Abstract
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-HT(3)AB 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 A(R92) (- face), A(L126)(+), A(N128)(+), A(I139)(-), A(Q151)(-) and A(T181)(+), and these receptors displayed further changes when the sulphydryl modifying reagent methanethiosulfonate-ethylammonium (MTSEA) was applied. Modifications of A(R92C)(-)- and A(T181C)(+)-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, A(S206C)(-)(/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-HT(3) receptors, and explain why the competitive pharmacologies of these two receptors are identical.
Highlights
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
Binding sites in the homomeric 5-HT3A receptor are composed of A+A– interfaces, but binding sites in the heteromeric 5-HT3AB receptor could be located at A+A, A+B, B+A– or B+B– interfaces
Given the inconsistencies of 5-HT3AB stoichiometries in the literature, we have performed a range of experiments on cysteine-substituted residues to clarify which subunit interfaces are present in the human heteromeric 5-HT3AB receptor ligand binding site
Summary
Methanethiosulfonate (MTS) reagents were obtained from Biotium (Hayward, CA, USA). Serotonin The mean ± standard error of the mean (SEM) of the normalised responses for a series of oocytes were plotted against agonist concentration and iteratively fitted to the following equation: IA. Methanethiosulfonate-ethylammonium (MTSEA) solutions were made up immediately before each experiment, and applied at a concentration of 2 mM for 2 min, followed by a 2 min wash. Pre-treatment values were recorded as two responses at an EC50 or maximal concentration of 5-HT. Oocytes were treated with a maximal concentration of agonist (1 mM 5-HT for 30 s) or competitive antagonist (1 mM d-TC for 60 s) before and during a 2 min treatment with MTSEA or MTSEA–biotin. Thereafter, 10 s treatments were used immediately prior to each of the 5-HT applications This was performed as described previously (Price & Lummis, 2004).
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