Ilyaskin AV , Diakov A , Korbmacher C , Haerteis S .

	Figure 1. ASIC1a‐mediated whole‐cell currents elicited by pH 5.5 were significantly increased in the presence of t‐DCA but were unaffected by t‐DCA pretreatment. (A, C, E), Representative whole‐cell current traces recorded in human ASIC1a expressing oocytes. Bath pH was 7.4 except for time intervals indicated by open bars during which bath pH was switched to 5.5. t‐DCA (500 μmol/L) was present in the bath solution as indicated by black bars. In the control experiment shown in C the 3rd and 4th pH 5.5 responses were elicited using a solution from a different reservoir to confirm the reproducibility of the responses. (B, D, F), Summary of results from similar experiments as shown in A (n = 34; N = 6), C (n = 15; N = 3), and E (n = 25; N = 2), respectively. Lines connect data points obtained in the same experiment. To quantify current responses, the integral of the inward current elicited by pH 5.5 below baseline was determined for each response. For the summary data shown in B and D mean current integral values of the 3rd and 4th response and of the 5th and 6th response were normalized to the mean current integral of the first two responses (Normalized current integral). For the summary data shown in F the current integral value of the 2nd response (after t‐DCA) was normalized to the current integral of the 1st response (before t‐DCA). *** p < 0.001; n.s. not significant; one‐way repeated measures ANOVA with Bonferroni post hoc test was used in B and D; Student's t‐test was used in F.
	Figure 2. Concentration‐dependent potentiation of proton‐activated ASIC1a currents by t‐DCA. (A–E), Representative whole‐cell current traces recorded in human ASIC1a expressing oocytes exposed to pulses of pH 5.5 in the absence and presence of t‐DCA in concentrations as indicated; data from similar experiments are summarized to the right of each trace. Bath pH was 7.4 except for the time intervals indicated by open bars during which bath pH was switched to 5.5. Lines connect data points obtained in the same experiment. The current integral value of the 2nd response (with t‐DCA) was normalized to the current integral of the 1st response (without t‐DCA). (F), Concentration–response relationship of the stimulatory effect of t‐DCA on proton‐activated ASIC1a currents. Data (mean ± SE) from the same experiments as shown in A–E were fitted to equation  (1) (n = 10; N = 2). ***P < 0.001; Student's t‐test.
	Figure 3. pH dependence of ASIC1a activation and steady‐state desensitization under control conditions and in the presence of t‐DCA. (A), Representative whole‐cell current traces recorded in human ASIC1a expressing oocytes demonstrating the pH‐dependent channel activation. Changes of bath pH from 7.4 to different values ranging from 6.9 to 5.5 are indicated by open bars and were performed under control conditions (upper panel) or in the presence of 500 μmol/L t‐DCA as indicated by black bars (lower panel). (B), Representative whole‐cell current traces recorded in human ASIC1a expressing oocytes demonstrating the pH‐dependent steady‐state desensitization of the channel under control conditions (upper panel) and in the presence of 500 μmol/L t‐DCA as indicated by black bars (lower panel). Changes of bath pH are indicated by open bars. After a 2 min exposure to pH 8.0 a first pulse of pH 5.5 was applied followed by a 2 min exposure to pH 7.4, 7.2, or 7.0 and a second pulse of pH 5.5 as indicated. (C), pH‐dependent ASIC1a activation curve under control conditions (●) or in the presence of t‐DCA () and pH‐dependent ASIC1a steady‐state desensitization curve under control conditions (■) or in the presence of t‐DCA (). For activation curves the current integral values (Q) elicited by pulses of pH were normalized to the maximal value (Q max) observed in the corresponding recording. Q max was observed at pH 5.5 or at pH 6.0 in the absence or presence of t‐DCA, respectively. For the steady‐state desensitization curve the current integral value of the 2nd pulse of pH 5.5 (Q, after incubation at different pH values ranging from 7.4 to 7.0) was normalized to the current integral value of the 1st pulse of pH 5.5 (Q max, after incubation at pH 8.0). Data (mean ± SE) were fitted using equation (1) (6 ≤ n ≤ 20, N = 3 for activation curve; 10 ≤ n ≤ 12; N = 3 for desensitization curve).
	Figure 4. t‐DCA increased proton‐activated ASIC1a single‐channel activity in outside‐out patches and slightly reduced single‐channel current amplitude. (A), Representative single‐channel current recording obtained at a holding potential of −70 mV from an outside‐out patch of an ASIC1a expressing oocyte. Bath pH was 7.4 except for three time intervals indicated by open bars during which bath pH was switched to 5.5. t‐DCA (500 μmol/L) was present in the bath solution as indicated by the black bar. The current level at which all channels are closed (C) was determined at pH 7.4. The insets (1, 2, and 3) show the indicated segments of the continuous current trace on an expanded time scale. Binned current amplitude histograms are shown on the right side of the insets and were obtained from the corresponding parts of the trace to calculate single‐channel current amplitude (i). Dotted lines in the insets indicate channel open levels. To quantify the responses, the current integral (Q) of the single‐channel currents activated by pH 5.5 was determined for each response. (B, C), Summary of results from similar experiments as shown in A. Lines connect data points obtained in the same experiment. In B (n = 12; N = 4) values were normalized to the initial response to pH 5.5 (Normalized current integral). In C the effect of t‐DCA on single‐channel current amplitude is summarized (n = 11; N = 4).*P < 0.05; ***P < 0.001; n.s., not significant; one‐way repeated measures ANOVA with Bonferroni post hoc test.
	Figure 5. Molecular docking approach using the open state crystal structure of ASIC1 predicted binding of t‐DCA to the channel pore. (A), Representative whole‐cell current trace recorded in an oocyte expressing human ASIC1a. The oocyte was exposed to pH 5.5 and n‐dodecyl‐β‐D‐maltoside (MALT, 10 μmol/L) as indicated by open and black bars, respectively. Data from similar experiments are summarized to the right of the trace like in Figure 1B and D as normalized current integrals (n = 20; N = 3). (B), Molecular surface representation of the transmembrane domains of chicken ASIC1 (Baconguis et al. 2014) viewed from the extracellular side with a t‐DCA molecule (orange color) positioned at the site predicted by molecular docking approach. In the docking model shown the putative interaction site of t‐DCA corresponds to the site where maltoside was cocrystallized with ASIC1 (Jasti et al. 2007). The arrows indicate the position of glycine residue 432 (G432; highlighted in green) in each ASIC1a subunit. G432 is the amino acid residue with the highest contribution to the total energy of interaction between ASIC1 and t‐DCA. (C), Sequence alignment of chicken (g.ASIC1) and human (h.ASIC1a) corresponding to the first part of the TM2. The homologous amino acid residues G432 in g.ASIC1 and G433 in h.ASIC1a are indicated by bold characters highlighted in red.
	Figure 6. Substituting the glycine residue G433 by cysteine or serine abolished or reduced the stimulatory effect of t‐DCA on proton‐activated ASIC1a currents, respectively. (A, B, C), Left panels: Representative whole‐cell current traces recorded in oocytes expressing the G433C (A and B) or G433S (C) mutant ASIC1a. Experiments were performed and analyzed as described for the experiments shown in Figure 1A–D. Right panels: Summary of data from similar experiments as shown in the left panel: A (n = 22; N = 3), B (n = 13; N = 3), C (n = 22; N = 3).

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