Coupling of M2 muscarinic receptor to L-type Ca channel via c-src kinase in rabbit colonic circular smooth muscle.
OBJECTIVE
The L-type Ca(2+) channel is a major pathway for Ca(2+) 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 Ca(2+) 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 Ca(2+) channel was determined by a GST pull-down assay.


RESULTS
Methacholine (10 micromol/L) enhanced Ca(2+) 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 Ca(2+) 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 Ca(2+) channel. In M(2) receptor transfected HEK293 cells, currents were enhanced 2-fold by carbachol.


CONCLUSIONS
These studies demonstrate stimulation of Ca(2+) current in colonic smooth muscle cells by M2 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.
Department of Physiology, University of Oklahoma Health Science Center, Oklahoma City, Oklahoma 73104, USA.
Xiaochun
X
Jin
Nemat
N
Morsy
Fouzia
F
Shoeb
Joelle
J
Zavzavadjian
Hamid I
HI
Akbarali
D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D020746:Calcium Channels, L-Type / Q000378:metabolism* / Q000502:physiology; D002460:Cell Line; D003106:Colon / Q000378:metabolism*; D004553:Electric Conductivity; D006801:Humans; D009130:Muscle, Smooth / Q000378:metabolism*; D020033:Protein Isoforms / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000502:physiology*; D011817:Rabbits; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic / Q000378:metabolism*; D014162:Transfection; D019061:src-Family Kinases
D020746:Calcium Channels, L-Type; D020033:Protein Isoforms; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human