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：
8849511
负责人：
Richard Aldrich
金额：
$ 54.03万
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 2017-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8849511
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起着重要作用，包括对可激发细胞和组织中电信号的生成，加工和调节。对离子通道功能的详细知识对于理解正常和病理生理学至关重要，这将促进各种疾病（例如癫痫，疼痛和心血管疾病）的新疗法。在此提案中，我们着重于钙激活的钾（SK）通道（KCA2或KCNN2）的小导体。 SK通道对于广泛的生理系统很重要，并且参与了小脑共济失调，癫痫以及学习和记忆。 SK通道使用无处不在的钙结合蛋白钙调蛋白来感知细胞内钙的变化。钙结合与组成型相关的钙调蛋白打开SK通道孔的分子机制知之甚少。我们的目标是对这一过程有准确的定量理解。在这个重要问题上进一步进展的关键障碍是缺乏同时测量配体结合和通道激活的方法。我们将将通道激活的电生理测量与一种新型光谱法（“条件结合”）相结合，以量化与SK通道的钙结合。我们的条件结合方法使用发光灯笼探针离子之间的能量转移来评估同一钙调蛋白分子内两个EF手动钙结合位点的占用。对这一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隔离结合信号，条件结合测量是必需的，并且足以估算显微镜，位点特异性结合亲密关系和合作相互作用。自由钙调蛋白和SK/CAM复合物上的初步结合测量结果证明了我们开发SK通道功能定量模型的方法的可行性。结合和门控测量是在切除的膜斑块中的功能SK通道上同时执行的，从而确保了派生的模型参数与通道与体内行为的相关性。我们的条件结合方法可以应用于任何分子系统，其中包含配对的EF手的结合位点，这是生物学中最大的钙结合蛋白类别。这一巨大组包括大多数钙调节的离子通道，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了解重要的离子通道的功能，以及离子通道门控，钙/钙调节蛋白调节机制的一般原理以及蛋白质功能的变构控制。