FUNCTIONAL-CHARACTERIZATION OF RENAL CHLORIDE CHANNEL, CLCN5, MUTATIONS ASSOCIATED WITH DENTS(JAPAN)-DISEASE

Background. The annual urinary screening of Japanese children above th ree years of age has identified a progressive renal tubular disorder c haracterized by low molecular weight proteinuria, hypercalciuria and n ephrocalcinosis, and this represents a variant of Dent's disease. Hith erto, 12 mutations of the X-linked renal specific chloride channel, CL CN5, have been reported in the Dent's(Japan) variant. To further ident ify such CLCN5 mutations and to define the structure-function relation ships of this channel, we have investigated five unrelated, non-consan guinous Japanese families with this disorder. Methods. Leukocyte DNA f rom probands was used with CLCN5 primers for PCR amplification of the coding region, and the DNA sequences of the products determined. Funct ional studies were performed by expressing the mutants in Xenopus oocy tes. Results. Five CLCN5 mutations consisting of two nonsense (R648X a nd R704X), two missense (S270R and L278F) and one acceptor splice site mutation (ag-->cg) in intron 4 were identified. The missense and spli ce site mutations represent novel abnormalities. Heterologous expressi on in Xenopus oocytes of wild-type and the missense mutants demonstrat ed that the mutations, which were translated, either abolished or mark edly reduced chloride conductance. Conclusions. These results expand t he spectrum of CLCN5 mutations associated with this renal disorder and provide insight into possible structure-function relationships. For e xample, both the missense mutations are located within a short putativ e loop between two transmembrane domains, and our results suggest that this region may have an important functional role in the regulation o f channel activity.