Fig 2: HDAC6-mediated channel activation by deacetylation of Lys-140 on PANX1.a, b Basal and PE-stimulated whole-cell currents in a1D-AR-transfected HEK293T cells expressing the indicated Arg-substituted PANX1 constructs. c Summary data (mean ± s.e.m) reveal that Arg substitution at Lys-140, but not at Lys-409, increased basal PANX1 current and occluded PE effects. n = 9, 7, or 9 cells examined over 3 independent experiments. Two-way ANOVA (F2,22 = 9.924, p = 0.0008) with Bonferroni’s multiple comparisons test (p values from comparisons are shown). d, e Effect of HDAC6 on whole-cell current in HEK293T cells expressing hPANX1(TEV) or hPANX1(TEV)-K(140/409)Q. f, g Summary data reveal that HDAC6 (f) or DAD (g) did not activate basal whole-cell current in cells expressing Gln-substituted hPANX1(TEV) channels. n = 10 or 11 (f), 12 or 9 (g) cells examined over 6 independent experiments. Data are presented as mean ± s.e.m. Two-tailed unpaired t-test (f t = 6.425, df = 19; g t = 2.118, df = 19). h Grouped results showing tubacin (2 µM), but not DMSO, reduced basal whole-cell currents in cells expressing hPANX1(TEV) channels and wild-type HDAC6 proteins. n = 7 or 6 cells examined over 3 independent experiments. Data are presented as mean ± s.e.m. Two-tailed unpaired t-test (t = 3.858, df = 11). i Inside-out patch recordings of hPANX1(TEV) and hPANX1(TEV)-K(140/409)Q during exposure to HDAC6 and TEV protease. j Unitary I-Vs for each of the indicated PANX1 channel constructs after activation by C-terminal cleavage with caspase 3 or TEV protease; lines indicate fits by linear regression to estimate the unitary slope conductance (?) for each group (green circles: hPANX1-eGFP, caspase: ?~101 pS; black triangles: hPANX1(TEV), TEVp: ?~91 pS; blue squares: hPANX1(TEV)-K(140/409)Q), TEVp: ?~94 pS; the n values provided are for biologically independent patches). PE phenylephrine, TEVp Tobacco Etch Virus protease.

Fig 3: Probenecid decreases proximity ligation assay (PLA) between Panx1EGFP and Crmp2. (A) Representative confocal maximum intensity projections of vehicle- (left) and probenecid-treated (right) Panx1-EGFP-expressing N2a cells demonstrating PLA-positive Crmp2-Panx1 clusters (white). (B) Probenecid treatment significantly decreased mean fluorescence intensity of the PLA signal (vehicle: 26.0 ± 2.7 arb.u. per 100 µm2 vs. probenecid: 17.1 ± 2.1 arb.u. per 100 µm2, p = 0.0037, n = 77 cells and n = 70 cells, respectively, Mann-Whitney test). (C) Controls for PLA included (i) cells were incubated with a-GFP anti-mouse and a-GFP anti-rabbit to serve as a positive control. (ii) No primary antibodies were included to serve as a negative control. Hoechst 33342 was used as a nuclear counterstain. Scale bars, 10 µm.

Fig 4: Increased network ensembles and altered Ca2+ dynamics in Panx1 KO cortical neurons. A, Representative analyses for functional connectivity in WT and Panx1 KO primary cortical neuron cultures. Ca2+ imaging data were collected using confocal microscopy in DIV12-14 primary cortical neurons using Fluo-4-AM. A MATLAB based program called FluoroSNNAP was used to determine network ensemble properties. Ai, Confocal micrographs of exemplary FOVs of WT and Panx1 KO (labeled KO) demonstrating Fluo-4-derived Ca2+ activity from low and high activity frames (as indicated), along with the FluoroSNNAP output ?F/F (middle) and inferred spikes (bottom) from the identified WT (75) and KO (85) cells. Aii, Percentage of active neurons in each frame from the example FOVs (top). The red line indicates the threshold for a statistically significant number of coactive cells in a frame used by FluoroSNNAP (3 SD). Raster plots of WT and KO example FOVs (bottom) generated from thresholded spike probability data. Spikes from cells participating in a network ensemble are shown in red. The exemplary high activity frames and cells from A are also highlighted in red. B, Network ensemble data from WT and Panx1 KO DIV12-14 primary neuron cultures. Bi, The mean number of network ensembles was increased in Panx1 KO cultures (WT: 4.0 ± 0.6, KO: 7.6 ± 0.7 network ensembles; t(13) = 4.1, p = 0.0014a1; n = 7–8 coverslips from 3 independent cultures; **p < 0.01). Bii, The number of cells involved in network ensembles was also increased in Panx1 KO neurons (WT: 5.0 ± 0.6, KO: 8.5 ± 0.6 cell per ensemble; t(13) = 4.4, p < 0.0001a2; n = 20–21 network ensembles from 3 independent cultures; ****p < 0.0001). C, Core network ensemble data from WT and Panx1 KO DIV12-14 primary neuron cultures. Ci, The mean number of core ensembles (co-activated neurons participating in more than one ensemble) was increased in Panx1 KO cultures (WT: 1.2 ± 0.3, KO: 2.7 ± 0.5 core ensembles; t(39) = 2.8, p = 0.0071b1; n = 20–21 network ensembles from 3 independent cultures; **p < 0.01). Cii, The number of cells forming a core ensemble was not significant different between the analyzed groups (WT: 3.1 ± 0.3, KO: 3.7 ± 0.3 cells per core ensemble; t(30) = 1.3, p = 0.1968b2; n = 12–20 core ensembles from 3 independent cultures; n.s., not significant). D, Distributions and violin plots of resting and total change (maximum minus minimum) of Fluo-4 fluorescence intensities in DIV12-14 primary cortical neuronal cultures. Di, Frequency distributions of Fluo-4 Ca2+ indicator dye fluorescence intensities of WT (red) and Panx1 KO (blue) revealed a right shift toward higher median Ca2+ levels at baseline (defined the as raw median fluorescence intensity value for each neuron; WT median = 37, n = 1017 cells; KO median = 58.50, n = 1089 cells; p < 0.0001c; Mann–Whitney U = 316,969; data compiled from 7 to 8 coverslips from 3 independent cultures per condition; ****p < 0.0001). Dotted lines represent the mean of each distribution; a.u., arbitrary units. E, Similarly, the difference between the maximum and minimum fluorescence intensity values (?F, fluorescence intensity range) was right-shifted and significant larger in Panx1 KO neurons (WT median = 16, n = 1017 cells; KO median = 25, n = 1089 cells; p < 0.0001d; Mann–Whitney U = 294,294; data compiled from a total of 7–8 coverslips across 3 independent cultures per condition; ****p < 0.0001). Dotted lines represent the mean of each distribution; a.u., arbitrary units. F, WT and Panx1 KO cortical neuronal cultures have a similar cell-type composition. Fi, Representative images of WT and Panx1 KO cortical neurons labeled with the pan-neuronal marker MAP2, interneuron marker Gad67, and the astrocytic marker GFAP. Scale bar, 100 µm. Fii, The proportion of excitatory neurons, inhibitory neurons, and astrocytes was similar between groups (WT excitatory neurons = 81.4% ± 1.3%, KO excitatory neurons = 79.8% ± 1.6%, p = 0.9702e8; WT inhibitory neurons = 17.1% ± 1.3%, KO inhibitory neurons = 15.2% ± 1.0%, p = 0.7500e9; WT astrocytes = 1.5% ± 0.4%, KO astrocytes = 4.9% ± 1.0%, p = 0.1026e10; simple-effect ANOVA with Bonferroni’s multiple-comparison test, n = 16 FOV from 2 independent cultures; n.s., not significant). G, WT and Panx1 cortical neurons exhibited similar cell viability. Conversion of MTT to formazan (absorbance measured at 540 nm) was not significant between groups (WT = 100% ± 2.5%, KO = 98.62% ± 8.5%; p = 0.9089f; t(4) = 0.128; n = 3 independent cultures; n.s., not significant). Data are presented as mean ± SEM.

Fig 5: PANX1 regulates the cytosolic free Mg2+ level, but not the externalization of phosphatidylserine, in apoptotic cells.(A) HeLa cells expressing Halo-tag were loaded with Halo-TMR and a magnesium indicator MGH(AM), followed by induction of apoptosis. Each trace represents a time course of normalized MGH/TMR fluorescence ratios of individual cell from three biological replicates. Timing of the initiation of cell shrinkage was defined as time = 0. Reddish and bluish traces represent wild-type cells (n = 22) and PANX1-KO2 cells (n = 20), respectively, from three biological replicates. Traces from different biological replicates were shown in different color codes. (B) Apoptosis of HeLa cells was induced by anti-FAS and cycloheximide in the presence of Alexa647-annexinV and propidium iodide. Fluorescence intensity of Alexa647-annexinV of each cell that does not show propidium iodide fluorescence was quantified using a fluorescent microscopy at 6 hr after induction of apoptosis (359 [WT] and 322 [PANX1-KO2] cells from three biological replicates).Figure 6—source data 1.Numerical data used for panel B.