ClC-3 is an essential molecular component of endogenous volume -sensitive osmolyte and anion channels (VSOACs) in Xenopus laevis oocytes

Volume-sensitive osmolyte and anion channels (VSOACs) are activated upon cell swelling in most vertebrate cells. Native VSOACs are believed to be a major pathway for regulatory volume decrease (RVD) through efflux of chloride and organic osmolytes. ClC-3, a member of the ClC superfamily of chloride channels, has been proposed to be a molecular candidate for VSOACs in some cell types.;To directly establish the link between native VSOACs and ClC-3 three approaches were taken.;The first approach used a commercially available anti-ClC-3 antibody (Ab), as a selective pharmacological tool, to determine the relationship between native VSOACs and ClC-3. Intracellular dialysis of anti-ClC-3 Ab functionally abolished transiently expressed guinea-pig ClC-3 currents (I gpClC-3) in NIH3T3 cells, native VSOACs in guinea pig cardiac myocytes, canine pulmonary artery smooth muscle cells and injection of anti-ClC-3 Ab significantly reduced native VSOACs in Xenopus laevis oocytes.;The second approach used ClC-3 antisense to determine the relationship between ClC-3, native VSOACs, and cell volume regulation in HeLa cells and oocytes. In situ hybridization in HeLa cells, semiquantitative and real-time PCR, and immunoblot studies in HeLa cells and oocytes demonstrated the presence of ClC-3 mRNA and protein, respectively. Exposing both cell types to hypotonic solutions induced cell swelling and activated native VSOACs. Transient transfection of HeLa cells with ClC-3 antisense oligonucleotide or oocytes injected with antisense cRNA abolished native ClC-3 mRNA transcript, protein, and significantly reduced the density of native VSOACs activated by hypotonic induced cell swelling.;The third approach demonstrated the functional re-expression and characterization of the native ClC-3 chloride channel (xClC-3) in oocytes. A full-length cDNA encoding native ClC-3 from Xenopus laevis oocyte cDNA (xClC-3) was cloned. The protein was re-expressed in defolliculated oocytes six days post surgery, when native ClC-3 protein and native VSOAC amplitudes were significantly reduced. Fluorescent micrographs illustrated that native ClC-3 and re-expressed xClC-3 are localized to the plasma membrane of oocytes. xClC-3 re-expressed oocytes exhibited Cl- current densities an order of magnitude larger than native VSOACs in control folliculated oocytes. (Abstract shortened by UMI.).