The 5-hydroxytryptamine type 3 (5-HT₃) receptor reveals a novel determinant of single-channel conductance

5-HT3 (5-hydroxytryptamine type 3) receptors are cation-selective ion channels of the Cys-loop transmitter-gated ion channel superfamily. Two 5-HT3 receptor subunits, 5-HT3A and 5-HT3B, have been characterized in detail, although additional putative 5-HT3 subunit genes (HTR3C, HTR30 and HTR3E) have recently been reported. 5-HT3 receptors function as homopentameric assemblies of the 5-HT3 subunit, or heteropentamers of 5-HT3A and 5-HT3B subunits of unknown stoichiometry. The single-channel conductances of human recombinant homomeric and heteromeric 5-HT3 receptors are markedly different, being <1 and approx. 16 pS respectively. Paradoxically, from the results of studies performed on the closely related nicotinic acetylcholine receptor, the channel-lining M2 domain of the 5-HT 3A subunit is predicted to enhance cation conduction, whereas that of the 5-HT3B subunit would not. The present study describes a novel determinant of single-channel conductance, outwith the M2 domain, which accounts for this anomaly. Utilizing a panel of chimaeric 5-HT3A and 5-HT3B subunits, a profound determinant of single-channel conductance was traced to a putative amphipathic helix (the 'HA stretch') within the large cytoplasmic loop of the receptor. Replacement of three arginine residues (R432, R436 and R440) unique to the HA stretch of the 5-HT3A subunit with the aligned residues (Q395, D399 and A403) of the 5-HT3B subunit increased the single-channel conductance 28-fold. Significantly, from ultrastructural studies of the Torpedo nicotinic acetylcholine receptor, the key residues may be components of narrow openings within the inner vestibule of the channel, located in the cytoplasm, which contribute to the permeation pathway. Our findings indicate an important and hitherto unappreciated function for the HA stretch in the Cys-loop family of transmitter-gated ion channels.