BK CHANNEL MODULATION BY BETA SUBUNITS

描述（由申请人提供）：大电导、电压和Ca 2+激活的BK钾通道是膜兴奋性和细胞质Ca 2+的调节剂。这些通道通过两个附加输入打开，一个是激活电压传感器的膜电位的去极化变化，另一个是增加[Ca 2 +]IN，这增加了Ca 2+结合位点的占据。这些输入通过通道复合物结构的传播变化联系在一起，这是一种由变构动力学模型充分描述的机制。不同的结构在激活和失活状态的电压传感器域，在被占领和未被占领的状态的Ca 2 +-结合域，更广泛地在开放和封闭的状态是稳定的相互作用，必须在一定程度上不同的氨基酸残基参与。这些在不同状态下不同的相互作用是机器的活塞和齿轮。这是真实的四聚体复合物的BK a单独形成电压和Ca 2+门控通道。这也适用于四个a和四个<$1的复合物，其中<$1亚基调节a亚基的功能。1作为a的配体，其结合位点在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留物水平上表征a和1之间的界面，并确定那些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取代的半胱氨酸（Cys）之间的成对接近度来实现这些目标，基于它们在BK通道的闭合和开放状态中的二硫键形成的程度和速率，并通过使用这些接近度作为BK通道α和β 1复合物在其不同功能状态中建模的约束。这将是不切实际的，除非BK通道a亚基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Kv1.2/Kv2.1通道同源，对于嵌合Kv1.2/Kv2.1通道，存在高分辨率结构。此外，BK a含有第七个TM螺旋S 0，这是没有先例的。此外，四种亚基中没有任何一种亚基的3D结构。0和1，然而，是足够小，它们与保守的S1-S6域的相互作用，可以有效地其特征在于通过中等分辨率的方法和建模。我们建议，系统地和广泛地取代，对半胱氨酸和1）排名的Cys的接近度的基础上，其程度和二硫键形成的速率常数，2）以确定交联的功能后果，并反过来3）以确定交联是否依赖于功能状态。我们已经成功地使用这种方法来定位S 0的细胞外末端，以及在四聚体通道结构中相对于彼此和相对于S1-S6的<$1、TM 1和TM 2的两个TM螺旋的细胞外末端。我们现在建议对一个S 0和一个TM 1和TM 2的胞内末端及其胞内N-和C-末端尾部做同样的事情。

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.