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. 2022 May 24;11:e75122. doi: 10.7554/eLife.75122

Figure 5. Electrophysiological analysis of human transmembrane protein 175 (hTMEM175)-transfected HEK293T cells.

Representative whole-cell electrical recordings and current-voltage relationships of hTMEM175-transfected (A), I46V-transfected (B), and I271V-transfected (C) HEK293T cells in the absence and presence of inhibitor AP-6, in bi-ionic conditions of 150 mM Cs+ (intracellular) and 150 mM Na+ (extracellular). Current amplitudes in the current-voltage relationship plots were gained by averaging current values in the shaded time window. (D) A schematic of whole-cell patch clamp performed in (A–C and E). (E) Current-voltage relationships of six independent whole-cell patch clamp recordings of hTMEM175 WT-, I46V-, and I271V-transfected cells in bi-ionic conditions of 150 mM Cs+ (intracellular) and 150 mM Na+ (extracellular).

Figure 5—source data 1. Source data for Figure 5.
Figure 5—source data 2. Source data for Figure 5.

Figure 5.

Figure 5—figure supplement 1. Characterization of transmembrane protein 175 (TMEM175) inhibition by AP-6.

Figure 5—figure supplement 1.

(A) Current-voltage relationships of whole-cell patch clamp recordings of human TMEM175 (hTMEM175)-transfected cells under various concentrations of AP-6. (B) Dose-response curve of Cs+ current at +100 mV from three independent titrations. Means and SEM are shown.
Figure 5—figure supplement 2. Spectroscopy of AP-6.

Figure 5—figure supplement 2.

1H-NMR, 13C{1H}-NMR, and IR spectrum of AP-6.
Figure 5—figure supplement 3. 4-Aminopyridine (4-AP) and AP-6 sensitivity of transmembrane protein 175 (TMEM175) mutants.

Figure 5—figure supplement 3.

Mean currents recorded from HEK293T cells transfected with human TMEM175 (hTMEM175) (blue), I46V (red), I271V (green), I46A (purple), I271A (orange), and non-transfected (black) at +100 mV: raw currents before applying 1 mM 4-AP (upper left), 4-AP-sensitive current (bottom left), raw currents before applying 2 mM AP-6 (upper right), and AP-6-sensitive current (bottom right). Number of experiments for each mutant are presented in parentheses.
Figure 5—figure supplement 4. 4-Aminopyridine (4-AP) insensitivity of I271V mutant.

Figure 5—figure supplement 4.

4-AP insensitivity of I271V mutant. Representative whole-cell electrical recordings and current-voltage relationships of I271V-transfected HEK293T cells in the absence or presence of 4-AP in bi-ionic conditions of 150 mM Cs+ (intracellular) and 150 mM Na+ (extracellular). Current amplitudes in the current-voltage relationship plots were gained by averaging current values in the shaded time window.