title: Conformational mapping of the cytosolic linker between domains III and IV of the cardiac Na+ channel protein and binding studies with a site-directed channel modifying antibody.
abstract: By combining antibody binding studies with conformational mapping using synthetic peptides, the structure of the cytosolic linker between domains III and IV of the cardiac Na+ channel alpha-subunit was analyzed. Inside-out patch clamp experiments with isolated cardiac Na+ channels from neonatal rat cardiocytes confirmed that a polyclonal antibody against amino acids 1490-1507 of the cardiac Na+ channel recognizes the linker in situ since Na+ inactivation became significantly retarded. Epitope fine mapping with a series of overlapping peptides identified the sequence YYNAMKKLG (corresponding to amino acids 1496-1504 of the cardiac sodium channel alpha-subunit) as the binding locus of the site directed antibody, an interesting result with respect to structure-function relationships because the functionally important hydrophobic amino-acid cluster in position 1487-1489 is not included. Circular dichroism measurements of synthetic 20-mer peptides in hydrophilic and lipophilic environments provided indications for a notable alpha-helical content only for segment GGQDIFMTEEQKKYYNAMKK. This sequence corresponds to amino acids 1483-1502 in the linker and adopts a highly ordered pattern of charge distribution due to this helical conformation. Ordered structure and helix dipole moment represent physical properties which may be important in a refined model for explaining the function of the linker in terminating the open channel configuration.
authors.affiliation: Institut für Organische Chemie, Universität Tübingen, Germany.
authors.forename: W
authors.initials: W
authors.lastname: Beck
authors.forename: G
authors.initials: G
authors.lastname: Jung
authors.forename: W G
authors.initials: WG
authors.lastname: Bessler
authors.forename: I
authors.initials: I
authors.lastname: Benz
authors.forename: M
authors.initials: M
authors.lastname: Kohlhardt
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D000906:Antibodies / Q000276:immunology*; D002942:Circular Dichroism; D003600:Cytosol / Q000737:chemistry; D008969:Molecular Sequence Data; D009206:Myocardium / Q000737:chemistry; D010452:Peptide Biosynthesis; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D051381:Rats; D015222:Sodium Channels / Q000737:chemistry* / Q000276:immunology
chemical_list: D000906:Antibodies; D010455:Peptides; D015222:Sodium Channels