Tryptophan 86 of the α Subunit in the Torpedo Nicotinic Acetylcholine Receptor Is Important for Channel Activation by the Bisquaternary Ligand Suberyldicholine

Abstract

Suberyldicholine, a bisquaternary compound, is a potent nicotinic acetylcholine receptor agonist. Previously, we suggested that at least some of the unusual binding properties of this ligand may be a consequence of its ability to cross-link two binding "subsites" within each of the high-affinity agonist binding domains [Dunn, S. M. J., and Raftery, M. A. (1997) Biochemistry 36, 3846-3853]. Tryptophan 86 of the alpha subunit has previously been implicated in the binding of agonist to this receptor. However, on the basis of the crystal structure of a homologous acetylcholine binding protein, this residue is predicted to lie 15-20 A from the high-affinity site, i.e., a distance that approximates the interonium distance of suberyldicholine. Tryptophan 86 was mutated to either an alanine or a phenylalanine, and the mutated subunit was coexpressed with wild-type beta, gamma, and delta subunits in Xenopus oocytes. Although the alanine mutation resulted in a loss of receptor expression, the alphaW86F mutant receptor was expressed on the oocyte surface, albeit with a much reduced efficiency. Acetylcholine-evoked currents of the alphaW86F receptor were not significantly different from those of the wild type with respect to the concentration dependence of channel activation, receptor desensitization, or d-tubocurarine inhibition. In contrast, the EC(50) for suberyldicholine-mediated activation of the alphaW86F receptor was increased by approximately 500-fold. Furthermore, suberyldicholine-evoked currents in the mutant receptor did not desensitize and were insensitive to block by d-tubocurarine. Thus, tryptophan 86 of the Torpedo receptor alpha subunit may be part of a subsite for recognition of suberyldicholine and other bisquaternary ligands.

Structural answers and persistent questions about how nicotinic receptors work

“…Long, bisquaternary dicholine agonists like suberyldicholine (18.7 Å between quaternary nitrogens) appeared to bind to multiple sites on muscle-type nAChRs, suggesting that the extent of an agonist binding site depends on the agonist (198,199). For the simpler alkyltrimethylammonium functional group of choline, length requirements of alkylthiol derivatives of the minimalist agonist tetramethylammonium tethered to cysteines supported the sufficiency of the single agonist binding site formed by aromatic side chains in the AChBP structure (200).…”

“…Additional interactions between epibatidine and the desensitized ligand binding site were suggested from ligand binding to chimeras between γ and δ subunits (the binding site at the αγ interface binds epibatidine more tightly than does the site at the αδ interface) (194). Photochemical labeling of Torpedo nAChRs (195,196) with a benzoylcholine partial agonist might lead to understanding the basis for low efficacy of partial agonists and the development of photochemically-active full agonists with well-defined photoreactive intermediates (197).Long, bisquaternary dicholine agonists like suberyldicholine (18.7 Å between quaternary nitrogens) appeared to bind to multiple sites on muscle-type nAChRs, suggesting that the extent of an agonist binding site depends on the agonist (198,199). For the simpler alkyltrimethylammonium functional group of choline, length requirements of alkylthiol derivatives of the minimalist agonist tetramethylammonium tethered to cysteines supported the sufficiency of the single agonist binding site formed by aromatic side chains in the AChBP structure (200).…”