Analysis of human renal chloride channel (hCLC-5) mutations based on a three-dimensional model, suggests a structural-functional relationship
F Wu1, P Roche2, PT Christie1, NY Loh1, AAC Reed1, RM Esnouf2 & RV Thakker1
Dent's disease is an X-linked renal tubular disorder characterised by low molecular weight proteinuria, hypercalciuria, and nephrolithiasis. The disease is caused by inactivating mutations of a renal-specific chloride channel, hCLC-5, that belongs to the family of mammalian voltage-gated chloride channels (CLCs). Heterologous expression of wild-type CLC-5 in Xenopus oocytes results in chloride (Cl-) conductance, which is markedly reduced or abolished by the mutant hCLC-5s associated with Dent's disease. Two bacterial CLC X-ray crystal structures have been characterised recently, and we have used these to further examine the role of the hCLC-5 mutations. We aligned the hCLC-5 sequence with the Salmonella typhimurium (StCLC) and Escherichia coli (EcCLC) sequences, and the level of conservation was sufficient to enable the use of the StCLC structure as a model for hCLC-5. This revealed that hCLC-5, like StCLC and EcCLC, could be considered to be a homodimer with each subunit consisting of 18 helices. Each subunit contains its own Cl- pore in its centre, and forms an extensive interface region with the other subunit. We selected the 15 reported hCLC-5 mutations (one inframe insertion and 14 missense mutations) that are predicted to form full-length channels, and mapped these on the hCLC-5 model. None of the mutations involved the Cl- selectivity filter, but 12 of the 15 mutations were clustered at the interface of the two subunits. Six of these mutations occurred in two of the helices that either form part of the interface or lie in close proximity to the interface, while three other mutations associated with residual channel activity were at the periphery of the interface. Thus, our results demonstrate a crucial role for the interaction between the two subunits at the interface of the homodimeric hCLC-5, and help to further elucidate the structural consequences of the hCLC-5 mutations associated with Dent's disease.