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(申请人提供)：在心脏，钙离子通过Cav通道进入，启动兴奋-收缩偶联，它们的失调在心力衰竭中起重要作用。选择性C内流对钙通道的功能至关重要，因为细胞外钠浓度高达70倍，但钙选择性的结构基础尚不清楚。在我们确定了Nav和Cav通道的共同祖先Navab的高分辨率结构的基础上，我们现在通过在Navab中构建钙特定的离子选择性过滤器，首次确定了钙选择孔的三维结构。这种实验方法将允许对钙结合、选择性、渗透和堵塞的结构决定因素进行高分辨率分析。用于治疗高血压、心绞痛和心律失常的钙拮抗剂药物可阻断哺乳动物的Cav通道。苯基烷基胺、苯并硫氮卓和二氢吡啶与三个特征明确的受体结合。值得注意的是，钙拮抗剂也阻断了CavAb，这为广泛应用于心血管疾病治疗的药物阻断钙通道提供了一个结构模板。为了确定CAV通道的孔功能和药理学的结构基础，我们将解决三个具体目标。1.我们将确定CavAb中钙结合和选择性的结构基础。我们将测量Ca/Na对Navab、CavAb和中间突变体的渗透比。我们将在无钙和有钙的情况下确定Navab/CavAb系列突变体的结构，确定孔中钙的结合部位，并估计钙与Navab和CavAb中的位点的相对亲和力。孔中特定结合位点的亲和力将与电生理研究估计的亲和力值相关联，并将离子电导测量与钙结合、选择性和渗透的生物物理模型相适应。2.我们将确定CavAb孔阳离子封闭的结构基础。大分子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钙渗透的亲和力，确定它们在孔中的结合位置，并比较通过X射线结晶学鉴定的结合到特定位置的亲和力与阻止钙渗透的亲和力。3.探讨钙拮抗剂阻断CavAb的结构基础。我们将测定结合钙拮抗剂药物的CavAb的结构，以便在高分辨率下了解通道抑制的分子基础。我们将研究在有无钙存在的情况下CavAb晶体中的药物结合，以了解钙结合和药物阻断之间的复杂相互作用。我们将创建人源化的CavAb嵌合体，以确定高亲和力结合和阻断人CAV通道的结构基础。我们将把观察到的晶体结构中的钙和药物结合与药物阻断钙电导的电生理分析联系起来，并通过将动力学和平衡结合参数与电生理数据进行拟合来检验生物物理模型。我们的结果将使我们首次对CAV通道的结构、功能和药理学有更高的了解。