Binding of [ 3H]batrachotoxinin A-20-α-benzoate and [ 3h]saxitoxin to receptor sites associated with sodium channels in trout brain synaptoneurosomes

Abstract

1. [ 3H]Batrachotoxinin A-20-α-benzoate ([ 3H]BTX-b) and [ 3H]saxitoxin ([ 3H]STX), radioligands that bind to distinct sites on the voltage-sensitive sodium channel, were bound specifically to saturable sites in rainbow trout ( Oncorhynchus mykiss) brain synaptoneurosomes. 2. Specific [ 3H]BTX-B binding was temperature dependent with highest levels of specific [ 3H]BTX-B binding observed at 7°C. Specific binding was inversely correlated with assay temperature at temperatures above 7°C. 3. Saturating concentrations of scorpion ( Leiurus quinquestriatus) venom (ScV) stimulated specific [ 3H]BTX-B binding at 27°C, but not at 7°C. The dihydropyrazole insecticide RH 3421 inhibited specific [ 3H]BTX-B binding at 7°C but had no effect on specific binding at 27°C. The sodium channel activators veratridine and aconitine and the local anesthetic dibucaine inhibited specific [ 3H]BTX-B binding at both 7°C and 27°C. 4. Displacement experiments in the presence of ScV at 27°C gave an equilibrium dissociation constant ( K d ) for [ 3H]BTX-B of 710 nM and a maximal binding capacity ( B max) of 11.3 pmol/mg protein. Kinetic experiments established the rates of association (1.17 × 10 5min −1 nM −1) and dissociation (0.0514min −1) of the ligand-receptor complex. 5. The binding of [ 3H]STX reached apparent saturation at 7.5 nM. Scatchard analysis of the saturation data indicated a K d of 3.8nM and a B max of 1.9 pmol/mg protein. 6. These studies provide evidence for high affinity, saturable binding sites for [ 3H]BTX-B and [ 3H]STX in trout brain preparations. Whereas certain neurotoxins modified the specific binding of [ 3H]BTX-B in trout brain synaptoneurosomes in a predictable fashion, other compounds known to affect specific [ 3H]BTX-B binding in mammalian brain preparations had no effect on specific [ 3H]BTX-B binding in the trout.