Physicochemical characterization of the .alpha.-peptide of the sodium channel from rat brain

Abstract

The alpha-peptide of the rat brain sodium channel of apparent molecular weight 260K has been purified to homogeneity in order to determine its structural and chemical properties. By negative-stain electron microscopy, the molecule morphology of the solubilized channel protein appears as a stack of disks or rouleaux whose dimensions are 40 A X 200 A. Measurement of the secondary structure by circular dichroism shows that the alpha-peptide is a conformationally flexible polypeptide that contains mostly beta-sheet and random-coil in mixed detergent-phospholipid micelles and folds into a conformation that has approximately 65% alpha-helix after reconstitution into phosphatidylcholine vesicles. Preparative polyacrylamide gel electrophoresis was used to obtain a chemically homogeneous peptide to analyze the amino acid and carbohydrate composition. The amino acid composition shows a reasonably high content of acidic amino acids with no striking excess of hydrophobic amino acids, while carbohydrate analyses show that carbohydrate is 31% by weight of the protein with sialic acid representing over 50% of the total carbohydrates. The high alpha-helical content, the amino acid composition, and the large carbohydrate mass are similar to those of the eel electroplax sodium channel and appear to be general features of the sodium channels which have been analyzed structurally and chemically to date.

Neurotoxin-modulated uptake of sodium by highly purified preparations of the electroplax tetrodotoxin-binding glycopeptide reconstituted into lipid vesicles

“…This step results in the loss of 40-60% of TTX-binding activity, as well as much of the purified protein (Talvenheimo et al, 1982;Weigele & Barchi, 1982;Rosenberg et al, 1984a;Elmer et al, 1985). The functional yields of sodium channel protein in all of these studies ranged from 3-12% of the initially extracted channel proteins (as measured with TTX binding) after reconstitution.…”

“…Although these results suggest the necessity of this peptide for channel function, such experiments are subject to the criticism that any conditions used to disrupt intermolecular bonds between peptides might also disrupt vital intramolecular bonds on the large glycopeptide, thus denaturing it. Moreover, Elmer et al (1985) have recently reported isolating the large glycopeptide from rat brain in the absence of other subunits, while still retaining the ability of the protein to bind tetrodotoxin.…”

Sodium channel expression detected with antibody 7493 in A2B5+ and A2B5− astrocytes from rat optic nerve in vitro

“…In the present study, we examine sodium channel expression in astrocytes from developing rat optic nerve maintained in culture for up to 28 days with polyclonal antibody 7493, which was generated against purified rat brain sodium channel protein (Elmer et al, 1985). We demonstrate that sodium channels, as determined by 7493 immunoreactivity, are expressed by some astrocytes in culture for as long as 28 days, despite the absence of contact with neurons.…”

“…The expression of sodium channels in astrocytes cultured from PO and P7 rat optic nerves was examined immunocytochemically with polyclonal antibody 7493, which was generated against purified rat brain sodium channels (Elmer et al, 1985). This antibody immunoblots and immunoprecipitates from labeled membrane protein a 260 kDa polypeptide corresponding to the alpha subunit of the rat brain sodium channel, and has been shown to immunostain both the nodal axon membrane and astrocytes in situ (Black et al, 1989a,b).…”

Distribution and lateral mobility of voltage-dependent sodium channels in neurons [published erratum appears in J Cell Biol 1989 May;108(5):preceding 2001]

“…This distribution of channels will determine some of the differential electrical properties of one part versus another part of a given excitable cell. A number of laboratories have recently isolated and characterized the sodium channel protein as an extremely carbohydrate-rich polypeptide of Mr 260 kD (9,22,26,36). Although localization of important receptors and some morphological and electrophysiological evidence on sodium channel distribution has been described (4-6, 17, 20, 43, 57), little is known concerning the mechanisms that regulate and maintain their surface localization.…”