Monoclonal antibodies against the voltage-sensitive Na+ channel from mammalian skeletal muscle

J. M. Casadei, R. D. Gordon, L. A. Lampson, D. L. Schotland, R. L. Barchi

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Abstract

A panel of 13 monoclonal antibodies against the voltage-sensitive Na+ channel of rat skeletal muscle has been characterized. Each of these antibodies reacted with the purified Na+ channel protein in a solid-phase radioimmunoassay. Nine antibodies specifically immunoprecipitated the Na+ channel in a form that retained its characteristic high affinity for saxitoxin, and 11 recognized the channel in a crude mixture of solubilized membrane proteins separated on a Sepharose CL-6B column. Six antibodies specifically labeled skeletal muscle in immunofluorescence techniques. In each case, antibody was localized only to the surface membranes of the muscle fibers. Eleven antibodies produced detectable reaction on immunoblot transfers of sarcolemmal membrane proteins; each of these bound to a diffuse 160- to 200-kDa band that comigrated with the large glycoprotein subunit of the purified Na+ channel. Further studies were carried out with one of these antibodies, L/D3. In immunoblots of a glycoprotein fraction prepared from muscle that had been homogenized rapidly in a solution containing detergent, EGTA, and protease inhibitors, L/D3 recognized only a single 260-kDa band. Incubation of solubilized muscle proteins at 4°C for 24 hr without EGTA prior to isolation of the glycoprotein fraction resulted in partial conversion of this 260-kDa component to a smaller component between 160 and 200 kDa that comigrated with the principal immunoreactive component of sarcolemma. Based on its immunoreactivity with monoclonal antibodies, the large subunit of the rat skeletal muscle Na+ channel appears to be ≃260 kDa in its native state but may be sensitive to proteolysis during the isolation of sarcolemmal membranes

Original languageEnglish (US)
Pages (from-to)6227-6231
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume81
Issue number19 I
DOIs
Publication statusPublished - Dec 1 1984

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