K ⁺ suppresses TMEM175-mediated H ⁺ conductance. Whole cell plasma membrane currents were recorded from TMEM175-transfected HEK293T cells using gap-free recording with a ramp from −120 to +120 mV (before LJP correction) at 400 ms, every 10 s (holding potential V_h = 0 mV) with pipette solution containing 150 mM K⁺, pH 7.2 ([H⁺] = 0.063 μM), and bath with varying [K⁺] and pH. Each cell was first recorded in a pH 7.2 bath containing no K⁺, followed a K⁺-free bath with pH 4.5 ([H⁺] = 31.62 μM), and baths containing varying amount of K⁺ as indicated. (A–C) Left: Representative traces of gap-free recording in bath with varying [K⁺] and pH as indicated. Right: Current–voltage (I–V) relationships obtained from ramps at time points indicated in the traces. (D) Amplitudes of inward (left, at −100 mV) and outward (right, at +100 mV) currents obtained using baths with varying [K⁺] normalized to that obtained with the immediately preceding pH 4.5 K⁺-free bath. For inward currents, the amplitudes of currents obtained with 1 mM K⁺-, 10 mM K⁺-, and 50 mM K⁺-containing baths normalized to that obtained with K⁺-free bath are 0.54, 0.54, and 2.69, respectively (all with the same [H⁺]/pH). Since the current amplitude of 50 mM K⁺-containing bath is 5.0× (=2.69/0.54) that with 10 mM K⁺-containing bath, one can estimate that, with 50 mM K⁺ in the bath, the fraction of K⁺ currents is 80% (= [5.0–1]/5.0). This estimate assumes that all the current obtained with 10 mM K⁺-containing bath is from H⁺ and the H⁺ current remained the same size in 50 mM K⁺-containing bath, which likely overestimates the contribution by H⁺ and underestimates the fraction of K⁺ currents. LJP was adjusted offline. Error bars are SEM and the number in parenthesis is the number of recordings in each condition. Mock (empty-vector)-transfected HEK293T cells had minimal inward currents under similar recording conditions with pH 4.5 bath containing 0 mM K⁺ (−9.8 ± 2.5 pA at −100 mV, n = 6), 1 mM K⁺ (−19.1 ± 4.8 pA, n = 5), or 50 mM K⁺ (−39.5 ± 10.8 pA, n = 4).