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-HT_3A and 5-HT_3B, have been characterized in detail, although additional putative 5-HT₃ subunit genes (HTR3C, HTR3D and HTR3E) have recently been reported. 5-HT₃ receptors function as homopentameric assemblies of the 5-HT₃ subunit, or heteropentamers of 5-HT_3A and 5-HT_3B 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-HT_3B 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-HT_3A and 5-HT_3B 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-HT_3A subunit with the aligned residues (Q395, D399 and A403) of the 5-HT_3B 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.