Enhancement of Current through Trek1 Two Pore Domain Channels by Flufenamic Acid
Enhancement of Current through Trek1 Two Pore Domain Channels by Flufenamic Acid
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氟芬那酸增强通过 Trek1 两个孔域通道的电流
DOI：
10.1016/j.bpj.2013.11.4121
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发表时间：
2014
影响因子：
3.4
通讯作者：
Al-Moubarak E
中科院分区：
文献类型：
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作者：
Al-Moubarak E
1华盛顿大学医学院，圣路易斯，密苏里州，美国；2伊利诺伊大学厄巴纳-香槟分校物理系和活细胞物理中心，厄巴纳，伊利诺州，美国；3霍华德休斯医学研究所，厄巴纳，伊利诺州，美国。离子通道受到多种电、化学和机械刺激的门控，因此在许多生理过程中起着关键作用。静态晶体结构和动态电生理研究已经为通道门控提供了宝贵的分子见解，但构象变化的时间轨迹一直无法获得。KirBac1。1是三个主要K频道族之一的模型。在本研究中，我们成功地(1)在特定位置用单个FRET供体和受体荧光团对标记纯化的四聚体蛋白分子；(2)将蛋白功能重构为脂质体；(3)利用单分子FRET技术研究了脂质体通道结构的动力学。我们的研究结果表明，KirBac1具有两亲性的“滑动螺旋”。在PIP2存在的情况下，1个围绕在膜下面的通道门周围的分子相互移动以缩小孔，从而关闭通道；细胞质结构域驻留在两个主要的结构状态，但在PIP2抑制下显示出从孔轴的整体转移；KirBac1的细胞外环。1，与选择性滤波器相邻，结构刚性，在PIP2选通期间不移动。当通道被PIP2抑制时，滑动螺旋和细胞质结构域结构都变得不那么动态，这意味着PIP2作为一个“锁”来限制通道蛋白的结构波动。我们进一步探讨了KirBac1的相对运动。1跨膜结构域和细胞质结构域。结果表明，在PIP2抑制期间，整个细胞质结构域从跨膜结构域移开，这可能是由于刚体运动耦合TM2弯曲造成的。总之，我们提供了脂质膜环境中离子通道动态结构的直接观察，揭示了动态结构重排KirBac1。1在PIP2抑制作用上。
1Washington University School of Medicine, St. Louis, MO, USA, 2Department of Physics and the Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, IL, USA, 3Howard Hughes Medical Institute, Urbana, IL, USA. Ion channels are gated by a multitude of electrical, chemical and mechanical stimuli, and thereby play key roles in many physiological processes. Static crystal structures and dynamic electrophysiological studies have generated invaluable molecular insights into channel gating, but the time trajectory of conformational changes has been unattainable. KirBac1. 1 is a model for one of the three main K channel families. In the present study, we have successfully (1) labeled purified tetrameric protein molecules with single FRET donor and acceptor fluorophore pairs at specific locations;(2) functionally reconstituted the proteins into liposomes;(3) examined the dynamics of channel structure in liposomes with single molecule FRET techniques. Our results indicate that the amphipathic’slide helices’ of KirBac1. 1 that surround the channel gate immediately below the membrane, move toward each other to narrow the pore in the presence of PIP2, which closes the channel; the cytoplasmic domain resides at two major structural states, but demonstrates an overall shift away from the pore axis upon PIP2 inhibition; the extracellular loop of KirBac1. 1, adjacent to the selectivity filter, is structurally rigid and does not move during PIP2 gating. Both slide helix and cytoplasmic domain structures become less dynamic when the channel is inhibited by PIP2, which implies that PIP2 acts as a’lock’to confine the structural fluctuations of the channel protein. We further probed the relative motions of the KirBac1. 1 transmembrane domain versus the cytoplasmic domain. The results indicate that the entire cytoplasmic domain moves away from the transmembrane domain during PIP2 inhibition, which may result from a rigid body motion coupled to TM2 bending. In summary, we provide direct observations of dynamic structures of an ion channel within a lipid membrane environment, revealing dynamic structural rearrangements KirBac1. 1 upon PIP2 inhibition.