Thompson AJ , Price KL , Lummis SC .

081925\Z\07\Z Wellcome Trust

	Figure 1. Residues that were mutated in this studyA, sequence alignment of human 5-HT3A and 5-HT3B subunits with Torpedo californica nACh receptor α and γ subunits, human GABAA 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 receptorsA, 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 receptorsAsterisks 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 receptorsThe EC50 or Imax 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 modificationA, AR92C and AR92CB 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 ABQ92C receptors. R92 is a loop D residue located on the A subunit complementary (−) face; Q92 is the equivalent B subunit residue. B, AT181C and AT181CB 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 ABK181C 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 AR92C (C) or ABQ92C (D) receptors are also shown. 5-HT application (200 μm for AR92C or 30 μm for ABQ92C) 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 (AS206C/E229C) in the C and F loopsA, 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 AS206C/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 AS206C/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.

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