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

 描述(申请人提供)：人钾通道KCNQ1是一种多位-螺旋膜蛋白。KCNQ1在上皮组织和心脏组织中均有表达。在心脏，KCNQ1与KCNE1共同介导了导致心脏动作电位复极化的Iks电流。导致KCNQ1功能丧失的突变会导致一种称为长QT综合征(LQTS)的先天性疾病。先天性LQTS易导致心律失常，并可导致猝死。这一建议关注的是单个突变S225L，该突变足以通过显著改变KCNQ1的电压响应而导致LQTS。S225L的电导-电压(G-V)关系相对于野生型发生了漂移，因此需要更高的电压来实现沟道电导。同源模型表明，随着KCNQ1从封闭状态转变为开放状态，S225经历了显著的环境变化，从疏水环境转移到更极性的环境。虽然目前的同源模型足以产生一个可检验的假说，但KCNQ1与其他钾通道结构之间的低序列保守性(~20%)突显了实验确定的KCNQ1结构的必要性。这一建议的中心假设是，KCNQ1电压传感器的闭合状态是由S225L取代的疏水特性稳定的。这一假设将通过两种方式进行检验。首先，通过在225位引入一系列不同电荷、大小和疏水性的氨基酸取代。预计封闭态的稳定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KCNQ1通道和隔离的电压敏感器域(VSD)中进行评估。利用核磁共振波谱可以测量VSD在无电场情况下的开闭平衡。已发表的状态锁定突变将用于为每个状态提供均匀的核磁共振参考谱，从而允许直接测量给定VSD突变的开闭平衡。使用平面贴片cAMP方法可以测量KCNQ1通道的G-V曲线。这种方法将允许在完整渠道的背景下评估突变系列的影响。它 预计观察到的隔离VSD的变化将与全通道的变化相关。其次，使用状态锁定突变的VSD的闭合状态结构将通过核磁共振来确定。电生理学、生物物理特性和闭态结构的结合将建立LQTS诱导的S225L突变的生化基础。VSD的闭合状态结构将是对该领域的重大贡献，有助于深入了解开放-关闭过渡的机制。

 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.