title: Adjacent pore-lining residues within sodium channels identified by paired cysteine mutagenesis.
abstract: 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 micro1 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 Y401C (domain I) spontaneously formed a disulfide bond 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.
authors.affiliation: Section of Molecular and Cellular Cardiology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: J P
authors.initials: JP
authors.lastname: Bénitah
authors.forename: G F
authors.initials: GF
authors.lastname: Tomaselli
authors.forename: E
authors.initials: E
authors.lastname: Marban
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002104:Cadmium / Q000494:pharmacology; D003545:Cysteine; D004229:Dithiothreitol / Q000494:pharmacology; D004594:Electrophysiology; D005260:Female; D005978:Glutathione / Q000494:pharmacology; D015640:Ion Channel Gating; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000502:physiology; D017354:Point Mutation; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015222:Sodium Channels / Q000096:biosynthesis / Q000737:chemistry / Q000502:physiology*; D014982:Xenopus laevis; D015032:Zinc / Q000494:pharmacology
chemical_list: D011994:Recombinant Proteins; D015222:Sodium Channels; D002104:Cadmium; D005978:Glutathione; D015032:Zinc; D003545:Cysteine; D004229:Dithiothreitol