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

- 5-HT and mCPBG exhibit the same efficacy at both 5-HT3A and 5-HT3AB receptors.
- The B and D recognition loops differ between the two subunits.
- B loop W of the 5-HT3A subunit imparts mCPBG activation to both receptors.
- D loop triplet RQY of the 5-HT3A subunit imparts 5-HT activation to both receptors.
- Signature D loop RQY on 5-HT3B subunit background may allow 5-HT activation.

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.