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

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.

Figure 1. Residues that were mutated in this study

A, sequence alignment of human 5-HT3A and 5-HT3B subunits with Torpedo californica nACh receptor α and γ subunits, human GABA_A receptor α1 subunit, and Lymnaea stagnalis AChBP. Residues mutated in this study are shown in bold on a grey background. The positions of the six binding loops A–F are indicated by black lines. B, a homology model of the 5-HT₃A receptor showing the location of the A subunit residues (stick representation) mutated in this study. Note that the numbering of the A and B residues in panel A corresponds to the human 5-HT₃ receptor, but the number in panel B is according to the mouse numbering used in this paper. Accession numbers for the alignment are: 5-HT3A P46098, 5-HT3B O95264, nACh α P02710, nACh γ P02714, GABA α1 P02710, AChBP P58154.

Figure 2. 5-HT concentration–response and PTX concentration–inhibition curves for A and AB receptors

A, 5-HT concentration–response curves. Wild-type receptors were unaltered by MTSEA (comparisons are also shown in Fig. 4). The calculated EC₅₀ values and Hill slopes can be found in Table 1. Typical EC₅₀ 5-HT responses are shown next to each curve. B, PTX concentration–inhibition curves. The presence of the B subunit is confirmed by a rightward shift in the PTX concentration–inhibition curve in heteromeric receptors. The mutants shown are examples, and are the same as those in Fig. 5.

Figure 3. Relative EC₅₀ values of wild-type and mutant receptors

Asterisks denote statistically different from wild-type. NF: non-functional at 100 μm 5-HT, or with an EC₅₀ too high to be accurately determined. Relative values are shown as the differences ± SED. Data from Table 1. The dotted line represents the difference between wild-type 5-HT₃A and wild-type 5-HT₃AB responses.

Figure 4. The effect of MTSEA on wild-type and mutant receptors

The EC₅₀ or I_max obtained after MTSEA treatment is compared to that obtained before treatment. BLOCK: complete inhibition by MTSEA. Asterisks denote statistically different from wild-type. NF: non-functional at 100 μm 5-HT. Relative values are expressed as the difference ± SED. Data from Table 1.

Figure 5. Agonist (5-HT) and antagonist (d-TC) protection of mutant receptors from MTS modification

A, A_R92C and A_R92CB receptors are completely protected from MTSEA–biotin modification by the presence of either agonist (5-HT) or antagonist (d-TC). There is no effect of MTSEA–biotin on AB_Q92C receptors. R92 is a loop D residue located on the A subunit complementary (−) face; Q92 is the equivalent B subunit residue. B, A_T181C and A_T181CB receptors are completely protected from MTSEA modification by the presence of either agonist (5-HT) or antagonist (d-TC). There is no effect of MTSEA on AB_K181C receptors. T181 is a loop B residue located on the A subunit principal (+) face; K181 is the equivalent B subunit residue. Typical current traces from oocytes expressing A_R92C (C) or AB_Q92C (D) receptors are also shown. 5-HT application (200 μm for A_R92C or 30 μm for AB_Q92C) is denoted by a black bar above the trace. Arrows indicate applications of MTSEA–biotin (2 mm with or without 1 mm 5-HT) for 2 min, followed by wash for 2 min (see methods for details). E, structures of MTSEA and MTSEA–biotin.

Figure 6. The effects of DTT on homomeric and heteromeric receptors containing A subunit double cysteine mutations (A_S206C/E229C) in the C and F loops

A, 5-HT-induced currents are only seen after application of DTT (10 mm for 1 min). A subsequent application of MTSEA (2 mm for 1 min) inhibits this response; this inhibition can be reversed by DTT. B, DTT treatment is also required for 5-HT-induced responses in heteromeric A_S206C/E229CB receptors. C, the locations of S206 and E229 on a 5-HT₃ receptor homology model (template PDB ID; 2PGZ). D, Concentration–response curves for homomeric and heteromeric receptors containing the A subunit double cysteine mutant. Parameters derived from these curves (Table 2) are consistent with those expected for homomeric and heteromeric responses. E, Disulphide bonds spontaneously reform in A_S206C/E229CB mutant receptors. Following removal of DTT, peak current responses to 5-HT (100 μm) decline with an exponential time course (τ = 0.17 ± 0.01 min⁻¹), but recover following several 10 s DTT applications (arrows). All traces are representative of ≥4 separate experiments.