Purification and functional reconstitution of the voltage-sensitive sodium channel from rabbit T-tubular membranes

S. D. Kraner, J. C. Tanaka, R. L. Barchi

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Abstract

The voltage-sensitive sodium channel has been purified from rabbit T-tubular membranes and reconstituted into defined phospholipid vesicles. Membranes enriched in T-tubular elements (specific [3H]nitrendipine binding = 41 ± 9 pmol/mg of protein, n = 7) were isolated from fast skeletal muscle. After solubilization with Nonidet P-40, the sodium channel protein was purified to > 95% of theoretical homogeneity based on the specific activity of [3H]saxitoxin binding. Two subunits of M(r) ~ 260,000 and 38,000 were found; these bands co-distributed with the peak of [3H]saxitoxin binding on sucrose gradients. The purified protein was reconstituted into egg phosphatidylcholine vesicles and retained the ability to gate specific 22Na+ influx in response to activation by batrachotoxin or veratridine. All activated fluxes were blocked by saxitoxin and tetrodotoxin. On sucrose gradients, the distribution of protein capable of functional channel activity paralleled the distribution of specific [3H]saxitoxin binding and of the M(r) 260,000 and 38,000 components. The cation selectivity for the reconstituted, batrachotoxin-activated channel was Na+ > K+ > Rb+ > Cs+, with flux ratios of 1:0.13:0.02:0.008. Nine of 25 monoclonal antibodies raised against the rat sarcolemmal sodium channel cross-reacted with the rabbit T-tubular sodium channel in a solid-phase radioimmunoassay. Six of these antibodies showed specific binding to immunoblot transfers of T-tubular membrane proteins. Each labeled a single band at M(r) ~ 260,000 corresponding in mobility to the large subunit of the sodium channel.

Original languageEnglish (US)
Pages (from-to)6341-6347
Number of pages7
JournalJournal of Biological Chemistry
Volume260
Issue number10
StatePublished - 1985
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Biochemistry
  • Cell Biology

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