Probing sodium channel cytoplasmic domain structure. Evidence for the interaction of the rSkM1 amino and carboxyl termini.
Epitopes for monoclonal antibodies directed against the purified adult rat skeletal muscle sodium channel (rSkM1) were localized using channel proteolysis and fusion proteins. The interactions between these and other monoclonal antibodies with site-specific polyclonal antibodies were used to investigate the spatial relationships among rSkM1 cytoplasmic segments. Competition. between antibodies for binding was performed using a solution-phase assay in which solubilized channel protein retains many of the biophysical characteristics of the rSkM1 protein in vivo. Our results support a model in which: 1) the amino terminus assumes a rigid structure having a fixed orientation with respect to other intracellular segments; 2) the interdomain 2-3 region is centrally located on the cytoplasmic surface of the channel, extends farther into the cytoplasm, and has an intermediate degree of flexibility; 3) the beginning of the amino terminus and end of the carboxyl terminus specifically interact with each other; and 4) domains 1 and 4 are adjacent. The sequences responsible for the interaction of the amino and carboxyl termini were identified by demonstrating the specific binding of a synthetic peptide encompassing the first 30 residues of the rSkM1 amino terminus to a fusion protein containing the rSkM1 carboxyl terminus.
Sun
W
W
Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
Barchi
R L
RL
Cohen
S A
SA
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001667:Binding, Competitive; D003593:Cytoplasm / Q000648:ultrastructure; D018604:Epitope Mapping; D008969:Molecular Sequence Data; D009132:Muscles / Q000648:ultrastructure*; D010455:Peptides / Q000737:chemistry / Q000276:immunology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000276:immunology; D015222:Sodium Channels / Q000737:chemistry / Q000276:immunology / Q000648:ultrastructure*
D000911:Antibodies, Monoclonal; D010455:Peptides; D011993:Recombinant Fusion Proteins; D015222:Sodium Channels