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

Abstract

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.

Keywords: H+ leak; TMEM175; glycocalyx; lysosome acidification; proton channel.