title: Effects of magnesium ion on the interaction of atrial muscarinic acetylcholine receptors and GTP-binding regulatory proteins.
abstract: Muscarinic acetylcholine receptors (mAChR) purified from porcine atrium were reconstituted into lipid vesicles with GTP-binding regulatory proteins (G proteins, Gi, Go, or Gn) purified from porcine cerebrum. Apparent affinities of the reconstituted mAChR and G proteins for carbachol and GDP, respectively, were estimated from the effects of these ligands on the binding of [3H]-L-quinuclidinyl benzilate ([3H]QNB) to mAChR and [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTP gamma S) to G proteins in the presence of different concentrations of MgCl2. A total of 30-35% of reconstituted mAChRs exhibited low affinity for carbamylcholine, irrespective of the presence or absence of guanine nucleotides, and the remainder of the mAChRs showed high affinities for carbamylcholine in the absence of GTP or GDP and a low affinity in their presence. The affinity for carbamylcholine in the absence of guanine nucleotides, but not in their presence, increased with increases in MgCl2 concentration. Apparent Kd's for carbamylcholine were estimated to be approximately 100 microM in the presence of guanine nucleotides, 1.5 microM in the absence of guanine nucleotide and Mg2+ (< 0.1 microM), and 0.1 microM in the absence of guanine nucleotide and the presence of MgCl2 (10 mM). These results indicate that mAChRs may assume at least three different conformations that are characterized by different affinities for agonists. Furthermore, the data suggest that MgCl2 is not necessary for the formation of the mAChR-G protein complex, but can induce a conformational change in the complex. On the other hand, the presence of MgCl2 was necessary for carbamylcholine to influence the binding of guanine nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry, Faculty of Medicine, University of Tokyo, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Shiozaki
authors.forename: T
authors.initials: T
authors.lastname: Haga
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D002217:Carbachol / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000187:drug effects* / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006325:Heart Atria / Q000378:metabolism*; D066298:In Vitro Techniques; D008274:Magnesium / Q000494:pharmacology*; D011813:Quinuclidinyl Benzilate / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000187:drug effects* / Q000378:metabolism; D013552:Swine
chemical_list: D011976:Receptors, Muscarinic; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D011813:Quinuclidinyl Benzilate; D006160:Guanosine Triphosphate; D002217:Carbachol; D019204:GTP-Binding Proteins; D008274:Magnesium