Lysosomal TMEM175 is not a H ⁺ -selective channel. (A) Schematic for whole organelle recording from lysosomes dissected from TMEM175-transfected HEK293T cells. The definitions of membrane potential (ΔΨ or Ψ) and current (I) followed a previously proposed nomenclature (Bertl et al., 1992). Ψ (= V_cytosol − V_lumen) is the voltage across the lysosomal membrane with lumen used as a reference. “Inward” cation current denotes cations leaving the lumen into the cytosol (bath), while “outward” cation current denotes entering the lumen from the cytosol (bath). I-Ψ relationships were obtained with a ramp protocol from −120 to +120 mV in 1 s, Ψ_h = 0 mV (before LJP adjustment). Pipette and bath solutions had pH 4.6 and pH 7.2, respectively. (B–D) Currents recorded with bath solutions containing 0 mM K⁺ (NMDG-containing) or 150 mM K⁺ and pipette solutions containing 0 mM (B), 5 mM (C), or 50 mM (D) K⁺. For each pipette condition, representative traces recorded with NMDG-containing and 150 mM K⁺-containing baths from the same lysosome are shown on the left. Summaries of the inward (at −100 mV) and outward (at +100 mV) current amplitudes are on the middle and right, respectively. Each circle represents data from one lysosome. The number of lysosomes in each pipette condition are n = 5 for B and C and n = 6 for D. The pink arrow in B indicates the Ψ_rev for 150 mM K⁺-containing bath if the P_H/P_K were 48,000, as previously reported by Hu et al., while the purple arrow indicates the measured Ψ_rev. LJP was adjusted offline. (E) Average reversal potentials obtained with 150 mM K⁺-containing bath and pipette (lumen) solutions containing 0, 5, or 50 mM K⁺. The number of lysosomes for determination of Ψ_rev for each pipette conditions containing 0 and 5 mM K⁺ are n = 5 and n = 6 for pipette containing 50 mM K⁺. The red dots and dashed red line indicate predicted reversal potentials under each condition if the channel were a H⁺-selective channel with P_H/P_K of 48,000, as proposed by Hu et al. (2022). Error bars are SEM.