| Project/Area Number |
15390067
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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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 | Kansai Medical University |
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Principal Investigator |
MATSUDA Hiroko Kansai Medical University, Faculty of Medicine, Professor, 医学部, 教授 (10181736)
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| Co-Investigator(Kenkyū-buntansha) |
TAKETO Megumi Kansai Medical University, Faculty of Medicine, Assistant, 医学部, 助手 (50298189)
HAYASHI Mikio Kansai Medical University, Faculty of Medicine, Assistant, 医学部, 助手 (10368251)
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| Project Period (FY) |
2003 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥11,700,000 (Direct Cost: ¥11,700,000)
Fiscal Year 2005: ¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2004: ¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2003: ¥5,700,000 (Direct Cost: ¥5,700,000)
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| Keywords | Inwardly rectifying K^+ channel / K ion |
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| Research Abstract |
To know the significance of the negative charges in the extracellular loop of the inwardly rectifying K^+ (Kir2.1) channels, single point mutants (D112N, D114N, D152N and E153Q) and double points mutants (D112N/D114N and D152N/E153Q) were constructed and transfected into COS-1 and HEK-293 cells. Single-channel currents similar to those through wild-type Kir2.1 channels were recorded from cells transfected with each single point mutant and D112N/D114N. Cells transfected with D152N/E153Q did not show inwardly rectifying K^+ currents, although fluorescence images confirmed that channel proteins produced by D152N/E153Q were transported to the cell surface. Tandem tetramers with one E153Q subunit and three double mutant subunits, E153Q-(D152N/E153Q)3, expressed Kir2.1 channels. Tandem tetramers with one D152N subunit and three double mutant subunits, D152N-(D152N/E153Q)3, did not express Kir2.1 channels but those with two D152N subunits and two double mutant subunits, (D152N)2-(D152N/E153Q)2, expressed Kir2.1 channels. In addition, outward currents through wild-type and D172N/E224S channels were recorded from outside-out macropatches in the absence of external K^+. These results suggest that one negative charge of D152 or two negative charges of E153 and K^+ either in the external solution or coming from the inside of cells are required for Kir2.1 channels to function. These negative charges may be involved in stabilizing a half hydrated K^+ ion near the external entrance to the selectivity filter revealed by the structural study of the KcsA K^+ channel.
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