Characterization of the role of TMEM175 in an invitro lysosomal H+ fluxes model

Lysosome acidification is a dynamic equilibrium of H+ influx and efflux across the membrane, which is crucial for cell physiology. The vacuolar H+ ATPase (V‐ATPase) is responsible for the H+ influx or refilling of lysosomes. TMEM175 was identified as a novel H+ permeable channel on lysosomal membranes, and it plays a critical role in lysosome acidification. However, how TMEM175 participates in lysosomal acidification remains unknown. Here, we present evidence that TMEM175 regulates lysosomal H+ influx and efflux in enlarged lysosomes isolated from COS1 treated with vacuolin‐1. By utilizing the whole‐endolysosome patch‐clamp recording technique, a series of integrated lysosomal H+ influx and efflux signals in a ten‐of‐second time scale under the physiological pH gradient (luminal pH 4.60, and cytosolic pH 7.20) was recorded from this in vitro system. Lysosomal H+ fluxes constitute both the lysosomal H+ refilling and releasing, and they are asymmetrical processes with distinct featured kinetics for each of the H+ fluxes. Lysosomal H+ fluxes are entirely abolished when TMEM175 losses of function in the F39V mutant and is blocked by the antagonist (2‐GBI). Meanwhile, lysosomal H+ fluxes are modulated by the pH‐buffering capacity of the lumen and the lysosomal glycosylated membrane proteins, lysosome‐associated membrane protein 1 (LAMP1). We propose that the TMEM175‐mediated lysosomal H+ fluxes model would provide novel thoughts for studying the pathology of Parkinson's disease and lysosome storage disorders.