Fig 2: Long and truncated variants of ASIC2b.(A) 5' terminal sequence of rat Asic2b cDNA (Accn1, transcript variant MDEG2, NM_012892, black) and of the short cDNA cloned from CC-PAN CCD (red, GenBank KP294334). The short sequence contains a putative translation initiation codon in frame with the ASIC2 ATG (in bold). (B) N-ter sequence of rat ASIC2b (black) and its putative truncated variant (red). The underlined sequence shows the first transmembrane domain. (C) Western blot analysis of ASIC2 expression in OKP and HEK cells transiently transfected with Asic2b or its truncated variant (t-Asic2b) or an empty vector. ASIC2b, acid-sensing ion channel 2b; CCDs, cortical-collecting ducts; CC, corticosteroid-clamped.

Fig 3: Expressions of ASIC subunits in rat cardiomyocytes.(a) Expressions of ASIC1, 2a and 3 proteins in rat cultured cardiomyocytes. Individual ASIC subunits peptides were added as negative controls. The blots were cropped from Supplementary Fig. S1a. The representative full-length blot for ASIC2a was shown in Supplementary Fig. S2. (b) Expressions of ASIC1, 2 and 3 transcripts in cultured rat ventricular myocytes. GAPDH transcript was used as control. M: marker; 1, 4: cardiomyocytes; 2: cortex; 3: negative control. (c) Double - labeling fluorescence of ASICs (green) and nucleus (blue, marker: Hoechst 33258) in cultured ratcardiomyocytes. NC: pretreatment with immunogenic peptide as negative control. Scale bars: 20 µm. All data were represented from at least three similar independent experiments.

Fig 4: The contribution of acid-sensing ion channels to the bystander effect.(a,b) ASIC1 (a) and ASIC2a (b) expression in CA1. (c) Change in membrane resistance during amiloride (ASIC antagonist) application (lilac-shaded region) (n = 6–18 cells, 9 mice, two-way ANOVA for interaction between time and drug treatment (F (12,314) = 2.617, p = 0.0018, asterisks indicate significant time points after Dunnet’s multiple comparison test). Grey data points: control experiment in which no amiloride was applied (n = 4–16, 6 mice) (no significant change from baseline). (d) Change in bystander current during amiloride application (F (12,314) = 2.473, p = 0.0032, asterisks indicate significant time points). No significant change from baseline for untreated control cells. (e) Examples bystander current at baseline baseline (dark red), after 20 mins amiloride (pink), and after 30 mins of washout (lilac). (f) Example ChR2 and eArch3.0 bystander currents in 500 µM acetazolamide (pale traces indicate baseline recordings, dark traces indicate 15 mins acetazolamide exposure). (g) Change in bystander current magnitude after acetazolamide application (thick line represents group mean) (ChR2, n = 7 cells, 3 mice, paired t-test: t = 1.313, df = 6, p = 0.2372; eArch3.0, n = 7 cells, 3 mice, paired t-test: t = 6.177, df = 6, p = 0.0008). (h) Occasionally, acetazolamide caused eArch3.0 bystander neurons to exhibit inward ASIC-like currents during green light. (i) Extracellular pH measurements in CA1 (contralateral to site of opsin injection) in acute slices using a solid state metal wire oxide pH sensor (100 µm diameter, 5X magnification). (j) pH change in response to three minutes of light stimulation (470 nm for ChR2 and YFP-controls, 560 nm for eArch3.0). For ChR2, n = 24 recording sites, 13 slices, 3 animals. For eArch3.0, n = 21 recording sites, 11 slices, 3 animals. For YFP controls, n = 22 recording sites, 12 slices, 2 animals. (k) Zoom-in of last minute of baseline pH recording and first two minutes of light stimulation.

Fig 5: Functional expression of ASIC2 in X. laevis oocyte.(A) ASIC2b coexpression attenuates ASIC2a desensitization. Original traces of whole-cell current (holding potential = –70 mV) in oocytes expressing ASIC2a alone or with ASIC2b, or t-ASIC2b, as indicated below the traces. Inward currents were induced by a rapid extracellular acidification from pH 7.4 to pH 4.0 (indicated by the horizontal bar). (B) Mean transient-induced (peak) and residual-induced (plateau) currents (corrected by currents recorded in control oocytes) in oocytes expressing ASIC2a alone (black bars) or coexpressing ASIC2b (red) or t-ASIC2b (blue). (C) Acid-induced plateau currents (normalized to maximal value achieved at pH 4) at different extracellular pH; colors as above. (D) Effect of amiloride: acid-induced (pH 4) plateau currents (normalized to the value measured in the absence of inhibitor) in the presence of 100 μM amiloride. Data are shown as mean ± SEM (each value represents an oocyte, n is shown in italic in each figure). Comparison between groups was performed by variance analysis (1-way ANOVA) followed by post hoc multiple comparison Tukey’s test. P < 0.05. ASIC2, acid-sensing ion channel; t-ASIC2b, truncated variant of ASIC2b.