Structural Basis for Calcium Selectivity and Drug Block of Cav Channels

描述（由申请人提供）：在心脏中，Ca通过Cav通道进入启动兴奋-收缩偶联，其失调在心力衰竭中很重要。选择性C进入对于Ca通道的功能至关重要，因为细胞外Na以70倍高的浓度存在，但Ca选择性的结构基础是未知的。基于我们对Nav和Cav通道的共同祖先NavAb的高分辨率结构的确定，我们现在通过在NavAb中构建Ca特异性离子选择性过滤器首次确定了Ca选择性孔的三维结构。这种实验方法将允许高分辨率分析的结构决定因素的钙结合，选择性，渗透，和块。哺乳动物Cav通道被用于治疗高血压、心绞痛和心律失常的Ca拮抗剂药物阻断。苯基烷基胺，苯并硫氮杂卓，二氢吡啶结合在三个良好的特点受体网站。值得注意的是，Ca拮抗剂药物也阻断CavAb，因此其为理解广泛用于治疗心血管疾病的药物阻断Ca通道提供了结构模板。为了确定Cav通道的孔功能和药理学的结构基础，我们将讨论三个特定目标。1.我们将确定Ca结合和选择性CavAb的结构基础。我们将测量NavAb、CavAb和中间突变体的Ca/Na渗透率比。我们将确定在不存在和存在Ca的情况下NavAb/CavAb系列突变体的结构，鉴定孔中Ca的结合位点，并估计Ca对NavAb和CavAb中位点的相对亲和力。孔中特定结合位点的亲和力将与从电生理学研究和拟合离子电导测量到Ca结合、选择性和渗透的生物物理模型估计的亲和力值相关。2.我们将确定CavAb孔的阳离子块的结构基础。大的二价和三价阳离子以高亲和力阻断哺乳动物Cav通道和CavAb。我们将测量这些阳离子对Ca渗透阻断的亲和力，确定它们在孔中的结合位点，并将结合到通过X射线晶体学鉴定的特定位点的亲和力与Ca渗透阻断进行比较。3.我们将探索钙拮抗剂药物阻断CavAb的结构基础。我们将确定结合有Ca拮抗剂药物的CavAb的结构，以了解高分辨率下通道抑制的分子基础。我们将研究药物结合在CavAb晶体中的存在和不存在的钙，以了解钙结合和药物阻断之间的复杂相互作用。我们将创建CavAb的人源化嵌合体，以确定高亲和力结合和阻断人Cav通道的结构基础。我们将在我们的晶体结构与钙电导的药物块的电生理学分析的钙和药物结合的观察相关联，我们将测试生物物理模型拟合动力学和平衡结合参数的电生理数据。我们的研究结果将首次对Cav通道的结构、功能和药理学提供高分辨率的见解。

DESCRIPTION (provided by applicant): In the heart, Ca entry through Cav channels initiates excitation-contraction coupling, and their dysregulation is important in heart failure. Selective C entry is crucial for function of Ca channels because extracellular Na is present at 70-fold higher concentration, yet the structural basis for Ca selectivity is unknown. Building on our determination of the high-resolution structure of a common ancestor of Nav and Cav channels, NavAb, we have now determined the three-dimensional structure of a Ca-selective pore for the first time by constructing a Ca-specific ion selectivity filter in NavAb. This experimental approac will allow high-resolution analysis of structural determinants of Ca binding, selectivity, permeation, and block. Mammalian Cav channels are blocked by Ca antagonist drugs used in therapy of hypertension, angina pectoris, and cardiac arrhythmia. Phenylalkylamines, benzothiazepines, and dihydropyridines bind at three well-characterized receptor sites. Remarkably, Ca antagonist drugs also block CavAb, which therefore provides a structural template for understanding block of Ca channels by drugs that are widely used in treatment of cardiovascular disease. To define the structural basis for pore function and pharmacology of Cav channels, we will address three Specific Aims. 1. We will determine the structural basis for Ca binding and selectivity in CavAb. We will measure the permeability ratio of Ca/Na for NavAb, CavAb, and intermediate mutants. We will determine the structures of the NavAb/CavAb series of mutants in the absence and presence of Ca, identify the binding sites for Ca in the pore, and estimate the relative affinity of Ca for sites in NavAb and CavAb. The affinity for specific bindin sites in the pore will be correlated with affinity values estimated from electrophysiological studies and fitting ion conductance measurements to a biophysical model of Ca binding, selectivity, and permeation. 2. We will determine the structural basis for cation block of the CavAb pore. Large divalent and trivalent cations block mammalian Cav channels and CavAb with high affinity. We will measure the affinities of these cations for block of Ca permeation, identify their binding sites in the pore, and compare affinity for binding to specific sites identiied by x-ray crystallography with block of Ca permeation. 3. We will explore the structural basis for block of CavAb by Ca antagonist drugs. We will determine the structure of CavAb with Ca antagonist drugs bound in order to understand the molecular basis for channel inhibition at high resolution. We will examine drug binding in CavAb crystals in the presence and absence of Ca in order to understand the complex interactions between Ca binding and drug block. We will create humanized chimeras of CavAb to define the structural basis for high- affinity binding and block of human Cav channels. We will correlate observations of Ca and drug binding in our crystal structures with electrophysiological analysis of drug block of Ca conductance, and we will test biophysical models by fitting kinetics and equilibrium binding parameters to electrophysiological data. Our results will give the first high-resolution insights into Cav channe structure, function, and pharmacology.