Coupling of M2 muscarinic receptor to L-type Ca2+ channel via c-src kinase in rabbit colonic circular smooth muscle

Background & Aims: The L-type Ca2+ channel is a major pathway for Ca2+ influx in colonic smooth muscle and is modulated by endogenous levels of nonreceptor tyrosine kinase, c-src. Tyrosine kinases are also activated by G-protein-coupled receptors (GPCR). This study determined whether muscarinic receptor couples to Ca2+ channels via c-src kinase. Methods: Currents were measured in rabbit colonic smooth muscle cells and in transfected HEK293 cells by patch-clamp technique. Tyrosyl phosphorylated proteins were detected by Western blots and the interaction of c-src with the c-terminus of alpha subunit of Ca2+ channel was determined by a GST pull-down assay. Results: Methacholine (10 mumol/L) enhanced Ca2+ channel currents by 30% under conditions whereby the M-3 receptor pathway was blocked by either 4-DAMP or by intracellular dialysis with anti-Galphaq antibody. Similar effects were observed by blocking intracellular Ca2+ release with heparin. Enhancement was abolished by intracellular anti-Galphai antibody and by the c-src inhibitor, PP2 but unaffected by the inactive analog PP3. Immunoblot with anti-src antibody revealed increased src phosphorylation by muscarinic receptor stimulation. Purified c-src directly associated with the c-terminus of alpha1c subunit of the Ca2+ channel. In M-2 receptor transfected HEK293 cells, currents were enhanced 2-fold by carbachol. Conclusions: These studies demonstrate stimulation of Ca2+ current in colonic smooth muscle cells by M-2 receptor coupled to Galphai-G protein and c-src activation. They also suggest a central role of c-src kinase in the cross-talk between tyrosine kinase receptor and GPCR.