Proline 308 in the mouse 5-HAT3A receptor links binding and function N. Meeus & S.C.R. Lummis, Dept of Biochemistry, Tennis Court Road, Cambridge, CB2 1AG, UK.

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TThe 5-HT₃ receptor is a member of the Cys-loop family of ligand-gated ion channels, proteins which possess a number of highly conserved proline (P) residues. As these residues impose considerable restraint on protein folding, they are often critical for the structure and/or function of the molecule. We have previously shown that 6 conserved proline residues are critical for binding and/or function of the 5-HT₃ receptor. One of these is in the M2-M3 loop, a region that has been shown to be critical for linking binding to function. Here we examine the other role of the other proline residue in the M2-M3 loop, which is conserved in cation selective receptors. A recent report (Miyazawa et al., 2003) suggests that in the related nicotinic acetylcholine receptor this proline forms a critical bend at the apex of the loop. To examine the role of this proline residue in the 5-HT₃ receptor, we have changed it to a range of alternate amino acid residues and characterised the mutant receptors.

Amino acid residues in the mouse 5-HT_3A(b) receptor subunit subunit were mutagenised using the Kunkel method (Kunkel, 1985) in the expression vector pcDNA3 (Clontech), and transfected into HEK 293 cells using calcium phosphate precipitation (Chen & Okayama, 1988). They were then examined using radioligand binding assays, immunocytochemistry, and function using fluorescent imaging (Baxter et al., 2002; Hargreaves et al., 1994; Spier & Lummis, 2000).

Radioligand binding data is shown in Table 1 and no mutant receptors were significantly different to wild type. Immunocytochemistry showed that all receptors were localised to the cell membrane as expected but no functional responses were observed in any of the mutant receptors.

Table 1.[³H]granisetron binding affinities for 5-HT₃ receptor mutants

Values are mean + s.e. mean, n=3-4.

The data suggest that the mutant receptor are correctly synthesised, assembled and targeted but that loss of proline 308 ablates function. Thus we suggest that this proline forms a critical link between binding and gating of the receptor.

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