Modulation of voltage-dependent Ca2+ channels in rabbit colonic smooth muscle cells by c-Src and focal adhesion kinase

There is emerging evidence indicating that smooth muscle contraction and Ca2+ influx through voltage-dependent L-type Ca2+ channels are regulated by tyrosine kinases; however, the specific kinases involved are largely unknown, In rabbit colonic muscularis mucosae cells, tyrosine-phosphorylated proteins of similar to 60 and 125 kDa were observed served in immunoblots using an anti-phosphotyrosine antibody and were identified as c-Src and focal adhesion kinase (FAK) by immunoblotting with specific antibodies, FAK co-immunoprecipitated with c-Src, and the phosphorylation of the c-Src.FAK complex was markedly enhanced by platelet-derived growth factor (PDGF) BE. The presence of activated c-Src in unstimulated cells was identified in cell lysates by immunoblotting with an antibody recognizing the autophosphorylated site (P416Y). In whole-cell patch-clamp studies, intracellular dialysis of a Src substrate peptide and anti-c-Src and anti-FAK antibodies suppressed Ca2+ currents by 60, 62, and 43%, respectively, In contrast, intracellular dialysis of an anti-mouse IgG or anti-Kv1.5 antibody did not inhibit Ca2+ currents, Co-dialysis of anti-cSrc and anti-FAK antibodies inhibited Ca2+ currents (63%) equivalent to dialysis with the anti-c-Src antibody alone, PDGF-BB enhanced Ca2+ currents by 43%, which was abolished by the anti-c-Src and anti-FAK antibodies, Neither the MER inhibitor PD 098059 nor an anti-Ras antibody inhibited basal Ca2+ currents or PDGF-stimulated Ca2+ currents, The alpha(1C) subunit of the L-type Ca2+ channel co-immunoprecipitated with anti-c-Src and anti-phosphotyrosine antibodies, indicating direct association of c-Src kinase with the Ca2+ channel, These data suggest that c-Src and FAK, but not the Ras/mitogen-activated protein kinase cascade, modulate basal Ca2+ channel activity and mediate the PDGF-induced enhancement of L-type Ca2+ currents in differentiated smooth muscle cells.