Mechanisms of sodium/calcium selectivity in sodium channels probed by cysteine mutagenesis and sulfhydryl modification

A conserved lysine residue in the 'P loop' of domain III renders sodium channels highly selective. Conversion of this residue to glutamate, to mimic the homologous position in calcium channels, enables Ca²⁺ to permeate sodium channels. Because the lysine-to-glutamate mutation converts a positively charged side chain to a negative one, it has been proposed that a positive charge at this position suffices for Na⁺ selectivity. We tested this idea by converting the critical lysine to cysteine (K1237C) in μ1 rat skeletal sodium channels expressed in Xenopus oocytes. Selectivity of the mutant channels was then characterized before and after chemical modification to alter side-chain charge. Wild-type channels are highly selective for Na⁺ over Ca²⁺ (P(Ca)/P(Na) < 0.01). The K1237C mutation significantly increases permeability to Ca²⁺ (P(Ca)/P(Na) = 0.6) and Sr²⁺. Analogous mutations in domains I (D400C), II (E755C), and IV (A1529C) did not alter the selectivity for Na⁺ over Ca²⁺, nor did any of the domain IV mutations (G1530C, W1531C, and D1532C) that are known to affect monovalent selectivity. Interestingly, the increase in permeability to Ca²⁺ in K1237C cannot be reversed by simply restoring the positive charge to the side chain by using the sulfhydryl modifying reagent methanethiosulfonate ethylammonium. Single-channel studies confirmed that modified K1237C channels, which exhibit a reduced unitary conductance, remain permeable to Ca²⁺, with a P(Ca)/P(Na) of 0.6. We conclude that the chemical identity of the residue at position 1237 is crucial for channel selectivity. Simply rendering the 1237 side chain positive does not suffice to restore selectivity to the channel.