2016 Fiscal Year Final Research Report
Imaging of quantitative changes in surface expression of ion channels using isolated perfused hearts
| Project/Area Number |
26670118
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| Research Category |
Grant-in-Aid for Challenging Exploratory Research
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| Allocation Type | Multi-year Fund |
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| Research Field |
General pharmacology
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| Research Institution | Yamagata University |
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Principal Investigator |
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| Project Period (FY) |
2014-04-01 – 2017-03-31
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| Keywords | Kチャネル / イメージング / 心臓 / GFP変異体 / pH |
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| Outline of Final Research Achievements |
KCNQ1 is the pore-forming subunit of the slowly activating delayed rectifier K+ channel that plays an important role in repolarization of cardiac action potential. This study was designed to develop the imaging method to visualize changes of surface expression of KCNQ1 in the isolated perfused animal hearts.
1.pHluorin (pHl), the GFP mutant with higher pH sensitivity, was fused to the extracellular region of KCNQ1 (KCNQ1-pHl). α1 adrenergic receptor (α1AR) was co-expressed with KCNQ1-pHl in HEK293 cells, and α1AR was stimulated to cause endocytosis of KCNQ1-pHl. Since inside of early endosome is acidic, pHl fluorescence was expected to be diminished. However, activation of α1AR did not lead to significant changes in pHl fluorescence.
2.To obtain a GFP mutant that has pH sensitivity higher than pHl, we started screening of cDNA clones using the E. coli protein expression system. Unfortunately, we could not obtain such a GFP mutant so far.
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| Free Research Field |
循環薬理学
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