Primary structure and expression of a sodium channel characteristic of denervated and immature rat skeletal muscle

The alpha subunit of a voltage-sensitive sodium channel characteristic of denervated rat skeletal muscle was cloned and characterized. The cDNA encodes a 2018 amino acid protein (SkM2) that is homologous to other recently cloned sodium channels, including a tetrodotoxin (TTX)-sensitive sodium channel from rat skeletal muscle (SkM1). The SkM2 protein is no more homologous to SkM1 than to the rat brain sodium channels and differs notably from SkM1 in having a longer cytoplasmic loop joining domains 1 and 2. Steady-state mRNA levels for SkM1 and SkM2 are regulated differently during development and following denervation: the SkM2 mRNA level is highest in early development, when TTX-insensitive channels predominate, but declines rapidly with age as SkM1 mRNA increases; SkM2 mRNA is not detectable in normally innervated adult skeletal muscle but increases greater than 100-fold after denervation; rat cardiac muscle has abundant SkM2 mRNA but no detectable SkM1 message. These findings suggest that SkM2 is a TTX-insensitive sodium channel expressed in both skeletal and cardiac muscle.     

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.     

Analysis of protease-sensitive regions in the skeletal muscle sodium channel in vitro and implications for channel tertiary structure.

The tertiary structure of the rat skeletal muscle sodium channel was probed in vitro by determining regions of sensitivity to V-8 protease, trypsin, and chymotrypsin. Resultant channel fragments were identified with antibodies to defined sequences distributed along the primary structure. The temporal pattern of proteolysis was followed with channel protein in either detergent-phospholipid micelles or membrane fragments as well as with channel exposed to sodium dodecyl sulfate. Proteolysis in micelles and membranes occurred in discrete, reproducible steps that were similar in both systems. Although the size of intermediates varied slightly, their sequence of appearance was similar for all enzymes, suggesting that the observed pattern was determined by the relative accessibility of selected sites in the tertiary structure. No major change in channel organization appeared to occur after solubilization of membranes in nonionic detergents. Highly accessible sites in the native structure included the carboxyl terminus and the region linking the second and third internal repeat domains, while the amino terminus and the repeat domains themselves were relatively resistant to proteolysis unless the protein was denatured. Kinetically, interdomain II-III was the most readily cleaved; interdomains I-II and especially III-IV were less easily accessible. While domains I and IV appeared to remain intact throughout our experiments, limit fragments for epitopes associated with domains II and III suggest that cleavage eventually occurs at sites between the putative S5 and S6 helices in these domains.     

Muscle surface membranes: preparative methods affect apparent chemical properties and neurotoxin binding.

Considerable disagreement exists between results reported by various authors for lipid composition and enzyme activity in purified muscle membrane fractions presumed to be sarcolemma, although an explanation for these discrepancies has not been presented. We have prepared muscle light surface membrane fractions of comparable density (1.050--1.120) by a low-salt sucrose method and by an LiBr-KCl extraction procedure and compared them for density profile, total lipid and cholesterol content, protein composition and ATPase activity. In addition, sodium channels characteristic of excitable membranes have been quantitated in each preparation using [3H]saxitoxin binding assays, and the density of acetylcholine receptors determined in fractions from control and denervated muscle using alpha-[125I]bungarotoxin. Although both fractions contain predominantly surface membrane, the LiBr fraction consistently shows the higher specific activity of p-nitrophenylphosphatase, higher free cholesterol content, and higher density of sodium channels and acetylcholine receptors. The density distribution of sodium channels appears uniform throughout both fractions. Quantitative differences were seen between sodium dodecyl sulfate polyacrylamide gel electrophoresis patterns of membrane proteins from the two preparations although most bands are represented in both. A majority of the low-salt sucrose light membrane proteins were accessible in varying degrees to labelling with diazotized diiodosulfanylic acid in intact muscle. These results suggest that light surface membrane fractions may be mixtures of sarcolemma and T-tubular membranes. Using our preparative methods, the LiBr fraction may contain predominantly sarcolemma while low-salt sucrose light membranes may be enriched in T-tubular elements.