PROTEIN-COUPLED RECEPTOR;
REGULATORY VOLUME DECREASE;
GREEN FLUORESCENT PROTEIN;
CELL-SURFACE MICRODOMAINS;
SINGLE-MOLECULE TRACKING;
PLASMA-MEMBRANE;
LIVING CELLS;
TRANSIENT CONFINEMENT;
CRYSTAL-STRUCTURE;
SUBUNIT;
D O I:
10.1074/jbc.M109.039974
中图分类号:
Q5 [生物化学];
Q7 [分子生物学];
学科分类号:
071010 ;
081704 ;
摘要:
We have directly observed the trafficking and fusion of ion channel containing vesicles and monitored the release of individual ion channels at the plasma membrane of live mammalian cells using total internal reflection fluorescence microscopy. Proteins were fused in-frame with green or red fluorescent proteins and expressed at low level in HL-1 and HEK293 cells. Dual color imaging revealed that vesicle trafficking involved motorized movement along microtubules followed by stalling, fusion, and subsequent release of individual ion channels at the plasma membrane. We found that KCNQ1-KCNE1 complexes were released in batches of about 5 molecules per vesicle. Toelucidate the properties of ion channel complexes at the cell membrane we tracked the movement of individual molecules and compared the diffusive behavior of two types of potassium channel complex (KCNQ1-KCNE1 and Kir6.2-SUR2A) to that of a G-protein coupled receptor, the A1 adenosine receptor. Plots of mean squared displacement against time intervals showed that mobility depended on channel type, cell type, and temperature. Analysis of the mobility of wild type KCNQ1-KCNE1 complexes showed the existence of a significant immobile subpopulation and also a significant number of molecules that demonstrated periodic stalling of diffusive movements. This behavior was enhanced in cells treated with jasplakinolide and was abrogated in a C-terminal truncated form (KCNQ1(R518X)-KCNE1) of the protein. This mutant has been identified in patients with the long QT syndrome. We propose that KCNQ1-KCNE1 complexes interact intermittently with the actin cytoskeleton via the C-terminal region and this interaction may have a functional role.
机构:
Fourth Mil Med Univ, Xijing Hosp, Dept Cardiol, Xian 710032, Peoples R ChinaFourth Mil Med Univ, Xijing Hosp, Dept Pathol & Pathophysiol, Xian 710032, Peoples R China
Sun, Dong-Dong
Wang, Hai-Chang
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机构:
Fourth Mil Med Univ, Xijing Hosp, Dept Cardiol, Xian 710032, Peoples R ChinaFourth Mil Med Univ, Xijing Hosp, Dept Pathol & Pathophysiol, Xian 710032, Peoples R China
Wang, Hai-Chang
Wang, Xiao-Bin
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机构:
Fourth Mil Med Univ, Xijing Hosp, Dept Pathol & Pathophysiol, Xian 710032, Peoples R ChinaFourth Mil Med Univ, Xijing Hosp, Dept Pathol & Pathophysiol, Xian 710032, Peoples R China
Wang, Xiao-Bin
Luo, Ying
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机构:
Fourth Mil Med Univ, Xijing Hosp, Dept Pathol & Pathophysiol, Xian 710032, Peoples R ChinaFourth Mil Med Univ, Xijing Hosp, Dept Pathol & Pathophysiol, Xian 710032, Peoples R China
Luo, Ying
Jin, Zhen-Xiao
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机构:
Fourth Mil Med Univ, Xijing Hosp, Dept Cardiovasc Surg, Xian 710032, Peoples R ChinaFourth Mil Med Univ, Xijing Hosp, Dept Pathol & Pathophysiol, Xian 710032, Peoples R China
Jin, Zhen-Xiao
Li, Zhi-Chao
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机构:
Fourth Mil Med Univ, Xijing Hosp, Dept Pathol & Pathophysiol, Xian 710032, Peoples R ChinaFourth Mil Med Univ, Xijing Hosp, Dept Pathol & Pathophysiol, Xian 710032, Peoples R China
Li, Zhi-Chao
Li, Gui-Rong
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机构:
Univ Hong Kong, Dept Med, Li Ka Shing Fac Med, Hong Kong, Hong Kong, Peoples R ChinaFourth Mil Med Univ, Xijing Hosp, Dept Pathol & Pathophysiol, Xian 710032, Peoples R China
Li, Gui-Rong
Dong, Ming-Qing
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机构:
Fourth Mil Med Univ, Xijing Hosp, Dept Pathol & Pathophysiol, Xian 710032, Peoples R ChinaFourth Mil Med Univ, Xijing Hosp, Dept Pathol & Pathophysiol, Xian 710032, Peoples R China