| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 1999: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1998: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1997: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
ATP receptor/channel (P2X receptor) was expressed in Xenopus oocytes using molecular biological techniques, and its structure-function relationship was investigated. In the first year, the effects of dopamine and 5-hydroxytryptamine (serotonin), which are neurotransmitters in the brain, and divalent cations (ZnィイD12+ィエD1 and CdィイD12+ィエD1) were compared among the four subclasses of ATP receptor/channel (P2X1 - P2X2). Based on the results from the comparison, P2X2 receptor was modified by site-directed mutagenesis, and the modifications by these substances were clarified. In addition, it was shown that trivalent cations including LaィイD13+ィエD1 are potent inhibitors of P2X receptors. In the second year, investigation was made on basic properties of ATP receptor/channel such as the sensitivity to ATP and the mechanisms underlying ion permeation. It was demonstrated that an aspartic acid residue at the position 315 contributes to the sensitivity to ATP, and that an asparagine residue at the
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position 333 is involved in the ion permeation mechanisms in P2X2 receptor. In the final year, examinations were made on the position presumably contributing the binding of ATP molecules and the effects of drugs on the channel pore using mutant receptor/channels. A region including highly conserved glycine residues exists in the extracellular segment of all the seven members of P2X receptors. When these glycine residues were replaced with an alanine or valine residue, the responsiveness to ATP was attenuated or abolished with the replacement of glycine residues at the positions 247 and 248. The results have raised the possibility that this region is indispensable for the binding of ATP molecules. As for the examination on the effects of 5-hydroxytryptamine and imipramine on the channel pore, the effects were different between the wild type P2X2 receptor and its pore mutants, suggesting that the channel pore is the target of these compounds. From these results, it has been demonstrated that this expression system with molecular biological techniques is highly useful for pharmacological research. Less
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