Activation of TMEM175 lysosomal ion channels by CysLT1 receptor antagonists

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 variants in TMEM175 have been identified as genetic risk factors for Parkinson's disease, highlighting the potential of small-molecule activators as a novel therapeutic strategy for this disease. We developed a high-throughput screening (HTS) assay using HEK-293 cells stably overexpressing TMEM175 at the cell surface and screened 960 Food and Drug Administration (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, (2-butyl-6,7-dichloro-2-cyclopentyl-indan-1-on-5-yl) oxybutyric acid (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.NEW & NOTEWORTHY TMEM175 regulates lysosomal voltage and pH and is genetically linked to Parkinson's disease. Our study identifies zafirlukast and related drugs as activators, defines their AKT dependency, and maps structural determinants of gating. These results open new avenues for targeting TMEM175 in neurodegenerative diseases.