Importance of recognition loops B and D in the activation of human 5-HT₃ receptors by 5-HT and meta-chlorophenylbiguanide

Abstract

The 5-HT₃ receptor is a cation selective member of the pentameric Cys-loop ligand-gated ion channels. While five subunits are known to exist, only two receptor subtypes have been significantly characterized: the homomeric receptor consisting of five A subunits and the heteromeric receptor containing both A and B subunits. The agonist recognition and activation of these receptors is orchestrated by six recognition loops three, A-C, on the principal subunit, and three, D-F, on the complementary subunit. In this study we have focused on the B loop of the principal subunit and loop D of the complementary subunit where aligned amino acids differ between the two subunits. A mutational analysis has been carried out using both 5-HT and m-chlorophenylbiguanide (mCPBG) to characterize receptor activation in the mutant receptors using two-electrode voltage clamp in Xenopus oocytes. The results show that the B loop W178I mutation of the 5-HT3A subunit markedly reduces the efficacy of mCPBG in both the homomeric and heteromeric receptors, while activation by 5-HT remains intact. Replacement of the D loop amino acid triplet RQY of the 5-HT3A subunit, with the aligned residues from the 5-HT3B subunit, QEV, converts 5-HT to a weak partial agonist in both the homomer and heteromer, but does not compromise activation by mCPBG. Exchange of the RQY triplet for the 5-HT3B subunit homologue, QEV, increases the Hill coefficient and decreases the EC₅₀ of this mutant when expressed with the wild type 5-HT3A subunit.

Keywords: 5-HT; 5-HT(3)A receptor; 5-HT(3)AB receptor; 5-HT(3)ABR; 5-HT(3)AR; 5-hydroxytryptamine; 5-hydroxytryptamine type 3A receptor; 5-hydroxytryptamine type 3AB receptor; Receptor activation; mCPBG; meta-chlorophenylbiguanide; nAChR; nicotinic acetylcholine receptor; wild type; wt.