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，处理和调制。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，这将促进对各种疾病（如癫痫、疼痛和心血管疾病）的新治疗。在本研究中，我们重点研究了小电导、钙活化钾（SK）通道（KCa2，或KCNN2）。SK通道在许多生理系统中都很重要，涉及小脑共济失调、癫痫和学习记忆。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。钙与组成相关的钙调蛋白结合打开SK通道孔的分子机制尚不清楚。我们的目标是对这一过程进行准确、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测量和一种新的光谱方法（“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镧系探针离子之间的能量转移来评估两个EF手钙结合位点同时在同一个钙调素分子内的占用情况。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合作相互作用是必要和充分的。对游离钙调素和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。结合和门控测量同时在切除的膜斑块中的功能性SK通道上进行，确保了导出的模型参数与通道在体内行为的相关性。我们的条件结合方法可以应用于任何含有配对EF-hand结合位点的分子系统，这是生物学中最大的钙结合蛋白类别。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，如钙调素依赖性激酶和钙泵。我们的研究将有助于理解一类重要的离子通道的功能，离子通道门控的一般原理，钙/钙调素调节机制，以及蛋白质功能的变构控制。

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.