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

Journal Article · Thu Jan 01 04:00:00 EST 1987 · Proc. Natl. Acad. Sci. U.S.A.; (United States)

DOI:https://doi.org/10.1073/pnas.84.1.308· OSTI ID:5605773

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

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. The membrane topology of the peptide recognized by this antiserum was proved in binding studies using oriented electroplax membrane vesicles. These vesicles were 98% right-side-out as determined by (/sup 3/H)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. The data imply that the region of the sodium channel primary sequence near the C terminus that is recognized by the anitserum is localized on the cytoplasmic side of the membrane; this localization provides some further constraints on models of sodium channel tertiary structure.

• Research Organization: Univ. of Pennsylvania School of Medicine, Philadelphia
• OSTI ID: 5605773
• Journal Information: Proc. Natl. Acad. Sci. U.S.A.; (United States), Journal Name: Proc. Natl. Acad. Sci. U.S.A.; (United States) Vol. 84:1; ISSN PNASA
• Country of Publication: United States
• Language: English

Related Subjects

550601* -- Medicine-- Unsealed Radionuclides in Diagnostics
62 RADIOLOGY AND NUCLEAR MEDICINE
ALKALI METAL COMPOUNDS
AMINO ACID SEQUENCE
ANIMALS
ANTIGENS
AQUATIC ORGANISMS
BETA DECAY RADIOISOTOPES
BIOSYNTHESIS
CARBOHYDRATES
CATIONS
CELL CONSTITUENTS
CELL MEMBRANES
CHARGED PARTICLES
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
EEL
ELECTRON CAPTURE RADIOISOTOPES
ELECTRON MICROSCOPY
FISHES
GLYCOSIDES
IMMUNOASSAY
IMMUNOLOGY
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
IONS
ISOTOPE APPLICATIONS
ISOTOPES
LABELLED COMPOUNDS
LIQUID COLUMN CHROMATOGRAPHY
MATERIALS
MEMBRANE TRANSPORT
MEMBRANES
MICROSCOPY
MOLECULAR STRUCTURE
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
PEPTIDES
PERMEABILITY
PROTEINS
RADIOASSAY
RADIOIMMUNOASSAY
RADIOIMMUNOLOGY
RADIOISOTOPES
SAPONINS
SEPARATION PROCESSES
SODIUM COMPOUNDS
STRUCTURAL MODELS
SYNTHESIS
TOXIC MATERIALS
TOXINS
TRACER TECHNIQUES
TRANSMISSION ELECTRON MICROSCOPY
TRITIUM COMPOUNDS
VERTEBRATES