCO₃ and 5 µg/ml gentamicin (Gibco Cat#15710-064) and digested in 0.78 mg/ml papain (Roche Cat#10108014001) in 5 ml of the same buffer at 37°C for 30 min, in an incubator containing 5% CO₂ and 95% air. Digested hippocampal tissue was washed with neuron medium twice, and triturated in the medium. The medium used for washes, trituration and culture of neurons consists of 1× B-27 supplement, 1× Glutamax, 5% FBS, and 1 µg/ml gentamicin in Neurobasal medium (as stated in Experimental model and subject details). 15,000 neurons were plated per well in 24-well plates on coverslips coated with poly-DL-ornithine (Sigma-Aldrich Cat#P0671) and laminin (Corning Cat#354232) and cultured in an incubator at 37°C and 5% CO₂ and 95% air.

Single-channel recording – determination of overall channel activity (N × Po)

Single-channel recording was performed at room temperature on hippocampal neurons on DIV10–15 using the cell-attached configuration at an Olympus IX50 inverted microscope as before (Patriarchi et al., 2016; Qian et al., 2017). The membrane potential was fixed at ~0 mV using a high K⁺ external solution. The external (bath) solution contained (in mM) 145 KCl, 10 NaCl, 10 HEPES, and 30 D-glucose (pH 7.4 with NaOH, 325–330 mOsM). The internal (pipette) solution contained 110 mM BaCl₂, 20 mM tetraethylammonium chloride (TEA-Cl), 10 mM HEPES, 500 nM BayK 8644 (Tocris Cat#1546; 200 nM used for Figures 1 and 2; 500 nM used for Figure 3), and 1 µM each of ω-conotoxins MVIIC and GVIA (China Peptides, custom synthesized) (pH 7.2 with TEA-OH, 325–330 mOsM). 3.5–5.5 MΩ resistance pipettes were used. The concentrations of kinase inhibitors, α₁AR receptor blocker and agonist are as indicated in the figure legends. Neurons were preincubated with kinase inhibitors or receptor blocker in culture medium for 10 min prior to the experiment, and kinase inhibitors and receptor blocker were, where relevant, present in the external solution during recording. In experiments where isradipine was used, it was placed in the pipette solution, and no pre-incubation with isradipine was performed. Recordings started within 10 min of placing coverslips in the recording chamber in a bath solution with or without PHE (Sigma-Aldrich Cat#P6126), PMA (Merck Millipore Cat#524400), and different kinase inhibitors and receptor blocker. Currents were sampled at 100 kHz and low-pass filtered at 2 kHz using an Axopatch 200B amplifier (Axon Instruments) and digitized using Digidata 1440A digitizer (Axon Instruments). Step depolarizations of 2-s duration (one sweep) were elicited to the patch from −80 to 0 mV at a start-to-start stimulation interval of 7 s. Typically, 100 sweeps were recorded per neuron and only cells with more than 70 sweeps recorded were analyzed. The single-channel search event detection algorithm of Clampfit 10.7.0.3 (Axon Instruments) was used to analyze single-channel activities. Ensemble average traces were computed by averaging all sweeps from one neuron and averaging the averaged traces from all neurons in each group.

Single-channel recording – determination of Po

To specifically determine unitary channel open probability Po, borosilicate pipettes with a resistance of 7–12 MΩ were used. Only patches with no more than 4 channels (k ≤ 4) were included in the Po analysis to not overinterpret Po (Horn, 1991). Data were corrected by the number of channels (k = 1) as previously described (Bartels et al., 2018; Turner et al., 2020). Unitary LTCC events from hippocampal neurons (DIV15–25) where isolated through blocking N/P/Q-type calcium channels by using 1 µM each of ω-conotoxins MVIIC and GVIA (China Peptides, custom synthesized) in the patch pipette and recorded as above at room temperature by step depolarizations from −80 to 0 mV. Extracellular bath solution contained 125 mM K-glutamate, 25 mM KCl, 2 mM MgCl₂, 1 mM CaCl₂, 1 mM ethylene glycol bis(2-aminoethyl)tetraacetic acid (EGTA), 10 mM HEPES, 10 mM glucose, and 1 mM Na-ATP, pH 7.4 with KOH. Depolarizing pipette solution contained 110 mM BaCl₂ and 10 mM HEPES, adjusted to a pH of 7.4 with TEA-OH. Data acquisition was performed at a sampling frequency of 10 kHz with an interpulse time of 5 s and data were low pass filtered at 2 kHz. The positive identification of LTCC activity was consequently tested by bolus application of either the dihydropyridine (DHP) BayK8644 (10 µM), which promotes L-type channel opening, or the channel-blocking DHPs isradipine (10 µM) or nimodipine (10 µM) to the bath solution at the end of each experimental run.

Drugs

were prepared as stock solution in dH₂O, freshly on the day of experiment respectively, 40 mM prazosin–HCl (Sigma-Aldrich), 20 mM NE bitartrate (Sigma-Aldrich) and 20 mM BK acetate (Sigma-Aldrich). Stocks were diluted again 1:10 or 1:100 and as a bolus directly applied to the bath solution during the recordings.

Co-immunoprecipitation of Ca<sub>V</sub>1.2 with Pyk2 and Src

Brains and hearts were homogenized with a Potter tissue homogenizer in 10 ml of a homogenization buffer containing 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 5 mM EGTA pH 7.4, 10 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100, 25 mM NaF, 25 mM sodium pyrophosphate, 1 mM 4-nitrophenyl phosphate, 2 μM microcystin and protease inhibitors (1 μg/ml leupeptin [Millipore Cat#108975], 2 μg/ml aprotinin [Millipore Cat#616370], 10 μg/ml pepstatin A [Millipore Cat#516481] and 200 nM phenylmethylsulfonyl fluoride [PMSF]). High-speed centrifugation was performed at 40,000 rpm for 30 min at 4°C. 500 μg of total brain or heart lysate extracts were incubated with 15 μl of Protein-A Sepharose beads (CaptivA protein resin, Repligen, Cat#CA-PRI-0100) and 2 μg of anti-Ca_V1.2 α1-subunit or control rabbit IgG antibody. Samples were incubated at 4°C for 4 hr before being washed three times with ice-cold wash buffer (0.1% Triton X-100 in 150 mM NaCl, 10 mM EDTA, 10 mM EGTA, 10 mM Tris, pH 7.4). Samples were then resolved by SDS–PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes before IB with anti-Ca_V1.2 α1-subunit (J.W. Hell lab), -Pyk2 (Millipore Cat#05-488; RRID:AB_2174219), and -Src (J.S. Brugge lab) antibodies. The antibody dilutions used are listed in Supplementary file 2.

GST pulldown assay

Fragments of intracellular loops of Ca_V1.2 α₁-subunit (Supplementary file 1; Davare et al., 2000; Gao et al., 2001) were expressed in Escherichia coli strain BL21 as GST fusion proteins, purified, and integrity verified by IB essentially as previously described (Bennin et al., 2002; Frangioni and Neel, 1993; Hall et al., 2013; Hall et al., 2006). Overnight cultures from single colonies of the corresponding plasmids were cultured initially in 50 ml of LB medium containing ampicillin (100 μg/ml) with aeration until saturation. Incubation temperature was optimized for each expression construct to optimize translation and stability and varied between 28 and 37°C. After a 1:10 dilution into the same medium, cultures were grown for about 2–4 hr until an A600 of about 0.8 was reached when isopropyl-β-D-thiogalactopyranoside was added for induction. After 4–5 hr bacteria were collected by centrifugation (5000 rpm, SLA 3000 rotor, Thermo Fisher Cat#07149) for 15 min and resuspended by gentle trituration in ice-cold 50 ml of Tris-buffered saline (TBS) Buffer (150 mM NaCl, 15 mM Tris-Cl, pH 7.4) containing protease inhibitors 1 μg/ml pepstatin A, 1 μg/ml leupeptin, 1 μg/ml aprotinin, and 200 nM PMSF. 0.1 mg/ml lysozyme was added to lyse cell walls. The mixture was kept on ice for 30 min before addition of Sarcosyl (1.5% final concentration), β-mercaptoethanol (10 mM), and DNAse (50 U) for 15 min to fully solubilize the fusion proteins. In order to neutralize Sarcosyl, Triton X-100 was then added to a final concentration of 5%. Insoluble material was removed by ultracentrifugation (1 hr, 4°C, 40,000 rpm, Ti70 rotor, Beckman Coulter Cat#337922). The fragments were immobilized onto glutathione Sepharose (Millipore/Cytiva Cat#17-5132-02) for 3 hr, washed three times with Buffer A (0.1% TX-100, 10 mM Tris–HCl, pH. 7.4) and incubated with affinity-purified His-tagged Pyk2 separately expressed in E. coli (3 hr, 4°C). Beads were washed three times in Buffer A and bound proteins were eluted and denatured in SDS sample buffer, resolved by SDS–PAGE and transferred to a nitrocellulose membrane. IB with anti-Pyk2 antibody (Millipore Cat#05-488; RRID:AB_2174219) was used to detect Pyk2 binding during pulldown.

Analysis of phosphorylation in PC12 cells

Drugs were used at the following concentrations: 2 µM PMA (Merck Millipore Cat#524400), 1 µM BK (Sigma-Aldrich Cat#05-23-0500), 3 µM PF-431396 (Tocris Cat#4278), 10 µM PP2 (Sigma-Aldrich Cat#P0042), 10 µM PP3 (Tocris Cat#2794), and 10 µM SU6656 (Sigma-Aldrich Cat#S9692).

For phospho-tyrosine analysis PC12 cells were washed after drug treatment twice in ice-cold phosphate-buffered saline (PBS) containing 1 mM pervanadate and 25 mM NaF. Samples were collected in PBS containing pervanadate, NaF, and protease inhibitors (see above), sonicated and extracted with SDS dissociation buffer (50 mM Tris–HCl, 1% SDS) at 65°C for 10 min. The SDS was neutralized with a fivefold excess of Buffer A containing phosphatase and protease inhibitors. 500 μg of total protein from PC12 cell extracts were incubated over night at 4°C with 2 μg of the phospho-tyrosine 4G10 (Sigma-Alrich/Upstate Cat# 05-321; RRID:AB_2891016) or mouse control antibody (Jackson Immunoresearch Cat#015-000-003) and 15 μl of Protein-G Sepharose (Millipore/Cytiva Cat#GE-17-0618-05), washed three times in ice-cold wash buffer (0.1% Triton X-100 in 150 mM NaCl, 10 mM EDTA, 10 mM EGTA, 10 mM Tris, pH 7.4), resolved by SDS–PAGE and transferred onto PVDF membranes for IB. The antibody dilutions used are listed in Supplementary file 2. For re-probing, blots were stripped in 62.5 mM Tris-Cl, 20 mM dithiothreitol (DTT), and 2% SDS at 50°C for 30 min. Chemiluminescence immunosignals were quantified using ImageJ (Rueden et al., 2017) by multiple film exposures of increasing length to ensure signals were in the linear range (Davare and Hell, 2003; Hall et al., 2006). Variations in total amounts of α₁1.2, Pyk2, and Src in the different PC12 cell lysates were monitored by direct IB of lysate aliquots. Lysate signals were used to correct α₁1.2 signals after 4G10 IP for such variations by dividing the latter by the former.

Lentiviral constructs for shRNA to Pyk2 and Src

A list of all shRNA target sequences is provided in Supplementary file 3. The shRNA sequence Sh1 against Pyk2 has been validated for Pyk2 knockdown (Sayas et al., 2006). The Sh1 sequence was cloned in the reverse orientation into the MfeI site of the lentiviral transfer vector pVETL-eGFP (Bartos et al., 2010; Boudreau and Davidson, 2012; Harper et al., 2006) for expression of Pyk2 shRNA and GFP to visualize infection. All HIV plasmids (HIV-GFP-Pyk2shA-D; HIV-GFP-SrcshA-D) for knocking down rat Pyk2 or Src as well as the scrambled, non-silencing hairpin control (HIV-GFP-shscr) were obtained from Origene (Cat# TL710108 and #TL711639). All expression plasmids (listed in Supplementary file 4) were confirmed by DNA sequencing.

Production of lentivirus for Pyk2 and Src knockdown

HEK293T/17 (ATCC Cat# CRL-11268, RRID:CVCL_1926) cells were plated onto 10 cm dishes at 1.8 × 10⁶ cells per dish and maintained until confluency (60–90%). Cells were transiently transfected with viral expression constructs using the calcium phosphate precipitation method (Jordan et al., 1996). For FIV virus production, cells were transfected in a 3:2:1 ratio of parental vector (pVETL, FIV 3.2): pCPRD-Env: pCI-VSVG for a total of 24 μg of DNA per plate. For production of HIV viral particles targeting Src and Pyk2, cells were transfected at a ratio of 5:2:2:2 of parental vector (e.g., pGFP-Pyk2-shC-Lenti:pCI-VSVG:pMDL g/p RRE:pRSV-REV) according to the manufacturer’s guidelines (OriGene). Media was exchanged 16 hr after transfection. Media containing the packaged recombinant virus was collected at 48 and 72 hr, filtered through 0.45 μm filters, and concentrated by centrifugation (7400 × g for 16 hr at 4°C). The viral pellet was resuspended in ice-cold PBS, aliquoted and stored at −80 °C. Before use for transduction of PC12 cells all viral particle solutions were titered in the HT-1080 cell line (ATCC Cat#CCL-121; RRID:CVCL 0317) by seeding 12-well plates at 5 × 10⁴ cells per well 1 day before infection. 1, 5, and 10 μl of solutions containing concentrated HIV or FIV particles was added to each well and expression of GFP was monitored for 72 hr post-infection before titer was calculated.

Slice preparation and electrophysiology

After decapitation, brains were removed from 13- to 18-week-old mice. 400-μm-thick transverse slices were made using a vibratome in cold, oxygenated (95% O₂ and 5% CO₂) dissection buffer (in mM: 87 NaCl, 2.5 KCl, 1.25 NaH₂PO₄, 26.2 NaHCO₃, 25 glucose, 0.5 CaCl₂, 7 MgCl₂, 50 sucrose). Slices were allowed to recover at room temperature for at least 1 hr in oxygenated artificial cerebrospinal fluid (ACSF) (in mM: 119 NaCl, 3 KCl, 2.5 CaCl₂, 1.25 NaH₂PO₄, 1.3 MgSO₄, 26 NaHCO₃, 11 glucose). Following recovery, slices were transferred to a recording chamber and maintained at 32–33°C in oxygenated ACSF. Field excitatory postsynaptic potential (fEPSP) was evoked by stimulating the Schaffer collateral pathway using bipolar electrode, and synaptic responses were recorded with ACSF-filled microelectrodes (1–10 MΩ) placed in the stratum radiatum of CA1 region. Recordings were acquired using an Axoclamp-2B amplifier (Axon Instruments) and a Digidata 1332 A digitizer (Axon Instruments). Baseline responses were collected at 0.07 Hz with a stimulation intensity that yielded 40–50% of maximal response. LTP was induced by four episodes of 200 Hz stimulation (0.5 s) with 5-s intervals. To measure LTCC-mediated LTP, 50 μM D-APV (Tocris Cat#0106) was included in ACSF. When used the inhibitors [10 μM nimodipine (Bayer Charge: BXR4H3P), 1 μM prazosin, 1 μM PF-719 (Tse et al., 2012), and 10 μM PP2] were added to ACSF from the start of the recording. PHE (10 μM) was added after at least 15 min of stable baseline, and LTP was induced ~10 min after the addition of PHE.

Quantification and statistical analysis

Statistical analyses were performed using Prism 5 or 9 (GraphPad). Data are presented as mean ± standard error of the mean. Sample sizes, p values, and statistical tests are indicated in the figure legends. For analysis of channel NPo (Figures 1—3, Figure 2—figure supplement 1, Figure 3—figure supplement 1), first outliers were identified using iterative Grubb’s method inbuilt in Prism. Then, statistical significance was determined using one-way analysis of variance (ANOVA) with post hoc Holm–Sidak’s multiple comparisons test. For analysis of specifically Po (Figures 4—6), data were tested either by an unpaired or paired Student’s t-test. For significance testing with more than two groups, a one-way ANOVA with additional Bonferroni correction was applied, p < 0.05%. For analysis of protein phosphorylation, statistical significance was determined using ANOVA with post hoc Bonferroni’s multiple comparisons test (Figures 8—10). For analysis of LTP (Figure 11), a one-way ANOVA was applied followed by Bonferroni correction.

Additional information

Competing interests

No competing interests declared

Author contributions

Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing – review and editing

Data curation, Formal analysis, Validation, Visualization, Methodology, Writing – review and editing

Resources, Data curation, Formal analysis, Funding acquisition, Validation, Investigation, Methodology

Data curation, Formal analysis, Validation, Visualization, Methodology

Data curation, Formal analysis, Validation, Investigation, Methodology

Data curation, Formal analysis, Investigation, Methodology

Data curation, Methodology

Data curation, Methodology, Writing – review and editing

Data curation, Funding acquisition, Methodology, Writing – review and editing

Conceptualization, Resources, Data curation, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing – review and editing

Conceptualization, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing – review and editing

Ethics

All procedures followed NIH guidelines and had been approved by the Institutional Animal Care and Use Committees (IACUC) at UC Davis (Protocol #20673 and #22403).

Additional files

Supplementary file 1.Amino acid residues of fragments of intracellular loops of Ca<sub>V</sub>1.2 α<sub>1</sub>-subunit used in GST pulldown studies.

Supplementary file 2.Antibody dilutions used for immunoblotting.

Supplementary file 3.shRNA sequences targeting rat Pyk2 and Src.

Supplementary file 4.Sequencing primers for validation of knockdown constructs.

MDAR checklist

Data availability

Raw datasets are available on Dryad (https://doi.org/10.25338/B86G9K).

The following dataset was generated:

ManKBartelsPHendersonPBKimKShiMZhangMHoSNieves-CintronMNavedoMFHorneMCHellJW2023Raw data for Manuscript entitled "Alpha-1 adrenergic receptor - PKC - Pyk2 - Src signaling boosts L-type Ca2+ channel Cav1.2 activity and long-term potentiation in rodents"Dryad Digital Repository10.25338/B86G9K

Acknowledgements

We thank Dr. Stephen Strittmatter (Yale University) for providing PF-719. Dr. K Man executed the electrophysiological recordings shown in Figures 1—3; Dr. P Bartels executed the electrophysiological recordings shown in Figures 4—6; Drs. Mei Shi and Mingxu Zhang performed the biochemical analysis in Figure 7; Dr. Peter Henderson performed the biochemical analysis in Figures 8—10; Dr. Karam Kim performed the LTP measurements in Figure 11. FUNDING This work was supported by National Institutes of Health grant R01 NS123050 (JWH), National Institutes of Health grant RF1 AG055357 (JWH), National Institutes of Health grant R01 MH097887 (JWH), National Institutes of Health grant R01 HL098200 (MFN), National Institutes of Health grant R01 HL121059 (MFN), and National Institutes of Health grant T32 GM099608 (PBH).

References

Avraham

H

Park

SY

Schinkmann

K

Avraham

S

2000

RAFTK/Pyk2-mediated cellular signalling

Cellular Signalling12123133

10.1016/s0898-6568(99)00076-5

10704819

Balijepalli

RC

Foell

JD

Hall

DD

Hell

JW

Kamp

TJ

2006

From the cover: localization of cardiac L-type Ca2+ channels to a caveolar macromolecular signaling complex is required for Beta2-adrenergic regulation

PNAS10375007505

10.1073/pnas.0503465103

16648270

Bari

A

Robbins

TW

2013

Noradrenergic versus dopaminergic modulation of Impulsivity, attention and monitoring behaviour in rats performing the stop-signal task: possible relevance to ADHD

Psychopharmacology23089111

10.1007/s00213-013-3141-6

23681165

Bartels

P

Yu

D

Huang

H

Hu

Z

Herzig

S

Soong

TW

2018

Alternative splicing at N terminus and domain I modulates Ca(V)1.2 inactivation and surface expression

Biophysical Journal115

163

10.1016/j.bpj.2018.06.001

29972807

Bartos

JA

Ulrich

JD

Li

H

Beazely

MA

Chen

Y

Macdonald

JF

Hell

JW

2010

Postsynaptic clustering and activation of Pyk2 by PSD-95

The Journal of Neuroscience30449463

10.1523/JNEUROSCI.4992-08.2010

20071509

Bean

BP

Nowycky

MC

Tsien

RW

1984

Beta-adrenergic modulation of calcium channels in frog ventricular heart cells

Nature307371375

10.1038/307371a0

6320002

Bence-Hanulec

KK

Marshall

J

Blair

LA

2000

Potentiation of neuronal L calcium channels by IGF-1 requires phosphorylation of the Alpha1 subunit on a specific tyrosine residue

Neuron27121131

10.1016/s0896-6273(00)00014-3

10939336

Bennin

DA

Don

ASA

Brake

T

McKenzie

JL

Rosenbaum

H

Ortiz

L

DePaoli-Roach

AA

Horne

MC

2002

Cyclin G2 Associates with protein phosphatase 2A catalytic and regulatory B' subunits in active complexes and induces nuclear aberrations and a G1/S phase cell cycle arrest

The Journal of Biological Chemistry2772744927467

10.1074/jbc.M111693200

11956189

Berkefeld

H

Sailer

CA

Bildl

W

Rohde

V

Thumfart

JO

Eble

S

Klugbauer

N

Reisinger

E

Bischofberger

J

Oliver

D

Knaus

HG

Schulte

U

Fakler

B

2006

BKca-Cav channel complexes mediate rapid and localized Ca2+-activated K+ signaling

Science314615620

10.1126/science.1132915

17068255

Berman

DE

Dudai

Y

2001

Memory extinction, learning anew, and learning the new: Dissociations in the molecular machinery of learning in cortex

Science29124172419

10.1126/science.1058165

11264539

Berridge

CW

Shumsky

JS

Andrzejewski

ME

McGaughy

JA

Spencer

RC

Devilbiss

DM

Waterhouse

BD

2012

Differential sensitivity to psychostimulants across prefrontal cognitive tasks: Differential involvement of noradrenergic α1- and α2-receptors

Biological Psychiatry71467473

10.1016/j.biopsych.2011.07.022

Bhat

S

Dao

DT

Terrillion

CE

Arad

M

Smith

RJ

Soldatov

NM

Gould

TD

2012

Cacna1C (Cav1.2) in the pathophysiology of psychiatric disease

Progress in Neurobiology99114

10.1016/j.pneurobio.2012.06.001

22705413

Bkaily

G

Wang

S

Bui

M

Menard

D

1995

ET-1 stimulates Ca2+ currents in cardiac cells

Journal of Cardiovascular Pharmacology26S293S296

10.1097/00005344-199506263-00088

Bloodgood

BL

Sabatini

BL

2007

Nonlinear regulation of unitary synaptic signals by Cav(2.3) voltage-sensitive calcium channels located in dendritic spines

Neuron53249260

10.1016/j.neuron.2006.12.017

17224406

Bolshakov

VY

Siegelbaum

SA

1994

Postsynaptic induction and presynaptic expression of hippocampal long-term depression

Science26411481152

10.1126/science.7909958

7909958

Boric

K

Muñoz

P

Gallagher

M

Kirkwood

A

2008

Potential adaptive function for altered long-term potentiation mechanisms in aging hippocampus

The Journal of Neuroscience2880348039

10.1523/JNEUROSCI.2036-08.2008

18685028

Boudreau

RL

Davidson

BL

2012

Generation of hairpin-based RNAi vectors for biological and therapeutic application

Methods in Enzymology507275296

10.1016/B978-0-12-386509-0.00014-4

22365779

Cahill

L

Prins

B

Weber

M

McGaugh

JL

1994

Beta-adrenergic activation and memory for emotional events

Nature371702704

10.1038/371702a0

7935815

Carter

ME

Yizhar

O

Chikahisa

S

Nguyen

H

Adamantidis

A

Nishino

S

Deisseroth

K

de Lecea

L

2010

Tuning arousal with optogenetic modulation of locus coeruleus neurons

Nature Neuroscience1315261533

10.1038/nn.2682

21037585

Catterall

WA

2000

Structure and regulation of voltage-gated ca²⁺ channels

Annual Review of Cell and Developmental Biology16521555

10.1146/annurev.cellbio.16.1.521

11031246

Cavuş

I

Teyler

T

1996

Two forms of long-term potentiation in area Ca1 activate different signal transduction cascades

Journal of Neurophysiology7630383047

10.1152/jn.1996.76.5.3038

8930253

Chao

JT

Gui

P

Zamponi

GW

Davis

GE

Davis

MJ

2011

Spatial association of the Cav1.2 calcium channel with α5β1-integrin

American Journal of Physiology. Cell Physiology300C477C489

10.1152/ajpcell.00171.2010

21178109

Cheng

TH

Chang

CY

Wei

J

Lin

CI

1995

Effects of endothelin 1 on calcium and sodium currents in isolated human cardiac myocytes

Canadian Journal of Physiology and Pharmacology7317741783

10.1139/y95-242

8834492

Cheng

JJ

Chao

YJ

Wang

DL

2002

Cyclic strain activates redox-sensitive proline-rich tyrosine kinase 2 (Pyk2) in endothelial cells

The Journal of Biological Chemistry2774815248157

10.1074/jbc.M110937200

12368297

Choi

J-H

Sim

S-E

Kim

J

Choi

DI

Oh

J

Ye

S

Lee

J

Kim

T

Ko

H-G

Lim

C-S

Kaang

B-K

2018

Interregional synaptic maps among engram cells underlie memory formation

Science360430435

10.1126/science.aas9204

Clifton

DR

Fields

KA

Grieshaber

SS

Dooley

CA

Fischer

ER

Mead

DJ

Carabeo

RA

Hackstadt

T

2004

A chlamydial type III translocated protein is tyrosine-phosphorylated at the site of entry and associated with recruitment of actin

PNAS1011016610171

10.1073/pnas.0402829101

15199184

Corvol

J-C

Valjent

E

Toutant

M

Enslen

H

Irinopoulou

T

Lev

S

Hervé

D

Girault

J-A

2005

Depolarization activates ERK and proline-rich tyrosine kinase 2 (Pyk2) independently in different cellular compartments in hippocampal slices

The Journal of Biological Chemistry280660668

10.1074/jbc.M411312200

15537634

Dai

S

Hall

DD

Hell

JW

2009

Supramolecular assemblies and localized regulation of voltage-gated ion channels

Physiological Reviews89411452

10.1152/physrev.00029.2007

19342611

Davare

MA

Dong

F

Rubin

CS

Hell

JW

1999

The A-kinase anchor protein Map2B and cAMP-dependent protein kinase are associated with class C L-type calcium channels in neurons

The Journal of Biological Chemistry2743028030287

10.1074/jbc.274.42.30280

10514522

Davare

MA

Horne

MC

Hell

JW

2000

Protein phosphatase 2A is associated with class C L-type calcium channels (Cav1.2) and Antagonizes channel phosphorylation by cAMP-dependent protein kinase

The Journal of Biological Chemistry2753971039717

10.1074/jbc.M005462200

10984483

Davare

MA

Avdonin

V

Hall

DD

Peden

EM

Burette

A

Weinberg

RJ

Horne

MC

Hoshi

T

Hell

JW

2001

A β2 adrenergic receptor signaling complex assembled with the Ca2+ channel Cav1.2

Science29398101

10.1126/science.293.5527.98

11441182

Davare

MA

Hell

JW

2003

Increased phosphorylation of the neuronal L-type Ca(2+) channel Ca(V)1.2 during aging

PNAS1001601816023

10.1073/pnas.2236970100

14665691

Deyo

RA

Straube

KT

Disterhoft

JF

1989

Nimodipine facilitates associative learning in aging rabbits

Science243809811

10.1126/science.2916127

2916127

Dikic

I

Tokiwa

G

Lev

S

Courtneidge

SA

Schlessinger

J

1996

A role for Pyk2 and SRC in linking G-protein-coupled receptors with MAP kinase activation

Nature383547550

10.1038/383547a0

8849729

Dikic

I

Dikic

I

Schlessinger

J

1998

Identification of a new Pyk2 Isoform implicated in chemokine and antigen receptor signaling

The Journal of Biological Chemistry2731430114308

10.1074/jbc.273.23.14301

9603937

Disterhoft

JF

Gispen

WH

Traber

J

Khachaturian

ZS

1994

Calcium hypothesis of aging and dementia

Ann N Y Acad Sci747

11

10.1111/j.1749-6632.1994.tb44398.x

Dittmer

PJ

Dell’Acqua

ML

Sather

WA

2014

Ca(2+)/calcineurin-dependent inactivation of neuronal L-type Ca(2+) channels requires priming by AKAP-anchored protein kinase A

Cell Reports714101416

10.1016/j.celrep.2014.04.039

24835998

Dodge-Kafka

KL

Langeberg

L

Scott

JD

2006

Compartmentation of cyclic nucleotide signaling in the heart: the role of A-kinase anchoring proteins

Circulation Research989931001

10.1161/01.RES.0000218273.91741.30

16645149

Dolmetsch

RE

Pajvani

U

Fife

K

Spotts

JM

Greenberg

ME

2001

Signaling to the nucleus by an L-type calcium channel-calmodulin complex through the MAP kinase pathway

Science294333339

10.1126/science.1063395

11598293

Döşemeci

A

Dhallan

RS

Cohen

NM

Lederer

WJ

Rogers

TB

1988

Phorbol ester increases calcium current and simulates the effects of angiotensin II on cultured neonatal rat heart myocytes

Circulation Research62347357

10.1161/01.res.62.2.347

2448059

Eiki

J

Saeki

K

Nagano

N

Iino

T

Yonemoto

M

Takayenoki-Iino

Y

Ito

S

Nishimura

T

Sato

Y

Bamba

M

Watanabe

H

Sasaki

K

Ohyama

S

Kanatani

A

Nagase

T

Yada

T

2009

A selective small molecule glucagon-like peptide-1 secretagogue acting via depolarization-coupled Ca(2+) influx

The Journal of Endocrinology201361367

10.1677/JOE-08-0528

19332449

Endoh

T

2005

Involvement of SRC tyrosine kinase and mitogen-activated protein kinase in the Facilitation of calcium channels in rat nucleus of the tractus solitarius by angiotensin II

The Journal of Physiology568851865

10.1113/jphysiol.2005.095307

16123104

Ferreira

MAR

O’Donovan

MC

Meng

YA

Jones

IR

Ruderfer

DM

Jones

L

Fan

J

Kirov

G

Perlis

RH

Green

EK

Smoller

JW

Grozeva

D

Stone

J

Nikolov

I

Chambert

K

Hamshere

ML

Nimgaonkar

VL

Moskvina

V

Thase

ME

Caesar

S

Sachs

GS

Franklin

J

Gordon-Smith

K

Ardlie

KG

Wellcome Trust Case Control ConsortiumGabriel

SB

Fraser

C

Blumenstiel

B

Defelice

M

Breen

G

Gill

M

Morris

DW

Elkin

A

Muir

WJ

McGhee

KA

Williamson

R

MacIntyre

DJ

MacLean

AW

St Clair

D

Robinson

M

Van Beck

M

Pereira

ACP

Kandaswamy

R

McQuillin

A

Collier

DA

Bass

NJ

Young

AH

Lawrence

J

Nicol Ferrier

I

Anjorin

A

Farmer

A

Curtis

D

Scolnick

EM

McGuffin

P

Daly

MJ

Corvin

AP

Holmans

PA

Blackwood

DH

Gurling

HM

Owen

MJ

Purcell

SM

Sklar

P

Craddock

N

2008

Collaborative genome-wide Association analysis supports a role for Ank3 and Cacna1C in bipolar disorder

Nature Genetics4010561058

10.1038/ng.209

Frangioni

JV

Neel

BG

1993

Solubilization and purification of enzymatically active glutathione S-transferase (pGEX) fusion proteins

Analytical Biochemistry210179187

10.1006/abio.1993.1170

8489015

Fu

Y

Westenbroek

RE

Scheuer

T

Catterall

WA

2013

Phosphorylation sites required for regulation of cardiac calcium channels in the fight-or-flight response

PNAS1101962119626

10.1073/pnas.1319421110

Fu

Y

Westenbroek

RE

Scheuer

T

Catterall

WA

2014

Basal and beta-adrenergic regulation of the cardiac calcium channel Cav1.2 requires phosphorylation of Serine 1700

PNAS1111659816603

10.1073/pnas.1419129111

25368181

Fuller

MD

Emrick

MA

Sadilek

M

Scheuer

T

Catterall

WA

2010

Molecular mechanism of calcium channel regulation in the fight-or-flight response

Science Signaling3

ra70

10.1126/scisignal.2001152

20876873

Gao

T

Cuadra

AE

Ma

H

Bünemann

M

Gerhardstein

BL

Cheng

T

Eick

RT

Hosey

MM

2001

C-terminal fragments of the α1C(CaV1.2) subunit associate with and regulate L-type calcium channels containing C-terminal-truncated α1CSubunits

Journal of Biological Chemistry2762108921097

10.1074/jbc.M008000200

Ghosh

D

Syed

AU

Prada

MP

Nystoriak

MA

Santana

LF

Nieves-Cintrón

M

Navedo

MF

2017

Calcium channels in vascular smooth muscle

Advances in Pharmacology784987

10.1016/bs.apha.2016.08.002

28212803

Giustino

TF

Maren

S

2018

Noradrenergic modulation of fear conditioning and extinction

Frontiers in Behavioral Neuroscience12

43

10.3389/fnbeh.2018.00043

29593511

Green

EK

Grozeva

D

Jones

I

Jones

L

Kirov

G

Caesar

S

Gordon-Smith

K

Fraser

C

Forty

L

Russell

E

Hamshere

ML

Moskvina

V

Nikolov

I

Farmer

A

McGuffin

P

Wellcome Trust Case Control ConsortiumHolmans

PA

Owen

MJ

O’Donovan

MC

Craddock

N

2010

The bipolar disorder risk allele at CACNA1C also confers risk of recurrent major depression and of schizophrenia

Molecular Psychiatry1510161022

10.1038/mp.2009.49

Grover

LM

Teyler

TJ

1990

Two components of long-term potentiation induced by different patterns of afferent activation

Nature347477479

10.1038/347477a0

1977084

Gui

P

Wu

X

Ling

S

Stotz

SC

Winkfein

RJ

Wilson

E

Davis

GE

Braun

AP

Zamponi

GW

Davis

MJ

2006

Integrin receptor activation triggers converging regulation of Cav1.2 calcium channels by C-SRC and protein kinase A pathways

The Journal of Biological Chemistry2811401514025

10.1074/jbc.M600433200

16554304

Hahn

B

Stolerman

IP

2005

Modulation of nicotine-induced attentional enhancement in rats by adrenoceptor antagonists

Psychopharmacology177438447

10.1007/s00213-004-1969-5

15252705

Hall

DD

Feekes

JA

Arachchige Don

AS

Shi

M

Hamid

J

Chen

L

Strack

S

Zamponi

GW

Horne

MC

Hell

JW

2006

Binding of protein phosphatase 2A to the L-type calcium channel Cav1.2 next to Ser1928, its main PKA site, is critical for Ser1928 Dephosphorylation

Biochemistry4534483459

10.1021/bi051593z

16519540

Hall

DD

Davare

MA

Shi

M

Allen

ML

Weisenhaus

M

McKnight

GS

Hell

JW

2007

Critical role of cAMP-dependent protein kinase anchoring to the L-type calcium channel Cav1.2 via A-kinase anchor protein 150 in neurons

Biochemistry4616351646

10.1021/bi062217x

17279627

Hall

DD

Dai

S

Tseng

PY

Malik

Z

Nguyen

M

Matt

L

Schnizler

K

Shephard

A

Mohapatra

DP

Tsuruta

F

Dolmetsch

RE

Christel

CJ

Lee

A

Burette

A

Weinberg

RJ

Hell

JW

2013

Competition between Α-Actinin and Ca²⁺-Calmodulin controls surface retention of the L-type Ca²⁺ channel Ca(V)1.2

Neuron78483497

10.1016/j.neuron.2013.02.032

23664615

Harper

SQ

Staber

PD

Beck

CR

Fineberg

SK

Stein

C

Ochoa

D

Davidson

BL

2006

Optimization of feline immunodeficiency virus vectors for RNA interference

Journal of Virology8093719380

10.1128/JVI.00958-06

16973543

He

JQ

Pi

Y

Walker

JW

Kamp

TJ

2000

Endothelin-1 and Photoreleased Diacylglycerol increase L-type Ca2+ current by activation of protein kinase C in rat ventricular myocytes

The Journal of Physiology524 Pt 3807820

10.1111/j.1469-7793.2000.00807.x

10790160

Hell

JW

Westenbroek

RE

Warner

C

Ahlijanian

MK

Prystay

W

Gilbert

MM

Snutch

TP

Catterall

WA

1993a

Identification and differential subcellular localization of the neuronal class C and class D L-type calcium channel A1 subunits

The Journal of Cell Biology123949962

10.1083/jcb.123.4.949

8227151

Hell

JW

Yokoyama

CT

Wong

ST

Warner

C

Snutch

TP

Catterall

WA

1993b

Differential Phosphorylation of two size forms of the neuronal class C L-type calcium channel A1 subunit

The Journal of Biological Chemistry2681945119457

8396138

Hell

JW

Yokoyama

CT

Breeze

LJ

Chavkin

C

Catterall

WA

1995

Phosphorylation of Presynaptic and postsynaptic calcium channels by cAMP-dependent protein kinase in hippocampal neurons

The EMBO Journal1430363044

10.1002/j.1460-2075.1995.tb07306.x

7621818

Hell

JW

Westenbroek

RE

Breeze

LJ

Wang

KKW

Chavkin

C

Catterall

WA

1996

N-methyl-D-aspartate receptor-induced proteolytic conversion of postsynaptic class C L-type calcium channels in hippocampal neurons

PNAS9333623367

10.1073/pnas.93.8.3362

8622942

Hoogland

TM

Saggau

P

2004

Facilitation of L-type Ca2+ channels in Dendritic spines by activation of Beta2 adrenergic receptors

The Journal of Neuroscience2484168427

10.1523/JNEUROSCI.1677-04.2004

15456814

Horn

R

1991

Estimating the number of channels in patch recordings

Biophysical Journal60433439

10.1016/S0006-3495(91)82069-0

Hu

XQ

Singh

N

Mukhopadhyay

D

Akbarali

HI

1998

Modulation of voltage-dependent Ca2+ channels in Rabbit Colonic smooth muscle cells by C-SRC and focal adhesion kinase

Journal of Biological Chemistry27353375342

10.1074/jbc.273.9.5337

Hu

H

Real

E

Takamiya

K

Kang

MG

Ledoux

J

Huganir

RL

Malinow

R

2007

Emotion enhances learning via norepinephrine regulation of AMPA-receptor trafficking

Cell131160173

10.1016/j.cell.2007.09.017

Huang

Y-Q

Lu

W-Y

Ali

DW

Pelkey

KA

Pitcher

GM

Lu

YM

Aoto

H

Roder

JC

Sasaki

T

Salter

MW

MacDonald

JF

2001

Cakbeta/Pyk2 kinase is a signaling link for induction of long-term potentiation in Ca1 hippocampus

Neuron29485496

10.1016/S0896-6273(01)00220-3

Hvoslef-Eide

M

Oomen

CA

Fisher

BM

Heath

CJ

Robbins

TW

Saksida

LM

Bussey

TJ

2015

Facilitation of spatial working memory performance following intra-Prefrontal cortical administration of the adrenergic Alpha1 agonist phenylephrine

Psychopharmacology23240054016

10.1007/s00213-015-4038-3

Jacquemet

G

Baghirov

H

Georgiadou

M

Sihto

H

Peuhu

E

Cettour-Janet

P

He

T

Perälä

M

Kronqvist

P

Joensuu

H

Ivaska

J

2016

L-type calcium channels regulate Filopodia stability and cancer cell invasion downstream of integrin signalling

Nature Communications7

13297

10.1038/ncomms13297

27910855

Jinnah

HA

Yitta

S

Drew

T

Kim

BS

Visser

JE

Rothstein

JD

1999

Calcium channel activation and self-biting in mice

PNAS961522815232

10.1073/pnas.96.26.15228

10611367

Jordan

M

Schallhorn

A

Wurm

FM

1996

Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation

Nucleic Acids Research24596601

10.1093/nar/24.4.596

8604299

Kamp

TJ

Hell

JW

2000

Regulation of cardiac L-type calcium channels by protein kinase A and protein kinase C

Circulation Research8710951102

10.1161/01.res.87.12.1095

11110765

Klauck

TM

Faux

MC

Labudda

K

Langeberg

LK

Jaken

S

Scott

JD

1996

Coordination of three signaling enzymes by AKAP79, a mammalian scaffold protein

Science27115891592

10.1126/science.271.5255.1589

8599116

Lacerda

AE

Rampe

D

Brown

AM

1988

Effects of protein kinase C Activators on cardiac ca²⁺ channels

Nature335249251

10.1038/335249a0

2457814

Lakkakorpi

PT

Bett

AJ

Lipfert

L

Rodan

GA

Duong

LT

2003

Pyk2 Autophosphorylation, but not kinase activity, is necessary for adhesion-induced association with C-SRC, Osteoclast spreading, and bone Resorption

The Journal of Biological Chemistry2781150211512

10.1074/jbc.M206579200

12514172

Leitch

B

Szostek

A

Lin

R

Shevtsova

O

2009

Subcellular distribution of L-type calcium channel subtypes in rat hippocampal neurons

Neuroscience164641657

10.1016/j.neuroscience.2009.08.006

19665524

Lemke

T

Welling

A

Christel

CJ

Blaich

A

Bernhard

D

Lenhardt

P

Hofmann

F

Moosmang

S

2008

Unchanged beta-adrenergic stimulation of cardiac L-type calcium channels in ca V 1.2 Phosphorylation site S1928A mutant mice

The Journal of Biological Chemistry2833473834744

10.1074/jbc.M804981200

18829456

Leonard

AS

Hell

JW

1997

Cyclic AMP-dependent protein kinase and protein kinase C Phosphorylate N-methyl-D-aspartate receptors at different sites

The Journal of Biological Chemistry2721210712115

10.1074/jbc.272.18.12107

9115280

Lev

S

Moreno

H

Martinez

R

Canoll

P

Peles

E

Musacchio

JM

Plowman

GD

Rudy

B

Schlessinger

J

1995

Protein tyrosine kinase Pyk2 involved in Ca(2+)-Induced regulation of ion channel and MAP kinase functions

Nature376737745

10.1038/376737a0

7544443

Li

X

Dy

RC

Cance

WG

Graves

LM

Earp

HS

1999

Interactions between two cytoskeleton-associated tyrosine Kinases: calcium-dependent tyrosine kinase and focal adhesion tyrosine kinase

The Journal of Biological Chemistry27489178924

10.1074/jbc.274.13.8917

10085136

Li

H

Pink

MD

Murphy

JG

Stein

A

Dell’Acqua

ML

Hogan

PG

2012

Balanced interactions of calcineurin with Akap79 regulate Ca2+-Calcineurin-NFAT signaling

Nature Structural & Molecular Biology19337345

10.1038/nsmb.2238

Li

B

Tadross

MR

Tsien

RW

2016

Sequential ionic and conformational signaling by calcium channels drives neuronal gene expression

Science351863867

10.1126/science.aad3647

Liu

YP

Lin

YL

Chuang

CH

Kao

YC

Chang

ST

Tung

CS

2009

Alpha adrenergic modulation on effects of norepinephrine transporter inhibitor Reboxetine in five-choice serial reaction time task

Journal of Biomedical Science16

72

10.1186/1423-0127-16-72

19678962

Liu

G

Papa

A

Katchman

AN

Zakharov

SI

Roybal

D

Hennessey

JA

Kushner

J

Yang

L

Chen

B-X

Kushnir

A

Dangas

K

Gygi

SP

Pitt

GS

Colecraft

HM

Ben-Johny

M

Kalocsay

M

Marx

SO

2020

Mechanism of adrenergic Cav1.2 stimulation revealed by proximity Proteomics

Nature577695700

10.1038/s41586-020-1947-z

31969708

Ma

H

Groth

RD

Cohen

SM

Emery

JF

Li

B

Hoedt

E

Zhang

G

Neubert

TA

Tsien

RW

2014

gammaCaMKII Shuttles Ca(2+)/Cam to the nucleus to trigger CREB Phosphorylation and gene expression

Cell159281294

10.1016/j.cell.2014.09.019

25303525

Magee

JC

Johnston

D

1997

A synaptically controlled, associative signal for hebbian plasticity in hippocampal neurons

Science275209213

10.1126/science.275.5297.209

8985013

Man

KNM

Navedo

MF

Horne

MC

Hell

JW

2020

Β₂ adrenergic receptor complexes with the L-type ca²⁺ channel ca_V1.2 and AMPA-type glutamate receptors: paradigms for pharmacological targeting of protein interactions

Annual Review of Pharmacology and Toxicology60155174

10.1146/annurev-pharmtox-010919-023404

31561738

Marrion

NV

Tavalin

SJ

1998

Selective activation of ca²⁺-Activated K⁺ channels by Co-localized ca²⁺channels in hippocampal neurons

Nature395900905

10.1038/27674

9804423

Marshall

MR

Clark

JP

IIIWestenbroek

R

Yu

FH

Scheuer

T

Catterall

WA

2011

Functional roles of a C-terminal signaling complex of Cav1 channels and A-kinase anchoring protein 15 in brain neurons

Journal of Biological Chemistry2861262712639

10.1074/jbc.M110.175257

McHugh

D

Sharp

EM

Scheuer

T

Catterall

WA

2000

Inhibition of cardiac L-type calcium channels by protein kinase C Phosphorylation of two sites in the N-terminal domain

PNAS971233412338

10.1073/pnas.210384297

11035786

Mikami

A

Imoto

K

Tanabe

T

Niidome

T

Mori

Y

Takeshima

H

Narumiya

S

Numa

S

1989

Primary structure and functional expression of the cardiac dihydropyridine-sensitive calcium channel

Nature340230233

10.1038/340230a0

2474130

Minzenberg

MJ

Watrous

AJ

Yoon

JH

Ursu

S

Carter

CS

2008

Modafinil shifts human locus coeruleus to low-tonic, high-Phasic activity during functional MRI

Science32217001702

10.1126/science.1164908

Mogilnicka

E

Czyrak

A

Maj

J

1987

Dihydropyridine calcium channel antagonists reduce immobility in the Mouse behavioral despair test; antidepressants facilitate nifedipine action

European Journal of Pharmacology138413416

10.1016/0014-2999(87)90480-8

3622617

Mogilnicka

E

Czyrak

A

Maj

J

1988

BAY K 8644 enhances immobility in the Mouse behavioral despair test, an effect blocked by nifedipine

European Journal of Pharmacology151307311

10.1016/0014-2999(88)90813-8

Moosmang

S

Haider

N

Klugbauer

N

Adelsberger

H

Langwieser

N

Müller

J

Stiess

M

Marais

E

Schulla

V

Lacinova

L

Goebbels

S

Nave

K-A

Storm

DR

Hofmann

F

Kleppisch

T

2005

Role of hippocampal Cav1.2 Ca2+ channels in NMDA receptor-independent synaptic plasticity and spatial memory

The Journal of Neuroscience2598839892

10.1523/JNEUROSCI.1531-05.2005

16251435

Murphy

JG

Sanderson

JL

Gorski

JA

Scott

JD

Catterall

WA

Sather

WA

Dell’Acqua

ML

2014

AKAP-anchored PKA maintains neuronal L-type calcium channel activity and NFAT transcriptional signaling

Cell Reports715771588

10.1016/j.celrep.2014.04.027

Mustafa

T

Walsh

J

Grimaldi

M

Eiden

LE

2010

Pac1Hop receptor activation facilitates catecholamine secretion selectively through 2-APB-sensitive Ca(2+) channels in Pc12 cells

Cellular Signalling2214201426

10.1016/j.cellsig.2010.05.005

20471475

Navedo

MF

Amberg

GC

Votaw

VS

Santana

LF

2005

Constitutively active L-type Ca2+ channels

PNAS1021111211117

10.1073/pnas.0500360102

16040810

Navedo

MF

Nieves-Cintrón

M

Amberg

GC

Yuan

C

Votaw

VS

Lederer

WJ

McKnight

GS

Santana

LF

2008

Akap150 is required for Stuttering persistent Ca2+ Sparklets and angiotensin II-induced hypertension

Circulation Research102e1e11

10.1161/CIRCRESAHA.107.167809

18174462

Nyegaard

M

Demontis

D

Foldager

L

Hedemand

A

Flint

TJ

Sørensen

KM

Andersen

PS

Nordentoft

M

Werge

T

Pedersen

CB

Hougaard

DM

Mortensen

PB

Mors

O

Børglum

AD

2010

Cacna1C (Rs1006737) is associated with schizophrenia

Molecular Psychiatry15119121

10.1038/mp.2009.69

Oliveria

SF

Dell’Acqua

ML

Sather

WA

2007

Akap79/150 anchoring of calcineurin controls neuronal L-type Ca(2+) channel activity and nuclear signaling

Neuron55261275

10.1016/j.neuron.2007.06.032

17640527

Panichello

MF

Buschman

TJ

2021

Shared mechanisms underlie the control of working memory and attention

Nature592601605

10.1038/s41586-021-03390-w

33790467

Park

SY

Avraham

HK

Avraham

S

2004

RAFTK/Pyk2 activation is mediated by Trans-acting Autophosphorylation in a SRC-independent manner

Journal of Biological Chemistry2793331533322

10.1074/jbc.M313527200

Patriarchi

T

Qian

H

Di Biase

V

Malik

ZA

Chowdhury

D

Price

JL

Hammes

EA

Buonarati

OR

Westenbroek

RE

Catterall

WA

Hofmann

F

Xiang

YK

Murphy

GG

Chen

C-Y

Navedo

MF

Hell

JW

2016

Phosphorylation of Cav1.2 on S1928 Uncouples the L-type Ca2+ channel from the Β2 adrenergic receptor

The EMBO Journal3513301345

10.15252/embj.201593409

27103070

Puumala

T

Riekkinen

P

SrSirviö

J

1997

Modulation of vigilance and behavioral activation by Alpha-1 adrenoceptors in the rat

Pharmacology, Biochemistry, and Behavior56705712

10.1016/s0091-3057(96)00408-x

9130297

Qian

H

Matt

L

Zhang

M

Nguyen

M

Patriarchi

T

Koval

OM

Anderson

ME

He

K

Lee

H-K

Hell

JW

2012

Β2 adrenergic receptor supports prolonged Theta tetanus - induced LTP

Journal of Neurophysiology10727032712

10.1152/jn.00374.2011

Qian

H

Patriarchi

T

Price

JL

Matt

L

Lee

B

Nieves-Cintrón

M

Buonarati

OR

Chowdhury

D

Nanou

E

Nystoriak

MA

Catterall

WA

Poomvanicha

M

Hofmann

F

Navedo

MF

Hell

JW

2017

Phosphorylation of Ser1928 mediates the enhanced activity of the L-type Ca2+ channel Cav1.2 by the Beta2-adrenergic receptor in neurons

Science Signaling10

eaaf9659

10.1126/scisignal.aaf9659

28119465

Ramos

BP

Arnsten

AFT

2007

Adrenergic pharmacology and cognition: focus on the Prefrontal cortex

Pharmacology & Therapeutics113523536

10.1016/j.pharmthera.2006.11.006

17303246

Reuter

H

1983

Calcium channel modulation by neurotransmitters, enzymes and drugs

Nature301569574

10.1038/301569a0

6131381

Robbins

TW

2002

The 5-choice serial reaction time task: behavioural pharmacology and functional neurochemistry

Psychopharmacology163362380

10.1007/s00213-002-1154-7

12373437

Roskoski

R

2015

Src protein-tyrosine kinase structure, mechanism, and small molecule inhibitors

Pharmacological Research94925

10.1016/j.phrs.2015.01.003

25662515

Rueden

CT

Schindelin

J

Hiner

MC

DeZonia

BE

Walter

AE

Arena

ET

Eliceiri

KW

2017

Imagej2: Imagej for the next generation of scientific image data

BMC Bioinformatics18

529

10.1186/s12859-017-1934-z

29187165

Sabri

A

Govindarajan

G

Griffin

TM

Byron

KL

Samarel

AM

Lucchesi

PA

1998

Calcium- and protein kinase C-dependent activation of the tyrosine kinase Pyk2 by angiotensin II in vascular smooth muscle

Circulation Research83841851

10.1161/01.RES.83.8.841

Sanderson

JL

Dell’Acqua

ML

2011

AKAP signaling complexes in regulation of excitatory synaptic plasticity

The Neuroscientist17321336

10.1177/1073858410384740

21498812

Sayas

CL

Ariaens

A

Ponsioen

B

Moolenaar

WH

2006

GSK-3 is activated by the tyrosine kinase Pyk2 during Lpa1-mediated Neurite retraction

Molecular Biology of the Cell1718341844

10.1091/mbc.e05-07-0688

Shankar

S

Teyler

TJ

Robbins

N

1998

Aging Differentially alters forms of long-term potentiation in rat hippocampal area Ca1

Journal of Neurophysiology79334341

10.1152/jn.1998.79.1.334

9425202

Shi

CS

Kehrl

JH

2004

Pyk2 Amplifies epidermal growth factor and C-SRC-induced Stat3 activation

The Journal of Biological Chemistry2791722417231

10.1074/jbc.M311875200

14963038

Sinnegger-Brauns

MJ

Hetzenauer

A

Huber

IG

Renström

E

Wietzorrek

G

Berjukov

S

Cavalli

M

Walter

D

Koschak

A

Waldschütz

R

Hering

S

Bova

S

Rorsman

P

Pongs

O

Singewald

N

Striessnig

J

2004

Isoform-specific regulation of mood behavior and Pancreatic beta cell and cardiovascular function by L-type ca 2+ channels

The Journal of Clinical Investigation11314301439

10.1172/JCI20208

15146240

Smoller

JW

2013

Disorders and borders: psychiatric Genetics and Nosology

American Journal of Medical Genetics. Part B, Neuropsychiatric Genetics162B559578

10.1002/ajmg.b.32174

24132891

Snutch

TP

Leonard

JP

Gilbert

MM

Lester

HA

Davidson

N

1990

Rat brain expresses a heterogeneous family of calcium channels

PNAS8733913395

10.1073/pnas.87.9.3391

1692134

Snutch

TP

Tomlinson

WJ

Leonard

JP

Gilbert

MM

1991

Distinct calcium channels are generated by alternative splicing and are Differentially expressed in the mammalian CNS

Neuron74557

10.1016/0896-6273(91)90073-9

1648941

Sorokin

A

Kozlowski

P

Graves

L

Philip

A

2001

Protein-tyrosine kinase Pyk2 mediates Endothelin-induced P38 MAPK activation in glomerular mesangial cells

The Journal of Biological Chemistry2762152121528

10.1074/jbc.M008869200

11278444

Splawski

I

Timothy

KW

Sharpe

LM

Decher

N

Kumar

P

Bloise

R

Napolitano

C

Schwartz

PJ

Joseph

RM

Condouris

K

Tager-Flusberg

H

Priori

SG

Sanguinetti

MC

Keating

MT

2004

Ca(V)1.2 calcium channel dysfunction causes a Multisystem disorder including arrhythmia and autism

Cell1191931

10.1016/j.cell.2004.09.011

15454078

Strauss

O

Mergler

S

Wiederholt

M

1997

Regulation of L-type calcium channels by protein tyrosine kinase and protein kinase C in cultured rat and human retinal pigment epithelial cells

FASEB Journal11859867

10.1096/fasebj.11.11.9285484

9285484

Taylor

SC

Green

KN

Carpenter

E

Peers

C

2000

Protein kinase C evokes Quantal catecholamine release from Pc12 cells via activation of L-type Ca2+ channels

Journal of Biological Chemistry2752678626791

10.1016/S0021-9258(19)61444-4

Thibault

O

Landfield

PW

1996

Increase in single L-type calcium channels in hippocampal neurons during aging

Science27210171020

10.1126/science.272.5264.1017

8638124

Tigaret

CM

Olivo

V

Sadowski

JHLP

Ashby

MC

Mellor

JR

2016

Coordinated activation of distinct Ca(2+) sources and Metabotropic glutamate receptors Encodes Hebbian synaptic plasticity

Nature Communications7

10289

10.1038/ncomms10289

26758963

Tigaret

CM

Lin

T-CE

Morrell

ER

Sykes

L

Moon

AL

O’Donovan

MC

Owen

MJ

Wilkinson

LS

Jones

MW

Thomas

KL

Hall

J

2021

Neurotrophin receptor activation rescues cognitive and synaptic abnormalities caused by hemizygosity of the psychiatric risk gene cacna1c

Molecular Psychiatry2617481760

10.1038/s41380-020-01001-0

Tse

KWK

Lin

KBL

Dang-Lawson

M

Guzman-Perez

A

Aspnes

GE

Buckbinder

L

Gold

MR

2012

Small molecule inhibitors of the Pyk2 and FAK Kinases modulate Chemoattractant-induced migration, adhesion and AKT activation in follicular and marginal zone B cells

Cellular Immunology2754754

10.1016/j.cellimm.2012.03.002

22507871

Turner

M

Anderson

DE

Bartels

P

Nieves-Cintron

M

Coleman

AM

Henderson

PB

Man

KNM

Tseng

PY

Yarov-Yarovoy

V

Bers

DM

Navedo

MF

Horne

MC

Ames

JB

Hell

JW

2020

Α-Actinin-1 promotes activity of the L-type ca²⁺ channel ca_V 1.2

The EMBO Journal39

e102622

10.15252/embj.2019102622

31985069

Voelker

TL

Del Villar

SG

Westhoff

M

Costa

AD

Coleman

AM

Hell

JW

Horne

MC

Dickson

EJ

Dixon

RE

2023

Acute Phosphatidylinositol 4,5 Bisphosphate depletion Destabilizes sarcolemmal expression of cardiac L-type ca²⁺ channel ca_V1.2

PNAS120

e2221242120

10.1073/pnas.2221242120

36976770

Walter

HJ

McMahon

T

Dadgar

J

Wang

D

Messing

RO

2000

Ethanol regulates calcium channel subunits by protein kinase C Delta -Dependent and -Independent mechanisms

Journal of Biological Chemistry2752571725722

10.1074/jbc.M910282199

Wang

H

Ardiles

AO

Yang

S

Tran

T

Posada-Duque

R

Valdivia

G

Baek

M

Chuang

Y-A

Palacios

AG

Gallagher

M

Worley

P

Kirkwood

A

2016

Metabotropic glutamate receptors induce a form of LTP controlled by translation and arc signaling in the hippocampus

The Journal of Neuroscience3617231729

10.1523/JNEUROSCI.0878-15.2016

Ward

SG

Reif

K

Ley

S

Fry

MJ

Waterfield

MD

Cantrell

DA

1992

Regulation of Phosphoinositide Kinases in T cells evidence that Phosphatidylinositol 3-kinase is not a substrate for T cell antigen receptor-regulated tyrosine Kinases

The Journal of Biological Chemistry267

23869

1385426

Wheeler

DG

Groth

RD

Ma

H

Barrett

CF

Owen

SF

Safa

P

Tsien

RW

2012

Cav1 and Cav2 channels engage distinct modes of Ca2+ signaling to control CREB-dependent gene expression

Cell14911121124

10.1016/j.cell.2012.03.041

22632974

White

JA

McKinney

BC

John

MC

Powers

PA

Kamp

TJ

Murphy

GG

2008

Conditional forebrain deletion of the L-type calcium channel ca V 1.2 disrupts remote spatial memories in mice

Learning & Memory1515

10.1101/lm.773208

Whitlock

JR

Heynen

AJ

Shuler

MG

Bear

MF

2006

Learning induces long-term potentiation in the hippocampus

Science31310931097

10.1126/science.1128134

Wu

X

Davis

GE

Meininger

GA

Wilson

E

Davis

MJ

2001

Regulation of the L-type calcium channel by α5β1 integrin requires signaling between focal adhesion proteins

Journal of Biological Chemistry2763028530292

10.1074/jbc.M102436200

Xu

H

Ginsburg

KS

Hall

DD

Zimmermann

M

Stein

IS

Zhang

M

Tandan

S

Hill

JA

Horne

MC

Bers

D

Hell

JW

2010

Targeting of protein phosphatases Pp2A and Pp2B to the C-terminus of the L-type calcium channel ca V1.2

Biochemistry491029810307

10.1021/bi101018c

Yang

L

Liu

G

Zakharov

SI

Morrow

JP

Rybin

VO

Steinberg

SF

Marx

SO

2005

Ser1928 is a common site for Cav1.2 phosphorylation by protein kinase C isoforms

Journal of Biological Chemistry280207214

10.1074/jbc.M410509200

Yang

S

Roselli

F

Patchev

AV

Yu

S

Almeida

OFX

2013

Non-receptor-tyrosine kinases integrate fast glucocorticoid signaling in hippocampal neurons

Journal of Biological Chemistry2882372523739

10.1074/jbc.M113.470146

Zamponi

GW

Striessnig

J

Koschak

A

Dolphin

AC

2015

The physiology, pathology, and pharmacology of voltage-gated calcium channels and their future therapeutic potential

Pharmacological Reviews67821870

10.1124/pr.114.009654

26362469

Zhao

M

Finlay

D

Zharkikh

I

Vuori

K

2016

Novel role of SRC in priming Pyk2 phosphorylation

PLOS ONE11

e0149231

10.1371/journal.pone.0149231

26866924

10.7554/eLife.79648.sa0

Editor's evaluation

Goda

Yukiko

Reviewing Editor

https://ror.org/02qg15b79

Okinawa Institute of Science and Technology

Japan

This study reports of a new signaling pathway in hippocampal neurons by which α₁ receptors for norepinephrine regulate Cav1.2 calcium channels; activation of α₁ receptors enhances a form of long-lasting synaptic plasticity that is dependent on L-type calcium channels. The experiments are comprehensive and well-executed, and the main conclusions are compellingly supported by the data shown. The work has significance for the field of neuroscience in general and for cellular mechanisms of neuroregulation in particular.

10.7554/eLife.79648.sa1

Decision letter

Goda

Yukiko

Reviewing Editor

https://ror.org/02qg15b79

Okinawa Institute of Science and Technology

Japan

Our editorial process produces two outputs: i) public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Decision letter after peer review:

Thank you for submitting your article "α1 adrenergic receptor -PKC -Pyk2 -Src signaling boosts L-type ca²⁺ channel Cav1.2 activity and long-term potentiation in rodents" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Gary Westbrook as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers consider the study to be important in identifying a novel signaling pathway involving norepinephrine with consequences on synaptic plasticity, although some key points needing further clarifications that may require some additional experiments and/or modification of the conclusions. The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1) Please provide further clarification of whether and how the linker domains II and III act in mediating Pyk2 and Src activation of Cav1.2, for example, by identifying the phosphorylation site and demonstrating its necessity by introducing a mutation.

2) Please confirm that the same pathway is targeted between PC12 cells and hippocampal neurons by testing whether bradykinin elicits the same response in hippocampal neurons.

3) Please provide additional evidence for the claim that alpha1-AR agonist increases Po by ruling out an increase in N caused e.g. by a rapid channel insertion to the membrane surface.

4) Clarify the basis for an apparent baseline modulation of channel activity by the alpha1-AR pathway that is revealed by the effects of inhibitors (Figures 2B, 2D).

In addition, please fully address all the comments raised by the three reviewers involving quantification of data, replacing of immunoblot in Figure 4B, addition of a schematic figure, and clarification/editing of the text.

Reviewer #1 (Recommendations for the authors):

1) My main critique would be that the study, while very well executed and rigorous, is fragmented, consisting of three parts that each feel incomplete: i, hippocampal neuron studies, mainly single channel recordings; ii, biochemical studies mainly in PC12 cells, using a different agonist bradykinin, and iii, the examination of LTP in young mice.

2) Does Norepinephrine activate this pathway in hippocampal neurons? This should be testable given the high affinity for NE of the α1-AR (line 487).

3) The single channel recordings cannot distinguish between L-type channels cav1.2 and cav1.3. This would require the use of selective knockout mice.

4) Patches identified to contain only one channel should be used to determine conclusively whether N and/or Po is increased by the α1-AR agonist Phe. This could be done by BayK8644 application at the end of the experiment, for example, as well as data mentioned at line 116. At the moment this cannot be conclusively stated that Po is increased.

5) Line 147, it is unclear why other Gq coupled receptors do not affect this pathway in hippocampal neurons. This is problematic as the study goes on to use bradykinin, rather than an α1-AR agonist in PC12 cells to dissect the pathway (Figure 5 onwards). Does bradykinin stimulate this pathway in hippocampal neurons?

6) If the same pathway is present in PC12 cells then the α1-AR should be expressed in these cells, rather than using bradykinin.

7) It is unclear why PP2 inhibits NPo and ensemble average current far below the baseline control (Figures2B and 2D), whereas SU6656 does not. This suggests non-specificity.

8) The II-III linker is identified as a binding site for Pyk2; but where are the src phosphorylation sites on CaV1.2 specifically mediating this effect? This needs to be identified, otherwise it is possible that the effect is not direct.

9) A diagram of the pathway is important to add, preferably in each figure, to show where every drug is supposed to act.

Reviewer #2 (Recommendations for the authors):

Overall, the work is carefully carried out but there are a few pitfalls, where information lacking. I suggest the authors to address these issues.

1. The pathway from PKC/ca²⁺, Pyk2, to Src has been already reported more than 20 years ago (see, for example, Sabri et al., Circ. Res 1998 and reference therein). But in this paper, the mechanism how noradrenaline activates Pyk2 through PKC is not known. Also the authors stated that phosphorylation of alpha11.2 by PKC inhibits Cav1.2 activity (52). Why this pathway is not active here?

2. The mechanism how Src activates Cav1.2 is not clear. The authors should make more effort to identify the site and use a mutant to show that it abolishes the increase in the channel activity. They discussed possible tyrosine phosphorylation at Y2122 but they did not confirm this. Also, this residue is located at the very end of protein, far away from the channel. It is not clear how it modulates the channel activity, if it has any function.

Reviewer #3 (Recommendations for the authors):

The authors should consider adding experiments that show whether the linker between domains II and III is indeed the site of regulation.

Editorial Points

Lines 203-207. These sentences are a bit garbled. Please revise.

Lines 272-273. Better wording would be.."binds via phosphoY402 to the SH2 domain".

Line 503. Better wording would be "regulate through".

10.7554/eLife.79648.sa2

Author response

Essential revisions:

1) Please provide further clarification of whether and how the linker domains II and III act in mediating Pyk2 and Src activation of Cav1.2, for example, by identifying the phosphorylation site and demonstrating its necessity by introducing a mutation.

The relevance of Pyk2 binding to loop II/III lies in the general concept that frequently kinase signaling cascades depend on close proximity of the kinase with the target, often through direct binding. The exact binding sequence might not be close to the location of the phosphorylation site in the linear sequence because the 3-dimensional folding could bring binding and phosphorylation sites close together even if far apart in the linear sequence. Accordingly, loop II/III might not harbor the phosphorylation site, which could be anywhere in the primary sequence of the channel. Thus, identification of the phosphorylation site would likely take significantly longer than one year and without a reasonable level of guarantee of success.

However, along the lines of the Reviewers’ notion to try and define more precisely binding sites and mechanisms for further mechanistic work on Pyk2 interacting with Cav1.2 and specifically loop II/III, we ordered six overlapping fluorescein-tagged peptides that covered the whole loop II/III segment. We attempted binding studies based on fluorescence polarization, as has worked for a number of other binding sites for us in the past. Unfortunately, none of the peptides showed specific binding. Perhaps the binding region is more complex than a simple ~ 20 residue long linear segment, possibly being significantly longer or consisting of multiple short, discontinuous attachment sites that are more than several residues apart but brought together by the three-dimensional folding of loop II/III.

2) Please confirm that the same pathway is targeted between PC12 cells and hippocampal neurons by testing whether bradykinin elicits the same response in hippocampal neurons.

We used the PC12 cell system for our biochemical analysis because such biochemical analysis in hippocampal cultures is impossible due to material limitation. We simply would not be able to grow enough primary hippocampal cultures for this biochemical analysis. PC12 cells have a very strong presence of both, Cav1.2 and Pyk2. They, thus, constitute a perfect system for the biochemical analysis of Pyk2 signaling to Cav1.2. In fact, Pyk2 was first identified and characterized in PC12 cells (e.g., Lev et al., 2005: Nature 376, 737-745; Dikic et al., 2006: Nature 383, 547-549).

The rationale behind analyzing different GPCRs is that different cell types can harbor different sets of GPCRs that are coupled to Gq-PKC signaling. Stimulating Gq – PKC ‘signaling modules’ like the Gq – PKC – Pyk2 – Src module should be transferable between cell types if they share this module even if it would be engaged by different GqPCRs, like the bradykinin receptor versus α 1 AR. In addition, we went to great length in our work to also directly stimulate PKC with the phorbol ester PMA in hippocampal neurons to observe and characterize the increase in LTCC activity and in PC12 cells to observe and characterize the increase in tyrosine phosphorylation. All of these effects were inhibited by Pyk2 and Src inhibitors.

Nevertheless, expression of the Gq-coupled bradykinin receptors is quite prominent in hippocampal neurons. We tested whether their activation would also augment LTCC activity in hippocampal neurons and now report that there is a clear and strong increase in Po in Figure 6.

In these experiments we added BayK8644 at the end of the recording (as suggested by Reviewer 1, #4), which is expected to further upregulate the currents under our cell-attached patch electrode if those are mediated by LTCCs, which was consistently the case (Figure 6). This approach allowed us to confirm the identity of the channels in the patches as L-type and aided in determining the number of channels N in each patch.

3) Please provide additional evidence for the claim that alpha1-AR agonist increases Po by ruling out an increase in N caused e.g. by a rapid channel insertion to the membrane surface.

We now provide more data that support an increase in Po versus N by first forming a cell-attached seal, which isolates the small surface area from which single-channel activity is recorded, and then apply PHE. This acute application of PHE avoids delays in recording as happening when PHE is bath applied before the recording pipette is attached to the cell during which time new channels could have been inserted (Figure 4).

The acute PHE application in these new experiments induced a fast increase in the activity of Cav1.2 channels isolated under the pre-formed patch, arguing that this increase is really due to an increase in Po rather than insertion of new channels into the patch, which appears unlikely in this configuration due to spatial restrains and the time course with which the increase in channel activity happens. Furthermore, we used a recoding pipette with a much smaller diameter than in our original work (resistance of pipets used in our original recordings was 3.5-5.5 MOhm and in these new experiments it was 7-12 MOhm). The reduction in diameter results in patches that contain typically <4 channels, which is required for reliably determining the channel number N whereas the original recordings often contained >4 channels and thus cannot truly be analyzed for N versus Po. To increase our confidence in our capability to count all channels we added BayK8644 at the end of the recordings in one set of experiments (i.e., the bradykinin stimulation; Figure 6).

At the same time, and cautioned by the Reviewers’ comments, we do not want to rule out that there is also an effect of alpha1 AR signaling on channel number N in our original experiments in which cells were pre-treated with PHE or PKC activators before forming the seal. We are now using a more carefully worded interpretation of these original experiments leaving open the possibility that in these initial experiments we also could have had an effect on N. However, we would like to re-emphasize that our new data leave little room for doubt that PHE augments channel activity at least in part by increasing Po.

4) Clarify the basis for an apparent baseline modulation of channel activity by the alpha1-AR pathway that is revealed by the effects of inhibitors (Figures 2B, 2D).

We now explicitly state in the Discussion: “Inhibitors of PKC, Pyk2, and Src reduce under nearly all conditions Cav1.2 baseline activity and also tyrosine phosphorylation of Cav1.2, Pyk2, and Src even when activators for alpha1 AR and PKC were present. Especially notable is the strong reduction of channel activity way below the control conditions by the Src inhibitor PP2 as well as the PKC inhibitor chelerythrine in Figure 2C. This effect is consistent with PP2 strongly reducing down below control conditions tyrosine phosphorylation of Src (Figure 8J), Pyk2 (Figure 8L), and Cav1.2 (Figure 9E) even with the PKC activator PMA present. These findings suggest that Pyk2 and Src experience significant although clearly by far not full activation under basal conditions as reflected by their own phosphorylation status, which translates into tyrosine phosphorylation of Cav1.2 under such basal conditions.” Because there are multiple ways Pyk2 and Src can be activated including Ca influx and cell-matrix interactions, defining the cause of this baseline activity has to remain beyond the scope of the current work.

In addition, please fully address all the comments raised by the three reviewers involving quantification of data, replacing of immunoblot in Figure 4B, addition of a schematic figure, and clarification/editing of the text.

Please note that association of Src with Cav1.2 had previously been described by several authors. To ensure that the reader understands that our Src coIP with Cav1.2 is only confirmatory we now state explicitly: “We also confirmed earlier work (Figure 7A bottom panel) that indicated association of Src with Ca_V1.2 in vitro (Bence-Hanulec et al., 2000; Endoh, 2005; Gui et al., 2006; Hu et al., 1998; Strauss et al., 1997; Wu et al., 2001) and in intact cells (Bence-Hanulec et al., 2000; Chao et al., 2011; Hu et al., 1998).” Also, we now provide the uncropped immunoblot for Figure 7B, which shows more convincingly that there is a clear band for the Src immunosignal.

Reviewer #1 (Recommendations for the authors):

1) My main critique would be that the study, while very well executed and rigorous, is fragmented, consisting of three parts that each feel incomplete: i, hippocampal neuron studies, mainly single channel recordings; ii, biochemical studies mainly in PC12 cells, using a different agonist bradykinin, and iii, the examination of LTP in young mice.

We would argue that both, the single-channel data and biochemical data, are at a level of completion that is by itself appropriate for eLife, each characterizing a multistep signaling pathway with agonists and antagonists and also shRNA addressing each step in the signaling cascade. The LTP studies are meant to put the signaling pathway we characterized in hippocampal neurons into a larger, network level context by testing effects of key treatment conditions as established for the single-channel and tyrosine phosphorylation data.

2) Does Norepinephrine activate this pathway in hippocampal neurons? This should be testable given the high affinity for NE of the α1-AR (line 487).

We now provide data that show that application of NE after seal formation and after establishing a baseline activity also augments LTCC Po to the same extent as PHE does (compare new Figure 4A-D with new Figure 4E-H). Cav1.2 activity can also be increased by beta2 AR signaling (but not beta1 AR signaling) (Qian et al., 2017: Sci Signal 10, eaaf9659; see also Patriarchi et al., 2016: EMBO J 35, 1330-1345). This upregulation by beta2 AR stimulation is strictly mediated by localized signaling from the b2 AR to Cav1.2 and cannot be engaged when the b2 adrenergic agonist is applied from the outside of the cell attached patch formed by the recoding electrode (Davare et al., 2001: Science 293, 98-101, as cited). Thus, the increase in Po upon bath application of NE after seal formation suggests that NE as the cognate ligand for alpha1 AR can stimulate Cav1.2 activity to the same degree as PHE.

3) The single channel recordings cannot distinguish between L-type channels cav1.2 and cav1.3. This would require the use of selective knockout mice.

It is possible that the alpha1AR signaling also regulates Cav1.3 but Cav1.3 only constitutes ~20% when Cav1.2 constitutes ~80% of all L-type channels in hippocampus (Hell et al., 1993: JCB 123, 949-962; Sinnegger-Brauns et al., 2004: J Clin Invest 113, 1030-1439). Accordingly, it seems hard to imagine that the observed effects on LTCC activity could be explained solely by upregulation of Cav1.3, which would have to be extremely high to solely explain an increase in Po by 3- to 4-fold as seen in Figure 1B. With Cav1.3 only contributing 20% of the activity, this activity would have to be increased by well over 10-fold to explain an overall increase of Po of all L-type channels by twofold if the effect is solely via Cav1.3. It would be very involving to obtain conditional Cav1.2 KO mice to further prove this point and likely take well beyond one year given that we would have to import appropriate floxed mice to UC Davis (which often takes 6 months by itself, given paperwork and regulations) and then set up the breeding scheme. A full KO of Cav1.2 would in theory be easier to set up but is embryonically lethal.

4) Patches identified to contain only one channel should be used to determine conclusively whether N and/or Po is increased by the α1-AR agonist Phe. This could be done by BayK8644 application at the end of the experiment, for example, as well as data mentioned at line 116. At the moment this cannot be conclusively stated that Po is increased.

To get patches with a single channel are very rare. Figures 1, 2, and 3 are based on a total of 295 recordings under the various conditions conducted over more than 3 years. Of all of these recordings, only 21 (7%) had a single channel active at any point in time perhaps reflecting that only a single activatable channel was present in the patch. The rest (93%) of all recordings had at least 2 and typically more channels. Accordingly, it is impossible to limit analysis to patches with single channels in these experiments.

The new experiments described in response to Essential revisions point #3 (i.e., PHE increases Po when acutely applied from outside the recording electrode; Figure 4) indicates that we can detect an increase in Po when PHE is applied after seal formation. This approach complements the previous experiments when PHE was pre-applied before seal formation when more channels could be inserted into the plasma membrane during the time period between the start of the drug treatment and seal formation. In addition, we used in these new experiments recording pipettes with a smaller diameter (resistance was increased from originally 3.5-5.5 to 7-12 MOhm in these experiments), which typically yields between 1-4 channels. To confirm that the new recording conditions did typically not yield more than 4 channels, BayK8644 was added at the end of the recordings in one set of experiments (Figure 6) to ensure that our quantification of channel number N is accurate and complete. Accordingly, this approach allowed us to define patches with <4 channels for which we can reliably extract N and thereby also Po. Thus, we can now be certain that PHE specifically augments Po and not more generally NPo.

5) Line 147, it is unclear why other Gq coupled receptors do not affect this pathway in hippocampal neurons. This is problematic as the study goes on to use bradykinin, rather than an α1-AR agonist in PC12 cells to dissect the pathway (Figure 5 onwards). Does bradykinin stimulate this pathway in hippocampal neurons?

We now document that bradykinin can augment Po of LTCC in hippocampal neurons (new Figure 6). Please see response to Essential revisions point #2 for additional details.

6) If the same pathway is present in PC12 cells then the α1-AR should be expressed in these cells, rather than using bradykinin.

Systematic radioligand binding studies and functional stimulation assays did not detect any evidence for the presence of any adrenergic receptor subtypes in PC12 cells (neither alpha1, alpha2 or β AR; Williams et al., 1998: J Biol Chem 273, 24624-24632). In fact, this publication and several subsequent studies reported the use of PC12 cells to heterologously express AR subtypes to study the signaling mechanisms and functional effects of individual subtypes (e.g., Zhong and Minnemann, 1999: J Neurochem 72, 2388-2396; Olli-Lähdesmäki et al., 1999: J Neurosci. 19, 9281-9288). This strategy speaks to the relevance of the concept of ‘transposable’ signaling modules between different GqPCRs, which can engage PKC-Pyk2-Src signaling with the trimeric Gq – phospholipase C β – PKC module being the common denominator that then triggers the rest of the cascade in some but may be not all contexts.

Because our main goal is to define how Cav1.2 is regulated in neurons, for the revision we opted to test Bradykinin in neurons, as suggested by the Reviewing Editor. Also, we had tested in both experimental systems the effect of direct PKC activation by the phorbol ester PMA and of the different Pyk2 and Src inhibitors with completely congruent results (for details, please see response to point #2 by the Reviewing Editor).

7) It is unclear why PP2 inhibits NPo and ensemble average current far below the baseline control (Figures2B and 2D), whereas SU6656 does not. This suggests non-specificity.

This differential effect of PP2 versus SU6656 could hint that Src is not the only Src family kinase (SFK) that is involved with other SFKs perhaps also playing a role as they have different sensitivities to PP2 and SU6656 (see, e.g., Blake et al., 2000: Mol Cell Biol. 20, 9018-9027; Bain et al., 2007: Biochem J 408, 297-315). However, this effect was not consistently observed. In detail, the PP2 effect is not as strong and the SU6656 stronger in Figure 3 compared to Figure 2 (although both inhibitors reduce Po below baseline levels in both figures). These observations are potentially reflecting some experimental or biological variability between experiments as these data were collected over a long time period (>3 years) because these experiments are very time consuming and dependent on, among other factors, obtaining good hippocampal cultures and having very low electric noise in the recording cage, both of which are not trivial. This variability provides an alternative explanation to the notion that another SFK member could be involved.

8) The II-III linker is identified as a binding site for Pyk2; but where are the src phosphorylation sites on CaV1.2 specifically mediating this effect? This needs to be identified, otherwise it is possible that the effect is not direct.

Although Gq-PKC-Pyk2-Src signaling clearly induces tyrosine phosphorylation of the Cav1.2 alpha1 subunit, we cannot exclude that our effects on Po are indirect, i.e., through phosphorylation of another subunit or channel component. However, we feel that the Po effects are of strong functional interest, whether due to direct or indirect phosphorylation. The biochemical work provides now impetus that justifies testing whether phosphorylation of Cav1.2 is mediating the functional effect (we are currently applying for funding for continuation of this project). However, given the already huge time commitment of the current work it seems outside the scope of the current project to determine the relevant phosphorylation sites, which can take years.

As stated in response to Essential revisions point #1 we were not able to define a synthetic peptide derived from loop II/III that would bind Pyk2 and thus cannot use a peptide displacement approach to acutely test the relevance of Pyk2 binding to Loop II/III.

9) A diagram of the pathway is important to add, preferably in each figure, to show where every drug is supposed to act.

We now added a scheme to each figure to make it easier for readers to follow the pathways and experiment design and data interpretation.

Reviewer #2 (Recommendations for the authors):

Overall, the work is carefully carried out but there are a few pitfalls, where information lacking. I suggest the authors to address these issues.

1. The pathway from PKC/ca²⁺, Pyk2, to Src has been already reported more than 20 years ago (see, for example, Sabri et al., Circ. Res 1998 and reference therein). But in this paper, the mechanism how noradrenaline activates Pyk2 through PKC is not known. Also the authors stated that phosphorylation of alpha11.2 by PKC inhibits Cav1.2 activity (52). Why this pathway is not active here?

In addition to Sabri et al., 1998, the PKC-Pyk2-Src signaling cascade was first identified in PC12 cells (Lev et al., 1995: Nature 376, 737-745; Dikic et al., 1996: Nature 383, 547-549) but no previous work linked this signaling cascade to Ca channel regulation. The primary importance of our work is to define whether and how alpha1 AR signaling regulates Cav1.2 in neurons because norepinephrine is a key neuromodulator that governs attention and, at higher levels, stress responses. The role of alpha1 AR in regulating neuronal Cav1.2 is indicated by our finding that the alpha1 AR-selective agonist PHE increases Po and that this increase is blocked by the alpha1 AR-selective antagonist prazosin. Another important aspect is to define regulation of Cav1.2 by the PKC-Pyk2-Src cascade with PKC, Pyk2, and Src forming a signaling complex with Cav1.2 on a functional and biochemical level as discussed in the manuscript.

As we now point out explicitly in the Introduction, the sites for direct phosphorylation of Cav1.2 by PKC (S27 and S31) that inhibit channel activity (McHugh et al., 2000) (former Ref 52) are located in a differentially spliced exon that is prominently expressed in heart but not neurons as further detailed by (Snutch et al., 1991) to explain why PKC does not inhibit Cav1.2 in neurons. We state: “T27/T31 are not present in the most prevalent brain isoform due to alternative splicing (Snutch et al., 1991), thus the inhibitory effect of PKC on LTCC currents is typically absent in neurons and neural crest-derived PC12 cells, or in vascular smooth muscle (Navedo et al., 2005; Taylor et al., 2000).”

2. The mechanism how Src activates Cav1.2 is not clear. The authors should make more effort to identify the site and use a mutant to show that it abolishes the increase in the channel activity. They discussed possible tyrosine phosphorylation at Y2122 but they did not confirm this. Also, this residue is located at the very end of protein, far away from the channel. It is not clear how it modulates the channel activity, if it has any function.

Please see response to Essential revisions point #1 and point #7 of Reviewer 2. Y2122 is only seen in rodents but not present in other mammals including humans. Y2122 is, thus, unlikely to be a general major regulatory site, which we now discuss more explicitly. Furthermore, as the Reviewer points out, it is very distal to the channel. Finally, perhaps we should emphasize that determining regulation of Cav1.2 by either cAMP/PKA or Gq/PKC signaling has been hampered for the last 3 decades in the many different labs that had been working on these issues by not being able to consistently and reproducibly being able to reconstitute either regulatory mechanism for Cav1.2 in HEK293 or other cell lines (mostly personal communications from multiple PIs but see some primary data and discussion of this issue in our review Dai, Hall, and Hell 2009: Physiol Rev 89, 411-452; p420, left column; see also Man, Bartels, Horne, and Hell, 2020: Sci Signal 13, eabc6438). Accordingly, we cannot readily express WT and mutant Cav1.2 (Iike Y2122F) in HEK293 cells and test whether regulation by PKC-Pyk2-Src is affected or not.

Reviewer #3 (Recommendations for the authors):

The authors should consider adding experiments that show whether the linker between domains II and III is indeed the site of regulation.

Please see response to Essential revisions point #1 and #8 by Reviewer 1. Briefly, we attempted but were not able to identify shorter loopII/III-derived peptides that would constitute the Pyk2 binding site.

Editorial Points

Lines 203-207. These sentences are a bit garbled. Please revise.

We revised this section mostly by simplifying the statement, which now reads: “Kinases and proteins that regulate kinase activity are often found in complexes with their ultimate target proteins (i.e., their ultimate substrates) including different ion channels for efficient and specific signaling (Dai et al., 2009; Dodge-Kafka et al., 2006).”

Lines 272-273. Better wording would be …"binds via phosphoY402 to the SH2 domain".

We revised this statement accordingly.

Line 503. Better wording would be "regulate through".

We revised this statement accordingly.