Monoclonal Antibodies Against the Voltage‐Sensitive Sodium Channel from Rat Skeletal Muscle: Mapping Antibody Binding Sites

Casadei, Jan; Barchi, Robert L.

doi:10.1111/j.1471-4159.1987.tb05584.x

Cited by 16 publications

(14 citation statements)

References 28 publications

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“…The catalytic subunit of cyclic AMP-dependent protein kinase from bovine heart (designated PKa in this report), forskolin, phosphoserine, phosphothreonine, and phosphotyrosine were obtained from Sigma Chemical. Other reagents and materials were obtained as detailed in earlier reports (Casadei et al, 1984;Kraner et al, 1985Kraner et al, , 1989.…”

Section: Methodsmentioning

confidence: 99%

Phosphorylation of the Rat Skeletal Muscle Sodium Channel by Cyclic AMP‐Dependent Protein Kinase

Yang

Barchi

1990

Journal of Neurochemistry

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Cyclic AMP-dependent phosphorylation of the rat brain sodium channel was reported to be restricted to five sites within an approximately 210 amino acid region of the primary sequence that is deleted in the homologous sodium channel from rat skeletal muscle. We find that, in spite of this deletion, the rat muscle sodium channel alpha-subunit is also an excellent substrate for phosphorylation by this kinase both in primary muscle cells in tissue culture and in vitro after isolation from adult muscle. Sodium channel protein purified from adult rat skeletal muscle was readily phosphorylated in vitro by the catalytic subunit of the bovine cyclic AMP-dependent protein kinase (PKa). Only the 260,000 MW alpha-subunit was labeled, with a maximum level of incorporation in vitro of approximately 0.5 mol [32P]phosphate per mole of channel protein. The beta-subunit of the channel is not phosphorylated under these conditions. In primary rat skeletal muscle cells in culture, incorporation of phosphate into the channel alpha-subunit is stimulated 1.3- to 1.5-fold by treatment of the cells with forskolin. Phosphorylation of the sodium channel isolated from these cells could also be demonstrated in vitro using PKa. This in vitro phosphorylation could be inhibited 80-90% by pretreatment of the cells in culture with forskolin, suggesting that the sites labeled in vitro by PKa were the same as those phosphorylated in the intact cells by the endogenous cyclic AMP-dependent kinase. In both the adult muscle channel and the channel from muscle cells in culture, phosphorylation by PKa was limited to serine residues.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Methodsmentioning

confidence: 99%

Phosphorylation of the Rat Skeletal Muscle Sodium Channel by Cyclic AMP‐Dependent Protein Kinase

Yang

Barchi

1990

Journal of Neurochemistry

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“…R. Barchi and S. Cohen of the University of Pennsylvania. This mAb has been shown to recognize a 200 kDa band in immunoblots of rat skeletal muscle (Casadei and Barchi, 1987), and to stain surface membrane in cross sections of rat muscle (Haimovich et al, 1987).…”

Section: Methodsmentioning

confidence: 99%

Aggregation of sodium channels during development and maturation of the neuromuscular junction

Lupa

Schaller

et al. 1993

J. Neurosci.

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The voltage-activated Na channel (NaCh) is an integral membrane protein that is enriched at the neuromuscular end plate. Using loose-patch voltage-clamp and immunofluorescence, we have found that the aggregation of NaChs occurs late, during maturation of the neuromuscular junction. A decline in expression of embryonic NaCh mRNA and increase in adult NaCh mRNA precedes the onset of aggregation, and the appearance of functional adult NaChs coincides with NaCh aggregation. We tested the possibility that only the adult NaCh subtype could aggregate during development and found that both the embryonic and adult isoforms become concentrated at the synapse. The NaCh is the first postsynaptic membrane protein shown to become clustered postnatally, and the mechanism producing this aggregation appears to be different from the process producing aggregation of other synaptic proteins.

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“…We have shown that A/B2 and L/D3 demonstrated competition with each other for binding to immobilized sodium channel protein (11). However, unlabeled F/C11 competed with neither A/B2 nor L/D3 while unlabeled B/D6 demonstrated partial competition with A/B2 and L/D3.…”

Section: Table II Summary Of Antibody Competition Resultsmentioning

confidence: 86%

“…This work also allowed us to map the location of epitopes for monoclonal antibodies we had previously generated against purified sodium channel protein. Our binding studies divided these antibodies into two large groups based on mutually exclusive competition (11). While epitopes in each group were typically clustered in similar regions of the channel sequence, in several cases we found monoclonals in the same group that recognized epitopes widely separated in the primary sequence but presumably brought together in the native protein by folding of the polypeptide backbone (6,12).…”

mentioning

confidence: 93%

Probing Sodium Channel Cytoplasmic Domain Structure

Sun

Barchi

Cohen³

1995

Journal of Biological Chemistry

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Epitopes for monoclonal antibodies directed against the purified adult rat skeletal muscle sodium channel (rSkM1) were localized using channel proteolysis and fusion proteins. The interactions between these and other monoclonal antibodies with site-specific polyclonal antibodies were used to investigate the spatial relationships among rSkM1 cytoplasmic segments. Competition between antibodies for binding was performed using a solution-phase assay in which solubilized channel protein retains many of the biophysical characteristics of the rSkM1 protein in vivo. Our results support a model in which: 1) the amino terminus assumes a rigid structure having a fixed orientation with respect to other intracellular segments; 2) the interdomain 2-3 region is centrally located on the cytoplasmic surface of the channel, extends farther into the cytoplasm, and has an intermediate degree of flexibility; 3) the beginning of the amino terminus and end of the carboxyl terminus specifically interact with each other; and 4) domains 1 and 4 are adjacent. The sequences responsible for the interaction of the amino and carboxyl termini were identified by demonstrating the specific binding of a synthetic peptide encompassing the first 30 residues of the rSkM1 amino terminus to a fusion protein containing the rSkM1 carboxyl terminus.In studies of voltage-dependent ion channels, a major goal is to correlate specific aspects of protein structure with channel function. In order to attain this goal, an accurate model of channel tertiary structure is required. Sequence information for a variety of voltage-dependent sodium channels is now available, providing the basis for several models of channel tertiary structure (1-5). All current models postulate the presence of four membrane-embedded homologous domains joined by cytoplasmic linking and terminal sequences. Although the models were initially based largely on theoretical considerations, various aspects have been tested using a variety of molecular, biochemical, and immunological techniques, and their general features have been validated.In previous studies, we used a combination of limited proteolysis and antibody binding to provide experimental support for the presence of four compact repeat domains in the skeletal muscle sodium channel, to identify the topography of the regions that join and flank these domains, and to probe the relative orientation of the large extramembrane cytoplasmic elements (6 -11). This work also allowed us to map the location of epitopes for monoclonal antibodies we had previously generated against purified sodium channel protein. Our binding studies divided these antibodies into two large groups based on mutually exclusive competition (11). While epitopes in each group were typically clustered in similar regions of the channel sequence, in several cases we found monoclonals in the same group that recognized epitopes widely separated in the primary sequence but presumably brought together in the native protein by folding of the polypeptide backbone (6, 12). Ident...

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Monoclonal Antibodies Against the Voltage‐Sensitive Sodium Channel from Rat Skeletal Muscle: Mapping Antibody Binding Sites

Cited by 16 publications

References 28 publications

Phosphorylation of the Rat Skeletal Muscle Sodium Channel by Cyclic AMP‐Dependent Protein Kinase

Phosphorylation of the Rat Skeletal Muscle Sodium Channel by Cyclic AMP‐Dependent Protein Kinase

Aggregation of sodium channels during development and maturation of the neuromuscular junction

Probing Sodium Channel Cytoplasmic Domain Structure

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