The interaction of acetylcholine receptors in porcine atrial membranes with three kinds of G proteins.
We developed a simple procedure to detect the interaction of muscarinic receptors in atrial membranes with exogenous GTP-binding proteins (G proteins). The procedure consists of mixing atrial membranes with G proteins in the presence of sodium cholate, diluting the mixture with a salt buffer and then measuring the ligand binding activity. The displacement by carbachol of [3H] QNB binding to muscarinic receptors in the atrial membranes was not affected by guanine nucleotides when the membranes had been treated at 60 degrees C for 30 min or with N-ethylmeleimide (NEM) and became affected by them after mixing the heat- or NEM-treated membranes with G proteins. The displacement curves in the presence of GTP were essentially the same irrespective of the presence or absence of G proteins. Those in the absence of GTP shifted to a lower concentration of carbachol with addition of a higher concentration of G proteins, indicating an increase in GTP-sensitive high affinity agonist binding sites. The highest affinity for carbachol was detected with membranes treated with NEM and then mixed with G proteins. The GTP-sensitive high affinity agonist binding could be detected with any one of three kinds of G proteins (Gi, Go, Gn) which were purified from porcine cerebrum, indicating that the muscarinic receptor m2 subtype may interact with and possibly activate these three kinds of G proteins.
Haga
T
T
Department of Biochemistry, Faculty of Medicine, University of Tokyo, Japan.
Ikegaya
T
T
Haga
K
K
D000818:Animals; D002217:Carbachol / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006325:Heart Atria / Q000187:drug effects / Q000378:metabolism; D006358:Hot Temperature; D008566:Membranes / Q000187:drug effects / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000378:metabolism*; D013552:Swine
D011976:Receptors, Muscarinic; D002217:Carbachol; D019204:GTP-Binding Proteins; D005033:Ethylmaleimide