Dysregulated PIKfyve-dependent inhibition of ClC-7 causes ammonium-dependent endolysosomal vacuolization and autophagic flux impairment: Rescue by glutaminase inhibition

ClC-7 is a lysosomal 2Cl − /1H + antiporter essential for normal lysosomal function. PIKfyve-generated PI(3,5)P 2 negatively regulates ClC-7 activity, and gain-of-function mutations (e.g., Y715C) that abolish this regulation cause severe genetic disorders. At the cellular level, these variants induce extensive endolysosomal vacuolization accompanied by impaired autophagic flux; however, the precise molecular mechanism driving vacuole enlargement remained elusive. Here, we report that prominent vacuole formation in cells expressing ClC-7 gain-of-function variants is closely associated with a marked intracellular ammonium accumulation. Furthermore, vacuole enlargement was attenuated by an ammonium scavenger and accelerated by exogenous glutamine or ammonium chloride, while pharmacological glutaminase inhibition suppressed both vacuole size and ammonium accumulation. Pharmacological inhibition of PIKfyve likewise induced vacuolization and ammonium accumulation, both of which were suppressed by ClC-7 knockout. Using a newly modified lipid-probe, we found that unlike PIKfyve inhibition, PI(3,5)P 2 is maintained on ClC-7 gain-of-function vacuoles. The formation of vacuoles induced by the overexpression of SNX10, which downregulates PI(3,5)P 2 synthesis, or by expression of a gain-of-function ClC-6 mutant was also accompanied by a marked accumulation of cellular ammonium. Additionally, cells overexpressing ClC-3 or ClC-5 exhibited vacuolization associated with cellular ammonium accumulation under the control of the accessory protein TMEM9. The impairment of autophagic flux triggered by either ClC-7 gain-of-function expression or PIKfyve inhibition was restored by glutaminase inhibition. Collectively, these findings demonstrate that glutamine-derived ammonium drives endolysosomal vacuolization and autophagic dysfunction downstream of the PIKfyve–ClC-7 axis, identifying glutaminase inhibition as a potential therapeutic strategy for associated genetic disorders.