The stability of the h5-HT3A receptor complex and the importance of C-terminus

Cellular and Molecular Neuropharmacology Research Group, School of Experimental and Clinical Medicine, The Medical School, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK The 5-HT3 receptor is a ligand-gated ion channel that mediates fast synaptic neurotransmission. We have previously shown that the C-terminus of the human (h) 5-HT3A subunit is important for the expression of the homomeric 5-HT3A receptor within the cell membrane, and suggested a role for the C-terminus in the promotion of receptor stability (Butler et al., 2008). In the present study, we further assess the stability of the h5-HT3A receptor in comparison with that arising when the C-terminal alanine is truncated (ΔAla455 [N-4TM-QY]; see Butler et al., 2008 for construct terminology). Expression of the h5-HT3A subunit and mutants/truncations by COS-7 cells and radioligand binding assays were performed as described previously (Butler et al., 2008). Briefly, COS-7 cells were transiently transfected with the appropriate cDNA via electroporation and harvested 48 h post-transfection to generate binding homogenates to assess the impact of urea upon specific [3H]granisetron binding. [3H]Granisetron specific binding (defined by ondansetron, 10 μM) arising from expression of the truncation mutant of the h5-HT3A subunit, N-4TM-QY (ΔAla455), was more susceptible to disruption by increasing concentrations of urea relative to the specific binding arising from expression of the wild-type h5-HT3A subunit (P<0.05, Mann Whitney U test). In contrast, there was no significant difference in the urea sensitivity of [3H]granisetron specific binding arising from expression of the wild-type h5-HT3A subunit and N-4TM-AAA (Gln453Ala and Tyr454Ala). The current study further supports the role of the C-terminus of the h5-HT3A subunit to promote stability of the arising 5-HT3 receptor complex. Butler, AS et al., 2008. Neuropharmacology (in press).