TMEM175 is an AKT-activated lysosomal potassium- and proton-permeable channel that functions to dissipate voltage and pH gradients generated by the V-type H + -ATPase. Loss-of-function (LOF) variants in TMEM175 have been identified as genetic risk factors for Parkinson’s disease (PD), highlighting the potential of small-molecule activators as a novel therapeutic strategy for this disease. We developed a high-throughput screening assay using HEK-293 cells stably overexpressing TMEM175 at the cell surface and screened 960 FDA-approved drugs for TMEM175 potentiators. The screen identified 71 activators, including the cysteinyl leukotriene 1 receptor (CysLT1R) antagonists, pranlukast and montelukast. Because HEK-293 cells lack CysLT1R expression, we suspected these drugs may be direct channel activators. Fluorescence and automated patch clamp assays were used to evaluate the dose-dependency of pranlukast, montelukast, zafirlukast, and the known TMEM175 activator, DCPIB. These experiments revealed rank-order potencies and efficacies of DCPIB ~ zafirlukast > montelukast >> pranlukast. DCPIB, zafirlukast, and pranlukast activated TMEM175 independently of AKT activation, whereas the AKT inhibitor MK2206 partially inhibited montelukast-dependent TMEM175 activation. Computer modeling revealed a conformation-dependent solvent-accessible cavity near T119 and H449 that could participate in drug-induced activation, prompting us to examine these sites with mutagenesis. Not only did T119A and H449A mutations decrease apparent potencies of DCPIB, zafirlukast, and montelukast, but the T119A mutation produced a constitutively open channel phenotype. This study adds zafirlukast to the short list of moderately potent TMEM175 activators and identifies a region of the channel that contributes to activation gating.
Li et al. (Wed,) studied this question.