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Evidence for distinct sites coupled to high affinity omega-conotoxin receptors in rat brain synaptic plasma membrane
P Feigenbaum1, M L Garcia, G J Kaczorowski
1Department of Membrane Biochemistry and Biophysics. Merck Sharp and Dohme Research Laboratories, Rahway, New Jersey 07065.
Abstract:
The neuronal Ca2+ channel blocker omega-conotoxin (GVIA) binds with very high affinity (Kd of 0.8 pM) to a single class of receptors in purified rat brain synaptic plasma membrane vesicles. Three types of agents have been found to modulate toxin binding. The affinity of omega-conotoxin is decreased by metal ions or organic cations which interact at the pore of voltage-dependent Ca2+ channels. Dynorphin A [1-13] and related peptides stimulate omega-conotoxin binding by increasing toxin affinity through a nonopiate allosteric mechanism. Venom of the spider Plectreurys tristes inhibits omega-conotoxin binding (IC50 of 30 ng protein/ml) by a noncompetitive allosteric mechanism. These results suggest that omega-conotoxin binding sites exist in a complex with distinct receptors for other agents, all of which may be functionally associated with neuronal Ca2+ channels.
Insights
Omega-conotoxin (GVIA), a neuronal calcium channel blocker, binds strongly to rat brain receptors. Its binding is modulated by metal ions, organic cations, dynorphin peptides, and spider venom, suggesting a complex association with neuronal calcium channels.
Area of Science:
- Neuroscience
- Pharmacology
- Molecular Biology
Background:
- Neuronal calcium channels are critical for neurotransmitter release.
- Omega-conotoxin (GVIA) is a potent blocker of neuronal voltage-dependent calcium channels.
- Understanding the regulation of omega-conotoxin binding provides insights into channel function.
Purpose of the Study:
- To characterize the binding of omega-conotoxin (GVIA) to neuronal receptors.
- To identify agents that modulate omega-conotoxin binding.
- To elucidate the functional association of omega-conotoxin binding sites with other neuronal receptors.
Main Methods:
- Binding assays using purified rat brain synaptic plasma membrane vesicles.
- Characterization of the affinity and mechanism of modulation by various agents.
- Analysis of allosteric interactions.
Main Results:
- Omega-conotoxin (GVIA) exhibits very high affinity binding (Kd = 0.8 pM) to a single class of receptors.
- Metal ions and organic cations decrease affinity by interacting with the channel pore.
- Dynorphin A [1-13] and related peptides increase affinity via a nonopiate allosteric mechanism.
- Spider venom (Plectreurys tristes) inhibits binding noncompetitively via allosteric mechanisms.
Conclusions:
- Omega-conotoxin binding sites are part of a complex with distinct receptors.
- These complexes are functionally associated with neuronal calcium channels.
- Modulation of omega-conotoxin binding reveals intricate regulatory mechanisms of calcium channel function.