Muscarinic M2 receptor stimulation of Cav1.2b requires phosphatidylinositol 3-kinase, protein kinase C, and c-Src.
This study investigated regulation of L-type calcium channels (Cav1.2b) by acetylcholine (ACh) in rabbit portal vein myocytes. Whole-cell currents were recorded using 5 mmol/L barium as charge carrier. ACh (10 micromol/L) increased peak currents by 40%. This effect was not reversed by the selective muscarinic M3 receptor antagonist 4-DAMP (100 nmol/L) but was blocked by the M2 receptor antagonist methoctramine (5 micromol/L). The classical and novel protein kinase C (PKC) antagonist calphostin C (50 nmol/L) abolished ACh responses, whereas the classical PKC antagonist Gö6976 (200 nmol/L) had no effect. ACh responses were also abolished by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (20 micromol/L), by the c-Src inhibitor PP2 (10 micromol/L) (but not the inactive analogue PP3), and by dialyzing cells with an antibody to the G-protein subunit Gbetagamma. Cells dialyzed with c-Src had significantly greater currents than control cells. Current enhancement persisted in the presence of LY294002, suggesting that c-Src is downstream of PI3K. Phorbol 12,13-dibutyrate (PDBu, 0.1 micromol/L) increased currents by 74%. This effect was abolished by calphostin C and reduced by Gö6976. The PDBu response was also reduced by PP2, and the PP2-insensitive component was blocked by Gö6976. In summary, these data suggest that ACh enhances Cav1.2b currents via M2 receptors that couple sequentially to Gbetagamma, PI3K, a novel PKC, and c-Src. PDBu stimulates the novel PKC/c-Src pathway along with a second pathway that is independent of c-Src and involves a classical PKC.
Callaghan
B
B
Department of Physiology and Cell Biology, University of Nevada, Reno, NV 89557, USA.
Koh
S D
SD
Keef
K D
KD
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D001464:Barium / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000502:physiology*; D002227:Carbazoles / Q000494:pharmacology; D002478:Cells, Cultured / Q000187:drug effects / Q000502:physiology; D002867:Chromones / Q000494:pharmacology; D058544:Class Ib Phosphatidylinositol 3-Kinase; D003959:Diamines / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D044387:GTP-Binding Protein beta Subunits / Q000037:antagonists & inhibitors / Q000502:physiology; D044388:GTP-Binding Protein gamma Subunits / Q000037:antagonists & inhibitors / Q000502:physiology; D007211:Indoles / Q000494:pharmacology; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D017136:Ion Transport / Q000187:drug effects; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000502:physiology*; D008297:Male; D009025:Morpholines / Q000494:pharmacology; D018721:Muscarinic Agonists / Q000494:pharmacology*; D018727:Muscarinic Antagonists / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000502:physiology; D009281:Naphthalenes / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010880:Piperidines / Q000494:pharmacology; D011169:Portal Vein / Q000166:cytology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000037:antagonists & inhibitors / Q000502:physiology*; D011743:Pyrimidines / Q000494:pharmacology; D011817:Rabbits; D043585:Receptor, Muscarinic M2 / Q000819:agonists / Q000037:antagonists & inhibitors / Q000502:physiology*; D043587:Receptor, Muscarinic M3 / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*
D000109:Acetylcholine; D001464:Barium; D020746:Calcium Channels, L-Type; D002227:Carbazoles; D002867:Chromones; D058544:Class Ib Phosphatidylinositol 3-Kinase; D003959:Diamines; D004791:Enzyme Inhibitors; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D007211:Indoles; D007527:Isoenzymes; D009025:Morpholines; D018721:Muscarinic Agonists; D018727:Muscarinic Antagonists; D009281:Naphthalenes; D015240:Phorbol 12,13-Dibutyrate; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010880:Piperidines; D011493:Protein Kinase C; D016392:Proto-Oncogene Proteins pp60(c-src); D011743:Pyrimidines; D043585:Receptor, Muscarinic M2; D043587:Receptor, Muscarinic M3; C412373:AG 1879; C118981:G-protein Beta gamma; C419385:L-type calcium channel alpha(1C); C081021:Go 6976; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C042375:4-diphenylacetoxy-1,1-dimethylpiperidinium; C058819:calphostin C; C054938:methoctramine