Cysteine modification reveals which subunits form the ligand binding site in human heteromeric 5-HT3AB receptors

Abstract

Nerve signals are transmitted across cell membranes by receptors that can consist of multiple different subunits. The 5-HT3 receptor is a pentamer which can function with A subunits alone, or with a mixture of A and B subunits. As 5-HT activates the receptor by binding at the interface of adjacent subunits, it is important to know which subunits are adjacent. Here we show that in both A-only and A+B receptors there is at least one A–A interface, without which the receptor cannot function. This knowledge is important for understanding the receptor mechanism, and also will allow the design of more specific drugs that act at the 5-HT binding site. The ligand binding site of Cys-loop receptors is formed by residues on the principal (+) and complementary (−) faces of adjacent subunits, but the subunits that constitute the binding pocket in many heteromeric receptors are not yet clear. To probe the subunits involved in ligand binding in heteromeric human 5-HT3AB receptors, we made cysteine substitutions to the + and − faces of A and B subunits, and measured their functional consequences in receptors expressed in Xenopus oocytes. All A subunit mutations altered or eliminated function. The same pattern of changes was seen at homomeric and heteromeric receptors containing cysteine substitutions at AR92 (− face), AL126(+), AN128(+), AI139(−), AQ151(−) and AT181(+), and these receptors displayed further changes when the sulphydryl modifying reagent methanethiosulfonate-ethylammonium (MTSEA) was applied. Modifications of AR92C(−)- and AT181C(+)-containing receptors were protected by the presence of agonist (5-HT) or antagonist (d-tubocurarine). In contrast modifications of the equivalent B subunit residues did not alter heteromeric receptor function. In addition a double mutant, AS206C(−)/E229C(+), only responded to 5-HT following DTT treatment in both homomeric and heteromeric receptors, indicating receptor function was inhibited by a disulphide bond between an A+ and an A– interface in both receptor types. Our results are consistent with binding to an A+A– interface at both homomeric and heteromeric human 5-HT3 receptors, and explain why the competitive pharmacologies of these two receptors are identical.