1989 Fiscal Year Final Research Report Summary
Study on brain calcium channel molecules with omega-conotoxin
| Project/Area Number |
63570052
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Neurophysiology and muscle physiology
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| Research Institution | Niigata University |
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Principal Investigator |
ABE Teruo Niigata University Brain Research Institute, Associate Professor, 脳研究所, 助教授 (50010103)
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| Project Period (FY) |
1988 – 1989
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| Keywords | omega-Conotoxin / Calcium channels |
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| Research Abstract |
[^3H] Propionyl derivative of omega-conotoxin GVIA was synthesized to examine the binding of the toxin to brain membrane fractions. The toxin specifically bound to two sites with dissociation constants (K_d) of 3 pM and 3.5 nM. Binding was not affected by dihydropyridines or verapamil. Diltiazem specifically blocked the binding to only the high-affinity sites. The omega-conotoxin GVIA (GVIA) receptor could be solubilized with various detergents. Digitonin gave best yield. Probably only the high-affinity site was recovered, as judged from the inhibition of toxin binding to the solubilized receptor by diltiazem.
To investigate the receptor molecule in the brain, monoclonal antibodies were used. A monoclonal antibody raised against the L-type calcium channel molecule (dihydropyridine receptor) purified,from rabbit skeletal muscle precipitated the DHP-binding activity solubilized from bovine and rabbit brains and bovine cardiac muscle by more than 80%. However, the antibody did not significantly precipitate the GVIA-binding activity. These results strongly suggest that the GVIA receptor molecules are mostly different from DHP receptor molecules in mammalian brains. It has been claimed that GVIA inhibits both L and N types of calcium channels in the nervous system. Our results indicate that GVIA does not inhibit L channels in the mammalian brains. To obtain monoclonal antibodies that precipitate the solubilized GVIA receptor, we immunized mice with synaptic membrane fractions from bovine and rat forebrains. Among the antibodies obtained two could precipitate some 1/3 of total solubilized GVIA receptor. However, simultaneous addition of these two antibodies increased the precipitation only a little, indicating that they recognize mostly the identical population of the GVIA receptor. These antibodies reacted with proteins of M_r, 36000 or 28000 on immunoblots. These proteins were highly concentrated in brains of various animals from chick to human.
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