Orientation of cobra alpha-toxin on the nicotinic acetylcholine receptor. Fluorescence studies

Four flourescein isothiocyanate (FITC) derivatives of Naja naja siamemsis 3 neurotoxin (alpha-toxin), labeled at the epsilon-amino groups of Lys-23, Lys-35, Lys-49, or Lys-69, and a tetramethylrhodamine isothiocyanate (TRITC) derivative, labeled at epsilon-amino group of Lys-23, were prepared and used to analyze the orientation of cobra alpha-toxin on the nicotinic acetylcholine receptor (AcChR) relative to both the plane of the membrane and the central ion channel. Fluorescence-quenching studies of the AcChR-bound FITC derivatives indicated significant solute accessibility to each site of labeling and suggested that none of the sites of FITC labeling is included in the binding surface of the alpha-toxin. Labeling of Lys-23 with TRITC did not affect the affinity of the alpha-toxin toward the AcChR and confirmed, contrary to some previous reports, a minimal role of Lys-23 in the binding surface of the alpha-toxin. Measurements of energy transfer between the lipid-membrane surface and the sites of labeling on receptor-bound alpha-toxin derivatives show that the relative distances of closest approach between the surface of the lipid membrane domain and the sites of labeling are in the order Lys-23 less than or equal to Lys-49 less than Lys-35 less than or equal to Lys-69. Energy transfer between AcChR tryptophans and the sites of labeling of bound derivatives was about 50% greater to Lys-49 than to Lys-23, Lys-35, or Lys-69, suggesting that Lys-49 is closer to receptor tryptophans and to the center of the extracellular domain of the receptor than Lys-23, Lys-35, or Lys-69. Combined with previous observations that the tip of the central loop of the alpha-toxin directly interacts with the AcChR, the above results suggest a model of the approximate orientation of the snake neurotoxins on the receptor. This model shows the tip of the central loop of the toxin directly interacting with the receptor surface and the major axis of the neurotoxin tilting from a perpendicular projection from the membrane. The surface of the alpha-toxin that includes Lys-23 projects away from the central ion channel and the surface that includes Lys-35 and Lys-69 faces the ion channel.