A9 fibroblasts transfected with the m3 muscarinic receptor clone express a Ca2+ channel activated by carbachol, GTP and GDP.
Muscarinic m3 receptor-mediated changes in cytosolic Ca2+ concentration ([Ca2+]i) occur by activation of Ca2+ release channels present in the endoplasmic reticulum membrane and Ca2+ entry pathways across the plasma membrane. In this report we demonstrate the coupling of m3 muscarinic receptors to the activation of a voltage-insensitive, cation-selective channel of low conductance (3.2 +/- 0.6 pS; 25 mM Ca2+ as charge carrier) in a fibroblast cell line expressing m3 muscarinic receptor clone (A9m3 cells). Carbachol (CCh)-induced activation of the cation-selective channel occurred both in whole cell and excised membrane patches (CCh on the external side), suggesting that the underlying mechanism involves receptor-channel coupling independent of intracellular messengers. In excised inside-out membrane patches from nonstimulated A9m3 cells GTP (10 microM) and GDP (10 microM) activated cation-selective channels with conductances of approximately 4.3 and 3.3 pS, (25 mM Ca2+ as charge carrier) respectively. In contrast, ATP (10 microM), UTP (10 microM) or CTP (10 microM) failed to activate the channel. Taken together, these results suggest that carbachol and guanine nucleotides regulate the activation of a cation channel that conducts calcium.
Singer-Lahat
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Laboratory of Cellular and Molecular Regulation, National Institute of Mental Health, Bethesda, MD 20892, USA.
Rojas
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Felder
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D000818:Animals; D015220:Calcium Channels / Q000096:biosynthesis / Q000187:drug effects / Q000502:physiology*; D002217:Carbachol / Q000494:pharmacology*; D005347:Fibroblasts; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology*; D006160:Guanosine Triphosphate / Q000494:pharmacology*; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D018408:Patch-Clamp Techniques; D051381:Rats; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic / Q000096:biosynthesis / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis; D013873:Thionucleotides / Q000494:pharmacology; D014162:Transfection
D015220:Calcium Channels; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic; D011994:Recombinant Proteins; D013873:Thionucleotides; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C023427:guanosine 5'-O-(2-thiodiphosphate); D006160:Guanosine Triphosphate; D002217:Carbachol