Fig. 2: Conservative mutations of 27 predicted pH sensors affect the ASIC1a pH dependence. | Communications Biology

Fig. 2: Conservative mutations of 27 predicted pH sensors affect the ASIC1a pH dependence.

From: Molecular mechanisms and hotspots of pH sensing in ASIC1a revealed by computational and functional analysis

Fig. 2

A Representative current traces of Xenopus laevis oocytes expressing ASIC1a WT, obtained by two-electrode voltage clamp to −60 mV to determine the pH dependence of activation (upper panel) and steady-state desensitization (SSD; lower panel). Channels were activated by a 10-s perfusion with stimulation solution of varying pH every 60-s. The conditioning solution was pH 7.4 between stimulations. The pH dependence of SSD was measured by applying conditioning solutions of varying pH for 50-s before the 10-s stimulation by pH5.0. B pH dependence curves of activation (filled symbols) and SSD (open symbols). The WT data (black) were obtained on the same days as the mutants, shown in color. Currents were normalized to the maximum peak current. The lines represent fits to the Hill equation (n = 7–15). C–H pH values for half-maximal activation (pH50) and half-maximal desensitization (pHD50) for WT and conservative mutations to non-titratable residues, obtained by fitting to the Hill equation, in the palm (C), AcP (D), finger and thumb (E), β-ball (F), knuckle (G) and transmembrane domains (H). Comparison of the mutants to WT was done by one-way ANOVA test followed by a Dunnett’s multiple comparisons test when > 75% of the groups showed a normal distribution and Kruskall-Wallis test followed by a Dunn’s multiple comparison test when ≥ 25% of the groups showed a non-normal distribution; *p < 0.05; **p < 0.01; ***p < 0.001; #p < 0.0001. Data are presented as Individual points and as mean ± SEM. The dotted lines represent the WT mean pH50 and pHD50 values. (I) Structure images of the human ASIC1a structural model (based on the open ASIC1a structure 4NTW), presenting residues whose conservative mutation to a non-titratable amino acid significantly shifted the pH50 (left panel) or pHD50 values (right panel). The color code highlights the amplitude of the shift, as indicated, with yellow to red for acidic, and blue to purple for alkaline shifts.

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