title: Depth asymmetries of the pore-lining segments of the Na+ channel revealed by cysteine mutagenesis.
abstract: We used serial cysteine mutagenesis to study the structure of the outer vestibule and selectivity region of the voltage-gated Na channel. The voltage dependence of Cd(2+) block enabled us to determine the locations within the electrical field of cysteine-substituted mutants in the P segments of all four domains. The fractional electrical distances of the substituted cysteines were compared with the differential sensitivity to modification by sulfhydryl-specific modifying reagents. These experiments indicate that the P segment of domain II is external, while the domain IV P segment is displaced internally, compared with the first and third domain P segments. Sulfhydryls with a steep voltage dependence for Cd(2+) block produced changes in monovalent cation selectivity; these included substitutions at the presumed selectivity filter, as well as residues in the domain IV P segment not previously recognized as determinants of selectivity. A new structural model is presented in which each of the P segments contribute unique loops that penetrate the membrane to varying depths to form the channel pore.
authors.affiliation: Departments of Medicine and Biochemical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
authors.forename: N
authors.initials: N
authors.lastname: Chiamvimonvat
authors.forename: M T
authors.initials: MT
authors.lastname: Pérez-García
authors.forename: R
authors.initials: R
authors.lastname: Ranjan
authors.forename: E
authors.initials: E
authors.lastname: Marban
authors.forename: G F
authors.initials: GF
authors.lastname: Tomaselli
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002104:Cadmium / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004563:Electrochemistry; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015222:Sodium Channels / Q000737:chemistry*; D013329:Structure-Activity Relationship; D014982:Xenopus laevis
chemical_list: D011994:Recombinant Proteins; D015222:Sodium Channels; D002104:Cadmium; D003545:Cysteine