Structure Determination of the Closed-State Human K+ Channel, KCNQ1, Voltage Senor Domain

 描述（由申请人提供）：人钾通道KCNQ 1是一种多位β-螺旋膜蛋白。KCNQ 1在上皮组织和心脏组织中都有表达。在心脏中，KCNQ 1与KCNE 1一起介导负责心脏动作电位复极化的Iks电流。导致KCNQ 1功能丧失的突变导致称为长QT综合征（LQTS）的先天性疾病。先天性LQTS使个体易患心律失常并可导致猝死。该建议集中在一个单一的突变，S225 L，这是足以导致LQTS显着改变KCNQ 1的电压响应。S225 L的电导-电压（G-V）关系相对于野生型偏移，使得沟道电导需要高得多的电压。同源性模型表明，S225经历了一个显着的变化，从一个疏水性更极性的环境，作为KCNQ 1从封闭到开放状态的过渡。虽然目前的同源性模型足以产生一个可测试的假设，KCNQ 1和其他钾通道结构之间的低序列保守性（~20%）突出了对实验确定的KCNQ 1结构的需要。该提议的中心假设是KCNQ 1电压传感器的闭合状态通过S225 L取代的疏水特性而稳定。这一假设将以两种方式进行检验。首先，通过在位置225处引入一系列改变电荷、大小和疏水性的氨基酸取代。预期闭合状态稳定化将与氨基酸取代的疏水性增加相关。这些突变的影响将在完整的KCNQ 1通道和孤立的电压传感器结构域（VSD）中进行评估。使用核磁共振（NMR）光谱学的VSD在没有电场的情况下的开-闭平衡可以被测量。已发表的状态锁定突变将用于为每个状态提供均匀的NMR参考光谱，从而允许直接测量给定VSD突变的开闭平衡。用平面贴片法测量了KCNQ 1通道的G-V曲线。这种方法将允许在完整通道的背景下评估突变系列的影响。它 预期孤立VSD观察到的变化将与全通道的变化相关。第二，VSD的闭合状态结构，使用状态锁定突变，将由NMR确定。电生理学、生物物理学表征和闭合状态结构的组合将建立LQTS诱导的S225 L突变的生化基础。VSD的闭合状态结构将代表对该领域的重大贡献，其提供了对开放-闭合转变机制的深入了解。

 DESCRIPTION (provided by applicant): The human potassium channel KCNQ1 is a polytopic -helical membrane protein. KCNQ1 is expressed in both epithelial and heart tissue. In the heart, KCNQ1, in association with KCNE1, mediates the Iks current responsible for the repolarization of the cardiac action potential. Mutations that cause a loss-of-function of KCNQ1 result in a congenital condition known as long-QT syndrome (LQTS). Congenital LQTS predisposes an individual to cardiac arrhythmia and can result in sudden death. This proposal focuses on a single mutation, S225L, which is sufficient to cause LQTS by significantly altering the voltage-response of KCNQ1. The conductance-voltage (G-V) relationship of S225L is shifted relative to wild type such that much higher voltages are required for channel conductance. Homology models suggest that S225 undergoes a significant change in environment, moving from a hydrophobic to a more polar environment, as KCNQ1 transitions from the closed- to open-state. While current homology models are sufficient to generate a testable hypothesis, low sequence conservation between KCNQ1 and other potassium channel structures (~20%) highlight the need for an experimentally determined KCNQ1 structure. The central hypothesis of this proposal is that the closed- state of the KCNQ1 voltage-senor is stabilized by the hydrophobic character of the S225L substitution. This hypothesis will be tested in two ways. First, by introducing a series of amino acid substitutions at position 225 that vary i charge, size, and hydrophobicity. It is expected that closed-state stabilization will correlate wit increasing hydrophobic character of the amino acid substitution. The effects of these mutations will be evaluated in both the full KCNQ1 channel, and in the isolated voltage-senor domain (VSD). Using nuclear magnetic resonance (NMR) spectroscopy the open-closed equilibrium of VSD in the absence of an electric field can be measured. Published state-locking mutations will be used to provide homogeneous NMR reference spectra for each state, thus allowing the open-closed equilibrium of a given VSD mutation to be directly measured. Using planar-patch-camp methods the G-V curve of the KCNQ1 channel can be measured. This approach will allow for the effects of the mutational series to be evaluated in the context of the complete channel. It is expected that changes observed for the isolated VSD will be correlated with that of the full channel. Second, the closed-state structure of the VSD, using state-locking mutations, will be determined by NMR. The combination of electrophysiology, biophysical characterization, and closed-state structure will establish the biochemical underpinnings of the LQTS-inducing S225L mutation. The closed-state structure of the VSD would represent a significant contribution to the field providing insight into the mechanism of the open- closed transition.