The functional role of the binding site aspartate in muscarinic acetylcholine receptors, probed by site-directed mutagenesis

Abstract

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-methyl-scopolamine, 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.