Topographical localization of the C-terminal region of the voltage-dependent sodium channel from Electrophorus electricus using antibodies raised against a synthetic peptide

R. D. Gordon, W. E. Fieles, D. L. Schotland, R. Hogue-Angeletti, R. L. Barchi

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

A peptide corresponding to amino acid residues 1783-1794 near the C terminus of the electric eel sodium channel primary sequence of the eel (Electrophorus electricus) sodium channel has been synthesized and used to raise an antiserum in rabbits. This antiserum specifically recognized the peptide in a solid-phase radioimmunoassay. Specificity of the antiserum for the native channel protein was shown by its specific binding to a 280-kDa protein in immunoblots of eel electroplax membrane proteins. The antiserum also specifically labeled the innervated membrane of the eel electroplax in immunofluorescent studies; noninnervated membrane was not labeled, consistent with the known distribution of sodium channels in this tissue. The membrane topology of the peptide recognized by this antiserum was probed in binding studies using oriented electroplax membrane vesicles. These vesicles were 98% 'right-side-out' as determined by [3H]saxitoxin binding. Binding of the antipeptide antiserum to this fraction was measured before and after permeabilization with 0.01% saponin. Specific binding to intact vesicles was low, but this binding increased 10-fold after permeabilization, implying a cytoplasmic orientation for the peptide. Confirmation for this orientation was then sought by localizing the antibody bound to intact electroplax cells with immunogold electron microscopy. Gold particles identifying the antibody were found almost exclusively associated with the cytoplasmic surface of the innervated membrane. Our data imply that the region of the sodium channel primary sequence near the C terminus that is recognized by our antiserum is localized on the cytoplasmic side of the membrane; this localization provides some further constraints on models of sodium channel tertiary structure.

Original languageEnglish (US)
Pages (from-to)308-312
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume84
Issue number1
DOIs
StatePublished - 1987
Externally publishedYes

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Electrophorus
Sodium Channels
Immune Sera
Peptides
Antibodies
Membranes
Saxitoxin
Saponins
Gold
Radioimmunoassay
Electron Microscopy
Membrane Proteins
Proteins
Cell Membrane
Rabbits
Amino Acids

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Topographical localization of the C-terminal region of the voltage-dependent sodium channel from Electrophorus electricus using antibodies raised against a synthetic peptide. / Gordon, R. D.; Fieles, W. E.; Schotland, D. L.; Hogue-Angeletti, R.; Barchi, R. L.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 84, No. 1, 1987, p. 308-312.

Research output: Contribution to journalArticle

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