Proton-selective conductance and gating of the lysosomal cation channel TMEM175

The lysosomal cation channel TMEM175 plays a key role in luminal pH homeostasis and lysosome function, with aberrant activity linked to Parkinson's disease. Although initially described as a K⁺-selective channel, TMEM175 exhibits substantial H⁺ permeability. Here, we dissect complex changes affecting human TMEM175 conductance and ionic properties of TMEM175-mediated current in response to pH shifts on the luminal side of the protein. A drop in pH from 7.4 to 4.7 on the side equivalent to the lysosomal lumen triggers a sustained increase in TMEM175-mediated inward and outward currents, which is accompanied by a transient shift in the reversal potential (E_rev) toward the theoretical equilibrium voltage for H⁺, yet remaining ~100 mV below the expected value even in the absence of K⁺. This discrepancy, along with low sensitivity of E_rev to the concentration gradient for K⁺, supports a model in which TMEM175-mediated H⁺ flux rapidly collapses the lysosomal pH-gradient. Molecular dynamics simulations identify H57 as a key residue on the luminal side of the open channel, which forms intra- and intersubunit salt bridges with D279 and E282. Supporting the functional importance of these interactions, the TMEM175 mutant H57Y displayed reduced H⁺- and K⁺-conductance and a reduced H⁺/K⁺ selectivity in whole-cell and lysosomal electrophysiological analyses. Our findings contribute to a better understanding of TMEM175's complex electrophysiological properties, thereby expanding the possibilities of understanding the channel's function in lysosomal physiology and pathophysiology.