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

Abstract

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 themore » 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.« less

Authors:
; ;  [1]
  1. (Univ. of Pennsylvania School of Medicine, Philadelphia (USA))
Publication Date:
OSTI Identifier:
5264670
Resource Type:
Journal Article
Resource Relation:
Journal Name: Journal of Biological Chemistry; (USA); Journal Volume: 264:22
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; PROTEINS; STRUCTURE-ACTIVITY RELATIONSHIPS; ANTIGEN-ANTIBODY REACTIONS; BIOCHEMICAL REACTION KINETICS; CARBOHYDRATES; CELL MEMBRANES; CONCANAVALIN; CYTOPLASM; IODINE 125; MEMBRANE PROTEINS; MOLECULAR WEIGHT; MONOCLONAL ANTIBODIES; MUSCLES; PEPTIDE HYDROLASES; PROTEOLYSIS; RATS; RECEPTORS; TRACER TECHNIQUES; ANIMALS; ANTIBODIES; BETA DECAY RADIOISOTOPES; CELL CONSTITUENTS; CHEMICAL REACTIONS; DAYS LIVING RADIOISOTOPES; DECOMPOSITION; ELECTRON CAPTURE RADIOISOTOPES; ENZYMES; HYDROLASES; INTERMEDIATE MASS NUCLEI; IODINE ISOTOPES; ISOTOPE APPLICATIONS; ISOTOPES; KINETICS; MAMMALS; MEMBRANES; NUCLEI; ODD-EVEN NUCLEI; ORGANIC COMPOUNDS; RADIOISOTOPES; REACTION KINETICS; RODENTS; VERTEBRATES; 550201* - Biochemistry- Tracer Techniques

Citation Formats

Kraner, S., Yang, J., and Barchi, R. Structural inferences for the native skeletal muscle sodium channel as derived from patterns of endogenous proteolysis. United States: N. p., 1989. Web.
Kraner, S., Yang, J., & Barchi, R. Structural inferences for the native skeletal muscle sodium channel as derived from patterns of endogenous proteolysis. United States.
Kraner, S., Yang, J., and Barchi, R. Sat . "Structural inferences for the native skeletal muscle sodium channel as derived from patterns of endogenous proteolysis". United States. doi:.
@article{osti_5264670,
title = {Structural inferences for the native skeletal muscle sodium channel as derived from patterns of endogenous proteolysis},
author = {Kraner, S. and Yang, J. and Barchi, R.},
abstractNote = {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.},
doi = {},
journal = {Journal of Biological Chemistry; (USA)},
number = ,
volume = 264:22,
place = {United States},
year = {Sat Aug 05 00:00:00 EDT 1989},
month = {Sat Aug 05 00:00:00 EDT 1989}
}
  • Voltage-dependent sodium channels are essential for normal membrane excitability and contractility in adult skeletal muscle. The gene encoding the principal sodium channel [alpha]-subunit isoform in human skeletal muscle (SCN4A) has recently been shown to harbor point mutations in certain hereditary forms of periodic paralysis. The authors have carried out an analysis of the detailed structure of this gene including delination of intron-exon boundaries by genomic DNA cloning and sequence analysis. The complete coding region of SCN4A is found in 32.5 kb of genomic DNA and consists of 24 exons (54 to >2.2 kb) and 23 introns (97 bp-4.85 kb). Themore » exon organization of the gene shows no relationship to the predicted functional domains of the channel protein and splice junctions interrupt many of the transmembrane segments. The genomic organization of sodium channels may have been partially conserved during evolution as evidenced by the observation that 10 of the 24 splice junctions in SCN4A are positioned in homologous locations in a putative sodium channel gene in Drosophila (para). The information presented here should be extremely useful both for further identifying sodium channel mutations and for gaining a better understanding of sodium channel evolution. 39 refs., 5 figs., 2 tabs.« less
  • The human growth hormone (GH) locus, a cluster of five genes, spans 47 kb on chromosome 17q22-q24. The skeletal muscle sodium channel {alpha}-subunit locus (SCN4A), a 32.5-kb gene, has previously been mapped to 17q23.1-q25.3. We demonstrate that both the GH gene cluster and the SCN4A gene colocalize to a single 525-kb yeast artificial chromosome (YAC) containing DNA derived from human chromosome 17. Restriction maps of two cosmids encompassing the 5{prime} terminus of the GH locus and including up to 40 kb of 5{prime}-flanking sequences demonstrate a perfect 20-kb overlap with previously published maps of the SCN4A gene. A 720-bp DNAmore » segment, encompassing sequences 32.3 to 31.6 kb 5{prime} to GH, was sequenced and found to be identical to exon 14 of SCN4A. These data demonstrate that the SCN4A gene and the entire GH gene cluster are contained within 100 kb on chromosome 17 and are separated by only 21.5 kb. Remarkably, this physical linkage between GH and SCN4A also reveals that multiple elements critical to tissue-specific transcriptional activation of the GH gene lie within the SCN4A gene. 48 refs., 5 figs.« less
  • A pro-inflammatory cytokine mixture (CM: interleukin (IL)-1β, tumor necrosis factor-α and interferon-γ) and IL-1β-induced matrix metalloproteinase (MMP)-3 activity have been shown to increase the proliferation of rat dental pulp cells and murine stem cell-derived odontoblast-like cells. This suggests that MMP-3 may regulate wound healing and regeneration in the odontoblast-rich dental pulp. Here, we determined whether these results can be extrapolated to human dental pulp by investigating the effects of CM-induced MMP-3 up-regulation on the proliferation and apoptosis of purified odontoblast-like cells derived from human skeletal muscle stem cells. We used siRNA to specifically reduce MMP-3 expression. We found that CMmore » treatment increased MMP-3 mRNA and protein levels as well as MMP-3 activity. Cell proliferation was also markedly increased, with no changes in apoptosis, upon treatment with CM and following the application of exogenous MMP-3. Endogenous tissue inhibitors of metalloproteinases were constitutively expressed during all experiments and unaffected by MMP-3. Although treatment with MMP-3 siRNA suppressed cell proliferation, it also unexpectedly increased apoptosis. This siRNA-mediated increase in apoptosis could be reversed by exogenous MMP-3. These results demonstrate that cytokine-induced MMP-3 activity regulates cell proliferation and suppresses apoptosis in human odontoblast-like cells. - Highlights: • Pro-inflammatory cytokines induce MMP-3 activity in human odontoblast-like cells. • Increased MMP-3 activity can promote cell proliferation in odontoblasts. • Specific loss of MMP-3 increases apoptosis in odontoblasts. • MMP-3 has potential as a promising new target for pupal repair and regeneration.« less
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