Aromatics at the murine nicotinic receptor agonist binding site: mutational analysis of the αY93 and αW149 residues

The adult muscle nicotinic acetylcholine (ACh) receptor (AChR) is a pentameric protein consisting of two α subunits, and one each of β, δ and e subunits. The receptor contains two agonist binding sites, which are formed by the α-δ and α-e subunit pairs (Blount & Merlie, 1989). Affinity labelling and mutagenesis studies have identified three regions of the α subunit that contribute to the stabilization of the bound ACh (for review see Devillers-Thiery et al. 1993). Residues in region A include aromatic residues in positions 86 (tryptophan) and 93 (tyrosine). Region B contains Trp149 and Tyr151, and finally, region C contains Tyr190, Tyr198, Asp200 and vicinal cysteines in positions 192 and 193. The high number of aromatic residues in the putative binding site is of interest. In another ACh binding protein, acetylcholinesterase, there is evidence that the quaternary ammonium portion of ACh interacts with an aromatic residue, tryptophan (Silman et al. 1994). For the nicotinic receptor, the data available are more controversial. Even though evidence exists to support the idea of a cation π-type interaction between a tryptophan residue (αW149) located within the putative binding site and the quaternary ammonium group of ACh (Zhong et al. 1998), other studies have proposed that different aromatic residues may contribute to stabilization of ACh in the binding pocket via interactions with the quaternary ammonium group (αY93, αY190 and αY198; Aylwin & White, 1994; Sine et al. 1994; Nowak et al. 1995). Furthermore, it has been proposed that in the AChR, the positively charged quaternary ammonium moiety of ACh does not interact with any of the aromatic residues, but rather with negatively charged residues on the δ and γ subunits (Asp180 in the δ and Asp174 in the γ subunit; Czajkowski & Karlin, 1995; Martin et al. 1996). In the adult-type mouse receptor, the two agonist binding sites have equal affinities for ACh (Akk & Auerbach, 1996; Wang et al. 1997; Salamone et al. 1999), suggesting that the microenvironment surrounding the ACh molecule is similar within the two sites. Similarity in affinities also suggests the absence of any binding cooperativity or interactions between the two binding sites. It is not clear, however, whether the two binding sites contribute equally to channel gating. It has been argued previously that, when the binding sites have different affinities for the ligand, the contributions to channel gating are different (Jackson, 1989). Even though the ligand binding sites in the adult-type receptor have equivalent affinities, there is evidence that the binding site mutation αD200N has differing effects on gating depending on which of the two α subunits it is in (Akk et al. 1996). In the present work, the kinetic properties of two putative binding site mutants, αY93F and αW149F, were investigated. Both mutations predominantly affect channel gating with smaller effect on the agonist binding properties of the receptor. In addition, the kinetic properties of hybrid receptors, i.e. receptors in which only one of the two a subunits contains the mutated residue, were examined. The results suggest that these residues at both binding sites contribute roughly equally to channel gating. For the αW149F site, the hybrid receptor activity can be descibed by one of the binding sites having wild-type-like affinity while the other has affinity close (a 1.1-fold difference) to what is observed in the pure mutant receptor. For the αY93F site, the KD of the mutated site in the hybrid receptor is similar (a 1.6-fold difference) to one in the pure mutant receptor, but the rate constants for agonist association and dissociation differ from the ones observed in the mutant receptor by 2- to 3-fold. Experiments on mutant receptors activated by tetramethylammonium (TMA) suggest that significant interaction takes place between the acetyl group of ACh and the αY93 site during channel gating.