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.
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.
Beck
W
W
Institut für Organische Chemie, Universität Tübingen, Germany.
Jung
G
G
Bessler
W G
WG
Benz
I
I
Kohlhardt
M
M
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
D000906:Antibodies; D010455:Peptides; D015222:Sodium Channels