The functional role of the binding site aspartate in muscarinic acetylcholine receptors, probed by site-directed mutagenesis.
Mutation of the Asp in transmembrane domain three of the muscarinic receptors to Asn (M1) or Glu (M1 and M2) strongly reduced the high-affinity component of agonist binding, and the M1 phosphoinositide response. Formation of the acetylcholine-receptor binary complex was also strongly inhibited. In contrast, binary complex formation by other agonists, as well as the antagonist (-)-N-methylscopolamine, was less affected. Ionic bonding between the carboxylate oxyanion and the positively-charged headgroup probably anchors acetylcholine when it is bound in its active conformation, but alternative, non-productive, binding modes, promoted by non-polar forces, may contribute to binary complex formation by other ligands.
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Division of Physical Biochemistry, Medical Research Council National Institute for Medical Research, London, UK.
Curtis
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Jones
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Hulme
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D000109:Acetylcholine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism*; D001665:Binding Sites; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D019204:GTP-Binding Proteins / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism; D008024:Ligands; D018721:Muscarinic Agonists / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010716:Phosphatidylinositols / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
D008024:Ligands; D018721:Muscarinic Agonists; D010716:Phosphatidylinositols; D011976:Receptors, Muscarinic; D001224:Aspartic Acid; D018698:Glutamic Acid; D019204:GTP-Binding Proteins; D000109:Acetylcholine