BK CHANNEL MODULATION BY BETA SUBUNITS

描述(申请人提供)：大电导、电压和钙离子激活的BK钾通道是膜兴奋性和细胞质钙离子的调节器。这些通道是由两种附加输入打开的，一种是膜电位的去极化变化，它激活电压传感器，另一种是[Ca~(2+)]IN的增加，它增加了钙结合部位的占有率。这些输入通过通道复合体结构的传播变化联系在一起，变构动力学模型很好地描述了这一机制。电压敏感结构域的激活和失活状态，钙结合结构域的占用和未占用状态，以及更广泛的开放和关闭状态的不同结构，是通过相互作用稳定的，这些相互作用必须在所涉及的氨基酸残基方面有所不同。这些在不同状态下不同的相互作用是机器的活塞和齿轮。BK a的四聚体复合体也是如此，它单独形成了一个电压和钙离子门控通道。这也适用于四个a和四个？1的复合体，其中？1亚单位调节a亚单位的功能。？1作为a的配体，其结合部位在a的不同功能状态下发生变化，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残基水平上表征a和？1之间的界面，并确定随着功能状态的变化而变化的那些相互作用。我们建议通过确定大量取代半胱氨酸(Cys)之间的两两接近度来实现这些目标，基于它们在BK通道闭合和开放状态下的二硫键形成的程度和速率，并将这些接近度作为约束条件来模拟BK通道不同功能状态下的a和？1复合体。这是不切实际的，除非BK通道a亚单位的通道形成和电压敏感部分由其跨膜(TM)螺旋S1-S6形成，与嵌合的Kv1.2/Kv2.1通道同源，对于嵌合Kv1.2/Kv2.1通道有高分辨率结构。此外，BKa含有第七个TM螺旋S0，这是没有先例的。此外，这四种亚基中的任何一种都没有三维结构。然而，S0和？1足够小，以至于它们与保守的S1-S6结构域的相互作用可以用中分辨率方法和建模来有效地表征。我们建议系统和广泛地替代半胱氨酸对，1)根据半胱氨酸的二硫键形成的程度和速率常数对半胱氨酸的接近程度进行排序，2)确定交联剂的功能结果，反之，3)确定交联剂是否依赖于官能态。我们已经成功地用这种方法在四聚体通道结构中定位了S0的胞外端，以及TM1和TM2的两个TM螺旋的胞外末端，它们相互之间以及相对于S1-S6。我们现在建议对S0和TM1和TM2的细胞内末端以及它们的细胞内N-末端和C-末端做同样的事情。 与公众健康相关：大电导、电压和钙离子激活的BK钾通道在许多不同类型的细胞中在控制兴奋性和细胞质钙浓度方面发挥关键作用。BK通道的功能增益突变与预防高血压、哮喘和癫痫的发病率增加有关，而功能丧失突变与高血压、哮喘和癫痫的发病率增加有关。因此，拟议的研究与公共卫生和美国国立卫生研究院发现人类疾病基本机制的任务有关。

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. PUBLIC HEALTH RELEVANCE: The large-conductance, voltage- and Ca2+-activated BK potassium channels play key roles in many different cell types in the control of excitability and cytoplasmic calcium concentration. Gain-in-function mutations in BK channels are associated with protection from, and loss-of-function mutations are associated with increased incidence of, hypertension, asthma, and epilepsy. Thus, the proposed research is relevant to public health and to the NIH mission to discover basic mechanisms underlying human disease.