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Roof and Floor of the Muscarinic Binding Pocket: Variations in the Binding Modes of Orthosteric Ligands

Division of Physical Biochemistry, MRC National Institute for Medical Research, Mill Hill, London, United Kingdom (E.C.H.); and GlaxoSmithKline, New Frontiers Science Park, Harlow, Essex, United Kingdom (J.A.G., C.J.L., B.T.)

Alanine substitution mutagenesis has been used to investigate residues that make up the roof and floor of the muscarinic binding pocket and regulate ligand access. We mutated the amino acids in the second extracellular loop of the M₁ muscarinic acetylcholine receptor that are homologous to the cis-retinal contact residues in rhodopsin, the disulfide-bonded Cys178 and Cys98 that anchor the loop to transmembrane helix 3, the adjoining acidic residue Asp99, and the conserved aromatic residues Phe197 and Trp378 in the transmembrane domain. The effects on ligand binding, kinetics, and receptor function suggest that the second extracellular loop does not provide primary contacts for orthosteric ligands, including acetylcholine, but that it does contribute to microdomains that are important for the conformational changes that accompany receptor activation. Kinetic studies suggest that the disulfide bond between Cys98 and Cys178 may contribute to structures that regulate the access of positively charged ligands such as N-methyl scopolamine to the binding pocket. Asp99 may act as a gatekeeper residue to this channel. In contrast, the bulkier lipophilic ligand 3-quinuclidinyl benzilate may require breathing motions of the receptor to access the binding site. Trp378 is a key residue for receptor activation as well as binding, whereas Phe197 represents the floor of the N-methyl scopolamine binding pocket but does not interact with acetylcholine or 3-quinuclidinyl benzilate. Differences between the binding modes of N-methyl scopolamine, 3-quinuclidinyl benzilate, and acetylcholine have been modeled. Although the head groups of these ligands occupy overlapping volumes within the binding site, their side chains may follow significantly different directions.

Address correspondence to: Dr. E. C. Hulme, Division of Physical Biochemistry, MRC National Institute for Medical Research, Mill Hill, London NW7 1AA. E-mail: ehulme{at}nimr.mrc.ac.uk

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