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

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 a-subunit was revealed using both gel slicing and fluorography techniques.Regions of the a-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.Amore extensive tryptic digest was obtained with a preparation in which the brevetoxin photolabel was incorporated into the a-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 a-subunit and implicate their region of interaction as an important component of the brevetoxin receptor site, Voltage-gated sodium channels from rat brain are complexes comprised of three glycoprotein subunits: a 260-kDa a-subunit covalently linked to a 33-kDa P2-subunit and noncovalently interacting with a 36-kDa pl-subunit (for review, see Catterall, 1992).Neurotoxins interact with at least five distinct receptor sites on the Na' channel, four of which have been shown to be present on the a-subunit.The primary structure of the rat brain Na' channel a-subunit contains four internally homologous domains, each having six putative transmembrane seg-