Figure 2. The TMEM175 channel is highly proton selective.

(A-B) Whole-cell currents recorded in an HEK293T cell overexpressing TMEM175 or a vector transfected (sham) cell. pH_C was set to 7.2 and extracellular pH (pH_E/L) varied as indicated. Unless otherwise indicated, all recording solutions (bath/extracellular and pipette/cytosolic) contained NMDG-MSA as the major ions. (C) Summary of whole-cell TMEM175 currents (I_TMEM175) at −120 mV normalized to current at pH_E/L = 4.6 (n = 6 cells, mean ± SEM, * p <0.05, *** p <0.001). Summary of whole-cell macroscopic conductance is shown in Supplemental Fig. S3A. (D) E_rev measured in an HEK293T cell overexpressing TMEM175 with pH_c set to 5.0 and pH_E/L varied as indicated. (E) E_rev as a function of ΔpH (= pH_E/L- pH_C) from experiments as in (D). The line fit to the data had a slope of −53 mV/ΔpH (n = 4 - 5, mean ± SEM). (F) E_rev of I_TMEM175 under “bi-ionic” conditions. The cytosolic solution contained K-MSA instead of NMDG-MSA as the major ions (pH_c = 7.2). (G) Analysis of pH-dependence of E_rev from experiments as in (F). The line is a least square linear regression fit to the averages of E_rev measured at pH_E/L 5.0 to 4,2 (n = 5 - 11, slope = −53 mV/ΔpH, mean ± SEM). (H) Summary of slope values in the pH-E_rev experiments when NMDG⁺ (as in D-E), Na⁺, and K⁺ (as in F-G) were used as the major ions (n = 4 - 6, mean ± SEM, NS, p > 0.05). (I) Analysis of relative H⁺ permeability over NMDG⁺, Na⁺, or K⁺ based on E_rev measurement (n = 6 - 13). The estimated P_K/P_H for the OTOP1 H⁺ channel, measured using the same recording conditions, is shown for comparison. Note that an incomplete block (by TEA⁺) of background K⁺ channels present in the HEK293T plasma membrane (PM) might make a contaminating contribution to P_K, hence leading to an underestimated P_H/P_K value for TMEM175 on the PM compared with on LELs. Additionally, the outward currents may also contain a contaminating component of H⁺, currents due to experimentally-induced cytosolic H⁺ accumulation (see Methods). (J) Average pHlourin fluorescence in TMEM175-transfected HEK293T cells (n > 30 cells per coverslip) in response to stimuli as indicated. Imaging solutions contained NMDG-MSA as the major ions. HOAc (pH 5.0) and NH₄Cl (pH 7.2), which freely enter the cytosol, served as positive and negative controls to acidify or alkalinize the cytosol, respectively. (K) Summary of relative pHluorin fluorescence intensity in response to pH_E/L 4.6 and HOAc (sham/TMEM175: n = 9/35, mean ± SEM, *** p <0.001). See Supplemental Fig. S3K–O for similar experiments using other imaging solutions. N represents the number of cells randomly selected from at least three independent biological replicates.