| Project/Area Number |
06404017
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General physiology
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| Research Institution | Okazaki National Research Institutes National Institute for Physiological Sciences |
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Principal Investigator |
OKADA Yasunobu National Institute for Physiological Sciences, Professor, 生理学研究所, 教授 (10025661)
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| Co-Investigator(Kenkyū-buntansha) |
富永 真琴 岡崎国立共同研究機構, 生理学研究所, 助手 (90260041)
挟間 章博 岡崎国立共同研究機構, 生理学研究所, 助手 (60218394)
樫原 康博 岡崎国立共同研究機構, 生理学研究所, 助手 (00161018)
老木 成稔 岡崎国立共同研究機構, 生理学研究所, 助教授 (10185176)
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| Project Period (FY) |
1994 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥34,800,000 (Direct Cost: ¥34,800,000)
Fiscal Year 1997: ¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 1996: ¥5,400,000 (Direct Cost: ¥5,400,000)
Fiscal Year 1995: ¥6,200,000 (Direct Cost: ¥6,200,000)
Fiscal Year 1994: ¥20,400,000 (Direct Cost: ¥20,400,000)
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| Keywords | cell volume regulation / C1^- channel / P-glycoprotein / ATP / cyclic AMP / actin / Patch-clamp / Cl^-チャネル / C1^-チャネル / Cキナーゼ / Mg / 容積感受性C1^-チャネル / パッチ・クランプ / ダブル・パッチ・クランプ |
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| Research Abstract |
Most mammalian cell types can readjust their volume after transient osmotic swelling or shrinkage even under anisotonic conditions. When cells are forced to swell by increased intracellular osmolality or decreased extracellular osmolality, they can exhibit a regulatory volume decrease (RVD). In many cell types, including most epithelial cells, RVD is attained by activation of K^+ and C1^- channels. In this study, the electrophysiological properties and molecular identity of this volume-sensitive C1^- channel were investigated in human intestinal epithelial Intestine 407 cells or human epidermoid KB cells by applying both patch-clamp and molecular biological techniques.
Single-channel studies, performed in 1994, showed that this channel has an intermediate unitary conductance and exhibits inactivation kinetics at large positive potentials in Intestine 407 cells. Whole-cell patch-clamp studies, performed in 1996, demonstrated that ATP,but not cyclic AMP,acts as an open-channel blocker in the Mg-free form by binding to the pore site toward which ATP is accessible from the extracellular side. Simultaneous measurements of cell size and whole-cell C1^- currents, performed in 1997, clearly showed that this channel is activated by cell volume expansion but not by membrane stretch.
Since P-glycoprotein (MDR1) was proposed to be the molecular identity of volume-sensitive C1^- channel by Varverde et al. (1992), we have examined a possible relation between the MDR1 expression and this channel activity. The antisense study, performed in 1995, definitely showed that abolition of endogenous MDR1 expression in Intestine 407 cells cannot abolish the C1^- channel activity. The study by exogenous MDR1 overexpression in KB cells, performed in 1997, indicated that the maximum amplitude of C1^- currents is not affected by overexpression of MDR1. Thus, it is concluded that both endogenously expressing and exogenously overexpressed MDR1 cannot be themselves the channel protein.
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