Mechanisms of Calcium-Calmodulin Mediated Ion Channel Gating

钙-钙调蛋白介导的离子通道门控机制

基本信息

批准号：
8471805
负责人：
Richard Aldrich
金额：
$ 50.39万
依托单位：
UNIVERSITY OF TEXAS AT AUSTIN
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-01 至 2016-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8471805
关键词：
Address Affinity Behavior Binding Binding Sites Biology Brain Ca(2+)-Transporting ATPase Calcium Calcium Binding Calcium-Binding Proteins Calmodulin Cardiovascular Diseases Cardiovascular system Categories Cell membrane Cells Cerebellar Ataxia Complex Coupled Coupling Data Development Disease Drug Targeting EF Hand Motifs Energy Transfer Ensure Epilepsy G-Protein-Coupled Receptors Generations Goals Heart Individual Inherited Ion Channel Ion Channel Gating Ions Kidney Knowledge Laboratory Research Lanthanoid Series Elements Learning Ligand Binding Ligands Lobe Measurement Measures Mediating Membrane Memory Methods Microscopic Modeling Molecular Molecular Conformation Movement Muscle Mutation Nuclear Receptors Optical Methods Optics Pain Phosphotransferases Physiological Physiology Play Principal Investigator Process Proteins Publishing Reaction Role SK potassium channel Signal Transduction Site Solutions Spectrum Analysis Structure System Testing Time Tissues Transducers Work base dimer experience improved in vivo instrumentation ligand gated channel luminescence mutant novel novel strategies patch clamp programs protein function research study sensor skeletal voltage gated channel

项目摘要

DESCRIPTION (provided by applicant): Ion channels play an important role in a wide range of cellular and system physiology, including generation, processing and modulation of electrical signals in excitable cells and tissues. Detailed knowledge of ion channel function is essential for an understanding of normal and pathological physiology that will facilitate new treatments for a wide variety of diseases such as epilepsy, pain, and cardiovascular disease. In this proposal we focus on the small-conductance, calcium-activated potassium (SK) channel (KCa2, or KCNN2). SK channels are important for a wide range of physiological systems, and are involved in cerebellar ataxia, epilepsy and learning and memory. SK channels use the ubiquitous calcium-binding protein calmodulin to sense changes in intracellular calcium. The molecular mechanism by which calcium binding to constitutively-associated calmodulin opens the SK channel pore is poorly understood. Our goal is to develop an accurate, quantitative understanding of this process. A critical barrier to further progress on this important problem is the lack of methods for measuring ligand binding and channel activation simultaneously. We will combine electrophysiological measurements of channel activation with a novel spectroscopic method ("conditional binding") for quantifying calcium binding to SK channels. Our conditional binding method uses energy transfer between luminescent lanthanide probe ions to assess the occupancy of two EF hand calcium binding sites within the same calmodulin molecule simultaneously. Extensive theoretical analysis of this phenomenon demonstrates that, by isolating the binding signals from a small subset of ligated configurations of the SK/CaM complex, conditional binding measurements are both necessary and sufficient for estimating microscopic, site-specific binding affinities and cooperative interactions. Preliminary binding measurements on free calmodulin and on SK/CaM complexes demonstrate the feasibility of our approach for developing quantitative models of SK channel function. The binding and gating measurements are performed simultaneously on functional SK channels in excised membrane patches, ensuring the relevance of the derived model parameters to the channels' in vivo behavior. Our conditional binding method can be applied to any molecular system containing paired EF-hand binding sites, the largest category of calcium binding proteins in biology. This enormous group includes the majority of calcium-modulated ion channels, as well as many other important effector proteins such as the calmodulin-dependent kinases and calcium pumps. Our studies will contribute to understanding the function of an important class of ion channel, and general principles of ion channel gating, calcium/calmodulin regulatory mechanisms, and allosteric control of protein function.

描述（由申请人提供）：离子通道在广泛的细胞和系统生理学中发挥着重要作用，包括可兴奋细胞和组织中电信号的生成、处理和调节。对离子通道功能的详细了解对于理解正常和病理生理学至关重要，这将有助于对癫痫、疼痛和心血管疾病等多种疾病进行新的治疗。在本提案中，我们重点关注小电导钙激活钾 (SK) 通道（KCa2 或 KCNN2）。 SK 通道对于多种生理系统都很重要，并参与小脑共济失调、癫痫以及学习和记忆。 SK 通道利用普遍存在的钙结合蛋白钙调蛋白来感知细胞内钙的变化。钙与组成型相关钙调蛋白结合打开 SK 通道孔的分子机制尚不清楚。我们的目标是对这一过程有一个准确、定量的理解。在这一重要问题上取得进一步进展的一个关键障碍是缺乏同时测量配体结合和通道激活的方法。我们将结合通道激活的电生理学测量和一种新颖的光谱方法（“条件结合”）来量化钙与 SK 通道的结合。我们的条件结合方法利用发光稀土探针离子之间的能量转移来同时评估同一钙调蛋白分子内两个 EF 手钙结合位点的占用情况。对这种现象的广泛理论分析表明，通过从 SK/CaM 复合物的一小部分连接构型中分离出结合信号，条件结合测量对于估计微观的、位点特异性的结合亲和力和协作相互作用是必要且充分的。对游离钙调蛋白和 SK/CaM 复合物的初步结合测量证明了我们开发 SK 通道功能定量模型的方法的可行性。在切除的膜片中的功能性 SK 通道上同时进行结合和门控测量，确保导出的模型参数与通道体内行为的相关性。我们的条件结合方法可应用于任何含有配对 EF-hand 结合位点的分子系统，这是生物学中钙结合蛋白的最大类别。这个庞大的群体包括大多数钙调节离子通道，以及许多其他重要的效应蛋白，例如钙调蛋白依赖性激酶和钙泵。我们的研究将有助于理解一类重要离子通道的功能，以及离子通道门控、钙/钙调蛋白调节机制和蛋白质功能的变构控制的一般原理。