Adjacent pore-lining residues within sodium channels identified by paired cysteine mutagenesis

The pores of voltage-gated ion channels are lined by protein loops that determine selectivity and conductance. The relative orientations of these ''P'' loops remain uncertain, as do the distances between them. Using site-directed mutagenesis, we introduced pairs of cysteines into the P loops of mu 1 rat skeletal muscle sodium channels and sought functional evidence of proximity between the substituted residues. Only cysteinyl residues that are in close proximity can form disulfide bonds or metal-chelating sites. The mutant Y4O1C (domain I) spontaneously formed a disulfide bend when paired with E758C in the P loop of domain II; the same residue, when coupled with G1530C in domain IV, created a high-affinity binding site for Cd2+ ions. The results provide the first specific constraints for intramolecular dimensions of the sodium channel pore.