Frey EN , Pavlovicz RE , Wegman CJ , Li C , Askwith CC .

R01 NS062967 NINDS NIH HHS , P30 NS045758 NINDS NIH HHS , T32 GM068412 NIGMS NIH HHS

	Figure 3. MTSET modification of desensitized L280C and L415C.A. Representative traces of calcium-dependent gating of unmodified L280C and L415C. Oocytes were exposed to the indicated pH (pH 6.8 for L280C or pH 6.9 for L415C) in either 5.0 mM Ca2+ or nominal Ca2+ -free solutions to foster ASIC1a transition to distinct gating states at the same pH. B. Quantification of calcium-dependent gating. The presence of 5.0 mM Ca2+ maintained L280C and L415C in the closed state when cells were treated with the conditioning pH (pH 6.8 for L280C or pH 6.9 for L415C) for ~2 minutes. This was evident by robust pH 5.0 evoked currents following conditioning with the mildly acidic pH (n = 6-8). Conditioning with the same pH in nominal Ca2+ induced steady-state desensitization of L280C and L415C (n = 6-8). C. Schematic of the experiment to assess accessibility. The closed state of ASIC1a was maintained by conditioning with pH 6.8 (L280) or 6.9 (L415) containing 5 mM Ca2+. The desensitized state was evoked by conditioning with the same pH in nominal calcium. 300 µM MTSET was applied in the conditioning pH for the indicated time and then excess MTSET was washed away with conditioning solution. Oocytes were then returned to pH 7.4 before activation with pH 5.0. D. Representative traces of L280C pH 5.0-evoked currents before (control) and after 6 minutes of MTSET incubation. E. Quantification of residual current of L280C after MTSET exposure for the indicated time (n = 4-6). F. Representative traces of L415C before (control) and after 60 second intervals of MTSET incubation. G. Quantification of residual current of L415C after MTSET exposure for the indicated time (n = 4-6). Curves represent one-phase association exponential fits of the data points. Data are mean ± SEM. “***” indicates p < 0.001 compared to 5.0 mM Ca2+ conditioning.
	Figure 2. MTSET modification of L280C, I307C, and L415C.A. Effect of MTSET on wildtype or mutant ASIC1a expressed in Xenopus oocytes. MTSET (300 µM) was applied at pH 7.4 for 3 minutes and removed by washing with pH 7.4 solutions. pH 5.0-evoked currents after MTSET incubation (“+ MTSET”) were compared to control currents in the same oocyte measured before MTSET application (“Control”). B–C. Quantification of (B) tau of inactivation (n = 6-8) and (C) residual current (n = 6-7). D. Representative recordings of steady-state desensitization (SSD) before and after MTSET modification. Oocytes were maintained at a basal of pH 7.9 and then incubated with pH 6.7 for 2 minutes to induce SSD (shaded bars) prior to activation with pH 5.0 (white bars). E. Quantification of MTSET-dependent changes in SSD (n = 5-6). F. Representative traces of MTSET exposure on pH-dependent activation. MTSET was applied as above and the response to pH 5.0 (white bars) or pH 6.5 (gray bars) from basal pH 7.4 was measured. G. Quantification of pH 6.5-mediated activation before and after MTSET modification (n = 6-8). Data are mean ± SEM. “**” and “***” indicate p-values < 0.01 and 0.001, respectively. Significance was determined with paired Student’s t-tests.
	Figure 4. FRRFa modulation of L280C.A. FRRFa increases residual current of wildtype and L280C. FRRFa (100 µM) was applied for 1 minute at pH 7.4 prior to activation with pH 5.0. For L280C, arrowheads highlight the FRRFa-induced increase in peak current amplitude. B. Quantification of residual current (n = 14-17). C. Quantification of FRRFa modulation on pH 5.0-evoked peak current amplitude. The percent change in amplitude was determined by subtracting the pH 5.0-evoked peak current amplitude of vehicle from the pH 5.0 evoked peak current amplitude evoked after FRRFamide modulation and normalizing to the vehicle peak current amplitude from the same cell (n = 17-25). D. Representative trace of FRRFa modulation on steady-state desensitization. 100 µM FRRFa or vehicle was applied for 1 minute at basal pH 7.4 and again during the 2 minute incubation with conditioning pH 6.7. Proton-gated current was evoked with pH 5.0 (white bar). E. Quantification of FRRFa modulation of steady-state desensitization (n = 4). F–G. FRRFamide concentration response curve for wildtype ASIC1a (F) and L280C (G). The effect of FRRFamide on residual current was assessed. Our data suggest that 100 µM FRRFa induced a maximal response on wildtype ASIC1a as 300 µM FRRFa was not significantly different from 100 µM FRRFa (n = 5, p = 0.69 paired Student’s t-test, difference between 100µM and 300µM was 10.42% ± 11.25%; data not shown). Based on this information, the calculated EC50 for wildtype ASIC1a was 20 ± 4 µM and 14 ± 4 µM for L280C (n = 6-8, p = 0.34). Data are mean ± SEM. “**” and “***” indicates p-value < 0.01 and 0.001 respectively.
	Figure 5. FRRFa impairs MTSET modification.A. Schematic of MTSET/peptide experimental design. 100 µM peptide or vehicle was applied for 1 minute before application of MTSET (in the presence of vehicle or peptide). Excess MTSET was removed by washing in peptide (or vehicle) containing solutions. Finally, peptide (or vehicle) was washed-out thoroughly and residual current was measured. B. Representative traces of L280C pH 5.0-evoked currents following 2.5 minutes of MTSET modification in the presence of vehicle, FRRFa, or FRRF (no amide). C. Quantification of residual current after exposure to MTSET in the presence of vehicle, 100 µM FRRFa, or 100 µM FRRF (no amide). Curves represent one-phase association exponential fits of the data points. FRRFa significantly altered MTSET modification (p < 0.001, 2-way ANOVA). No difference was observed between Vehicle and FRRF (no amide). Data are mean ± SEM, n = 3-22.
	Figure 6. FRRFa modulates L280C after MTSET modification.A. Schematic of experimental design and representative traces. Left Panel: L280C was activated with pH 5.0 with and without 100 µM FRRFa. Right Panel: L280C was modified with 300 µM MTSET for 3 minutes at pH 7.4. After treatment, MTSET was removed by washing with pH 7.4 and pH 5.0-evoked current was recorded in the absence of FRRFamide. Channels were then washed with pH 7.4 and allowed to recover for 1 minute. Then pH 7.4 solutions containing 100 µM FRRFa were applied for 1 minute. After application of FRRFa, channels were activated with pH 5.0. For quantification (B–D), % change was determined by subtracting the stated characteristic (peak amplitude, residual current, or τinact) with FRRFa from control (no peptide) and normalized to the no peptide response. B. Quantification of % change in peak current amplitude. The magnitude of the change in peak current amplitude evoked with FRRFa was independent of MTSET modification (n = 8-11, p = 0.6). C. Quantification of the % change in the rate of inactivation (τinact). FRRFa response on inactivation after MTSET was not significantly different from FRRFa response on unmodified L280C (n = 8-11, p = 0.9). D. Quantification of % change in residual current. After MTSET modification, FRRFa still increased residual current (n = 8, p = 0.02, paired Student’s t-test), but this was not as robust as FRRFa-induced residual current of unmodified L280C (n = 8-11, p = 0.03). Data are mean ± SEM. “*” indicates p < 0.05 and n.s. indicates no significant difference.
	Figure 1. Location and characteristics of L280C, I307C, and L415C in human ASIC1a. A–B. Human ASIC1a was modeled based on the chicken ASIC1 crystal structure (PDB ID: 3HGC). One subunit has been removed to show the inside of the central vestibule. The subunits are color-coded to highlight different regions of the ASIC1a structure. The boxed region is magnified in B to illustrate the positions of L280, I307, and L415. Images were rendered using the UCSF Chimera package [63,64]. C. Representative recordings of acid-activated currents in Xenopus oocytes expressing wild-type human ASIC1a, L280C, I307C, or L415C. Basal pH was maintained at pH 7.4 before application of pH 5.0 (white bars above trace). D. Quantification of the tau of inactivation (n = 10-14), calculated through a single exponential fit of the decay phase of the acid-evoked current. E. Quantification of proton-dependent activation (n = 6-9). I/Imax is the peak current amplitude evoked from test pH conditions normalized to peak current amplitude evoked with pH 5.0. F. Quantification of the proton-dependence of steady-state desensitization (n = 6-10). I/Imax. is the peak current amplitude of pH 5.0-evoked currents after conditioning in the test pH normalized to the pH 5.0-evoked current after conditioning in pH 7.9 (see methods). Data are mean ± SEM. “***” indicates a p-value < 0.001, respectively. “n.s.” indicates no significant difference.

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