BK CHANNEL MODULATION BY BETA SUBUNITS

描述（由申请人提供）：大电导，电压和Ca2+激活的BK钾通道是膜兴奋性和细胞质Ca2+的调节剂。这些通道是由两个附加输入打开的，一个是膜电位的去极化变化，激活电压传感器，另一个是[Ca2+] in的增加，增加Ca2+结合位点的占用。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很好地描述的机制。电压传感器结构域的激活和失活状态、Ca2+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，都是通过相互作用来稳定的，这些相互作用必须在一定程度上不同于所涉及的氨基酸残基。这些在不同状态下不同的相互作用就是机器的活塞和齿轮。bka的四聚体复合体确实如此，它单独形成电压和Ca2+门控通道。4个a和4个¿1的复合体也是如此，其中¿1亚基调节了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、不连续的。该项目的长期目标是在残留水平上表征a和¿1之间的接口，并识别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半胱氨酸（Cys）在BK通道关闭和打开状态下二硫键形成的程度和速率之间的成对接近度来实现这些目标，并将这些接近度作为BK通道a和¿1复合物在不同功能状态下建模的约束条件。这将是不切实际的，除非BK通道的通道形成和电压传感部分是由其跨膜（TM）螺旋S1-S6形成的亚基，与嵌合Kv1.2/Kv2.1通道同源，后者具有高分辨率结构。此外，BK a包含第7个TM螺旋，S0，这是没有先例的。此外，这四种亚基都没有三维结构。然而，S0和¿1足够小，它们与保守的S1-S6结构域的相互作用可以通过中分辨率方法和建模有效地表征。我们建议系统和广泛地替换半胱氨酸对，1)根据它们的范围和二硫键形成的速率常数对Cys的接近度进行排序，2)确定交联的功能后果，反过来3)确定交联是否依赖于功能状态。我们已经使用这种方法成功地定位了S0的胞外末端，以及¿1、TM1和TM2的两个TM螺旋在四聚体通道结构中的相对于彼此和相对于S1-S6的位置。我们现在建议对S0和¿1 TM1和TM2的细胞内末端及其细胞内N端和c端尾部进行同样的处理。

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.