3H-batrachotoxinin-A benzoate binding to voltage-sensitive sodium channels: inhibition by the channel blockers tetrodotoxin and saxitoxin

GB Brown
1986 Journal of Neuroscience  
The sodium channel blockers tetrodotoxin (TTX) and saxitoxin (STX) and the channel activator batrachotoxin (BTX) produce their effects by binding to separate and distinct sites on the channel protein. The fact that TTX-and STX-modified sodium channels are blocked to sodium flux has precluded drawing any direct conclusions regarding the effect of TTXSTX on BTX binding based on electrophysiological or 22Na flux measurements. Nevertheless, these sites have been presumed to be noninteracting. In
more » ... s study, 3H-batrachotoxinin-A benzoate (BTX-B), a tritiated congener of BTX, has been used to provide a direct assessment of these binding interactions. Equilibrium specific binding of 3H-BTX-B to sodium channels in vesicular preparations of mouse brain in the presence of scorpion toxin was measured using a filtration assay procedure. At 25°C both TTX and STX inhibit 3H-BTX-B binding in a concentrationdependent and noncompetitive manner. This inhibition is markedly temperature-dependent, being negligible at 37°C and maximal at WC, the lowest temperature investigated. Scatchard analysis of BTX-B binding isotherms at 25°C in the presence and absence of 1 PM TTX revealed that inhibition is due to a 3-fold decrease in the affinity of BTX-B binding with no change in the number of binding sites sites (B"). The concentration dependence for TTX inhibition of both specific 3H-STX and 3H-BTX-B binding is identical, suggesting that inhibition of 3H-BTX-B binding is due to a direct effect of TTXSTX binding at their specific sodium channel site. The channel blockers did not alter the binding of scorpion toxin under these assay conditions, nor did BTX-B affect the binding of 3H-STX. These findings support the conclusion that occupancy of the sodium channel TTX/STX binding site by the channel blockers can be accompanied by a conformational perturbation of the BTX site resulting in decreased BTX-B binding affinity. The voltage-sensitive sodium channel is responsible for the rapid rising phase of the action potential in a variety of excitable membranes. In order to mediate this transient process, it is clear that the ionophore must be conformationally mobile, able to assume different states. The conformational dynamics of the sodium channel are directly reflected in the voltage-dependent gating process by which, in response to a depolarizing pulse, the
doi:10.1523/jneurosci.06-07-02064.1986 pmid:2426426 fatcat:2464schjgrbs7mt3kwebzuemvi