SITE OF COVALENT LABELING BY A PHOTOREACTIVE BATRACHOTOXIN DERIVATIVENEAR TRANSMEMBRANE SEGMENT IS6 OF THE SODIUM-CHANNEL ALPHA-SUBUNIT

The binding site for batrachotoxin, a lipid soluble neurotoxin acting at Na+ channel receptor site 2, was localized using a photoreactive, r adiolabeled batrachotoxin derivative to covalently label purified and reconstituted rat brain Na+ channels. In the presence of the brevetoxi n 1 from Ptychodiscus brevis and the pyrethroid RU51049, positive allo steric enhancers of batrachotoxin binding, a protein with an apparent molecular mass of 240 kDa corresponding to the Na+ channel alpha subun it was specifically covalently labeled. The region of the alpha subuni t specifically photolabeled by the photoreactive batrachotoxin derivat ive was identified by antibody mapping of proteolytic fragments. Even after extensive trypsinization, an anti-peptide antibody recognizing a n amino acid sequence adjacent to Na+ channel transmembrane segment IS 6 was able to immunoprecipitate up to 70% of the labeled peptides. Ana lysis of a more complete digestion with trypsin or V8 protease indicat ed that the batrachotoxin receptor site is formed in part by a portion of domain I. The identification of a specifically immunoprecipitated photolabeled 7,3-kDa peptide containing transmembrane segment S6 from domain I restricted the site of labeling to residues Asn-388 to Glu-42 9 if V8 protease digestion was complete or Leu-380 to Glu-429 if diges tion was incomplete. These results implicate the S6 transmembrane regi on of domain I of the Na+ channel alpha subunit as an important compon ent of the batrachotoxin receptor site.