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.
Department of Pharmacology, University of Washington, Seattle 98195.
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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