Inhibition of VRAC by c-Src tyrosine kinase targeted to caveolae is mediated by the Src homology domains

We used the whole cell patch-clamp technique in calf pulmonary endothelial (CPAE) cells to investigate the effect of wild-type and mutant c-Src tyrosi ne kinase on I-Cl,I-swell, the swelling-induced Cl- current through volume- regulated anion channels (VRAC). Transient transfection of wild-type c-Src in CPAE cells did not significantly affect ICl, swell. However, transfectio n of c-Src with a Ser(3)Cys mutation that introduces a dual acylation signa l and targets c-Src to lipid rafts and caveolae strongly repressed hypotoni city-induced I-Cl,I-swell in CPAE cells. Kinase activity was dispensable fo r the inhibition of I-Cl,I-swell, since kinase-deficient c-Src Ser(3)Cys ei ther with an inactivating point mutation in the kinase domain or with the e ntire kinase domain deleted still suppressed VRAC activity. Again, the Ser( 3)Cys mutation was required to obtain maximal inhibition by the kinase-dele ted c-Src. In contrast, the inhibitory effect was completely lost when the Src homology domains 2 and 3 were deleted in c-Src. We therefore conclude t hat c-Src-mediated inhibition of VRAC requires compartmentalization of c-Sr c to caveolae and that the Src homology domains 2 and/or 3 are necessary an d sufficient for inhibition.