BK CHANNEL MODULATION BY BETA SUBUNITS

通过 Beta 子单元进行 BK 通道调制

基本信息

批准号：
8601759
负责人：
Arthur Karlin
金额：
$ 34.65万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-03-05 至 2016-01-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Large-conductance, voltage- and Ca2+-activated BK potassium channels are regulators of membrane excitability and of cytoplasmic Ca2+. These channels are opened by two additive inputs, a depolarizing change in membrane potential, which activates the voltage-sensors, and an increase in [Ca2+]IN, which increases occupation of the Ca2+ binding sites. These inputs are linked through propagated changes in the structure of the channel complex, a mechanism well-described by an allosteric kinetic model. The different structures in the activated and deactivated states of the voltage-sensor domains, in the occupied and unoccupied states of the Ca2+-binding domains, and more broadly in the open and closed states are stabilized by interactions that must differ to some extent in the amino acid residues involved. These interactions that differ in the different states are the pistons and gears of the machine. This is true of the tetrameric complex of BK a which alone forms a voltage- and Ca2+-gated channel. This is also true of the complex of four a and four ¿1, in which the ¿1 subunits modulate the function of the a subunits. ¿1 acts as a ligand of a, and its binding site changes in the different functional states of a. The binding interface is likely to be extensive and discontinuous. The long-term goals of this project are to characterize at the residue level the interface between a and ¿1 and to identify those interactions that change with the change in functional state. We propose to accomplish these goals by determining the pairwise proximities between a large number of substituted cysteines (Cys), based on their extents and rates of disulfide bond formation in the closed and open states of the BK channel, and by using these proximities as constraints in modeling the BK channel a and ¿1 complex in its different functional states. This would be impractical except that the channel-forming and voltage-sensing part of the BK channel a subunit, formed by its transmembrane (TM) helices S1-S6, is homologous to the chimeric Kv1.2/Kv2.1 channel, for which there is a high-resolution structure. In addition, BK a contains a seventh TM helix, S0, for which there is no precedent. Moreover, there are no 3D structures of any of the four types of ¿ subunits. S0 and ¿1, however, are small enough that their interactions with the conserved S1-S6 domain can be usefully characterized by medium-resolution methods and by modeling. We propose to substitute, systematically and extensively, pairs of cysteines and 1) to rank the proximities of the Cys based on their extents and rate constants of disulfide bond formation, 2) to determine the functional consequences of crosslinking, and conversely 3) to determine whether crosslinking is dependent on functional state. We have used this approach successfully to locate the extracellular ends of S0, and of the two TM helices of ¿1, TM1 and TM2, relative to each other and to S1-S6, in the tetrameric channel structure. We now propose to do the same with the intracellular ends of a S0 and of ¿1 TM1 and TM2 and their intracellular N- and C-terminal tails.

描述（由适用提供）：大传导，电压和Ca2+活化的BK钾通道是膜激发膜和细胞质CA2+的调节剂。这些通道由两个其他输入（膜电位的分裂变化）打开，它激活了电压传感器，并增加了[Ca2+] IN的增加，从而增加了Ca2+结合位点的发生。这些输入通过传播通道复合物的结构的传播变化链接，该机制由变构动力学模型很好地描述。电压传感器结构域的激活和停用状态的不同结构，在Ca2+结合域的被占用和未占用状态下，在开放和封闭状态下更广泛地稳定在氨基酸保留涉及的氨基酸中必须有所不同的相互作用。在不同状态下不同的相互作用是机器的活塞和齿轮。 BK a的四聚体配合物是正确的，该复合物单独形成电压和Ca2+定量的通道。四个a和四个€1的复合物也是如此，其中„ 1个亚基调节A亚基的功能。 „ 1充当A的配体，其结合位点在A的不同功能状态下变化。结合界面可能是广泛的和不连续的。该项目的长期目标是在居住水平上表征A和1之间的接口，并确定随着功能状态变化而变化的相互作用。我们建议通过确定大量替代的半胱氨酸（CY）之间的成对接近，根据其在BK通道的封闭状态和开放状态中的二硫键形成率和二硫键形成速率，以及将这些接近作为在其不同功能状态中建模的1个复合物来建模的约束。这将是不切实际的，除了由BK通道的通道形成和电压传感器部分A亚基由跨膜（TM）螺旋S1-S6形成，与嵌合KV1.2/KV2.1通道同源，为此有高分辨率结构。另外，BK A包含第七型TM螺旋，S0没有先例。此外，没有四种类型的亚基中的任何一个3D结构。但是，S0和»1足够小，以至于它们与配置的S1-S6域的相互作用可以通过中分辨率方法和建模来有效地表征。我们建议基于二硫键键形成的范围和速率常数，2）确定交联是否取决于功能状态，以系统和广泛的半胱氨酸对替代二对半胱氨酸，以及1）对CYS的近端进行排名。我们已经成功地使用了这种方法来定位S0的细胞外末端，以及相对于彼此的两个TM螺旋，相对于彼此的TM，TM1和TM2的两个TM螺旋，以及在四聚体通道结构中。现在，我们建议使用S0的细胞内末端以及1 TM1和TM2及其细胞内N-和C末端尾巴进行相同的操作。