BREVETOXIN-3 (PBTX-3) AND ITS DERIVATIVES MODULATE SINGLE TETRODOTOXIN-SENSITIVE SODIUM-CHANNELS IN RAT SENSORY NEURONS

Brevetoxin-3 (PbTx-3), produced by marine dinoflagellates (Ptychodiscu s brevis), is a lipophilic 11-ring polyether molecule that binds with high affinity to site 5 of the voltage-sensitive sodium (Na+) channel. The effects of PbTx-3 and its derivatives were studied in cell-attach ed membrane patches on neurons dissociated from neonatal rat nodose ga nglia by the path clamp technique, PbTx-3 (30-500 nM) produced a shift in activation to more negative membrane potentials whereby single-cha nnel activity was observed under steady-state conditions (maintained d epolarization at -50 mV). The unitary current-voltage relationship is linear, which exhibits a reversal potential of approximately +60 mV, T wo unitary current amplitudes could be observed in the presence of PbT x-3, with slope conductances of 10.7 pS and 21.2 pS. PbTx-3 inhibits t he inactivation of Na+ channels and prolongs the mean open time of the se channels, Unitary Na+ currents could be blocked by 1 mu M tetrodoto xin (TXX) added to the pipette solution, which indicates that the sing le-channel currents are caused by the opening of TTX-sensitive Na+ cha nnels. The PbTx-3 molecule is proposed to have multiple active centers (A-ring lactone, C-42 of R side chain) interacting with the Na+ chann el binding site, Modification of the molecular structure of PbTx-3 at these centers produced derivatives (PbTx-6, 2,3,41,43-tetrahydro-PbTx- 3, 2,3,27,28,41,43-hexahydro-PbTx-3 and 2,3-dihydro-PbTx-3 A-ring diol ), which were less potent than PbTx-3 in producing similar effects on Na+ channel kinetics. PbTx-3 and its derivatives may provide insight i nto the mechanics of voltage-sensitive Na+ channel gating.