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Transfection of either ClC-6Tyr553Cys/Glu200Ala or ClC-6Glu200Ala produced slightly increased vesicles, suggesting that p.Glu200Ala, previously involving infantile spasms and microcephaly, can be pathogenic. Bafilomycin treatment abrogated vacuole generation, indicating that H+-driven Cl- accumulation osmotically drives vesicle enlargement. Our work establishes mutations in CLCN6 associated with neurological diseases, whose spectral range of clinical features is dependent on the