Distinctions in agonist and antagonist specificity conferred by anionic residues of the nicotinic acetylcholine receptor.
Two anionic residues in the nicotinic acetylcholine receptor, Asp-152 in the alpha-subunit and Asp-174 in the gamma-subunit or the corresponding Asp-180 in the delta-subunit, are presumed to reside near the two agonist binding sites at the alphagamma and alphadelta subunit interfaces of the receptor and have been implicated in electrostatic attraction of cationic ligands. Through site-directed mutagenesis and analysis of state changes in the receptor elicited by agonists, we have distinguished the roles of anionic residues in conferring ligand specificity and ligand-induced state changes. alphaAsp-152 affects agonist and antagonist affinity similarly, whereas gammaAsp-174 and deltaAsp-180 primarily affect agonist affinity. Combining charge neutralization on the alpha subunit with that on the gamma and delta subunits shows an additivity in free energy changes for carbamylcholine and d-tubocurarine, suggesting independent contributions of these residues to stabilizing the bound ligands. Since both aromatic and anionic residues stabilize cationic ligands, we substituted tyrosines (Y) for the aspartyl residues. While the substitution, alphaD152Y, reduced the affinities for agonists and antagonists, the gammaD174Y/deltaD180Y mutations reduced the affinity for agonist binding, but surprisingly enhanced the affinity for d-tubocurarine. To ascertain whether selective changes in agonist binding stem from the capacity of agonists to form the desensitized state of the receptor, carbamylcholine binding was measured in the presence of an allosteric inhibitor, proadifen. Mutant nAChRs carrying alphaD152Q or gammaD174N/deltaD180N show similar reductions in dissociation constants for the desensitized compared with activable receptor state and a similar proadifen concentration dependence. Hence, these mutations influence ligand recognition rather than the capacity of the receptor to desensitize. By contrast, the alphaD200Q mutation diminishes the ratio of dissociation constants for two states and requires higher proadifen concentrations to induce desensitization. Thus, the contributions of alphaAsp-152, gamma/deltaAsp-174/180, and alphaAsp-200 in stabilizing ligand binding can be distinguished by the interactions between agonists and allosteric inhibitors.
Osaka
H
H
Department of Pharmacology 0636, University of California, San Diego, La Jolla, California 92093, USA.
Sugiyama
N
N
Taylor
P
P
D000595:Amino Acid Sequence; D001665:Binding Sites; D002217:Carbachol / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D002794:Choline / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018722:Nicotinic Agonists / Q000378:metabolism / Q000494:pharmacology*; D018733:Nicotinic Antagonists / Q000378:metabolism / Q000494:pharmacology*; D011335:Proadifen / Q000378:metabolism / Q000494:pharmacology; D011978:Receptors, Nicotinic / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014403:Tubocurarine / Q000378:metabolism / Q000494:pharmacology; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
D018722:Nicotinic Agonists; D018733:Nicotinic Antagonists; D011978:Receptors, Nicotinic; C009335:subecholine; D014443:Tyrosine; D002217:Carbachol; D011335:Proadifen; D002794:Choline; D014403:Tubocurarine