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. 2020 Mar 31;9:e53430. doi: 10.7554/eLife.53430

Figure 6. Permeation and selectivity through the isoleucine constriction.

(A) The isoleucine constriction is flanked by two layers of ordered water molecules. The cytosolic layer of waters is partially coordinated by Ser45, while the luminal layer is partially coordinated by Thr49 and Thr274. (B) Mean current recorded from HEK293T cells transfected with hTMEM175 (blue), S45A (red dashed), S45T (red), T274V (orange dashed), T274S (orange), I46M (green), I46M/I271M (magenta) and non-transfected (white) at +100 mV in a bi-ionic condition of 150 mM Cs+ (intracellular) and 150 mM Na+ (extracellular). (C) Normalized I-V relationship of whole-cell patch clamp of hTMEM175 transfected (blue), S45T transfected (red), T274S transfected (orange) and I46M transfected (green) HEK293T cells in a bi-ionic condition of 150 mM Cs+ (intracellular) and 150 mM Na+ (extracellular). All experiments were performed at least three times and error bars represent SEM. (D) Model for ion selectivity and gating in hTMEM175. In the open state, ions are transiently dehydrated through the isoleucine constriction, favoring permeation of K+ ions. In the closed state, the isoleucine constriction closes, preventing ion permeation.

Figure 6.

Figure 6—figure supplement 1. Functional analysis of hTMEM175 mutants.

Figure 6—figure supplement 1.

(A) Superose 6 fluorescence size exclusion chromatography traces of hTMEM175 (blue), S45A (red dashed), S45T (red), T274V (orange dashed), T274S (orange), I46M (green), I46M/I271M (magenta) and non-transfected (black). (B) Representative whole-cell electrical recordings of HEK293T cells transfected with S45A, S45T, T274V, T274S, I46M or I46M/I271M mutants in bi-ionic condition of 150 mM Cs+ (intracellular) and 150 mM Na+ (extracellular). Recording protocol is same as in Figure 1C.