Investigating Functional Consequences of Novel Disease-Causing Mutations of CLCN7 Gene

CLC-7 is an intracellular chloride-proton antiporter of the CLC protein family. In complex with its accessory protein Ostm-1, it localizes to the lysosomes where it is important for protein degradation and to the ruffled border of osteoclasts, where it is thought to play a critical role in the acidification of the resorption lacuna and in the osteoclast-mediated bone resorption. ClC-7 KO mouse phenotype revealed severe osteopetrosis, retinal degeneration and neurodegeneration associated with lysosomal storage. Consistently several mutations in the human CLCN7 gene were identified in patients with osteopetrosis, a disease characterized by dense and fragile bones associated with highly heterogeneous clinical symptoms. We investigated the effects of two undescribed CLC-7 mutations identified in patients diagnosed for osteopetrosis: a novel homozygous missense mutation associated with a clinical phenotype manifesting a severe neurodegeneration and a heterozygous missense mutation in a patient with a milder phenotype. We verified by confocal microscopy that the lysosomal co-localization of CLC-7 and Ostm1 was preserved for both CLC-7 mutants. Moreover, to determine the effects of CLC-7 mutations on transporter activity we performed patch-clamp recordings on a plasma-membrane-targeted CLC-7 that overcomes the limit of the lysosomal localization of the protein. Preliminary results surprisingly reveal that the CLC-7 homozygous mutation did not completely abolish chloride currents, but showed strongly reduced currents with slower kinetics of activation; instead the CLC-7 heterozygous mutation showed a behavior similar to the WT. We are currently studying whether the Cl -/H + coupling is affected by the mutations using an optical assay that employs the E 2GFP/DsRed Cl 2/pH sensor fused to the C-terminus of CLC-7