The role of a binding loop of residue in the function of the mouse 5-HT3A receptor N.L. Sullivan and S.C.R. Lummis, Dept of Biochemistry, Tennis Court Road, Cambridge, CB2 1AG, UK.

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The 5-HT₃ receptor (5-HT₃R) is a homo-pentameric member of the Cys-loop family of ligand-gated ion channels in which 6 binding loops (A-F) compose the binding site. The extracellular domain of 5-HT₃R has a similar structure to the recently crystallized Lymnaea acetylcholine binding protein (AChBP). A computer-generated model of the 5-HT₃ receptor structure based on the AChBP structure only places one residue from binding loop A (N128) within 5 Å of the docked 5-HT. In the preferred model of the 5-HT molecule in the binding site, the keto group of residue N128 potentially forms hydrogen bonds with the hydroxyl group of 5-HT (Reeves et al., 2003). To test the accuracy of the model, we therefore analyzed the role of residue N128 in the binding and function of the 5-HT₃ receptor by replacing it with a range of different amino acids.

N128 was mutated using the Kunkel method (Kunkel, 1985) in the expression vector pcDNA3 (Invitrogen), transfected into HEK 293 cells using calcium phosphate precipitation (Chen and Okayama, 1988) and stable cell lines were selected using geneticin (Life Technologies). Colonies with the highest expression of the 5-HT₃R were selected based on 2-point radioligand binding affinity Radioligand binding activity was characterized with the antagonist [³H]-granisetron (Spier et al., 2000) and receptor function in response to 5-HT was analysed using fluorescent imaging of changes in membrane potential (Baxter et al., 2002; Hargreaves et al., 1994).

Table I [³H]-granisetron K_^d values and 5-HT pEC₅₀ values

Values are mean ± s.e.mean for normalized data, n=4-11. Bold font indicates mutants that are significantly different from wild type (P<0.01, ANOVA). NR = no response up to 300µM.

Binding affinities for all mutants were similar to wild-type suggesting that N128 does not play a critical role in granisetron binding. Since E and V could substitute effectively for N in 5-HT-induced responses, but A, Q, R, and D resulted in an increased EC₅₀ or loss of function, neither the size nor the charge of this residue is critical. It is also unlikely that N128 forms a hydrogen bond with 5-HT because V is an effective substitute for N. These results suggest that N128 has an important but complex role in receptor function.

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