Structural Basis for Calcium Selectivity and Drug Block of Cav Channels

Cav 通道钙选择性和药物阻断的结构基础

• 批准号: 9195112
• 负责人: WILLIAM A CATTERALL
• 金额: $ 38.63万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9195112
• 关键词: Action Potentials; Address; Affinity; Amino Acids; Angina Pectoris; Anti-Arrhythmia Agents; Antihypertensive Agents; Arrhythmia; Binding; Binding Sites; Calcium; Calcium Channel; Calcium Channel Blockers; Calorimetry; Cardiovascular Diseases; Cations; Cell physiology; Cells; Charge; Chimera organism; Complex; Coupling; Crystallization; Cytosol; Data; Dihydropyridines; Divalent Cations; Drug usage; Electrophysiology (science); Electrostatics; Equilibrium; Extracellular Fluid; Family; Functional disorder; Generations; Heart; Heart failure; Human; Hypertension; Ions; Kinetics; Measurement; Measures; Mediating; Modeling; Molecular; Molecular Target; Monovalent Cations; Myocardial Contraction; Permeability; Pharmaceutical Preparations; Pharmacology; Physiological; Positioning Attribute; Process; Regulation; Research; Resolution; Safety; Series; Signal Transduction; Site; Structure; Testing; Therapeutic; Time; X-Ray Crystallography; base; bindin; biophysical analysis; biophysical model; design; extracellular; fighting; heart function; improved; insight; mutant; phenylalkylamine; public health relevance; radioligand; receptor; response; stoichiometry; three dimensional structure; voltage

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.

描述（由申请人提供）：在心脏中，Ca 通过 Cav 通道进入会引发兴奋-收缩耦合，并且它们的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 的浓度高出 70 倍，但 Ca 选择性的结构基础尚不清楚。基于我们对 Nav 和 Cav 通道共同祖先 NavAb 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。该实验方法将允许对 Ca 结合、选择性、渗透和阻断的结构决定因素进行高分辨率分析。哺乳动物的 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。苯烷基胺、苯并硫氮杂卓类药物和二氢吡啶类药物结合在三个已明确表征的受体位点。值得注意的是，Ca 拮抗剂药物也阻断 CavAb，因此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。为了定义 Cav 通道的孔功能和药理学的结构基础，我们将解决三个具体目标。 1. 我们将确定 CavAb 中 Ca 结合和选择性的结构基础。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 NavAb/CavAb 系列突变体在 Ca 不存在和存在下的结构，识别孔中 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通过电生理学研究和将离子电导测量拟合到 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阻止 Ca 渗透的亲和力，识别它们在孔中的结合位点，并将通过 X 射线晶体学鉴定的特定位点的结合亲和力与阻止 Ca 渗透进行比较。 3.我们将探讨Ca拮抗剂药物阻断CavAb的结构基础。我们将确定与 Ca 拮抗剂药物结合的 CavAb 的结构，以便以高分辨率了解通道抑制的分子基础。我们将在存在和不存在 Ca 的情况下检查 CavAb 晶体中的药物结合，以了解 Ca 结合和药物阻断之间的复杂相互作用。我们将创建 CavAb 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。我们将把晶体结构中 Ca 和药物结合的观察结果与 Ca 电导药物阻断的电生理分析联系起来，并且我们将通过将动力学和平衡结合参数拟合到电生理数据来测试生物物理模型。我们的研究结果将首次对 Cav 通道结构、功能和药理学提供高分辨率的见解。