Beta 2 subunits of sodium channels from vertebrate brain. Studies with subunit-specific antibodies.

D A Wollner,D J Messner,W A Catterall

doi:10.1016/s0021-9258(18)47853-2

D A Wollner, D J Messner + Show 1 more

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https://doi.org/10.1016/s0021-9258(18)47853-2

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Abstract

The sodium channel purified from rat brain is composed of three subunits: alpha (Mr 260,000), beta 1 (Mr 36,000), and beta 2 (Mr 33,000). alpha and beta 2 subunits are linked through disulfide bonds. Procedures are described for preparative isolation of the beta 1 and beta 2 subunits under native conditions. Pure beta 2 subunits obtained by this procedure were used to prepare a specific anti-beta 2 subunit antiserum. Antibodies purified from this serum by antigen affinity chromatography recognize only disulfide-linked alpha beta 2 complexes and beta 2 subunits in immunoblots, and immunoprecipitate 32P-labeled alpha subunits of purified sodium channels having intact disulfide bonds, but not those of sodium channels from which beta 2 subunits have been detached by reduction of disulfide bonds. These antibodies also immunoprecipitate 89% of the high affinity saxitoxin-binding sites from rat brain membranes, indicating that nearly all sodium channels in rat brain have disulfide-linked alpha beta 2 subunits. Approximately 22% of beta 2 subunits in adult rat brain are not disulfide-linked to alpha subunits. Anti-beta 2 subunit antibodies are specific for sodium channels in the central nervous system and will not cross-react with sodium channels in skeletal muscle or sciatic nerve. The brains of a broad range of vertebrate species, including electric eel, are shown to express sodium channels with disulfide-linked alpha beta 2 subunits.

Highlights

The sodium channel purified from rat brain is com- observed in studiesof rat brainsodium channels by two other posed of three subunits: a ( X 260,000), @1 (Mr groups [11, 12] and are not a component of purified sodium
Pure 82 subunits obtained by this procedurewere used to prepare a specific anti-82 subunit antiserum. Antibodies purified from this serum by antigen affinity chromatography recognize only disulfide-linked 4 2 complexes and 82 subunits in immunoblots, and immunoprecipitate 32P-labeled CI subunits of purified sodium channels having intact disulfide bonds,but not those of sodiumchannels from which 82 subunits have been detached by reduction of disulfide bonds
P2 subunits can be removed without effect (7,s)I.njection of mRNA encoding only the a subunits of the rat brain sodium channelinto Xenopus oocytes leads to expression of functional channels [13,14,15], indicating that this mRNA contains all the information necessary t o directsynthesis of functional sodium channels in the oocyte

Summary

Introduction

The sodium channel purified from rat brain is com- observed in studiesof rat brainsodium channels by two other posed of three subunits: a ( X 260,000), @1 These antibodies immunoprecipitate 89%of the high affinity saxitoxin-binding sites from rat brain membranes,indicating that most sodium channels in rat brain have disulfide-linked a@2subunits.

Results

Conclusion