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 tyrosine kinase on I(Cl,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 I(Cl,swell). However, transfection of c-Src with a Ser3Cys mutation that introduces a dual acylation signal and targets c-Src to lipid rafts and caveolae strongly repressed hypotonicity-induced I(Cl,swell) in CPAE cells. Kinase activity was dispensable for the inhibition of I(Cl,swell), since kinase-deficient c-Src Ser3Cys either with an inactivating point mutation in the kinase domain or with the entire kinase domain deleted still suppressed VRAC activity. Again, the Ser3Cys mutation was required to obtain maximal inhibition by the kinase-deleted 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 that c-Src-mediated inhibition of VRAC requires compartmentalization of c-Src to caveolae and that the Src homology domains 2 and/or 3 are necessary and sufficient for inhibition.
Trouet
D
D
Laboratory of Physiology, Catholic University of Leuven, Campus Gasthuisberg, B-3000 Leuven, Belgium.
Carton
I
I
Hermans
D
D
Droogmans
G
G
Nilius
B
B
Eggermont
J
J
D000215:Acylation; D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D002417:Cattle; D021941:Caveolae / Q000378:metabolism*; D002460:Cell Line; D048429:Cell Size; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology; D005347:Fibroblasts / Q000502:physiology; D017930:Genes, Reporter / Q000235:genetics; D015151:Immunoblotting; D018408:Patch-Clamp Techniques; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D018909:src Homology Domains / Q000502:physiology*; D019061:src-Family Kinases
D018118:Chloride Channels; D011993:Recombinant Fusion Proteins; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases