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Solubilization of the omega-conotoxin receptor associated with voltage-sensitive calcium channels from bovine brain
T Yamaguchi1, H Saisu, H Mitsui
1Department of Neurochemistry, Niigata University, Japan.
Abstract:
The omega-conotoxin receptor in brain membranes contains components of Mr approximately equal to 310,000, approximately equal to 230,000, and 37,000 as identified by photoaffinity labeling. The toxin specifically bound to two sites with apparent dissociation constants (Kd) of approximately 3 pM and 3.5 nM under the conditions employed. There was about 8 times more of the low affinity site than the high affinity site. Binding was not affected by dihydropyridines or verapamil. However, diltiazem stereospecifically inhibited the binding to the high affinity site. Dissociation of the toxin from the membranes was very slow and only partial. Among the detergents tested, digitonin solubilized the highest toxin-binding activity. The digitonin extract contained only a single class of binding sites with an apparent Kd of about 0.46 nM. Probably only the high affinity binding site was recovered in active form in digitonin extract. The properties of the toxin binding to digitonin extract were in good agreement with those of the binding to the high affinity site in the original membranes. Photoaffinity labeling of the digitonin extract indicated that the solubilized toxin receptor contained the two large components (Mr congruent 310,000 and approximately equal to 230,000) observed in the membranes.
Insights
Omega-conotoxin receptors in brain membranes exhibit high and low affinity binding sites. Digitonin successfully solubilized the high-affinity receptor, revealing its key protein components.
Area of Science:
- Neuropharmacology
- Molecular Biology
- Biochemistry
Background:
- Omega-conotoxins are peptide toxins that target neuronal calcium channels.
- Understanding the molecular properties of omega-conotoxin receptors is crucial for neuroscience research.
Purpose of the Study:
- To characterize the binding properties and molecular components of the omega-conotoxin receptor in brain membranes.
- To investigate the effects of various agents on toxin binding.
- To identify suitable methods for receptor solubilization and purification.
Main Methods:
- Photoaffinity labeling to identify receptor components.
- Radioligand binding assays to determine dissociation constants (Kd).
- Inhibition studies using calcium channel blockers (dihydropyridines, verapamil, diltiazem).
- Detergent solubilization and characterization of the purified receptor.
Main Results:
- The omega-conotoxin receptor in brain membranes has two binding sites with apparent Kd values of approximately 3 pM (high affinity) and 3.5 nM (low affinity).
- The low affinity site was approximately 8 times more abundant than the high affinity site.
- Diltiazem stereospecifically inhibited binding to the high affinity site, while dihydropyridines and verapamil had no effect.
- Digitonin was the most effective detergent for solubilizing the toxin-binding activity.
- The digitonin extract contained a single class of binding sites with an apparent Kd of approximately 0.46 nM, likely representing the high affinity site.
- Photoaffinity labeling of the digitonin extract identified components with Mr approximately equal to 310,000 and approximately equal to 230,000.
Conclusions:
- The omega-conotoxin receptor in brain membranes exhibits distinct high and low affinity binding characteristics.
- The high affinity binding site appears to be preferentially recovered in an active form after digitonin solubilization.
- The solubilized receptor retains key molecular components, suggesting successful isolation of the functional toxin-binding entity.