Interaction of atrial muscarinic receptors with three kinds of GTP-binding proteins

• Published in: Journal of molecular and cellular cardiology Vol. 22; no. 3; pp. 343 - 351
• Main Authors: Ikegaya, Takayoshi, Nishiyama, Terumasa, Haga, Kazuko, Haga, Tatsuya, Ichiyama, Arata, Kobayashi, Akira, Yamazaki, Noboru
• Format: Journal Article
• Language: English
• Published: Kent Elsevier Ltd 01.03.1990; Elsevier
• Subjects: acetylcholine; Acetylcholine - metabolism; Acetylcholine - pharmacology; Animals; atrium; Biological and medical sciences; Cell physiology; Fundamental and applied biological sciences. Psychology; G proteins; GTP-Binding Proteins - metabolism; GTPase; Heart Atria - metabolism; Heart Atria - ultrastructure; m2 subtype; Molecular and cellular biology; Muscarinic acetylcholine receptors; Receptors, Muscarinic - metabolism; Responses to growth factors, tumor promotors, other factors; Swine; m2 subtype; G proteins; GTPase; Muscarinic acetylcholine receptors; Atrium; Binding protein; Vertebrata; Mammalia; Reconstituted system; Molecular interaction; Muscarinic receptor; Artiodactyla; Ungulata; Pig; G protein
• ISSN: 0022-2828; 1095-8584
• DOI: 10.1016/0022-2828(90)91467-L

Purified porcine atrial muscarinic, acetylcholine receptors were reconstituted into lipid vesicles with three different G proteins (G i, G o and G n) 1 1 Abbreviations: G proteins, GTP-binding proteins; G i, G o, and G n, G proteins purified from porcine cerebrum and characterized by apparent molecular size of α subunit of 41, 39, 40 kDa, respectively; [ 3H]QNB, [ 3H]L-guinuclidinyl benzylate; GTPγS, guanosine 5′(3-O-thio)triphosphate. purified from porcine cerebrum. All the G proteins interacted with the receptor as evidenced by GTP-sensitive high affinity binding with acetylcholine, and stimulation by acetylcholine of GTPγS binding and GTPase activities. The curves of displacement by acetylcholine of [ 3H]QNB binding were explained by assuming two sites with the same affinity for [ 3H]QNB but different affinities for acetylcholine. The proportion of the high affinity site increased from 3 to 7% up to 82 to 83% of total binding sites with increasing G protein concentration, and essentially the same results were obtained with the three G proteins. The GTPase activities of G i, G o and G n in the reconstituted vesicles were 2.7-, 1.7- and 1.6-times higher, respectively, in the presence of 1 m m acetylcholine than those in the presence of 10 μ m atropine. An obvious enhancement by acetylcholine of the GTPγS binding was observed in the presence of 10 to 100 μ m GDP, while the enhancement was minimal, if at all, in the absence of GDP. When the molar ratios of reconstituted G i, G o and G n to muscarinic receptors were 54, 84 and 107, respectively, the acetylcholine-induced increase in the [ 35S]GTPγS binding was as much as 12, 35 and 27 mol with G i, G o and G n, respectively, per mole of the receptor molecule, indicating that the muscarinic receptors interact with G proteins catalytically. Atrial muscarinic receptors are considered to be composed solely of m2 subtype, and hence the present results suggest that a single muscarinic receptor subtype interacts with at least three kinds of G proteins.