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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. Image published in: Frey EN et al. (2013) Image reproduced on Xenbase with permission of the publisher and the copyright holder. Creative Commons Attribution license Larger Image Printer Friendly View

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