Molecular assembly and dynamics of fluorescent protein-tagged single KCa1.1 channel in expression system and vascular smooth muscle cells.
Molecular assembly and dynamics of fluorescent protein-tagged single KCa1.1 channel in expression system and vascular smooth muscle cells.
复制标题
DOI：
10.1152/ajpcell.00191.2011
复制
发表时间：
2012-04
期刊：
影响因子：
--
通讯作者：
H. Yamamura;Chikako Ikeda;Yoshiaki Suzuki;S. Ohya;Y. Imaizumi
中科院分区：
文献类型：
--
作者：
H. Yamamura;Chikako Ikeda;Yoshiaki Suzuki;S. Ohya;Y. Imaizumi
大电导Ca（2+）激活的K（+）（K（Ca）1.1，BK）通道在血管张力调节中起着关键作用。为了阐明BK通道及其功能偶联蛋白在膜表面上的分子动力学，我们使用全内反射荧光（TIRF）显微镜检查了质膜中荧光标记的BK亚基的单分子成像。用TIRF技术检测了人胚肾293（HEK）细胞表达的黄色荧光蛋白（YFP）标记的BKα亚基（BKα-YFP）在细胞膜上单个通道及其簇水平的动态迁移率，扩散系数为6.7 × 103 nm 2/s。当BKα-YFP与青色荧光蛋白（CFP）标记的BKβ1亚基（BKβ1-CFP）在HEK细胞中共表达时，迁移率降低了约50%。荧光图像分析表明，在血管平滑肌细胞（VSMCs）中低密度表达的绿色荧光蛋白（GFP）标记的BKα亚基（BKα-GFP）优先与天然BKα亚基形成异四聚体分子组装体，而不是同四聚体BKα-GFP。BKα-YFP在VSMC中的运动速度（0.29 × 103 nm 2/s）远比BKα-YFP/BKβ1-CFP在HEK细胞中的运动速度（2.5 × 103 nm 2/s）受到更大的限制。用细胞松弛素D预处理VSMCs，破坏肌动蛋白，似乎增加BKα-YFP的移动的行为，然后加入jasplakin显著降低BKα-YFP的行为。大多数BKα-YFP与小窝蛋白1（Cav 1）-CFP在VSMC中共定位，但出乎意料地在HEK细胞中不常见。荧光共振能量转移分析显示BKα-YFP与Cav 1-CFP之间存在直接的相互作用，尤其是在VSMCs中。通过活细胞中的单分子成像获得的这些结果表明，BKα分子在膜表面上的动力学受到其辅助β亚基、细胞骨架以及与VSMC中Cav 1的直接相互作用的强烈限制或调节。
The large-conductance Ca(2+)-activated K(+) (K(Ca)1.1, BK) channel has pivotal roles in the regulation of vascular tone. To clarify the molecular dynamics of BK channels and their functionally coupled protein on the membrane surface, we examined single-molecule imaging of fluorescent-labeled BK subunits in the plasma membrane using total internal reflection fluorescence (TIRF) microscopy. The dynamic mobility of yellow fluorescent protein (YFP)-tagged BKα subunit (BKα-YFP) expressed in human embryo kidney 293 (HEK) cells was detected in TIRF regions at the level of individual channels and their clusters on the plasma membrane with a diffusion coefficient of 6.7 × 10(3) nm(2)/s. When BKα-YFP was coexpressed with cyan fluorescent protein (CFP)-tagged BKβ1 subunit (BKβ1-CFP) in HEK cells, the mobility was reduced by ∼50%. Fluorescent image analyses suggest that green fluorescent protein (GFP)-tagged BKα subunit (BKα-GFP) expressed in vascular smooth muscle cells (VSMCs), at low density, preferentially formed a heterotetrameric molecular assembly with native BKα subunits, rather than homotetrameric BKα-GFP. Movement of BKα-YFP in VSMCs (0.29 × 10(3) nm(2)/s) was far more restricted than BKα-YFP/BKβ1-CFP in HEK cells (2.5 × 10(3) nm(2)/s). Actin disruption by pretreatment with cytochalasin D in VSMCs appeared to increase the mobile behavior of BKα-YFP, which was then significantly reduced by addition of jasplakinolide. Most BKα-YFP colocalized with caveolin 1 (Cav1)-CFP in VSMCs, but unexpectedly not frequently in HEK cells. Fluorescence resonance energy transfer analyses showed the direct interaction between BKα-YFP and Cav1-CFP, particularly in VSMCs. These results, obtained by single molecule imaging in living cells, indicate that the dynamics of BKα molecules on the membrane surface are strongly restricted or regulated by its auxiliary β-subunit, cytoskeleton, and direct interaction with Cav1 in VSMCs.