Structural inferences for the native skeletal muscle sodium channel as derived from patterns of endogenous proteolysis

Journal Article · Sat Aug 05 04:00:00 EDT 1989 · Journal of Biological Chemistry; (USA)

OSTI ID:5264670

Kraner, S; Yang, J; Barchi, R ^[1]

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1. Univ. of Pennsylvania School of Medicine, Philadelphia (USA)

The alpha subunit (Mr approximately 260,000) of the rat skeletal muscle sodium channel is sensitive to cleavage by endogenous proteases during the isolation of muscle surface membrane. Antisera against synthetic oligopeptides were used to map the resultant fragments in order to identify protease-sensitive regions of the channel's structure in its native membrane environment. Antibodies to the amino terminus labeled major fragments of Mr approximately 130,000 and 90,000 and lesser amounts of other peptides as small as Mr approximately 12,000. Antisera to epitopes within the carboxyl-terminal half of the primary sequence recognized two fragments of Mr approximately 110,000 and 78,000. Individual antisera also selectively labeled smaller polypeptides in the most extensively cleaved preparations. The immunoreactivity patterns of monoclonal antibodies previously raised against the purified channel were then surveyed. The binding sites for one group of monoclonals, including several that recognize subtype-specific epitopes in the channel structure, were localized within a 12-kDa fragment near the amino terminus. The distribution of carbohydrate along the primary structure of the channel was also assessed by quantitating {sup 125}I-wheat germ agglutinin and 125I-concanavalin A binding to the proteolytic peptides. Most of the carbohydrate detected by these lectins was located between 22 and 90 kDa from the amino terminus of the protein. No lectin binding was detected to fragments arising from carboxyl-terminal half of the protein. These results were analyzed in terms of current models of sodium channel tertiary structure. In its normal membrane environment, the skeletal muscle sodium channel appears sensitive to cleavage by endogenous proteases in regions predicted to link the four repeat domains on the cytoplasmic side of the membrane while the repeat domains themselves are resistant to proteolysis.

• OSTI ID: 5264670
• Journal Information: Journal of Biological Chemistry; (USA), Journal Name: Journal of Biological Chemistry; (USA) Vol. 264:22; ISSN JBCHA; ISSN 0021-9258
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMALS
ANTIBODIES
ANTIGEN-ANTIBODY REACTIONS
BETA DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
CARBOHYDRATES
CELL CONSTITUENTS
CELL MEMBRANES
CHEMICAL REACTIONS
CONCANAVALIN
CYTOPLASM
DAYS LIVING RADIOISOTOPES
DECOMPOSITION
ELECTRON CAPTURE RADIOISOTOPES
ENZYMES
HYDROLASES
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
MAMMALS
MEMBRANE PROTEINS
MEMBRANES
MOLECULAR WEIGHT
MONOCLONAL ANTIBODIES
MUSCLES
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
PEPTIDE HYDROLASES
PROTEINS
PROTEOLYSIS
RADIOISOTOPES
RATS
REACTION KINETICS
RECEPTORS
RODENTS
STRUCTURE-ACTIVITY RELATIONSHIPS
TRACER TECHNIQUES
VERTEBRATES