Neurotoxin binding to receptor sites associated with voltage-sensitive sodium channels in intact, lysed, and detergent-solubilized brain membranes.

[3H]Saxitoxin binds to a single class of receptor sites in rat brain synaptosomes and in broken membrane fractions derived from rat brain with a KD of approximately 2 11~ at 36°C. Batrachotoxin and scorpion toxin, which act at different receptor sites to activate sodium channels, have no effect on saxitoxin binding. Osmotic lysis and depolarization also have no effect on saxitoxin binding. These results show that saxitoxin binds equally well to resting, active, and inactivated sodium channels. Saxitoxin binding sites were concentrated in the crude mitochondrial fraction in subcellular fractionation experiments. Further separation of this fraction on discontinuous sucrose gradients showed that the receptor sites were enriched in fractions containing broken neuronal membranes, synaptosomes, and synaptic plasma membrane. Scorpion mono[‘251]iodotoxin also binds to a single class of receptor sites in synaptosomes. Comparison of saxitoxin and scorpion toxin binding to synaptosomes indicates that there are 3.7 saxitoxin receptor sites for each scorpion toxin receptor site. Lysis or depolarization causes a marked inhibition of scorpion toxin binding. Batrachotoxin, veratridine, and aconitine enhance scorpion toxin binding in intact synaptosomes and partially restore binding in lysed membrane fractions. Competitive interactions among these toxins in enhancing scorpion toxin binding in lysed membrane fractions indicate that they act at a common binding site. These results with lysed membrane fractions show that both the allosteric interactions between scorpion toxin and the lipid-soluble toxins and the competitive interactions among the three lipid-soluble toxins are retained in the absence of membrane potential and ion gradients. Dissolution of brain membranes with 1% Triton X100 results in solubilization of up to 40% of the saxitoxin receptor sites with no change in KD for saxitoxin. The soluble binding activity can be stabilized by addition of phosphatidylcholine and Ca’+. Analysis of the solubilized saxitoxin receptor by sucrose density gradient velocity sedimentation indicates a single peak of binding activity with a sedimentation coefficient of 10 S. Under similar conditions, scorpion toxin binding activity is lost irreversibly. Binding of scorpion toxin may depend upon the integrity of the bilayer membrane.