Identification of peptide components of the brevetoxin receptor site of rat brain sodium channels.

V.L Trainer,D.G Baden,W.A Catterall

doi:10.1016/s0021-9258(17)32105-1

V.L Trainer, D.G Baden + Show 1 more

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https://doi.org/10.1016/s0021-9258(17)32105-1

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Abstract

To identify the binding domain for brevetoxins, a family of lipid-soluble neurotoxins acting at Na+ channel receptor site 5, purified and reconstituted rat brain Na+ channels were photolabeled with p-azidobenzoyl tritium-labeled brevetoxin, and the labeled peptides were identified. A radiolabeled band with an apparent molecular mass of 250 kDa corresponding to the Na+ channel alpha-subunit was revealed using both gel slicing and fluorography techniques. Regions of the alpha-subunit specifically photolabeled by this ligand were then identified by antibody mapping of proteolytic fragments. Even after extensive proteolysis, anti-peptide antibodies recognizing amino acid sequences within or adjacent to Na+ channel transmembrane segments IS6 and IVS5 were each able to immunoprecipitate up to 40% of the labeled peptides. A more extensive tryptic digest was obtained with a preparation in which the brevetoxin photolabel was incorporated into the alpha-subunit of purified Na+ channel in detergent solution. The identification of a specifically immunoprecipitated 6-kDa peptide containing transmembrane segment S6 from domain I restricted the labeled peptide fragment to residues Thr-400 to Lys-443 if tryptic digestion was complete or Ala-396 to Lys-455 if tryptic cleavage was incomplete. Similarly, the identification of a specifically immunoprecipitated 6-kDa peptide from domain IV restricted the labeled peptide to residues Glu-1738 to Lys-1785 or Glu-1738 to Lys-1793 on the extracellular side of transmembrane segment S5. These results provide direct evidence for close association of transmembrane segments IS6 and IVS5 in the native conformation of the Na+ channel alpha-subunit and implicate their region of interaction as an important component of the brevetoxin receptor site.

Highlights

Brevetoxins, produced by the marine dinoflagellate Ptychodiscus brevis, are lipid-soluble polyether neurotoxins that act at Na’ channel receptor site 5 (Catterall and Risk,1981;Poliet al., 1986;Sharkeyet al., were each able to immunoprecipitate up to 40% of the 1987)
Site 5 on Solubilized Sodium Channels-The brevetoxins are unusually large, hydrophobic polyethers that might beexpected to have high affinity for detergent and phospholipids present in the solutions used in sodium channel purification
That both pore. A brevetoxin molecule (PbTx)-3 and p-AB PbTx-3 specifi- FIG6

Summary

To whom correspondence should be addressed

The abbreviations used are: PbTx, brevetoxin fromP. brevis; TPCKtrypsin, ~-l-tosylamido-2-phenylethyclhloromethyl ketone-treated trypsin; p-AB [3H]PbTx-3, p-azidobenzoyltritium-labeled brevetoxin; SP, sodium channel peptide; WGA, wheat germ agglutinin; PAGE, polyacrylamide gel electrophoresis; Tricine, N-[2-hydroxy-l,l-bis(hyogy, Mailstop SJ-30, University of Washington, Seattle, WA 98195. Brevis; TPCKtrypsin, ~-l-tosylamido-2-phenylethyclhloromethyl ketone-treated trypsin; p-AB [3H]PbTx-3, p-azidobenzoyltritium-labeled brevetoxin; SP, sodium channel peptide; WGA, wheat germ agglutinin; PAGE, polyacrylamide gel electrophoresis; Tricine, N-[2-hydroxy-l,l-bis(hyogy, Mailstop SJ-30, University of Washington, Seattle, WA 98195. A-Sepharose was swollen for 20 min a t room temp in 0.1 M sodium 4 "C as described above. One pl of containing 10 mM Tris, adjusted t o pH 6 with HCl, 150 mM NaCI, 1 mM serum was added per plof swollen protein A-Sepharoseand mixed by MgCl,, 1mM CaCl,, 0.05% SDS, was used in this hydrolysis reaction to rotation at room temperature for 30 min. SDS-PAGE a n d Gel Slicing-For analysis of p-AB PHIPbTx-3 bindpellet was resuspended in volume of buffer S and used for immuno- ing to purifiedNa' channels, a 7% porous reducing gel system was used precipitation of photolabeled Na' channel peptides. Purified samples containing 250-350pmoUmlNa' channel in Na,SO, mediuwmerreeconstitutienpdthoosphatidylcholine/

RESULTS

DISCUSSION

Findings

A Molecular Model of the Brevetoxin Receptor Site-Based