To identify a pharmacological strategy to distinguish Kv2/KvS heteromeric conductances from other endogenous neuronal conductances, we determined the Kv2/KvS selectivity of known Kv2 inhibitors. To test whether Kv2 inhibitors also inhibit Kv2/KvS heteromeric channels, we transfected KvS cDNA into a stable cell line which was subsequently induced to express Kv2.1 (Kv2.1-CHO) and later recorded whole cell currents. We previously found that 1 μM RY785 or 100 nM GxTX blocked almost all the voltage-gated K⁺ conductance of this Kv2.1-CHO cell line, with 1±2% or 0 ± 0.1% (mean ± SEM) current remaining respectively at 0 mV (Tilley et al., 2019; Marquis and Sack, 2022). To test the pharmacological response of KvS we began with Kv8.1, a subunit that creates heteromers with biophysical properties distinct from Kv2 homomers (Salinas et al., 1997a), and modulates motor neuron vulnerability to cell death (Huang et al., 2024). After transfection of Kv8.1 into Kv2.1-CHO cells, we find that a sizable component of the delayed rectifier current became resistant to 1 μM RY785 (Figure 1A). This RY785-resistant current was inhibited by 100 nM GxTX, suggesting that the GxTX-sensitivity arises from inclusion of Kv2.1 subunits in the channels underlying the RY785-resistant current. A simple interpretation is that RY785-resistant yet GxTX-sensitive currents are carried by Kv2.1/Kv8.1 heteromeric channels. The fraction of RY785-resistant current had a pronounced cell-to-cell variability (Figure 1D). Co-expression of KvS and Kv2 subunits can result in Kv2 homomers and Kv2/KvS heteromers (Pisupati et al., 2018), and we presume variability in the RY785-sensitive fraction results from cell-to-cell variability in the proportion of Kv8.1-containing channels.

As a control, we transfected Kv2.1-CHO cells with Navβ2, a transmembrane protein not expected to interact with Kv2.1. In Kv2.1-CHO cells transfected with Navβ2, 1 μM RY785 efficiently blocked Kv2.1 conductance, leaving 4 ± 0.6% (mean ± SEM) of current (Figure 1B and D). We also transfected Kv2.1-CHO cells with a member of the AMIGO family of Kv2-regulating transmembrane proteins. AMIGO1 promotes voltage sensor activation of Kv2.1 channels in these Kv2.1-CHO cells (Sepela et al., 2022). 1 μM RY785 blocked Kv2.1 conductances in cells transfected with AMIGO1, leaving 0.6 ± 1% (SEM) of current (Figure 1C and D). These control experiments indicate that transfection of a set of other transmembrane proteins did not confer resistance to RY785, suggesting that the RY785 resistance is not generically induced by overexpression of non-KvS transmembrane proteins.

To determine whether Kv8.1-containing channels are completely resistant to RY785, we performed an RY785 concentration-effect experiment. To pre-block Kv2.1 homomers, we began concentration-effect measurements at 0.35 μM RY785, which we expect to block 98% of homomers based on the estimated K_D of 6 nM RY785 for the Kv2.1 currents in these Kv2.1-CHO cells (Marquis and Sack, 2022). Notably, currents resistant to 0.35 μM RY785 were blocked by higher concentrations of RY785, with nearly complete block observed in 35 μM RY785 (Figure 2A). We quantified block of tail currents at –9 mV following a 200ms step to +71 mV, and normalized to current from the initial 0.35 μM RY785 treatment. This protocol revealed an IC₅₀ of 5±1 μM (SD; Figure 2B). The Hill coefficient of 1.2±0.2 is consistent with 1:1 binding to a homogenous population of RY785-inhibited channels. We had noted that solution exchanges can change current amplitudes, and interleaved time-matched solution exchange controls. These controls revealed variable current rundown of approximately 30% on average (Figure 2C). Time-matched control washes were followed by treatment with 35 μM RY785 to confirm that currents in these cells had similar RY785 sensitivity to those in our concentration-effect experiment. Following block by 35 μM RY785, washing with 0.35 μM RY785 caused increases in current amplitudes in every trial (Figure 2D). The run-down and incomplete wash-out indicates that the 5 μM IC₅₀ of RY785 for these resistant channels may be an underestimate. Kv2.1/Kv8.1 currents were unblocked in the first current test following RY785 washout (Figure 2—figure supplement 1). This rapid recovery indicates that unblocking of Kv2.1/Kv8.1 heteromers occurred during the less than 3 min wash time at –89 mV, or that RY785 unblocked on the millisecond time scale during activating voltage pulses. This is distinct from Kv2.1 homomers, where RY785 becomes trapped in deactivated channels and unblocks much more slowly, with a time constant of about 2 hr at –92 mV or 100 s at +28 mV (Marquis and Sack, 2022). The dramatically faster unblock from Kv2.1/Kv8.1 is consistent with the weaker affinity observed for RY785. Overall, the results suggest that Kv8.1-containing channels in these Kv2.1-CHO cells form a pharmacologically homogenous population with an affinity for RY785 ~3 orders of magnitude weaker than Kv2.1 homomers. Our estimates of the affinities of Kv2.1 homomeric and Kv2.1/Kv8.1 heteromeric channels for RY785 suggest that ~1 μM RY785 elicits nearly complete block of Kv2.1 homomer conductance while blocking little Kv2.1/KvS heteromer conductance.

We wondered whether RY785 block of Kv2 homomers could better reveal the gating of heteromers. Previous studies have identified that Kv2/Kv8.1 gating kinetics are distinct from Kv2 homomers (Salinas et al., 1997a), and we set out to identify whether RY785-resistant currents from Kv8.1 transfected CHO cells had similarly modulated kinetics. While concentrations of RY785 that partially block Kv2.1/Kv8.1 modified the kinetics of voltage-dependent gating, suggesting state-dependent block, we did not observe modification of kinetics with 3.5 μM or lower concentrations of RY785 (Figure 3). To study the biophysical properties of the Kv8.1 conductance in the Kv2.1-CHO cells, we analyzed currents in 1 μM RY785 to block the Kv2.1 homomers. For comparison, Kv2.1-CHO cells were transfected with a control plasmid and treated with a DMSO vehicle control. We stepped cells to –9 mV from a holding potential of –89 mV and fit the current rise with an exponential function (equation 1). Cells transfected with Kv8.1 and blocked with 1 μM RY785 had a significantly slower activation time constant and lower sigmoidicity (shorter relative activation delay) than those only expressing Kv2.1 (Figure 4A–C). Conductance-voltage relations were fit with a Boltzmann function (equation 2) revealing that the half-maximal conductance of Kv8.1-transfected cells is shifted positive relative to Kv2.1 alone (Figure 4D and E). We did not detect a significant difference in the steepness (z) of the conductance-voltage relation (Figure 4F). Currents from Kv8.1-transfected cells inactivated less during a 10 s step to –9 mV (Figure 4G and H). However, the steady-state inactivation of Kv8.1-transfected cells was shifted to more negative voltages and is less steep than Kv2.1-transfected cells (Figure 4I–K). We did not observe substantial changes in current amplitude in cells treated with DMSO vehicle control (Figure 4—figure supplement 1). Overall, the biophysical properties reported here are consistent with a previous report which identified that co-expression of Kv8.1 with Kv2.1 in Xenopus oocytes slows the rate of activation, reduces inactivation and shifts steady-state inactivation to more negative voltages (Salinas et al., 1997a). This previous report also identified a positive shift in the conductance-voltage relation when Kv8.1 is co-expressed with a Kv2 subunit (Kv2.2), similar to our findings with Kv2.1/Kv8.1. Together these results show that RY785-resistant currents in cells transfected with Kv8.1 are distinct from Kv2.1 homomer currents and have changes in gating consistent with prior reports of Kv8.1/Kv2 biophysics. This validates using RY785 block of Kv2 homomers as a method to reveal the biophysics of a Kv8.1-containing population.

To test if RY785 resistance and GxTX sensitivity of Kv8.1 is shared broadly by KvS subunits, we similarly assessed subunits of Kv5, Kv6, and Kv9 subtypes: Kv5.1, Kv6.4, and Kv9.3. Each of these KvS subunits create Kv2/KvS heteromers that have distinct biophysical properties (Kramer et al., 1998; Kerschensteiner and Stocker, 1999; Bocksteins et al., 2012). Kv5.1/Kv2.1 heteromers play an important role in controlling the excitability of mouse urinary bladder smooth muscle (Malysz and Petkov, 2020), mutations in Kv6.4 have been shown to influence human labor pain (Lee et al., 2020), and deficiency of Kv9.3 disrupts parvalbumin interneuron physiology in mouse prefrontal cortex (Miyamae et al., 2021). We transfected Kv5.1, Kv6.4, and Kv9.3, to determine whether they also produced delayed rectifier current resistant to 1 μM RY785 yet sensitive to 100 nM GxTX (Figure 5A–C). We observed that, in 1 μM RY785,>10% of the voltage-gated current remained in 12/13 Kv5.1, 9/14 Kv6.4, and 5/5 Kv9.3 transfected cells (Figure 5D). Like Kv8.1, the fraction of RY785-resistant current had pronounced cell-to-cell variability (Figure 5D) suggesting that the RY785-sensitive fraction could be due to different ratios of functional Kv2.1 homomers to Kv2.1/KvS heteromers. Addition of 100 nM GxTX blocked RY785-resistant current from cells transfected with each of these KvS subunits. A slightly higher fraction of Kv9.3 current remained in 100 nM GxTX, possibly due to Kv9.3 negatively shifting the midpoint of the conductance voltage relationship (Kerschensteiner and Stocker, 1999). While a fraction of KvS subunits appear to be retained intracellularly, immunofluorescence for Kv5.1, Kv9.3, and Kv2.1 also appeared localized to the perimeter of transfected Kv2.1-CHO cells (Figure 5—figure supplement 1). These results show that voltage-gated outward currents in cells transfected with members from each KvS subtype have decreased sensitivity to RY785 but remain sensitive to GxTX. While we did not test every KvS subunit, the ubiquitous resistance suggests that all KvS subunits may provide resistance to 1 μM RY785 yet remain sensitive to GxTX, and that RY785 resistance is a hallmark of KvS-containing channels.

We set out to assess whether RY785 together with GxTX could be a means of distinguishing endogenous Kv2/KvS heteromers from Kv2 channels in native neurons. We first designed experiments to test whether RY785 could inhibit endogenous Kv2 currents in mice, by studying neurons unlikely to express KvS subunits. Rat superior cervical ganglion (SCG) neurons have robust GxTX-sensitive conductances (Liu and Bean, 2014) yet transcriptomics have revealed little evidence of KvS expression (Sapio et al., 2020). We investigated whether SCG neurons have conductances consistent with Kv2/KvS heteromers. As functional characterization alone cannot be trusted to classify their channel mediators of conductances, we define conductances consistent with Kv2/KvS heteromers as 'KvS-like' and conductances consistent with Kv2 homomers as 'Kv2-like'. To help isolate Kv2-like and KvS-like currents, we bathed SCG neurons in a cocktail of Nav, Cav, Kv1, Kv3, and Kv4 inhibitors then recorded voltage-gated currents. We found that exposing SCG neurons to 1 μM RY785 inhibited most of the voltage-gated current, and subsequent addition of 100 nM GxTX to the same neurons inhibited little additional current (Figure 6A and B). To quantify inhibition, we analyzed tail currents 10ms after repolarizing to –45 mV. This time window was chosen because a hallmark of Kv2 currents is relatively slow deactivation (Thorneloe and Nelson, 2003; Zheng et al., 2019). Tail currents after application of 1 μM RY785 were decreased by 88 ± 5% (mean ± SEM) in SCG neurons (Figure 6B). Subsequent application of 100 nM GxTX had little further effect. To determine if the RY785-sensitive conductances are consistent with previous reports of Kv2 channels, we examined the biophysical properties of the Kv2-like (RY785-sensitive) currents defined by subtraction (Figure 6C). Current activation began to be apparent at –45 mV and had a conductance that was half maximal at –11 mV (Figure 6D). The faster component of deactivation of Kv2-like currents in SCG neurons had a time constant of 16ms±0.6 (mean ± SEM) at –45 mV (Figure 6E). These results are consistent with reported biophysical properties of Kv2 channels (Kramer et al., 1998; Liu and Bean, 2014; Tilley et al., 2019; Sepela et al., 2022). Together these results show that 1 µM RY785 almost completely inhibits endogenous Kv2-like conductances in these mouse neurons, consistent with mouse SCG neurons having few, if any, functional Kv2/KvS heteromers. We cannot rule out that the small amount of current remaining after RY785 (12% of the control) is due to Kv2/KvS heteromers, but its insensitivity to GxTX suggests that it may instead be current from a non-Kv2 channel remaining in the cocktail of K-channel inhibitors.

To determine if RY785/GxTX pharmacology could reveal endogenous KvS-containing channels, we next studied neurons likely to express KvS subunits. Mouse dorsal root ganglion (DRG) somatosensory neurons express Kv2 proteins (Stewart et al., 2024), have GxTX-sensitive conductances (Zheng et al., 2019), and express a variety of KvS transcripts (Bocksteins et al., 2009; Zheng et al., 2019), yet transcript abundance does not necessarily correlate with functional protein abundance. To record from a consistent subpopulation of mouse somatosensory neurons which has been shown to contain GxTX-sensitive currents and have abundant expression of KvS mRNA transcripts (Zheng et al., 2019), we used a Mrgprd^GFP transgenic mouse line which expresses GFP in nonpeptidergic nociceptors (Zylka et al., 2005; Zheng et al., 2019). Deep sequencing identified that mRNA transcripts for Kcnf1 (Kv5.1), Kcng2 (Kv6.2), Kcng3 (Kv6.3), and Kcns1 (Kv9.1) are present in GFP⁺ neurons of this mouse line (Zheng et al., 2019) and we confirmed the presence of Kcnf1 (Kv5.1) and Kcns1 (Kv9.1) transcripts in GFP⁺ neurons from Mrgprd^GFP mice using RNAscope (Figure 7—figure supplement 1). We investigated whether these neurons have conductances consistent with KvS-like pharmacology by performing whole cell voltage clamp on cultured DRG neurons that had clear GFP fluorescence. Voltage-clamped neurons were bathed in the same cocktail of channel inhibitors used on SCG neurons, plus the Nav1.8 inhibitor A-803467. Application of 1 μM RY785 inhibited outward currents somewhat, but unlike in SCG neurons, a prominent delayed-rectifier outward conductance with slow deactivation remained (Figure 7A left panel). Tail currents in 1 μM RY785 decreased 29 ± 3% (mean ± SEM) (Figure 7B left panel). Subsequent application of 100 nM GxTX decreased tail currents by 68 ± 5% (mean ± SEM) of their original amplitude before RY785. We do not know the identity of the outward current that remains in the cocktail of inhibitors + RY785+GxTX. We observed variable current run-up or run-down but no significant effect of vehicle in blinded, interleaved experiments, while RY785 significantly decreased tail currents relative to vehicle controls (Figure 7A and B right panel). We do not know what conductances the vehicle solution exchange affects, the changes appear to be time-dependent or due to the solution exchange itself. Concurrent application of 100 nM GxTX and 1 μM RY785 to neurons in vehicle decreased currents by 69 ± 5% (mean ± SEM).

To determine if the RY785- and GxTX-sensitive conductances in GFP⁺ neurons from Mrgprd^GFP mice are consistent with previous reports of Kv2 homomeric or Kv2/KvS heteromeric channels, we examined the biophysical properties of the Kv2-like (RY785-sensitive) and KvS-like (RY785-resistant, GxTX-sensitive) currents defined by subtraction (Figure 7C). Obvious Kv2-like and KvS-like channel conductances began at –44 mV and had half-maximal conductances around –19 mV (Figure 7D), consistent with Kv2 and many KvS-containing channels (Kramer et al., 1998; Richardson and Kaczmarek, 2000; Sano et al., 2002; Thorneloe and Nelson, 2003). While changes in inactivation are prominent with KvS subunits, we did not investigate inactivation in neurons because the lengthy depolarizations required often resulted in irreversible leak current increases that degraded the accuracy of RY785/GxTX subtraction current quantification. We did note that the KvS-like currents deactivated slower than Kv2-like currents (Figure 7E), consistent with the effects of several KvS subunits whose transcripts are expressed in nociceptor DRG neurons. Kv5.1, Kv6.3, and Kv9.1 all slow deactivation of Kv2 conductances in heterologous cells (Salinas et al., 1997b; Kramer et al., 1998; Sano et al., 2002), and in Kv2.1-CHO cells transfected with Kv5.1, we confirmed that RY785-resistant currents deactivate slower than Kv2.1 controls (Figure 7—figure supplement 2). Together, these results indicate that, in these mouse DRG neurons, RY785-sensitive currents are Kv2-like, while RY785-resistant yet GxTX-sensitive currents are KvS-like. Under these conditions, 58 ± 3% (mean ± SEM) of the delayed rectifier conductance was resistant to RY785 yet sensitive to GxTX (KvS-like; Figure 7F). We note that the ratio of KvS- to Kv2-like conductances is expected to vary with holding potential, as KvS subunits can change the degree and voltage-dependence of steady state inactivation (e.g. Figure 4I).

We also tested the other major mouse C-fiber nociceptor population, peptidergic nociceptors, to determine if this subpopulation also has conductances resistant to RY785 yet sensitive to GxTX. We voltage clamped DRG neurons from a Calca^GFP mouse line that expresses GFP in peptidergic nociceptors (Gong et al., 2003). Deep sequencing has identified mRNA transcripts for Kcng2 (Kv6.2), Kcng3 (Kv6.3), Kcnv1 (Kv8.1) and Kcns3 (Kv9.3) present in GFP⁺ neurons, an overlapping but distinct set of KvS subunits from the Mrgprd^GFP non-peptidergic population (Zheng et al., 2019). In GFP⁺ neurons from Calca^GFP mice, we found that a fraction of outward current was inhibited by 1 µM RY785 and additional current inhibited by 100 nM GxTX (Figure 7—figure supplement 3A-C). In these experiments, 58 ± 2% (mean ± SEM) was KvS-like (Figure 7—figure supplement 3D) identifying that KvS-like conductances are present in these peptidergic nociceptors. For Calca^GFP neurons we did not include the Kv1, Kv3, Kv4, Nav, and Cav channel inhibitor cocktail used for other neuron experiments, indicating that the cocktail of inhibitors is not required to identify KvS-like conductances. Overall, these results show that the unique pharmacology of RY785 and GxTX can reveal endogenous KvS-like conductances in the major subtypes of mouse C-fiber nociceptors.

Human DRG neurons express Kv2 proteins (Stewart et al., 2024), and express KvS transcripts (Ray et al., 2018) suggesting that they may have Kv2/KvS conductances. We performed whole-cell voltage clamp on cultured human DRG neurons, choosing smaller-diameter neurons and using the same solutions as Mrgprd^GFP mouse DRG neuron recordings. In human DRG neurons, a fraction of outward current was inhibited by 1 µM RY785 and additional current was inhibited by 100 nM GxTX, consistent with the presence of KvS-like conductances (Figure 8A, B and C). These RY785- or GxTX-sensitive conductances became apparent near –44 mV and were half-maximal between –14 and –4 mV (Figure 8D). Of the total conductance sensitive to RY785 +GxTX in these human DRG neurons, 76 ± 2% (mean ± SEM) was KvS-like (Figure 8E). Unlike mouse neurons, we did not detect a significant difference in tail currents of RY785 versus vehicle controls. However, RY785-subtracted currents always had Kv2-like biophysical properties whereas vehicle-subtraction currents had variable properties that precluded the same biophysical analysis. Overall, these results show that human DRG neurons can produce endogenous voltage-gated currents with pharmacology and gating consistent with Kv2/KvS heteromeric channels.

Human dorsal root ganglia (DRG) were obtained from Sierra Donor Services. The donor was a 58-year-old Asian Indian female and DRG were from the 1^st and 2^nd lumbar region (cause of death: Stroke). DRG were extracted 6 hr after aortic cross clamp and placed in an ice cold N-methyl-D-glucamine-artificial cerebral spinal fluid (NMDG-aCSF) solution containing in mM: 93 NMDG, 2.5 KCl, 1.25 NaH₂PO₄, 30 NaHCO₃, 20 HEPES, 25 Glucose, 5 L-Ascorbic acid, 2 Thiourea, 3 Na pyruvate, 10 MgSO₄ and 0.5 CaCl₂ pH adjusted to 7.4 with HCl. Human DRG were obtained from the organ donor with full legal consent for use of tissue for research in compliance with procedures approved by Sierra Donor Services.

The CHO-K1 cell line transfected with a tetracycline-inducible rat Kv2.1 construct (Kv2.1-CHO; Trapani and Korn, 2003) was cultured as described previously (Tilley et al., 2014). Transfections were achieved with Lipofectamine 3000 (Life Technologies, L3000001). 1 μl Lipofectamine was diluted, mixed, and incubated in 25 μl of Opti-MEM (Gibco, 31985062). Concurrently, 0.5 μg of KvS or AMIGO1 or Navβ2, 0.5 μg of pEGFP, 2 μl of P3000 reagent and 25 μl of Opti-MEM were mixed. DNA and Lipofectamine 3000 mixtures were mixed and incubated at room temperature for 15 min. This transfection cocktail was added to 1 ml of culture media in a 24-well cell culture dish containing Kv2.1-CHO cells and incubated at 37 °C in 5% CO₂ for 6 hr before the media was replaced. Immediately after media was replaced, Kv2.1 expression was induced in Kv2.1-CHO cells with 1 μg/ml minocycline (Enzo Life Sciences, ALX-380–109 M050), prepared in 70% ethanol at 2 mg/ml. Voltage clamp recordings were performed 12–24 hr later. We note that the expression method of Kv2/KvS heteromers used here is distinct from previous studies which show that the KvS:Kv2 mRNA ratio can affect the expression of functional Kv2/KvS heteromers (Salinas et al., 1997b; Pisupati et al., 2018). We validated the functional Kv2/KvS heteromer expression using voltage clamp to establish distinct channel kinetics and the presence of RY785-resistant conductance in KvS-transfected cells and using immunohistochemistry to label apparent surface localization of KvS subunits (Figure 1, Figure 5—figure supplement 1, Figures 4 and 5). During recordings, the experimenter was blinded as to whether cells had been transfected with KvS, or Navβ2 or AMIGO1. Human Kv5.1, human Kv6.4 and human Kv8.1, AMIGO1-YFP, and pEGFP plasmids were gifts from James Trimmer (University of California, Davis, Davis, CA). Human Kv9.1 and human Kv9.3 plasmids were purchased from Addgene. Human Navβ2 plasmid was a kind gift from Dr. Alfred George (Lossin et al., 2002).

Voltage clamp was achieved with a dPatch amplifier (Sutter Instruments) run by SutterPatch software (Sutter Instruments). Solutions for Kv2.1-CHO cell voltage-clamp recordings: CHO-internal (in mM) 120 K-methylsulfonate, 10 KCl, 10 NaCl, 5 EGTA, 0.5 CaCl₂, 10 HEPES, 2.5 MgATP pH adjusted to 7.2 with KOH, 289 mOsm. CHO-external (in mM) 145 NaCl, 5 KCl, 2 CaCl₂, 2 MgCl₂, 10 HEPES pH adjusted to 7.3 with NaOH, 298 mOsm. Osmolality was measured with a vapor pressure osmometer (Wescor, 5520). The liquid junction potential of –9 mV between these solutions was accounted for. The liquid junction potential was calculated according to the stationary Nernst–Planck equation (Marino.M and Brogioli, 2014) using LJPcalc (RRID:SCR_025044). For voltage-clamp recordings, Kv2.1-CHO cells were detached in a PBS-EDTA solution (Gibco, 15040–066), spun at 500 × g for 2 min and then resuspended in 50% cell culture media and 50% CHO-external recording solution. Cells were then added to a recording chamber (Warner, 64–0381) and were rinsed with the CHO-external patching solution after adhering to the bottom of the recording chamber. Transfected Kv2.1-CHO cells were identified by GFP fluorescence and were selected for whole cell voltage clamp. Thin-wall borosilicate glass recording pipettes (Sutter, BF150-110-10) were pulled with blunt tips, coated with silicone elastomer (Sylgard 184, Dow Corning), heat cured, and tip fire-polished to resistances less than 4 MΩ. Series resistance of 2–14 MΩ was estimated from the Sutterpatch whole-cell parameters routine. Series resistance compensation between 13 and 90% was used to constrain voltage error to less than 15 mV; compensation feedback lag was 6 µs for most experiments or 100 µs for concentration-effect experiments. Capacitance and ohmic leak were subtracted using a P/4 protocol. Output was low-pass filtered at 5 kHz using the amplifier’s built-in Bessel and digitized at 25 kHz or, for concentration-effect experiments, 1 and 10 kHz. Experiments were performed on Kv2.1-CHO cells with membrane resistance greater than 1 GΩ assessed prior to running voltage clamp protocols while cells were held at a membrane potential of –89 mV. RY785 (gift from Bruce Bean, Harvard, or Cayman, 19813) was prepared in DMSO as a 1 mM stock for dilutions to 1 µM or a 35 mM stock for concentration-effect experiments. Stocks of GxTX-1E Met35Nle (Tilley et al., 2014) in water were 10 µM. Stocks were stored frozen and diluted in recording solution just prior to application to cells. Solutions were flushed over cells at a rate of approximately 1 ml/min. Concentrated RY785 and GxTX stocks were stored at –20 °C. Kv2.1-CHO cells were given voltage steps from –89 mV to –9 mV for 200ms every 6 s during application of RY785 until currents stabilized. When vehicle control was applied to cells, –9 mV steps were given for a similar duration. All RY785 solutions contained 0.1% DMSO. Vehicle control solutions also contained 0.1% DMSO but lacked RY785. Perfusion lines were cleaned with 70% ethanol then doubly-deionized water. For concentration-effect experiments, changes in current amplitude due to solution exchange were controlled for by treating every other tested cell with multiple washes of the same, 0.35 μM RY785 solution instead of increasing concentrations of RY785. The timing and duration of these control washes was similar to that of the washes in concentration-effect experiments.

Whole cell recordings from Mrgprd^GFP mouse and human neurons were performed using the same methods as CHO cell recordings with the following exceptions. Voltage clamp was achieved with a dPatch amplifier run by SutterPatch software or an AxoPatch 200B amplifier (Molecular Devices) controlled by PatchMaster software (v2x91, HEKA Elektronik) via an ITC-18 A/D board (HEKA Instruments Inc). Solutions for voltage-clamp recordings: internal (in mM) 140 KCl, 13.5 NaCl, 1.8 MgCl₂ 0.09 EGTA, 4 MgATP, 0.3 Na₂GTP, 9 HEPES pH adjusted to 7.2 with KOH, 326 mOsm. The external solution contained (in mM) 3.5 KCl, 155 NaCl, 1 MgCl₂, 1.5 CaCl₂, 0.01 CdCl₂, 10 HEPES, 10 glucose pH adjusted to 7.4 with NaOH, 325 mOsm. The calculated liquid junction potential of –4 mV between these solutions was accounted for. For voltage-clamp recordings, neurons on cover slips were placed in the same recording chamber used for CHO cell recordings and were rinsed with an external patching solution. Neurons from Mrgprd^GFP mice with green fluorescence were selected for recordings. Human DRG neurons with cell capacitances between 22.5 and 60 pF were used. After whole-cell voltage clamp was established, non-Kv2/KvS conductances were suppressed by changing to an external solution containing a cocktail of inhibitors: 100 nM alpha-dendrotoxin (Alomone) to block Kv1 (Harvey and Robertson, 2004), 3 μM AmmTX3 (Alomone) to block Kv4 (Maffie et al., 2013; Pathak et al., 2016), 100 μM 4-aminopyridine to block Kv3 (Coetzee et al., 1999; Gutman et al., 2005), 1 μM TTX to block TTX sensitive Nav channels, and 10 μM A-803467 (Tocris) to block Nav1.8 (Jarvis et al., 2007). It is possible that off target effects of blockers may introduce errors in the quantification Kv2/KvS heteromer-mediated K⁺ currents. For example, 4-aminopyridine is expected to block a small fraction, 2%, of Kv2 homomers and have a lesser impact on Kv2/KvS heteromers (Post et al., 1996; Thorneloe and Nelson, 2003; Stas et al., 2015) which could result in a slight overestimation of the ratio of Kv2/KvS heteromers to Kv2 homomers. After addition of 1 μM RY785, neurons were given 10 steps to –24 mV for 500ms to allow for voltage dependent block of RY785. Thin-wall borosilicate glass recording pipettes were pulled with blunt tips, coated with silicone elastomer, heat cured, and tip fire-polished to resistances less than 2 MΩ. Series resistance of 1–4 MΩ was estimated from the whole-cell parameters circuit. Series resistance compensation between 55 and 98% was used to constrain voltage error to less than 15 mV. Ohmic leak was not subtracted. Neurons were held at a membrane potential of –74 mV to mimic a physiological resting potential. KvS subunits can profoundly shift the voltage-inactivation relation (Salinas et al., 1997a; Kramer et al., 1998; Kerschensteiner and Stocker, 1999) and this potential is likely insufficiently negative to relieve inactivation from all Kv2/KvS heteromeric channels. Also, the activation membrane potential is close to the half-maximal point of Kv2/KvS conductances. Thus the ratio of Kv2-like to KvS-like conductance is expected to vary with voltage protocols.

Whole cell recordings from Calca^GFP mice were made using the same methods as Mrgprd^GFP mice with the following exceptions. The internal solution contained in mM: 139.5 KGluconate, 1.6 MgCl₂, 1 EGTA, 0.09 CaCl₂, 9 HEPES, 14 creatine phosphate, 4 MgATP, 0.3 GTP pH adjusted to 7.2 with KOH. The external solution contained in mM: 155 NaCl, 3.5 KCl, 1 MgCl₂, 1.5 CaCl₂, 10 glucose, 10 HEPES pH adjusted to 7.4 with NaOH and did not contain the cocktail of inhibitors used for recordings with Mrgprd^GFP mice. The liquid junction potential of –13 mV between these solutions was accounted for. Ohmic leak was subtracted. After establishing whole-cell recording, cells were lifted and placed in front of a series of quartz fiber flow pipes for rapid solution exchange and application of RY785 and GxTX.

Whole cell recordings from mouse superior cervical ganglion neurons were performed using an Axon Instruments Multiclamp 700B Amplifier (Molecular Devices). Electrodes were pulled on a Sutter P-97 puller (Sutter Instruments) and shanks were wrapped with Parafilm (American National Can Company) to allow optimal series resistance compensation without oscillation. Voltage or current commands were delivered and signals were recorded using a Digidata 1321 A data acquisition system (Molecular Devices) controlled by pCLAMP 9.2 software (Molecular Devices). The internal solution was (in mM): 140 mM K aspartate, 13.5 mM NaCl, 1.8 mM MgCl₂, 0.09 mM EGTA, 9 mM HEPES, 14 mM creatine phosphate (Tris salt), 4 mM MgATP, 0.3 mM Tris-GTP, pH 7.2 adjusted with KOH. The base external solution was the same as for DRG recordings. The calculated liquid junction potential of –15 mV between these solutions was accounted for. After establishing whole-cell recording, the cell was lifted and placed in front of a series of quartz fiber flow pipes for rapid solution exchange and application of RY785 and GxTX. The external solution used for recording Kv2 currents used the same cocktail of inhibitors for sodium channels and other potassium channels as for the DRG recordings except that A-803467 was omitted because the sodium current in SCG neurons is all TTX sensitive (Toledo-Aral et al., 1997).

Activation kinetics were fit from 10% to 90% of current (IK) rise with the power of an exponential function:(1)IK=A1-e-tτactσ

where A is the maximum current amplitude, τact is the time constant of activation, σ is sigmoidicity, and t is time. The t = 0 mark was adjusted to 100 μs after the start of the voltage step from the holding potential to correct for filter delay and cell charging.

Conductance values were determined from tail current levels at –9 mV after 200ms steps to the indicated voltage. Tail currents were the mean current amplitude from 1 to 5ms into the –9 mV step. Conductance–voltage relations were fit with the Boltzmann function:(2)f(V)=A(1+e−(V−V12)zFRT)−1

where V is voltage, A is amplitude, z is the number of elementary charges, F is Faraday’s constant, R is the universal gas constant, and T is temperature (held at 295 K).

Deactivation kinetics were fit with a double exponential:(3)f(x)=y0+A1e−(t−t0)τ1+A2e−(t−t0)τ2

Where t is time, y0 is the initial current amplitude, t0 is the start time of the exponential decay, τ1 and τ2 are the time constants, and A1 and A2 are the amplitudes of each component.

In CHO cells peak currents were analyzed because outward currents seem to offer the best signal/noise. In neurons, voltage gated currents remained in the toxin cocktail +RY785 and GxTX, that were sometimes unstable. To minimize complications from these currents, we restricted analysis of RY785 and GxTX subtraction experiments to tail currents at elapsed times to minimize complications from non-Kv2 endogenous voltage-gated channels which deactivate more quickly. We note that the analysis of conductance activation by using tail currents is only accurate when dealing with non-inactivating conductances. We expect that inactivation of Kv2/KvS conductances during the 200ms pre-pulse is minimal (Salinas et al., 1997a; Kramer et al., 1998; Kerschensteiner and Stocker, 1999) and did not notice inactivation during the activation pulse. Also, deactivation kinetics can vary in a heterogenous population of Kv2/KvS heteromers. While analysis of tail currents could skew the quantification of total Kv2 like and KvS-like conductances, our data supports that mouse nociceptors and human neurons have tail currents that are resistant to RY785 and sensitive to GxTX consistent with the presence of Kv2/KvS heteromers.

The rKv2.1 homomer (PDB ID: 8SD3; Fernández-Mariño et al., 2023) was energy minimized using cryo-EM structure refinement with Rosetta (Wang et al., 2016). This energy-minimized model was used for generation of the hKv2.1/hKv8.1 heteromer using ColabFold (Mirdita et al., 2022) with 48 recycles, dropout enabled, and Amber minimization (Salomon‐Ferrer et al., 2013). To provide the same pore conformation as input between homomer and heteromer docking, the Kv8.1 subunit from ColabFold was superimposed to a Kv2.1 subunit from the energy-minimized model used for homomer docking; this heteromer model was then energy minimized by fixing the Kv2.1 backbone and sidechain chi torsion angles while allowing the Kv8.1 backbone and sidechain chi torsion angles to minimize with Rosetta FastRelax (Tyka et al., 2011) using the BetaNov16 scoring function (Pavlovicz et al., 2020).

RY785 was extracted as a structure data file from PubChem (Kim et al., 2023). With Avogadro (Hanwell et al., 2012), RY785 underwent bond correction, protonation at pH 7.4, and energy minimization using the Merck molecular force field (Halgren, 1996a; Halgren, 1996b; Halgren, 1996c; Halgren, 1996d; Halgren and Nachbar, 1996e; Halgren, 1999a; Halgren, 1999b). Next, using the Antechamber protocol of AmberTools (Case et al., 2023), the partial atomic charge, atom, and bond-type assignments for each ligand were AM1-BCC corrected. The input conformer was generated using the RosettaGenFF (Park et al., 2021) crystal structure prediction protocol, taking the lowest energy packing arrangement as input.

Docking was performed using Rosetta’s GALigandDock (Park et al., 2021) for ion channel docking as described previously (Harris et al., 2024). Briefly, GALigandDock in the flexible docking mode was used with a padding value of 7 Å, 20 generations with a pool of 100 poses, and the entire pool of poses as output. With this run mode, the entire pore cavity had the potential to be sampled with side chain flexibility. We ran GALigandDock 100 times, generating 10,000 total models for both the rKv2.1 homomer and hKv2.1/hKv8.1 heteromer. The top 10 models were selected for visual inspection by sorting the top 10% of models by total score, followed with the top 10 of the subset by interface score. Contact analysis was performed using UCSF ChimeraX (Pettersen et al., 2021) Clashes and Contacts tool and the H-Bonds tool with default distance cutoff and acceptance settings.

Kv2.1-CHO cells were fixed for 15 min at 4 °C in 4% formaldehyde prepared fresh from paraformaldehyde in PBS buffer pH 7.4. Cells were then washed 3x5 min in PBS, followed by blocking in blotto-PBS (PBS, pH 7.4 with 4% (w/v) non-fat milk powder and 0.1% (v/v) Triton-X100) for 1 hr. Cells were incubated for 1 hr with primary antibodies diluted in blotto-PBS and subsequently washed 3x5 min in PBS. Antibodies used were mAb K89/34 for Kv2.1 (NeuroMab, RRID:AB_1067225), rabbit pAb 5.1 C for Kv5.1 (in-house, RRID:AB_3076240), and rabbit anti-V5 for Kv9.3-V5 (Rockland, 600-401-378). For surface labeling of Kv5.1, non-permeabilized cells were incubated with Kv5.1 mAb (Santa Cruz Biotech, 81881) in blotto-PBS lacking Triton-X100. The cells were then incubated with mouse IgG subclass- and/or species-specific Alexa‐conjugated fluorescent secondary antibodies (Invitrogen) diluted in blotto-PBS for 45 min and washed 3x5 min in PBS. Cover glasses were mounted on microscope slides with Prolong Gold mounting medium (Thermo Fisher, P36930) according to the manufacturer’s instructions. Widefield fluorescence images were acquired with an AxioCam MRm digital camera installed on a Zeiss AxioImager M2 microscope with a 63×/1.40 NA Plan-Apochromat oil immersion objective and an ApoTome coupled to Axiovision software version 4.8.2.0 (Zeiss, Oberkochen, Germany).

A 6-week-old Mrgprd^GFP mouse was briefly anesthetized with 3–5% isoflurane and then decapitated. The spinal column was dissected, and the left and right L1 DRG were removed and drop fixed for 12 min in ice cold 4% paraformaldehyde in 0.1 M phosphate buffer (PB) pH adjusted to 7.4. The L1 vertebrae was identified by the 13^th rib. The DRG was washed 3×10 min each in PB and cryoprotected at 4 °C in 30% sucrose diluted in PB for 2 hr. The DRG were then frozen in Optimal Cutting Temperature (OCT) compound (Fisher, 4585) and stored at –80 °C until sectioning. Samples were cut into 20 μm sections on a freezing stage sliding microtome and were collected on Colorfrost Plus microscope slides (Thermo Fisher Scientific, 12-550-19). Sections were processed for RNA in situ detection using an RNAscope Fluorescent Detection Kit according to the manufacturer’s instructions (Advanced Cell Diagnostics) with the following probes: KCNF1 (508731, mouse) or KCNS1 (525941, mouse). TSA Vivid 650 Fluorophore was used to label probes (TSA Vivid, 7527). Following in situ hybridization, immunohistochemistry to label GFP was performed. Sections were incubated in vehicle solution (4% milk, 0.2% triton diluted in PB) for 1 hr at RT. Tissue was then incubated in a rabbit polyclonal anti-GFP antibody (Rockland 600-401-215S) diluted 1:1000 in vehicle overnight at 4 °C. Sections were washed three times in vehicle for 5 min per wash and then incubated in a goat anti-rabbit secondary antibody (Invitrogen, A-11008) diluted 1:1500 in vehicle. Sections were then mounted with Prolong Gold (Thermo Fisher, P36930) and #1.5 cover glass (Thermo Fisher Scientific, NC1776158).

Images were acquired with an inverted scanning confocal and airy disk imaging system (Zeiss LSM ⁸⁸⁰ Airyscan, ⁴¹⁰⁹⁰⁰-247-075) run by ZEN black v2.¹. Laser lines were 488 nm and 633 nm. Images were acquired with a ^0.8 NA 20 x objective (Zeiss, 420650–9901) details in figure legends.

All statistical tests were performed in Igor Pro software version 8 (Wavemetrics, Lake Oswego, OR). Independent replicates (n) are individual cells/neurons while biological replicates (N) are individual mice. All tests were two-tailed. Wilcoxon rank tests were used for two-sample comparisons. Dunnett tests were used for multiple comparisons.