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Molecular Mechanisms of Potassium Channel Permeation and Gating

钾通道渗透和门控的分子机制

基本信息

批准号：
10531935
负责人：
Crina M Nimigean
金额：
$ 41.53万
依托单位：
WEILL MEDICAL COLL OF CORNELL UNIV
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-10 至 2024-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10531935
关键词：
Affect Alzheimer&apos s Disease Asthma Binding Binding Proteins Binding Sites Biological Assay Calcium Calcium Binding Calcium-Activated Potassium Channel Cell membrane Cells Chemicals Circadian Rhythms Computer Analysis Confounding Factors (Epidemiology)Cryoelectron Microscopy Data Defect Dependence Disease Drug Targeting Edetic Acid Electrophysiology (science)Epilepsy Exhibits Functional disorder Goals Grant Hearing Homologous Gene Hypertension Impaired cognition Investigation Ion Channel Ions Kidney Kinetics Knowledge Length Ligand Binding Ligand Binding Domain Ligands Lipid Bilayers Lipids Liposomes Location Maintenance Mechanical Stress Membrane Membrane Lipids Membrane Potentials Membrane Proteins Modeling Molecular Molecular Conformation Molecular Sequence Alteration Movement Muscle Tonus Neurons Organism Pathway interactions Physiological Physiological Processes Physiology Play Potassium Potassium Channel Process Reporting Resolution Role Signal Transduction Sleep Disorders Stimulus Structure System Therapeutic Thick Thinness Variant Vascular Smooth Muscle Voltage-Gated Potassium Channel Water X-Ray Crystallography cell type circadian regulation design experimental study insight large-conductance calcium-activated potassium channels molecular dynamics nanodisk neurotransmitter release particle prevent reconstitution response sensor simulation voltage

项目摘要

ABSTRACT Potassium (K+) channels are major determinants of cell excitability and play crucial roles in physiological processes. Specifically, large conductance and Ca2+-activated K+ (BK) channels, have the ability to couple intracellular Ca2+ to membrane potential variations, play major physiological roles ranging from vascular smooth muscle tone maintenance and regulation of circadian rhythms, to hearing, regulating neuronal firing, and neurotransmitter release. BK channel dysfunction has been associated with many pathophysiological conditions, so understanding Ca2+-gating can have major therapeutic consequences. The overall objective of this grant is to understand molecular mechanisms of Ca2+-gating in K+ channels (opening, closing, and inactivation) by employing functional, structural, and computational analysis on a model BK channel, MthK, a close prokaryotic homolog. Unlike BK channels, where voltage-dependent gating is interfering with Ca2+-gating thus preventing structural determination of specific conformations, MthK is devoid of voltage sensors and thus a perfect system for investigating Ca2+-dependent gating alone. In addition, electrophysiology experiments suggested that MthK, just like BK, lacks inactivation at the selectivity filter. This is intriguing, because BK and MthK share high sequence and structure similarity in the filter with other “inactivating” K+ channels. However, in specific conditions of K+ and bilayer thickness, MthK does inactivate, raising the possibility that BK also inactivates under these conditions. This can have major physiological consequences, as knowing conditions that control activity will lead to new understanding of BK channels’ role in different cell types and cellular locations. Our first aim is to determine the molecular mechanism of Ca2+-activation. We propose to determine the structures of apo and Ca2+ bound MthK, from channels reconstituted in lipid nanodiscs, using single- particle cryo-EM. Lipid composition will be adjusted to yield an open state. MD simulations will be employed to refine the structures in the lipid membrane, simulate K+ flux, and uncover possible activation pathways. Our second aim is to determine the lipid bilayer-dependent inactivation mechanism. We will systematically investigate MthK activation and inactivation kinetics in liposomes of varying lipid thickness made by varying lipid lengths. Preliminary stopped-flow functional data revealed that thinner bilayers promote MthK inactivation. Using single-particle cryo-EM, we will determine structures of MthK in nanodiscs of different lipid composition and associate functional states directly from functional assays. MD simulations will refine these structures, as well as observe how changing membrane thickness affects conformation and conduction. In our third aim, to understand the more subtle changes that lead to inactivation in MthK, we propose to use X-ray crystallography of pore-only MthK together with MD simulations to reveal the sequence of molecular changes involving selectivity filter, lower gate, ions and water, by imposing conditions that promote inactivation in functional assays. The accomplishment of these aims will provide a comprehensive picture of Ca2+-gating in K+ channels.

摘要钾离子通道是细胞兴奋性的主要决定因素，在细胞的生理活动中起着至关重要的作用。流程.具体而言，大电导和Ca 2+激活的K+（BK）通道具有耦合的能力，细胞内Ca ~（2+）-膜电位变化，在血管内皮细胞和血管内皮细胞中起着重要的生理作用，平滑肌张力维持和昼夜节律调节，听觉，调节神经元放电，和神经递质的释放BK通道功能障碍与许多病理生理学因此，了解Ca 2+门控可能具有重要的治疗后果。的总体目标该基金旨在了解K+通道中Ca 2+门控的分子机制（打开，关闭，失活），通过对模型BK通道MthK，原核同源物。与BK通道不同，BK通道的电压依赖性门控干扰钙离子门控从而阻止特定构象的结构确定，MthK缺乏电压传感器，一个完美的系统，用于单独研究钙依赖性门控。此外，电生理学实验表明MthK，就像BK一样，在选择性过滤器处缺乏失活。这是耐人寻味的，因为BK和 MthK在过滤器中与其他“失活”K+通道具有高度的序列和结构相似性。但在在K+和双层厚度的特定条件下，MthK不起作用，提高了BK也在这种条件下，这可能会产生重大的生理后果，因为知道条件这种控制活性将导致对BK通道在不同细胞类型和细胞内作用的新理解，地点我们的第一个目标是确定Ca 2+激活的分子机制。我们建议确定 apo和Ca 2+结合的MthK的结构，来自脂质纳米盘中重构的通道，使用单- 粒子冷冻电镜将调整脂质组成以产生开放状态。MD模拟将用于细化脂膜结构，模拟K+通量，并揭示可能的激活途径。我们第二个目的是确定脂质双层依赖性失活机制。我们将系统地研究不同脂质厚度脂质体中MthK活化和失活动力学，脂质长度。初步停流功能数据显示，较薄的双层促进MthK失活。使用单颗粒冷冻EM，我们将确定不同脂质组成的纳米盘中MthK的结构并直接从功能测定中关联功能状态。MD模拟将完善这些结构，因为以及观察改变膜厚度如何影响构象和传导。我们的第三个目标是，为了了解导致MthK失活的更微妙的变化，我们建议使用X射线晶体学孔MthK与MD模拟一起揭示了分子变化的顺序，选择性过滤器，降低门，离子和水，通过施加条件，促进失活的功能测定。这些目标的实现将提供一个全面的图片Ca ~（2+）门控的K ~+通道。