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 re ceptors to Asn (M(1)) or Glu (M(1) and M(2)) strongly reduced the high -affinity component of agonist binding, and the M(1) phosphoinositide response. Formation of the acetylcholine-receptor binary complex was a lso 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 posi tively-charged headgroup probably anchors acetylcholine when it is bou nd in its active conformation, but alternative, non-productive, bindin g modes, promoted by non-polar forces, may contribute to binary comple x formation by other ligands.