Neurotoxin binding and allosteric modulation at receptor sites 2 and 5 on purified and reconstituted rat brain sodium channels.
Purified and reconstituted sodium channels have previously been shown to be functional in voltage-dependent ion conductance and in high affinity binding of tetrodotoxin and saxitoxin at neurotoxin receptor site 1 and alpha-scorpion toxins at receptor site 3, but high affinity binding of neurotoxins at receptor sites 2, 4, and 5 has not been demonstrated. The pyrethroid insecticide RU39568 enhances the specific binding of [3H]batrachotoxinin A 20-alpha-benzoate (BTX-B) to neurotoxin receptor site 2 on purified and reconstituted sodium channels up to 500-fold, reducing the Kd to 1.5 nM. Brevetoxins and alpha-scorpion toxins cause further allosteric enhancement of BTX-B binding. The pyrethroids deltamethrin and bifenthrin and the nonpyrethroid insecticide 2,2-bis(p-chlorophenyl)trichloroethane can partially substitute for RU39568 in enhancing BTX-B binding, but other pyrethroids are inactive. The brevetoxin PbTx-1 binds specifically to neurotoxin receptor site 5 on purified and reconstituted sodium channels with a Kd value of approximately 30 nM. Brevetoxin binding is enhanced up to 2-fold by the combination of batrachotoxin and RU39568. The allosteric enhancement of BTX-B binding by RU39568 is voltage dependent, decreasing progressively with depolarization to 0 mV. In contrast, PbTx-1 binding is not voltage dependent and PbTx-1 reduces the voltage dependence of the effect of RU39568. The results demonstrate restoration of high affinity binding and allosteric interactions of ligands at neurotoxin receptor sites 2 and 5 on purified and reconstituted sodium channels and provide an experimental approach to covalent labeling and identification of the peptide components of those receptor sites.
Trainer
V L
VL
Department of Pharmacology, University of Washington, Seattle 98195.
Moreau
E
E
Guedin
D
D
Baden
D G
DG
Catterall
W A
WA
D000494:Allosteric Regulation; D000818:Animals; D001496:Batrachotoxins / Q000378:metabolism; D001921:Brain / Q000378:metabolism*; D008387:Marine Toxins / Q000378:metabolism; D008564:Membrane Potentials; D015394:Molecular Structure; D009498:Neurotoxins / Q000378:metabolism*; D035964:Oxocins; D011722:Pyrethrins / Q000494:pharmacology; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism; D011950:Receptors, Cholinergic / Q000378:metabolism; D015222:Sodium Channels / Q000378:metabolism*; D000095702:Polyether Toxins
D001496:Batrachotoxins; D008387:Marine Toxins; D009498:Neurotoxins; D035964:Oxocins; D011722:Pyrethrins; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic; D015222:Sodium Channels; C041417:batrachotoxin receptor; C072089:brevetoxin receptor; C053342:brevetoxin; D000095702:Polyether Toxins