Structural Determinants of Ca(2+) Permeability and Conduction in the Human 5-Hydroxytryptamine Type 3A Receptor

Cation-selective cysteine (Cys)-loop transmitter-gated ion channels provide an important pathway for Ca(2+) entry into neurones. We examined the influence on Ca(2+) permeation of amino acids located at intra- and extracellular ends of the conduction pathway of the human 5-hydroxytryptamine type 3A (5-HT(3A)) receptor. Mutation of cytoplasmic arginine residues 432, 436, and 440 to glutamine, aspartate, and alanine (the aligned residues of the human 5-HT(3B) subunit (yielding 5-HT(3A)(QDA)) increased P(Ca)/P(Cs) from 1.4 to 3.7. The effect was attributable to the removal of an electrostatic influence of the Arg-436 residue. Despite its relatively high permeability to Ca(2+), the single channel conductance of the 5-HT(3A)(QDA) receptor was depressed in a concentration-dependent and voltage-independent manner by extracellular Ca(2+). A conserved aspartate, located toward the extracellular end of the conduction pathway and known to influence ionic selectivity, contributed to the inhibitory effect of Ca(2+) on macroscopic currents mediated by 5-HT(3A) receptors. We introduced a D293A mutation into the 5-HT(3A)(QDA) receptor (yielding the 5-HT(3A)(QDA D293A) construct) to determine whether the aspartate is required for the suppression of single channel conductance by Ca(2+). The D293A mutation decreased the P(Ca)/P(Cs) ratio to 0.25 and reduced inwardly directed single channel conductance from 41 to 30 pS but did not prevent suppression of single channel conductance by Ca(2+). The D293A mutation also reduced P(Ca)/P(Cs) when engineered into the wild-type 5-HT(3A) receptor. The data helped to identify key residues in the cytoplasmic domain (Arg-436) and extracellular vestibule (Asp-293) that markedly influence P(Ca)/P(Cs) and additionally directly demonstrated a depression of single channel conductance by Ca(2+).