Structural and electrostatic properties of the 5-HT3 receptor pore revealed by substituted cysteine accessibility mutagenesis

5-HT3 receptors are members of the Cys loop family of ligand-gated ion channels. We used the substituted cysteine accessibility method to identify amino acid residues in the channel forming domain, M2 that face the water-accessible surface and to locate their position in the ion conduction pathway. Cysteine was substituted for each residue, one at a time, in the M2 segment (Asp(274)-Asp(298)). 5-Hydroxytryptamine EC50 values for functional mutants did not vary from wild type (1.4 +/- 0.2 mum) by more than 10-fold, and five mutants were nonfunctional. Covalent modification of the mutant receptors with sulfydryl reagents revealed 11 residues to be water-accessible, with a pattern consistent with an a-helix except at Leu(285) and Leu(293). The data suggest that charge selectivity begins at a more cytoplasmic level than Val(291). Modification at some positions (Val(291) Leu(293), Ile(294), Leu(287), and Ser(280)) resulted in channels that were locked open. Reaction rates with accessible cysteines were voltage-dependent at some residues, suggesting that access occurs via the ion channel. Overall the data observed are similar but not identical to that reported for other members of the family and confirms the high degree of structural and functional homology between receptors in the Cys loop receptor family.