1993 Fiscal Year Final Research Report Summary
CLONING OF T-TYPE CALCIUM CHANNEL GENE
| Project/Area Number |
04670108
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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 |
General pharmacology
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| Research Institution | CHIBA UNIVERSITY |
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Principal Investigator |
GONOI Tohru CHIBA UNIVERSITY, PATHOGENIC FUNGI AND MICROBIAL TOXICOSES RESEARCH ASSOCIATE, 真核微生物研究センター, 助手 (30134365)
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| Co-Investigator(Kenkyū-buntansha) |
AKAO Mitutaro CHIBA UNIVERSITY, PATHOGENIC FUNGI AND MICROBIAL TOXICOSES PROFESSOR, 真核微生物研究センター, 教授 (30101356)
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| Project Period (FY) |
1992 – 1993
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| Keywords | T-type Ca channel / L-type Ca channel / Xenopus Oocyte / CHO cell / Somatostatin Receptor / PACAP receptor / Patch-clamp method |
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| Research Abstract |
A mouse myeloma cell line, PAI, which functionally expresses T-type Ca channel, but no L-type Ca channels, was used as a source material for cloning a T-type Ca channel gene. cDNA was made from the cells using several pairs of degenerate oligo DNA primers, which were designed based on L-type Ca channel gene sequences, were used for the polymerase chain reactions to amplify a possibly T-type Ca channel gene. We obtained a unique sequence which has homologous parts to already known L-type Ca channel genes but is different from any DNA sequences reported to date. Work of sequensing full length of the gene is now undertaken.
We have also made a Xenopus o oocyte-expression system to identify functional properties of the genes we cloned. We have isolated genes of 2 alpha-subunits (4A and 4B) and 3 beta-subunits of L-type Ca channel from rat brain and pancreas. The subunit genes were expressed in different combinations in frog g oocytes and Chinese hamster ovary cells, and properties of the channels were investigated electrophysiologically. Ca channels currents induced by 4A and beta2 genes showed extremely longer inactivation time than those of the other combinations of the genes. The injection of an alpha-subunit gene alone did not induce any detectable Ca channel current.
A gene of GTP-binding protein coupling membrane receptor protein has been closed by low-stringency screening of cDNA library of a mouse clonal cell line of a pancreatic beta-cell MIN6. The frog oocyte expression system was used to identify that the gene encodes the 3rd type of PACAP (pituitary adenylate cyclase-activating polypeptide) receptor.
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