Proton selective conductance and gating of lysosomal cation channel TMEM175

The lysosomal cation channel TMEM175 is crucial for maintaining lysosomal function and pH homeostasis, and its aberrant function is linked to Parkinson’s disease (PD). While TMEM175 activity was first interpreted in the context of its potassium (K ⁺ ) selective conductance, subsequent studies revealed also a substantial permeability to protons (H ⁺ ). Here we dissect the complex changes in TMEM175 conductance and current reversal voltages in response to pH jumps on the luminal side of the channel protein. In whole-cell patch clamp experiments with plasma membrane redistributed TMEM175 we show that a pH jump from symmetrical pH 7.4 to pH 4.7 on the luminal side triggers a continuous rise in inward and outward current, concomitant with a transient positive excursion of the reversal voltage (E _rev ). The peak E _rev shift remains almost 100 mV below the estimated equilibrium voltage for protons and shows little sensitivity to the K ⁺ gradient. The data are consistent with a scenario in which a TMEM175 mediated proton flux elicits a fast collapse of the pH gradient. In MD simulations we identify the luminal H57 as titratable partner for the formation of intra- and inter-subunit salt bridges with D279 and E282 for stabilizing the channel open state. This presumed gating function is confirmed by mutational studies and lysosomal patch-clamp experiments in which a H57Y mutant exhibits a reduced pH dependency of activation. Our findings contribute to a better comprehension of TMEM175’s complex electrophysiological properties and foster understanding of TMEM175 as a pharmacological target for neurodegenerative disease therapy.