Voltage-dependent chloride channels of the ClC family are important components in key cellular processes such as cell-membrane excitability, cell-volume regulation, transepithelial transport, and acidification of intracellular organelles. ClC channels function as dimers, although the precise membrane topology of the subunits has not been clearly defined. Specific members of this family have been found localized to either the cell membrane or intracellular vesicles. The importance of these channels is underscored in humans, where mutations in three ClC genes cause distinct diseases affecting either muscle or renal function. We are using a reverse genetic approach to study the functional properties of ClC channels in C. elegans . Here we present our results concerning one isoform, CLH-5. In C. elegans , six genes encode ClC channels ( clh genes). cDNAs for all isoforms have been isolated, and preliminary expression patterns have been defined using reporter gene fusions to green fluorescent protein (gfp) and lacZ 1, 2, 3, 4 . Our data indicate that CLH-5 is expressed in touch-sensitive neurons and that it contributes to their function. To determine the expression pattern for
clh-5 , we used two GFP reporter constructs: Pclh-5::gfp, in which GFP expression is under the control of the
clh-5 promoter, and clh-5D4::gfp , in which GFP is fused in-frame to the 3' end of a partial
clh-5 genomic fragment that includes the promoter, the N-terminus and transmembrane segments D1 trough D4. In both cases, transgenic animals express GFP in the mechanosensory neurons that mediate response to light touch (AVM, ALML/R, PVM, PVML/R) and in tail interneurons. Transgenic animals expressing the truncated CLH-5 protein tagged with GFP were insensitive to light touch. We hypothesize that this phenotype results from a dominant negative interaction between CLH-5::D4::GFP and wild-type CLH-5. Consistent with this model, we have obtained a Mec phenotype by expressing
clh-5 dsRNA under control of the
clh-5 promoter. CLH-5 may be required in a general way for the function of mechanosensory neurons, for example by contributing to the membrane potential. Alternatively, CLH-5 may directly mediate touch sensation through a mechanism involving stretch-sensitive gating. Other members of this ion channel family are volume regulated ( C. elegans CLH-3 5 and human ClC-2 6 ), and, although the detailed activation mechanisms are unknown, mechanical forces may be important in regulating these ClC channels. In the future, we are interested in determining the important factors that regulate CLH-5 activity, as well as how CLH-5 may interact with known components of the mechanosensory pathway. 1 Petalcorin, M.I.R., Oka, T., Koga, M., Ogura, K., Wada, Y., Oshima, Y., and Futai, M. 1999. J. Mol. Biol. 294: 347-355. 2 Schriever, A.M., Friedrich, T., Pusch, M., and Jentch, T.J. 1999. J. Biol. Chem. 274: 34238-34244. 3 Nehrke, K., Begenisich, T., Pilato, J., and Melvin, J.E. 2000. Am. J. Cell. Physiol. 279: C2052-C2066. 4 Bianchi, L., Miller, D.M., George, A.L. 2001. Neurosci. Lett. 299:177-80. 5 Rutledge, E., Bianchi, L., Christensen, M., Boehmer, C., Morrison, R., Broslat, A., Beld, A.M., George, A.L., Greenstein, D., Strange, K. 2001. Curr. Biol. 11:161-170. 6 Grunder, S., Thiemann, A., Pusch, M., Jentsch, T.J. 1992. Nature 360: 759-62.