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Figure 4. E18’ plays a direct role in selective Na+ conduction.(A) Relative permeability ratios for WT and mutant ASIC1a channels (mean ± SEM; n = 3–6). Δ: G10’A/S, A11’V/F, S12’G/A/C/T, T15’V and E18’A channels were non-functional (see Figure 4—figure supplement 1). (B) E18’ and €18’ side chains, proton-gated currents (pH 6.0) in extracellular NaCl, and current-voltage relationship in extracellular NaCl (orange) or KCl (blue; mean ± SEM, n = 3–4). Specific € incorporation illustrated in Figure 4—figure supplement 1. (C) Example and mean (± SEM, n = 5–6) single channel Na+ currents. (D) Relative permeability ratios for concatemeric channels carrying no mutations (WT-WT-WT) or one, two or three L7´A,S12’A or E18’Q mutations (mean ± SEM, n = 3–6). Δ: Concatemeric channels containing three L7’A- and three S12’A-mutated subunits non-functional (see Figure 4—figure supplement 2).DOI:
http://dx.doi.org/10.7554/eLife.24630.008Figure 4—figure supplement 1. Experimental identification of determinants of ion selectivity.(A) Example recordings of proton-gated current during voltage ramps in extracellular NaCl (orange), KCl (blue), CsCl (green) or LiCl (teal) at oocytes expressing selected mutant ASIC1a mRNA. (B) Mean (± SEM, n = 4–9) relative permeability ratios calculated after experiments depicted in (A) and half-maximal effective proton concentration values (‘pH50’) from experiments depicted in (C). Δ: At oocytes injected with G10’A/S, A11’V/F, S12’G/A/C/T, T15’V and E18’A mRNA, pH 5.0 elicited currents no greater than at uninjected oocytes, as evident in (C). (C) Example recordings of current responses to increased proton concentrations at uninjected oocytes or oocytes expressing selected ASIC1a mutants (scale bars: x, 10 s; y, 1 μA, except for E18’€ and E18’UAG (no €) as indicated). ‘E18’€” describes oocytes injected with E18’UAG mRNA + tRNA-€. ‘E18’UAG (no €)” describes oocytes injected with E18’UAG mRNA + tRNA, which allows only for non-specific incorporation of endogenous amino acids to this position and thus serves as a control for specific incorporation. Graph shows mean pH 6.0-gated current amplitude for both groups (***p<0.001, Student’s t test).DOI:
http://dx.doi.org/10.7554/eLife.24630.009 Image published in: Lynagh T et al. (2017) © 2017, Lynagh et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View |