Evaluating Candidate Mechanisms of A Disease-Causing Mutation in the Lysosomal Chloride Transporter CLC-7

Lysosomes are organelles containing hydrolytic enzymes important for macromolecule degradation. For these acid hydrolases to function, an appropriately low pH must be maintained in the lysosome's lumen. CLC-7 is a lysosomal Cl-/H+ antiporter believed play important, albeit controversial, role acidification. The majority of established defects this protein loss-of-function mutations which cause osteopetrosis sometimes associated with storage disorders. However, we recently reported novel disease resulting from gain-of-function CLCN7 variant, two unrelated children. Many phenotypes, including delayed myelination, albinism, and storage, but not osteopetrosis, mutation. Both children carry same mutation, Y715C, localizes interface between transmembrane cytoplasmic domains newly published ClC-7 structures. Notably, when sorting motifs mutated, heterologously expressed plasma membrane, allowing measurement transporter currents. When patient mutation introduced context, observe approximate three-fold increase outward rectifying current, faster activation kinetics, slower deactivation kinetics compared wildtype Xenopus oocyte expression system. Here sought evaluate candidate mechanisms mutation's course action by performing extensive mutagenic screen, testing, amongst other factors, roles side chain hydroxylation, aromaticity, charge, steric effects, hydrophobicity. Overall demonstrate importance tyrosine at position transporter. Further experiments explore possibility phosphorylation regulation activity.