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

PIP2 和钙调蛋白与 KCNQ (Kv7) K 离子通道的生物物理相互作用

• 批准号: 8838438
• 负责人: Crystal Rae Archer
• 金额: $ 3.07万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-30 至 2018-03-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8838438
• 关键词: Address; Affinity; Arrhythmia; Basic Amino Acids; Binding; Binding Sites; Biochemical; Biological Assay; Calcium; Calmodulin; Calmodulin 1; Calorimetry; Cell membrane; Charge; Competitive Binding; Complex; Cytoplasmic Protein; Data; Disease; Drug Targeting; Environment; Epilepsy; Goals; Health Sciences; Inherited; Ion Channel; Knowledge; Label; Lead; Location; Membrane Lipids; Membrane Potentials; Membrane Proteins; Methods; Molecular; Morbidity - disease rate; Muscarinic Acetylcholine Receptor; Mutation; Neurons; Peptides; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Physiological Processes; Potassium Channel; Protein Fragment; Publishing; Regulation; Research; Research Personnel; Research Training; Second Messenger Systems; Site; Solubility; System; Texas; Thermodynamics; Titrations; Universities; Work; analytical ultracentrifugation; base; biophysical techniques; deafness; innovation; inorganic phosphate; insight; novel strategies; novel therapeutics; prevent; public health relevance; research study; response; second messenger; sensor; skills; stoichiometry

 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.

 描述（由适用提供）：PIP2和Calmoulin与KCNQ（KV7）KION通道的生物物理相互作用 + KCNQ钾通道控制细胞令人兴奋，并且在这些膜蛋白的C-末端的遗传突变中，这些膜蛋白的近端可能会导致心脏情绪化，聋哑和聋哑人，聋哑和epilepsy。首先在交感神经元中观察到KCNQ通道产生的特征“ M-电流”，可以通过刺激GQ/11毒蕈碱受体来抑制。磷脂酰肌醇4、5-双磷酸（PIP2）和钙调蛋白（CAM）是GQ/11秒的Messenger分子，建议通过直接结合KCNQ通道的近端C末端来调节KCNQ通道功能。结果，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通道函数。可以很好地确定CAM可以同时结合KCNQ通道C端的A和B螺旋，并且先前的工作表明CAM可能始终与通道结合。 PIP2结合位点的确切位置不太清楚，但建议躺在两个不同的域中，富含碱性氨基酸，也位于KCNQ近端C末端。 PIP2和CAM如何调节KCNQ通道及其之间的相互作用的机制尚不确定。但是，这些结合位点彼此之间的紧密近端表明，这些分子可以进行丰富的串扰动态以调节KCNQ通道函数。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。这项研究提出了一种新的方法，以了解控制KCNQ通道调制的机制。最先进的生物物理方法将用于确定KCNQ通道的PIP2和CAM的生化结合亲和力及其精确的作用位点。为了全面了解结合亲和力，这项研究的实验员纯化的蛋白质片段对应于KCNQ C-terminus的一半，此外，除了与所提出的结合域相对应的短肽。我们的初步数据显示了KCNQ通道的钙调蛋白的结合亲和力和热力学参数的惊人差异。同样令人信服的是，这些初步结果暗示了每个KCNQ通道亚型上每个提出的域的PIP2亲和力的急剧差异。预计该项目的完成将对许多离子通道疾病产生重大影响，因为用于控制KCNQ通道的分子机制对于许多其他离子通道似乎很常见。随着申请人在德克萨斯大学健康科学中心的支持环境中的研究培训中继续进行，我们将提出更多的结果，以帮助定义PIP2的结构和分子机制以及在KCNQ渠道上的CAM动作。