Neurotoxin binding and allosteric modulation at receptor sites 2 and 5 on purified and reconstituted rat brain sodium channels

• Published in: The Journal of biological chemistry 1993-08, Vol.268 (23), p.17114-17119
• Main Authors: TRAINER, V. L, MOREAU, E, GUEDIN, D, BADEN, D. G, CATTERALL, W. A
• Format: Article
• Language: English
• Subjects: Allosteric Regulation; Animals; Batrachotoxins - metabolism; Biological and medical sciences; Brain - metabolism; Cell receptors; Cell structures and functions; Fundamental and applied biological sciences. Psychology; Marine Toxins - metabolism; Membrane Potentials; Miscellaneous; Molecular and cellular biology; Molecular Structure; Neurotoxins - metabolism; Oxocins; Pyrethrins - pharmacology; Rats; Receptors, Cell Surface - metabolism; Receptors, Cholinergic - metabolism; Sodium Channels - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(19)85309-7

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.