Structure and Function of Membrane Proteins

膜蛋白的结构和功能

基本信息

批准号：
10263022
负责人：
MIGUEL HOLMGREN
金额：
$ 312.88万
依托单位：
NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10263022
关键词：
2019-nCoV 4-Aminopyridine ATP1A3 gene Active Biological Transport Affect Amino Acids Apoptosis Architecture Barium Behavioral Binding Binding Sites Biochemical Biophysics Birth Brain COVID-19 Caspase Cations Cell membrane Cells Cerebellar Ataxia Chemicals Child Childhood Cysteine DNA Sequence Alteration Data Dependence Disease Dystonia Dystonia 12 Electrophysiology (science)Encephalopathies Environment Enzymes Equilibrium Event Eye Movements Family Fever Genome Goals Homeostasis Homologous Gene Human Pathology Hydrolysis Impaired cognition In Vitro Individual Inflammasome Integral Membrane Protein Ion Channel Ion Transport Ions Knock-in Mouse Link Location Membrane Membrane Potentials Membrane Proteins Membrane Structure and Function Modeling Modification Molecular Molecular Conformation Monitor Movement Mutation Na(+)-K(+)-Exchanging ATPase Names Nature Neurologic Nonstructural Protein Open Reading Frames Optic Atrophy Organelles Paralysed Pathogenesis Pathway interactions Phase Play Population Positioning Attribute Potassium Potassium Channel Preparation Property Protein Conformation Protein Dynamics Proteins Pump Recurrence Relaxation Resistance Role SARS coronavirus Scheme Sensorineural Hearing Loss Series Side Signal Transduction Site Site-Directed Mutagenesis Sodium Speed Standardization Structural Protein Structure Sulfhydryl Reagents Syndrome System Talipes cavus Techniques Thermodynamics Time Transmembrane Domain Travel Virus Virus Replication Work alternating hemiplegia biophysical properties conformational conversion experimental study extracellular in vivo inhibitor/antagonist member mutant nervous system disorder nonsynonymous mutation novel overexpression pathogenic virus response simulation squid axon voltage

2019-nCoV 4-Aminopyridine ATP1A3 gene Active Biological Transport Affect Amino Acids Apoptosis Architecture Barium Behavioral Binding Binding Sites Biochemical Biophysics Birth Brain COVID-19 Caspase Cations Cell membrane Cells Cerebellar Ataxia Chemicals Child Childhood Cysteine DNA Sequence Alteration Data Dependence Disease Dystonia Dystonia 12 Electrophysiology (science)Encephalopathies Environment Enzymes Equilibrium Event Eye Movements Family Fever Genome Goals Homeostasis Homologous Gene Human Pathology Hydrolysis Impaired cognition In Vitro Individual Inflammasome Integral Membrane Protein Ion Channel Ion Transport Ions Knock-in Mouse Link Location Membrane Membrane Potentials Membrane Proteins Membrane Structure and Function Modeling Modification Molecular Molecular Conformation Monitor Movement Mutation Na(+)-K(+)-Exchanging ATPase Names Nature Neurologic Nonstructural Protein Open Reading Frames Optic Atrophy Organelles Paralysed Pathogenesis Pathway interactions Phase Play Population Positioning Attribute Potassium Potassium Channel Preparation Property Protein Conformation Protein Dynamics Proteins Pump Recurrence Relaxation Resistance Role SARS coronavirus Scheme Sensorineural Hearing Loss Series Side Signal Transduction Site Site-Directed Mutagenesis Sodium Speed Standardization Structural Protein Structure Sulfhydryl Reagents Syndrome System Talipes cavus Techniques Thermodynamics Time Transmembrane Domain Travel Virus Virus Replication Work alternating hemiplegia biophysical properties conformational conversion experimental study extracellular in vivo inhibitor/antagonist member mutant nervous system disorder nonsynonymous mutation novel overexpression pathogenic virus response simulation squid axon voltage

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项目摘要

Voltage-activated potassium (KV) channels are potassium selective integral membrane proteins formed by the assembly of four homologous subunits. In response to a membrane depolarization, KV channels open allowing K+ to permeate. In some members of KV channels, sustain depolarization leads to inactivation caused by an N terminus gate. In shaker KV channels, the first 20 amino acids at the NH2 terminus of the protein are essential in enabling it to act as a gate. The tip of the NH2 terminus interacts with residues in the intracellular cavity of KV channels, blocking the permeation of K+. By nature of their tetrameric architecture, inactivating KV channels have four N terminus gates and a set of four sites of action in the intracellular cavity. Yet, N-type inactivation is produced by the binding of only one N terminus gate. Is the site of action in the pore specific to the subunit to which the bound N terminus belongs? To study the interactions between the N terminus gate and the site of action we are using thermodynamic cycle between positions in the N-terminus and in the T1 domain. In parallel we are performing molecular dynamic simulations to guide our experimental work. We also study the gating mechanisms of transporters, like the Na/K-ATPase. This enzyme, a member of the P-type family (named for their phosphorylated intermediates), harnesses the energy from the hydrolysis of one ATP to alternately export 3Na ions and import 2K ions against their electrochemical gradients. By performing this active transport, the Na/K pump plays an essential role in the homeostasis of intracellular Na and K that is crucial to sustaining cell excitability, volume, and Na-dependent secondary transport. On the basis of biochemical data accumulated during the decade following its discovery, the Na/K-ATPase was proposed to alternately transport Na and K ions according to a model known as the Post-Albers scheme. As ions are transported through the Na/K pump, they become temporarily occluded within the protein, inaccessible from either side, before being released. By restricting Na/K pumps to only the reversible transitions associated with deocclusion and extracellular release of Na+, it is possible to detect pre-steady state electrical signals accompanying those transitions. The signals arise because Na+ traverse a fraction of the membrane potential as they enter or leave their binding sites deep within the pump. At a fixed membrane potential and external sodium concentration, the populations of pumps with empty binding sites, and those with bound or occluded Na, reach a steady-state distribution. A sudden change of membrane voltage then shifts the Na-binding equilibrium, and initiates a redistribution of the pump populations towards a new steady state. The consequent change in Na-binding-site occupancy causes Na to travel between the extracellular environment and the pump interior. In so doing they generate a current. As the system approaches a new steady distribution, fewer Na move, and the current declines. The electrical signals therefore appear as transient currents. Using the squid giant axon preparation, which exploits axial current delivery to generate very fast membrane voltage steps, we previously identified three phases of relaxation in transient pump currents (Holmgren et al., 2000): fast (comparable to the voltage-jump time course), medium-speed (tm 0.2-0.5 ms), and slow (ts 1-10 ms). We suggested that each phase reflects a distinct Na-binding event (or release, depending on the direction of the voltage change) with its associated conformational transition (occlusion or deocclusion). In other words, the Na/K-ATPase undergoes dynamic rearrangements that open external gates to allow bound Na access to the extracellular environment immediately prior to release. We would like to understand how these gates operate, the precise dynamic relationships between the three events thatrelease individual Na+ from the Na/K-ATPase, the thermodynamic principles that govern these conformational changes, the structural movements underlying these events, as well as the type of structural dynamics associated with them. Recently, genetic mutations in the brain specific Na/K-ATPase (ATP1A3) have been linked to specific human pathologies, like Alternating Hemiplegia of Childhood (AHC), a devastating disease affecting over 120 unrelated children around the world. We have begun to study the functional consequences of some of these mutations, in particular D801N, E815K and G947R.These positions are located within the transmembrane region of the ATP1A3. We hypothesize that because of their location, they might influence ion binding transitions. In addition, we have acquired a knock-in mice for the most common ATP1A3 mutation to perform behavioral experiments. Recently, we have acquired a knock-in mice for the most common ATP1A3 mutation causing AHC (D801N). to perform behavioral experiments. The ion channel activity of the ORF3a protein from previous SARS-CoVs has been related to the activation of the inflammasome and apoptosis of infected cells, suggesting a relevance of this protein in the pathogenesis of COVID-19. Blocking the viroporin activity of ORF3a protein with potassium channel inhibitors, like 4-aminopyridine or barium ions, represses the caspase-dependent apoptosis induced by ORF3a overexpression for both in vitro and in vivo. Frequent non-synonymous mutations are detected in the ORF3a from SARS-CoV-2 with a possible gain or loss of channel function, which might have a novel influence on the virus pathogenicity. Our immediate goal is to characterize the biophysical properties of ORF3a protein from 2019-nCoV/USA-CA4/2020, a virus strain affecting the US population. We will determine its ion selectivity and its voltage dependence (if any). Once the biophysical properties of this specific ORF3a protein have been characterized, we will standardize them for comparison with functional properties from other mutants of ORF3a proteins observed in others SARS-CoV-2 strains.

电压激活的钾（KV）通道是由四个同源亚基组装形成的钾选择性整合膜蛋白。为了响应膜去极化，KV通道打开，允许K+渗透。在KV通道的某些成员中，维持去极化导致由N末端门引起的失活。在摇动KV通道中，该蛋白质NH2末端的前20个氨基酸对于使其充当栅极至关重要。 NH2末端的尖端与KV通道的细胞内腔中的残基相互作用，阻断了K+的渗透。从其四聚体结构的性质上，灭活的KV通道具有四个N末端大门，并且在细胞内腔中有四个作用部位。然而，N型失活是仅通过一个N末端门的结合而产生的。孔特有的孔中的作用部位是否特定于绑定的N末端所属的亚基？为了研究N末端门与作用部位之间的相互作用，我们使用了N末端和T1域中位置之间的热力学循环。同时，我们正在进行分子动态模拟，以指导我们的实验工作。我们还研究了转运蛋白的门控机制，例如Na/k-ATPase。这种酶是P型家族的成员（以其磷酸化的中间体命名），可利用一个ATP的水解到交替导出3NA离子并将2K离子进口到其电化学梯度上。通过执行这种主动运输，Na/k泵在细胞内Na和K的稳态中起着至关重要的作用，这对于维持细胞兴奋性，体积和NA依赖性二级转运至关重要。根据在发现后的十年中积累的生化数据，根据称为Albers后方案的模型，提出了Na/k-ATPase交替运输Na和k离子。当离子通过Na/k泵运输时，它们在蛋白质内暂时被遮住，在释放之前，它们都无法从任何一侧访问。通过将Na/K泵限制为仅与Na+的去概括和细胞外释放相关的可逆转变，可以检测伴随这些过渡的前稳态的状态电信号。之所以出现信号，是因为Na+在进入或将其结合位点留在泵内时遍历膜电位的一部分。在固定的膜电位和外部钠浓度下，具有空结合位点的泵的种群，以及具有结合或遮挡的NA的泵的种群达到稳态分布。然后，膜电压突然变化会移动NA结合平衡，并将泵种群重新分布到新的稳定状态。随之而来的NA结合位点占用率的变化导致NA在细胞外环境和泵内部之间行进。这样，它们会产生电流。随着系统接近新的稳定分布，NA移动的较少，并且当前的下降。因此，电信号作为瞬态电流。使用鱿鱼巨型轴突制备，利用轴向电流输送以产生非常快的膜电压步骤，我们先前鉴定出瞬态泵电流中放松的三个阶段（Holmgren等，2000）：快速（与电压 - 悬浮时间相当），中等速度，中等速度（TM 0.2-0.5 ms）（tm 0.2-0.5 ms）和慢速（慢速）。我们建议每个阶段都反映出一个独特的NA结合事件（或释放，取决于电压变化的方向），其相关构象转变（闭塞或去缩合）。换句话说，Na/k-ATPase经历了动态重排，该重排打开外部门，以允许在释放前立即进入细胞外环境。我们想了解这些门的运作方式，这三个事件之间的精确动态关系使单个Na+从Na/k-atpase中释放了na/k-atpase，控制这些构象变化的热力学原理，这些事件的结构运动以及与之相关的结构动力学类型。最近，大脑特异性NA/K-ATPase（ATP1A3）中的遗传突变与特定的人类病理相关，例如儿童偏瘫（AHC）（一种毁灭性的疾病），这种疾病影响了全球120多名无关的儿童。我们已经开始研究其中一些突变的功能后果，特别是D801N，E815K和G947R。这些位置位于ATP1A3的跨膜区域内。我们假设由于其位置，它们可能会影响离子结合的过渡。此外，我们为最常见的ATP1A3突变获得了一只敲门小鼠，以执行行为实验。最近，我们为最常见的ATP1A3突变（D801N）购买了一只敲门小鼠。进行行为实验。先前SARS-COV的ORF3A蛋白的离子通道活性与感染细胞的炎性体和凋亡的激活有关，这表明该蛋白在Covid-19的发病机理中的相关性。用钾通道抑制剂（如4-氨基吡啶或钡离子）阻止ORF3A蛋白的病毒蛋白活性抑制ORF3A过度表达的caspase依赖性凋亡对体外和体内的抑制。从SARS-COV-2中检测到ORF3A中频繁的非同义突变，可能会增加通道功能的增益或丧失，这可能对病毒致病性产生新的影响。我们的近期目标是表征2019-NCOV/USA-CA4/2020的ORF3A蛋白的生物物理特性，这是一种影响美国人群的病毒菌株。我们将确定其离子选择性及其电压依赖性（如果有）。一旦表征了该特定ORF3A蛋白的生物物理特性，我们将标准化它们，以与其他在其他SARS-COV-2菌株中观察到的ORF3A蛋白突变体的功能特性进行比较。