Mechanisms of Calcium-Calmodulin Mediated Ion Channel Gating

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重要作用，包括可兴奋细胞和组织中电信号的产生、处理和调节。离子通道功能的详细知识对于理解正常和病理生理学是必不可少的，这将有助于各种疾病的新治疗，如癫痫，疼痛和心血管疾病。在这个建议中，我们专注于小电导，钙激活钾（SK）通道（KCa 2，或KCNN 2）。SK通道对广泛的生理系统是重要的，并且参与小脑共济失调、癫痫以及学习和记忆。SK通道使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。钙结合到组成性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一个准确的、定量的了解。在这个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。我们将结合联合收割机的通道激活的电生理测量与一种新的光谱方法（“条件结合”）定量钙结合SK通道。我们的条件结合方法使用发光镧系元素探针离子之间的能量转移，以评估同一钙调素分子内的两个EF手钙结合位点的占用率。这种现象的广泛的理论分析表明，通过分离的SK/钙调素复合物的连接配置的一个小的子集的结合信号，有条件的结合测量是必要的和足够的估计微观，位点特异性结合亲和力和合作的相互作用。初步结合测量游离钙调素和SK/钙调素复合物证明了我们的方法的可行性SK通道功能的定量模型的发展。结合和门控测量同时进行功能SK通道在切除的膜补丁，确保导出的模型参数的通道的体内行为的相关性。我们的条件结合方法可以应用于任何含有配对EF-手结合位点的分子系统，这是生物学中最大的一类钙结合蛋白。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，如钙调蛋白依赖性激酶和钙泵。我们的研究将有助于了解一类重要的离子通道的功能，离子通道门控，钙/钙调素调节机制和蛋白质功能的变构控制的一般原理。

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.