The fifth subunit of the (α4ß2)2 ß2 nicotinic ACh receptor modulates maximal ACh responses.

BACKGROUND AND PURPOSE: The fifth subunit in the (α4ß2)2 α4 nicotinic ACh receptor (nAChR) plays a determining role in the pharmacology of this nAChR type. Here, we have examined the role of the fifth subunit in the ACh responses of the (α4ß2)2 ß2 nAChR type. EXPERIMENTAL APPROACH: The role of the fifth subunit in receptor function was explored using two-electrode voltage clamp electrophysiology, along with subunit-targeted mutagenesis and the substituted cysteine scanning method applied to fully linked (α4ß2)2 ß2 receptors. KEY RESULTS: Covalent modification of the cysteine-substituted fifth subunit with a thiol-reactive agent (MTS) caused irreversible inhibition of receptor function. ACh reduced the rate of the reaction to MTS, but the competitive inhibitor dihydro-ß-erythroidine had no effect. Alanine substitution of conserved residues that line the core of the agonist sites on α4(+)/ß2(-) interfaces did not impair receptor function. However, impairment of agonist binding to α4(+)/ß2(-) agonist sites by mutagenesis modified the effect of ACh on the rate of the reaction to MTS. The extent of this effect was dependent on the position of the agonist site relative to the fifth subunit. CONCLUSIONS AND IMPLICATIONS: The fifth subunit in the (α4ß2)2 ß2 receptor isoform modulates maximal ACh responses. This effect appears to be driven by a modulatory, and asymmetric, association with the α4(+)/ß2(-) agonist sites. LINKED ARTICLES: This article is part of a themed section on Nicotinic Acetylcholine Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.11/issuetoc.

Non-equivalent ligand selectivity of agonist sites in (α4ß2)2α4 nicotinic acetylcholine receptors: a key determinant of agonist efficacy.

The α4ß2 nicotinic acetylcholine receptor (nAChR) is the most abundant nAChR type in the brain, and this receptor type exists in alternate (α4ß2)2α4 and (α4ß2)2ß2 forms, which are activated by agonists with strikingly differing efficacies. Recent breakthroughs have identified an additional operational agonist binding site in the (α4ß2)2α4 nAChR that is responsible for the signature sensitivity of this receptor to activation by agonists, yet the structural mechanisms determining agonist efficacy at this receptor type are not yet fully understood. In this study, we characterized the ligand selectivity of the individual agonist sites of the (α4ß2)2α4 nAChR to determine whether differences in agonist selectivity influence agonist efficacy. Applying the substituted cysteine accessibility method to individual agonist sites in concatenated (α4ß2)2α4 receptors, we determined the agonist selectivity of the agonist sites of the (α4ß2)2α4 receptor. We show that (a) accessibility of substituted cysteines to covalent modification by methanesulfonate reagent depends on the agonist site at which the modification occurs and (b) that agonists such as sazetidine-A and TC-2559 are excluded from the site at the α4/α4 interface. Given that additional binding to the agonist site in the α4/α4 interface increases acetylcholine efficacy and that agonists excluded from the agonist site at the α4/α4 interface behave as partial agonists, we conclude that the ability to engage all agonist sites in (α4ß2)2α4 nAChRs is a key determinant of agonist efficacy. The findings add another level of complexity to the structural mechanisms that govern agonist efficacy in heteromeric nAChRs and related ligand-gated ion channels.