The binding site for batrachotoxin, a lipid-soluble neurotoxin acting at Na channel receptor site 2, was localized using a photoreactive, radiolabeled batrachotoxin derivative to covalently label purified and reconstituted rat brain Na channels. In the presence of the brevetoxin 1 from Ptychodiscus brevis and the pyrethroid RU51049, positive allosteric enhancers of batrachotoxin binding, a protein with an apparent molecular mass of 240 kDa corresponding to the Na channel subunit was specifically covalently labeled. The region of the subunit specifically photolabeled by the photoreactive batrachotoxin derivative was identified by antibody mapping of proteolytic fragments. Even after extensive trypsinization, an anti-peptide antibody recognizing an amino acid sequence adjacent to Na channel transmembrane segment IS6 was able to immunoprecipitate up to 70% of the labeled peptides. Analysis of a more complete digestion with trypsin or V8 protease indicated 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-429 if V8 protease digestion was complete or Leu-380 to Glu-429 if digestion was incomplete. These results implicate the S6 transmembrane region of domain I of the Na channel subunit as an important component of the batrachotoxin receptor site.

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