Studies reporting the Src-mediated phosphorylation of Tyr198 and Tyr308 in murine PANX1 occurred prior to the availability of PANX1 structures (Michalski et al., 2020; Deng et al., 2020; Qu et al., 2020; Jin et al., 2020; Mou et al., 2020; Ruan et al., 2020; Weilinger et al., 2016; DeLalio et al., 2019). We first mapped the corresponding residues, Tyr199 and Tyr309, into the apo state structure of hPANX1 (Figure 1A). Interestingly, both residues are located in positions close to the side tunnel of PANX1, an anion-selective permeation pathway responsible for the PANX1 basal current (Ruan et al., 2020). Tyr199 is part of the intersubunit interface of PANX1 and is partially buried, which may make it less accessible to Src kinase. Tyr309, on the other hand, is completely buried inside the protein. Therefore, phosphorylation of Tyr199 and Tyr309, if occurs, would require at least conformational changes or partial unfolding of the intracellular domain. Such structural alternations are unlikely to occur spontaneously without specific cellular conditions that favor this process. Accordingly, phosphorylation of either of these two residues is expected to cause major conformational changes of the intracellular domain due to the negative charges and the bulky size of the introduced phosphate group(s).

We next compared the sequences of PANX1 across different species (Figure 1B). Both Tyr199 and Tyr309, as well as several residues nearby, are conserved among different PANX1 orthologs (Figure 1B), suggesting that phosphotyrosine antibodies developed against one species are likely to cross-react with the others. Tyr199 is also conserved in PANX2 and PANX3, the other two members of the pannexin family. In contrast, Tyr309 is only conserved in PANX1 and PANX3, while PANX2 has a phenylalanine at this position and lacks a few residues preceding this residue (Figure 1B). Given the critical location of the two tyrosine residues, we decided to experimentally characterize their phosphorylation status in the presence of Src kinase.

To investigate the phosphorylation of hPANX1 by Src, we used two commercially available phosphor-specific antibodies that were used in previous studies to identify Tyr198 and Tyr308 as phosphorylation sites of murine PANX1 (Weilinger et al., 2016; Lohman et al., 2015; DeLalio et al., 2019) and were later reported to react with hPANX1 (Metz and Elvers, 2022). In addition to wild-type hPANX1, we tested phosphor-ablating mutants Y199F and Y309F (equivalent to mPANX1 Y198F and Y308F) as negative controls, which are expected to abrogate the antibody binding even in the presence of active Src kinase. As in the prior studies (Weilinger et al., 2016; DeLalio et al., 2019), we used mouse Src (mSrc), which has 99.4% pairwise sequence similarity to human Src. To increase the chance of detecting phosphorylated PANX1 and to introduce a negative control for mSrc, we generated two mSrc mutants, Y529F and K297M, which enhances and abolishes the kinase activity, respectively (Figure 2A). For the ease of fluorescence imaging, our hPANX1 and mSrc genes are tagged with GFP and mCherry tags, respectively (See Materials and Methods for detailed construct information), which allowed us to compare the in-gel fluorescence with the western blot signals.

From the non-transfected HEK293T cells, neither anti-PANX1-pY198 nor anti-PANX1-pY308 produced any signal, suggesting that the antibodies do not recognize endogenous proteins when hPANX1 and mSrc are absent (Figure 2B, lane 1). Similarly, from cells transfected with hPANX1 alone, including WT, Y199F, and Y309F, neither antibody produced any signal, despite the presence of clear GFP signals in the fluorescent gel indicating PANX1 expression (Figure 2B, lane 4–6). This is consistent with the notion that both antibodies do not recognize non-phosphorylated PANX1.

Unexpectedly, from another control sample of cells transfected with WT mSrc alone, anti-PANX1-pY198 yielded two prominent bands close to 75 kDa; anti-PANX1-pY308 also produced a band corresponding to the lower band of anti-PANX1-pY198, albeit much weaker (Figure 2B, lane 2). While the lower band corresponded to the fluorescent signal of mSrc (Figure 2B, lane 2), the upper band may represent endogenous proteins induced and/or phosphorylated by mSrc. The same signal pattern was detected with anti-PANX1-pY198 antibody from cells expressing the constitutively active mSrc-Y529F variant alone, although both bands were slightly upshifted (Figure 2B, lane 3). This upward shift was also observed in the fluorescent signal of mSrc-Y529F, suggesting that it may be due to a more extensive phosphorylation of mSrc-Y529F itself (lower band) and other endogenous proteins (upper band) associated with mSrc-Y529F (Figure 2B, lane 3). Interestingly, in addition to the band corresponding to the mSrc fluorescence signal, the anti-PANX1-pY308 antibody showed a major band at about 50 kDa (Figure 2B, lane 3), which may represent other sets of endogenous proteins induced and/or phosphorylated by mSrc. Taken together, our control experiments showed that both antibodies demonstrate promiscuous activity against mSrc and/or other unknown endogenous proteins, raising concerns as to whether these antibodies should be used to interpret the phosphorylation of PANX1.

Despite the caveats of the antibodies mentioned above, we proceeded to test whether the two antibodies could at least produce additional signals that may represent phosphorylated hPANX1 (when hPANX1 and mSrc are co-expressed). We designed several co-transfection experiments combining WT mSrc or its constitutively active variant (Y529F) with WT PANX1 or its phosphor-ablating mutants (Y199F and Y309F). All combinations yielded the same signal pattern as when mSrc was expressed alone (Figure 2B, lanes 7–9 vs 2, lanes 10–12 vs 3), although under some conditions the signals were weaker, which we attribute to the lower protein expression level indicated by the fluorescent signals (Figure 2B, lane 10). Even when the constitutively active mSrc was present, both antibodies failed to detect any signals corresponding to the GFP fluorescence signal of hPANX1 (Figure 2B, lanes 7–12). Furthermore, there was no difference in the signal pattern of WT hPANX1 and its two phosphor-ablating mutants, which is unexpected if these antibodies are indeed specific for phosphorylated PANX1.

Our hPANX1 construct contains a GFP tag at its very C-terminus, which is unlikely to affect PANX1 phosphorylation due to the flexibility of the C-terminus and its considerable distance from the proposed phosphorylation sites. Nevertheless, to thoroughly address this concern, we co-expressed a GFP-free version of hPANX1 with the constitutively active mSrc-Y529F, and performed western blot analysis. Our result showed that both anti-PANX1-pY198 and anti-PANX1-pY308 failed to detect hPANX1, regardless of the presence of the C-terminal GFP tag (Figure 2—figure supplement 1, lanes 3–6).

We next tested whether anti-PANX1-pY198 and anti-PANX1-pY308 antibodies could detect PANX1 phosphorylation in vitro, as demonstrated in the literature (DeLalio et al., 2019), using purified hPANX1 produced in-house and purified human Src with a C-terminal GST tag (Src-GST) purchased from a commercial source.

We first wanted to know whether purified hPANX1 could be phosphorylated by Src in an in vitro context. To this end, we used a pan-specific phosphotyrosine antibody, anti-pY100, which is expected to recognize a wide range of phosphorylated tyrosine sites. Our result clearly showed that anti-pY100 detected only Src (Figure 3A, lanes 7–12), which is known to be phosphorylated (Nada et al., 1991), but not PANX1 (Figure 3A, lanes 1–9), indicating that PANX1 is unlikely to be phosphorylated in our in vitro setting. Similar to anti-pY100, anti-PANX1-pY198 yielded a single band of the same size as the Src protein only in the presence of Src (Figure 3A, lanes 10–15). These data confirmed that anti-PANX1-pY198 recognizes only Src, but not PANX1.

Notably, anti-PANX1-pY308 appeared to detect both Src and hPANX1 (Figure 3A, lanes 16–18). However, the hPANX1 band was present even in the absence of Src (Figure 3A, lane 17), and was not detected by anti-pY100 (Figure 3A, lane 7–9). This led us to speculate that anti-PANX1-pY308 may recognize non-phosphorylated PANX1. To test this hypothesis, we performed in vitro phosphorylation experiments on hPANX1 mutants around Y309, including L306A, K307A, V308A, Y309F/A, E310A, I311A, and L312A (Figure 1B; Figure 3B and C). Our results showed that these mutations also abolished the detection of the hPANX1 band by anti-PANX1-pY308 (Figure 3B and C). Thus, our data indicate that the anti-PANX1-pY308 antibody recognizes a non-phosphorylated peptide epitope around Y309 of hPANX1.

We also speculate that anti-PANX1-pY308 prefers Src over hPANX1 as an epitope, because it recognized Src both in its purified form and in crude whole-cell lysate, whereas it only recognized hPANX1 in its purified form, but not in the whole-cell lysate (Figure 2B; Figure 3A–C). As a result, the Src signal appeared to dominate in the western blot using whole-cell lysate (Figure 2B). To test this hypothesis, we blotted whole-cell lysates from HEK293T cells expressing either the WT PANX1 or the Y308F variant. Anti-PANX1-pY308 indeed detect a weak band for WT PANX1, but not for the Y308F variant (Figure 3D).

Taken together, by using both cultured HEK293T cells and in vitro phosphorylation experiments, we demonstrated that the anti-PANX1-pY198 and anti-PANX1-pY308 antibodies are non-specific and may not be suitable for studying endogenous PANX1 protein phosphorylation.

Because commercially available phospho-specific antibodies failed to effectively recognize PANX1, we decided to use the Phos-tag gel to gauge the phosphorylation status of PANX1. The Phos-tag gel allowed for an effective separation of proteins based on their phosphorylation level (Nagy et al., 2018; Nishioka et al., 2021). We transiently transfected plasmids encoding hPANX1 and mSrc genes into HEK293T cells and analyzed the cell lysates by Phos-tag gel and western blot. Figure 4A clearly showed differential migration patterns of mSrc WT, Y529F, and K297M in Phos-tag gel. De-phosphorylating of mSrc by lambda protein phosphatase (λ-PPase) could partially convert the phosphorylated species into the non-phosphorylated protein (Figure 4—figure supplement 1), suggesting that Y529F and K297M mutation could affect the autophosphorylation of mSrc kinase itself (Figure 4A, lanes 3–5). The hPANX1, when expressed by itself, showed one prominent band in both Phos-tag gel and SDS-PAGE gel (Figures 4A and 3B, lane 2), which likely represents the non-phosphorylated hPANX1 due to the absence of mSrc. We next co-expressed WT hPANX1 with the three mSrc variants, and analyzed the cell lysates with Phos-tag gel to separate phosphorylated and unphosphorylated hPANX1. Contrary to our expectation, the GFP fluorescence only showed a monomeric band irrespective of mSrc activity, suggesting that hPANX1 is insensitive to phosphorylation by mSrc (Figure 4A, lanes 6–8 vs 2). To ensure that the expression levels of hPANX1 and mSrc were each comparable across the experimental conditions, we also analyzed the same cell lysate samples using regular SDS-PAGE gel followed by western blot and in-gel fluorescence and found no significant difference in protein expression (Figure 4B, lanes 2–8). Our PANX1 gene is tagged with GFP at its C-terminus. To investigate whether GFP would negatively affect the accessibility of the two tyrosine residues to mSrc, we also co-expressed WT hPANX1 without GFP with mSrc variants in HEK293T cells and analyze the cell lysate by Phos-tag gel and western blot analysis. No significant difference was observed between hPANX1 expressed alone or with mSrc variants in Phos-tag gel (Figure 4—figure supplement 2, lane 7–9 vs 3). Therefore, our Phos-tag gel analysis indicates that mSrc has no impact on hPANX1 phosphorylation.

However, the inability to detect phosphorylated hPANX1 by Phos-tag gel analysis may be due to the low level of phosphorylation. To address this possibility, we purified hPANX1 protein expressed alone or co-expressed with the constitutively active mSrc-Y529F variant, and conducted liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis on the samples, which offers high sensitivity for detecting phosphorylated species. While we unambiguously identified MS/MS spectra covered both Tyr199 and Tyr309 sites, the tyrosine residues are not phosphorylated even when hPANX1 is co-expressed with mSrc-Y529F (Figure 4—figure supplements 3–5). Interestingly, we observed hPANX1 Ser385 phosphorylation when mSrc-Y529F is present (Figure 4—figure supplement 6). However, out of 67 MS/MS spectra, only five spectra contain the phosphorylated Ser385 (~7.5%). We note that phosphorylated peptides may display a very different fragmentation and ionization efficiency compared to the non-phosphorylated counterparts (Dephoure et al., 2013). Therefore, the ratio of the MS/MS spectra does not represent the true phosphorylation level of hPANX1 protein. Our Phos-tag gel analysis suggests that the Ser385 phosphorylation could be at a very low level as we did not observe other major bands in Phos-tag gel when hPANX1 is co-expressed with mSrc-Y529F (Figure 4A). Nevertheless, our result clearly demonstrated that Tyr199 and Tyr309 of hPANX1 are not phosphorylated by mSrc, at least not to a detectable level, when transiently expressed in HEK293T cells.

Since our results conducted in HEK293T cells contradicted to what was known in the literature, we wanted to test whether our result was cell line dependent. To this end, we co-expressed hPANX1 and mSrc in Neuro2A cells, which were previously used as a model system to access the phosphorylation of Y308 by NMDAR activation (Weilinger et al., 2016). We noticed that hPANX1 protein expressed in Neuro2A cells was heavily glycosylated as indicated by its heterogeneous migration bands in regular SDS-PAGE gel (Figure 5A, lane 2). However, the multimeric bands can be reduced to a single band after PNGase F treatment, confirming that the heterogeneity is indeed due to N-linked glycosylation (Figure 5A, lane 3). Next, we tested the phosphorylation status of the samples using Phos-tag gel. Our result showed that deglycosylated hPANX1 migrates to the same location independent of mSrc, suggesting that Src does not alter the phosphorylation status of hPANX1 (Figure 5B, lanes 8, 10, 12 vs 3).

To rule out the possibility that hPANX1 may not be recognized by mouse Src, we repeated the transient transfection experiment using mPANX1 (Figure 6). Using Phos-tag gel, we failed to observe any difference between mPANX1 expressed alone or co-expressed with mSrc variants in HEK293T cells, indicating that mPANX1 behaves in the same way as hPANX1 and is not phosphorylated by mSrc (Figure 6A, lanes 6–8 vs 2). The expression levels of mPANX1 and mSrc are each comparable across different experimental conditions as judged by western blot and in-gel fluorescence image (Figure 6B).

We noted that the PANX1 antibody from Abcam (Catalogue number: ab124131) seems to detect other non-specific bands in our experiment, including a major band close to 100 kDa and a minor band at approximately 50 kDa (Figures 4B and 6B). To sort out the identity of the two bands, we carried out a de-glycosylation experiment on non-transfected HEK293T cells and hPANX1-expressing cells (Figure 6—figure supplement 1). Our result suggests that the 50 kDa band could be partially de-glycosylated and migrate to a location where overexpressed PANX1 protein is expected to stay, whereas the 100 kDa band is insensitive to PNGase F treatment (Figure 6—figure supplement 1). Thus, the lower band (50 kDa) likely represents endogenous PANX1 from HEK293T cells, while the 100 kDa band is a non-specific target of anti-PANX1 antibody. This result highlights the caveats/complications of solely relying on western blot to interpret the band identity. When combined with the fluorescence signal, it is possible to determine with more confidence which bands correspond to the full-length mPANX1 and mSrc protein in the SDS-PAGE gel.

To investigate whether hPANX1 channel activity is affected by the presence of active mSrc, we co-expressed GFP-tagged hPANX1 with the mCherry-tagged constitutively active mSrc-Y529F, and performed whole-cell patch clamp analysis. We targeted cells showing both green and red fluorescence, indicative of expression of both hPANX1 and mSrc proteins (Figure 7A). We did not observe any significant difference in the CBX-sensitive currents compared to the currents when hPANX1 was expressed alone (Figure 7B). This data strongly suggests that mSrc does not affect the gating of hPANX1 channel in HEK293T cells, further reinforcing the notion that PANX1 is not phosphorylated by Src.

Full-length human PANX1 in the pEGC Bacmam vector from our previous study was used (Ruan et al., 2020). Mouse PANX1 (UniprotID: Q9JIP4) was synthesized by GenScript and subcloned into the pEGC Bacmam vector (Goehring et al., 2014). The translated product contains the human or mouse PANX1 protein, a thrombin digestion site (LVPRGS), an enhanced GFP protein, and an 8 x His tag. pCMV5 mouse Src was a gift from Joan Brugge & Peter Howley (Addgene plasmid # 13663; http://n2t.net/addgene:13663; RRID:Addgene_13663). The mouse Src gene was subcloned into the pEGN Bacmam vector (Goehring et al., 2014). The translated product contains an mCherry protein, a thrombin digestion site, and the Src protein. Primers for site-directed mutagenesis were designed using QuikChange Primer Design website (https://www.agilent.com/store/primerDesignProgram.jsp) and synthesized by Eurofins Genomics. The QuikChange mutagenesis protocol was used to generate all the mutants of the study. Sanger sequencing was performed to identify positive clones.

HEK293T cells are purchased from Sigma-Aldrich (Catalogue Number: 96121229). Neuro2A cells are purchased from ATCC (Catalogue Number: CCL-131). The cells are authenticated and are mycoplasma contamination tested negative by the vendor.

Adherent HEK293T cells were grown in DMEM media supplemented with 10% fetal bovine serum. Transient transfection was conducted using lipofectamine-2000 by following the manufacturer’s protocol. Specifically, the cells were cultured in 60 mm Petri dishes until 80% confluency. Transfection solution was made by mixing 500 ng of plasmid DNA, 4 uL of lipofectamine-2000 reagent, and 100 uL Opti-MEM media. After 10 min incubation at room temperature, the DNA-lipid complexes were added to the cell culture and incubated at 37 °C. The next day, 10 mM sodium butyrate was added to the cells to boost protein expression. The cell culture was then grown at 30 °C for another day before harvesting. The cell pellet was flash-frozen with liquid nitrogen and stored at –80 °C. For co-transfection experiments, equal amounts of hPANX1 (250 ng) and mSrc (250 ng) plasmids were used in the transfection mixture. For Neuro2A cell culture, EMEM media is used instead of DMEM. All the other procedures described above are the same for expressing proteins in Neuro2A cells.

For the western blot experiment, the cell pellet was lysed in TBS buffer (20 mM Tris pH 8.0, 150 mM NaCl) with protease an inhibitor cocktail (1 mM phenylmethylsulfonyl fluoride, 2 mM pepstatin, 0.8 μM aprotinin and 2 μg/ml leupeptin), 1 mM sodium orthovanadate and 1% glyco-diosgenin detergent for 30 min on ice. The lysate was clarified by centrifugation at 21,000 rpm for 20 min and the soluble portions were mixed with 2 X SDS loading buffer supplemented with 5% 2-Mercaptoethanol. The samples were resolved in precast gradient Tris-Glycine gel (4–20%) or 7.5% PhosTag gel. A Chemidoc instrument was used to directly image the in-gel fluorescence signal after electrophoresis. Subsequently, the protein in the gel was transferred to the PVDF membrane using the semi-dry transfer buffer (48 mM Tris base, 39 mM glycine, 20% methanol). The membrane is blocked in the TBST buffer (20 mM Tris pH8.0, 150 mM NaCl, 0.1% Tween 80) with 4% non-fat milk for 1 h at room temperature. Afterward, primary antibodies (1:2000 dilution) were incubated with the membrane overnight at 4 °C. The next day, the membrane was washed with TBST buffer for four times, 10 min each before goat anti-rabbit/mouse IgG secondary antibodies were added (1:25000 dilution). After 1 hr, the secondary antibodies were discarded and the membrane was washed again with TBST for four times, 10 min each. The western blot signal was detected using ECL Pierce substrate and imaged using a Chemidoc instrument. A brightfield image was overlaid with the illuminance signal to visualize the position of protein markers in the membrane. The anti-PANX1 antibody is obtained from Abcam (Catalogue number: ab124131). The anti-Src (Catalogue number: 36D10) and anti-pY100 (Catalogue number: 9411) antibodies are obtained from Cell Signaling. The anti-PANX1-pY198 (Catalogue number: ABN1681) and anti-PANX1-pY308 (Catalogue number: ABN1680) antibodies were obtained from Millipore Sigma.

For de-glycosylation experiment, Neuro2A/HEK293T cells expressing designated genes were solubilized in TBS buffer with protease inhibitor cocktail (1 mM phenylmethylsulfonyl fluoride, 2 mM pepstatin, 0.8 μM aprotinin and 2 μg/ml leupeptin), 1 mM sodium orthovanadate and 1% glyco-diosgenin detergent for 30 min on ice. The samples were clarified by centrifugation at 21,000 rpm for 30 min. The de-glycosylation reaction was made by mixing 16 uL of the supernatant with 2 uL of PNGase F enzyme and 2 uL of GlycoBuffer 2 (10X). The control reaction replaced the PNGase F enzyme with water. The reaction was allowed to occur at room temperature overnight. The next day, the samples were mixed with 20 uL 2X SDS sample-loading buffer (BioRad) supplemented with 5% βME and resolved by SDS-PAGE. The gel was imaged in the ChemiDoc system by probing the GFP and mCherry fluorescence signal.

For the dephosphorylation experiment, mSrc-mCherry WT, Y529F, and K297M were expressed in HEK293T cells. The cells were lysed in TBS buffer (20 mM Tris pH8, 150 mM NaCl) with 1% glyco-diosgenin detergent for 30 min on ice. After clarifying the non-soluble debris by centrifugation at 21,000 rpm for 20 min, 20 μL supernatant was mixed with 2.5 μL of 10 X NEBuffer for Protein MetalloPhosphatases, 2.5 μL 10 mM MnCl₂ and 2 μL Lambda Protein Phosphatase (NEB). The dephosphorylation reaction was allowed to occur at 30 °C overnight. Control samples without adding Lambda Protein Phosphatase are placed on ice. The next day, the samples were mixed with 2X SDS sample-loading buffer (BioRad) supplemented with 5% βME and resolved by SDS-PAGE or Phos-tag gel. The gel was imaged in the ChemiDoc system by probing the mCherry fluorescence signal or western blot analysis.

For in vitro phosphorylation and mass-spectrometry analysis, the hPANX1 WT or mutants were expressed alone or co-expressed with mSrc Y529F mutant in HEK293T cells. Specifically, 50 μg of plasmid DNA (in the case of the co-expressing experiment, 25 μg of human PANX1 plasmid and 25 μg of mouse Src Y529F mutant plasmid was used) was mixed with 150 ug of PEI 25 K (Polysciences) in a and incubated at room temperature for 30 min. The DNA-PEI complex was then added to the suspension cell culture of HEK293T cells at a density of 2×10⁶ cells/ml in FreeStyle medium (Gibco). After growing at 37 °C for 8 hr, 10 mM sodium butyrate was added, and the temperature was changed to 30 °C for 40 hr. The cells were then harvested and stored at –80 °C until purification. The protein purification procedure was described in our previous study (Ruan et al., 2020).

The in vitro phosphorylation is conducted using the human Src-GST protein (Sigma-Aldrich, Catalogue number: S1076) according to the manufacturer’s protocol. Specifically, 1.5 μg of purified PANX1 protein is mixed with kinase assay buffer (25 mm MOPS, pH 7.2, 20 mM MgCl₂, 12.5 mM MnCl₂, 5 mM EGTA, 2 mM EDTA, 0.25 mM DTT), diluted 1:5 with 50 ng/μl bovine serum albumin (BSA). The reaction was supplemented with 0.25 mM ATP, and 0.3 μg recombinant human (active) Src-GST kinase. Samples were incubated for 1 h at 30 °C using a PCR thermocycler. After 1 hr, 2 x SDS loading buffer (BioRad) is mixed with the sample to stop the reaction. The sample is then resolved by an Tris-Glycine gel for subsequent western blot analysis.

Purified hPANX1 protein w- or w/o- co-expressing mSrc Y529F mutant was resolved in SDS-PAGE gel and the band corresponding to hPANX1 protein was cut and subjected to in-gel digestion with trypsin. Half of each digested sample was analyzed by nano LC-MS/MS with a Waters M-Class HPLC system interfaced with a Thermo Fisher Fusion Lumos mass spectrometer. Peptides were loaded on a trapping column and eluted over a 75 µm analytical column at 350 nL/min; both columns were packed with Luna C18 resin (Phenomenex). The mass spectrometer was operated in data-dependent mode, with the Orbitrap operating at 60,000 FWHM and 15,000 FWHM for MS and MS/MS, respectively. The instrument was run with a 3 s cycle for MS and MS/MS.

Data were searched using a local copy of Mascot (Matrix Science) with the following parameters. Enzyme: Trypsin/P; Database: SwissProt Human (concatenated forward and reverse plus common contaminants); Fixed modification: Carbamidomethyl (C) Variable modifications: Oxidation (M), Acetyl (N-term), Pyro-Glu (N-term Q), Deamidation (N/Q); Mass values: Monoisotopic; Peptide Mass Tolerance: 10 ppm; Fragment Mass Tolerance: 0.02 Da; Max Missed Cleavages: 2. Mascot DAT files were parsed into Scaffold (Proteome Software) for validation, filtering and to create a non-redundant list per sample. Data were filtered using 1% protein and peptide FDR and requiring at least two unique peptides per protein.

HEK293T cells were transfected using Lipofectamine 2000 (Thermo Fisher) according to the manufacturer’s protocol. The transfected cells were incubated at 37 °C for 18–24 hr before electrophysiological measurements. Whole-cell recordings were performed using a Multiclamp 700B (Axon Instruments) and Clampex software, with pipettes of 3–5 MOhm resistance filled with an internal solution containing 145 mM NaCl, 10 mM Hepes, 10 mM EGTA, pH adjusted to 7.4. The external bath solution contained 160 mM NaCl, 10 mM Hepes, 3 mM KCl, 2 mM CaCl₂, and 1 mM MgCl₂, also adjusted to pH 7.4. During the recordings, voltage steps ranging from −100 mV to +140 mV were applied, each lasting 100ms with a 20 mV increment between steps, and membrane currents were digitally recorded at 10 kHz and filtered at 2 kHz. To precisely measure the PANX1 channel’s current, carbenoxolone disodium salt (CBX), a blocker of the channel, was added to the bath solution at a final concentration of 0.1 mM, and CBX-sensitive currents were subsequently calculated by comparing the difference in current amplitude in a cell with and without the presence of CBX.