Localization of epitopes for antibodies that differentially label sodium sodium channels in skeletal muscle surface and T-tubular membranes.
We previously characterized two monoclonal antibodies, A/B2 and L/D3, that bind to the amino-terminus of the sodium channel but produce distinct immunocytochemical patterns in innervated adult skeletal muscle. Because these findings suggested the presence of several channel isoforms, we sought to define the epitopes for each antibody. Five peptides encompassing the amino-terminal 126 residues of the adult skeletal muscle sodium channel were synthesized and tested by radioimmunoassay against each antibody. Both monoclonals bound only to a peptide comprising residues 1-30 (I1-30). A nested set of peptides within this region was then synthesized and used to compete for antibody binding to I1-30. L/D3 binding was quantitatively inhibited by oligopeptides 1-30, 7-30, 13-30, and 19-30 but not 25-30, while binding of A/B2 was blocked only by the intact I1-30 peptide. This data implies that the epitope for L/D3 lies within residues 19-25 while the epitope for A/B2 is contained within residues 1-6. These tentative epitope localizations were confirmed using both proteolytic cleavage of I1-30 and immunoreactivity of a peptide corresponding to residues 1-12 with A/B2 but not L/D3. Therefore, epitopes for each monoclonal antibody are present in the SkM-1 sequence and are in close proximity in the amino-terminus of the protein. Their characteristic immunocytochemical labeling patterns may reflect differing accessibility of the epitopes in various membrane environments.
Cohen
S A
SA
Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104.
Barchi
R L
RL
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001667:Binding, Competitive; D002462:Cell Membrane / Q000276:immunology*; D000939:Epitopes / Q000276:immunology*; D003012:Microbial Collagenase; D008969:Molecular Sequence Data; D009132:Muscles / Q000276:immunology* / Q000648:ultrastructure; D051381:Rats; D015222:Sodium Channels / Q000276:immunology*; D014357:Trypsin
D000911:Antibodies, Monoclonal; D000939:Epitopes; D015222:Sodium Channels; D014357:Trypsin; D003012:Microbial Collagenase