Biophysical interactions of PIP2 and calmodulin with KCNQ (Kv7) K+ ion channels

 描述（由申请人提供）：PIP2和钙调蛋白与KCNQ（Kv7）K离子通道+KCNQ钾通道的生物物理相互作用控制细胞兴奋性，这些膜蛋白C-末端近端一半的遗传突变可导致心律失常、耳聋和癫痫。由KCNQ通道产生的签名"M电流"首先在交感神经元中观察到，并且可以通过刺激Gq/11毒蕈碱受体来抑制。磷脂酰肌醇4，5-二磷酸（PIP2）和钙调素（CaM）是Gq/11的第二信使分子，它们通过直接结合KCNQ通道的近端C末端来调节KCNQ通道的功能。因此，许多遗传突变可能通过破坏PIP2和CaM与通道的结合来干扰KCNQ通道功能。钙调素可以结合KCNQ通道C端的A和B螺旋，以前的工作表明钙调素可能与通道组成性结合。PIP2结合位点的精确位置不太清楚，但建议位于两个不同的富含碱性氨基酸的结构域上，这些结构域也位于KCNQ近端C末端。PIP2和CaM如何调节KCNQ通道以及它们之间的相互作用的机制还不确定。然而，这些结合位点彼此的紧密接近表明这些分子可能参与丰富的串扰动态来调节KCNQ通道功能。总体假设是PIP2和CaM之间的复杂相互作用指导KCNQ通道的功能。本研究为理解KCNQ通道的调控机制提供了一种新的方法。尖端的生物物理学方法将用于确定PIP2和CaM对KCNQ通道的生物化学结合亲和力，以及它们的精确作用位点。为了全面了解结合亲和力，本研究中的实验采用了纯化的蛋白质片段对应的近端一半的KCNQ C-末端，除了短肽对应于拟议的结合域。我们的初步数据显示惊人的差异，结合亲和力和热力学参数的钙调素KCNQ通道。同样令人信服的是，这些初步结果暗示了PIP2对每个KCNQ通道亚型上每个拟议结构域的亲和力的巨大差异。该项目的完成预计将对许多离子通道疾病产生重大影响，因为控制KCNQ通道的分子机制与许多其他离子通道相似。随着申请人在德克萨斯大学健康科学中心的支持性环境中继续进行研究培训，我们将提出更多的结果，这些结果将有助于确定PIP2和CaM对KCNQ通道作用的结构和分子机制。

 DESCRIPTION (provided by applicant): Biophysical interactions of PIP2 and calmodulin with KCNQ (Kv7) K ion channels + KCNQ potassium channels control cellular excitability, and inherited mutations in the proximal half of the C- terminus of these membrane proteins can result in cardiac arrhythmia, deafness and epilepsy. The signature "M-current" produced by KCNQ channels was first observed in sympathetic neurons, and can be inhibited by stimulation of Gq/11 muscarinic receptors. Phosphatidylinositol 4, 5-bisphosphate (PIP2) and calmodulin (CaM) are Gq/11 second messenger molecules suggested to modulate KCNQ channel function by directly binding the proximal C-terminus of KCNQ channels. As a result, many inherited mutations may interfere with KCNQ channel function by disrupting PIP2 and CaM binding to the channels. It is well established that CaM can bind both the A and B helices of the C-terminus of KCNQ channels, and previous work indicates that CaM may be constitutively bound to the channels. The precise locations of the PIP2 binding sites are less clear, but are suggested to lie on two distinct domains enriched with basic amino acids that also reside on the KCNQ proximal C-terminus. The mechanisms for how PIP2 and CaM modulate KCNQ channels, and their interactions between each other, are as yet uncertain. However, the close proximity of these binding sites to each other suggests that these molecules may engage in a rich crosstalk dynamic to modulate KCNQ channel function. The overarching hypothesis is that the complex interactions between PIP2 and CaM guide the function of KCNQ channels. This study presents a novel approach to understand the mechanisms controlling the modulation of KCNQ channels. Cutting edge biophysical methods will be used to determine the biochemical binding affinities of PIP2 and CaM for KCNQ channels, and their precise sites of action. In order to gain a comprehensive understanding of the binding affinities, the experiments in this study employ purified protein fragments corresponding to the proximal half of the KCNQ C-terminus, in addition to short peptides corresponding to the proposed binding domains. Our preliminary data show stunning differences in the binding affinities and thermodynamic parameters of calmodulin for KCNQ channels. Also compelling is that these preliminary results hint at drastic differences of PIP2 affinity for each of the proposed domains on each KCNQ channel subtype. The completion of this project is expected to provide a significant impact on many ion channel diseases, since the molecular mechanisms for controlling KCNQ channels appear common to many other ion channels. As the applicant continues to progress in her research training in the supportive environment at The University of Texas Health Science Center, we will present more results that should help define the structural and molecular mechanism of PIP2 and CaM actions on KCNQ channels.