Immunoaffinity isolation of Na+ channels from rat skeletal muscle. Analysis of subunits.
Polyclonal antiserum and monoclonal antibodies raised against the sodium channel from rat skeletal muscle sarcolemma have been immobilized on Sepharose and used to immunoaffinity purify this channel directly from skeletal muscle without the intervening purification of surface membranes. These antibodies isolate a approximately 260-kDa protein from whole muscle, although each purifies predominantly a 150-kDa component when isolated sarcolemmal membranes are used as starting material. A 45-kDa band is also found in the material purified from sarcolemma but not that obtained from whole muscle. In addition, these immunoaffinity columns isolate a 38-kDa band from both whole muscle and sarcolemma that copurifies with the 260-kDa protein. In some preparations this component appears as two closely spaced bands of 37 and 39 kDa. These small subunits coelute with the 260-kDa subunit when thiocyanate gradients are used to displace protein bound to the immunoaffinity columns and behave as integral components of the sodium channel. Estimates of stoichiometry were made for the large and small subunits of the muscle channel protein. After correction for labeling efficiency, values consistent with a ratio of one 260-kDa subunit to one 38-kDa subunit were obtained. We conclude that the rat skeletal muscle sodium channel contains a large alpha subunit of approximately 260 kDa that is sensitive to proteolytic nicking during the isolation of sarcolemmal membranes. In addition, at least one 38-kDa beta subunit is associated with each alpha subunit in the native channel.
J M
JM
Casadei
R D
RD
Gordon
R L
RL
Barchi
D000818:Animals; D000911:Antibodies, Monoclonal; D004591:Electrophoresis, Polyacrylamide Gel; D007106:Immune Sera; D007158:Immunologic Techniques; D007473:Ion Channels / Q000032:analysis*; D046911:Macromolecular Substances; D008970:Molecular Weight; D009132:Muscles / Q000032:analysis*; D011817:Rabbits; D051381:Rats; D012964:Sodium / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D007106:Immune Sera; D007473:Ion Channels; D046911:Macromolecular Substances; D012964:Sodium