Atomic basis for chloride channel and transporter gating and selectivity

氯离子通道和转运蛋白门控和选择性的原子基础

基本信息

批准号：
10319992
负责人：
Alessio Accardi
金额：
$ 32.35万
依托单位：
WEILL MEDICAL COLL OF CORNELL UNIV
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-01-10 至 2023-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10319992
关键词：
Address Affect Albers-Schonberg disease Amides Amino Acid Motifs Amino Acid Substitution Amino Acids Anion Transport Proteins Anions Architecture Bacteria Bartter Disease Binding Binding Sites Biochemical Bioinformatics Biological Bone Diseases Brain Bypass CLC Gene Carrier Proteins Cations Cell membrane Chloride Channels Chlorides Coupling Data Dent Disease Disease Electrophysiology (science)Electrostatics Epithelial Esters Event Family Formulation Foundations Functional disorder Future Genes Genetic Diseases Goals Hereditary Disease Homologous Gene Human Human Genome Impairment Intervention Investigation Ion Channel Ions Kidney Lysosomal Storage Diseases Malignant - descriptor Measurement Mediating Membrane Modeling Molecular Molecular Conformation Movement Muscle Muscle Contraction Mutagenesis Mutation Myotonia Congenita Neurons Organism Outcome Pathway interactions Pharmacology Phylogenetic Analysis Physiological Physiological Processes Physiology Plants Potassium Channel Process Property Proteins Resolution Role Route Side Sodium Chloride Structure Testing Therapeutic Time Tissues Vertebral column X-Ray Crystallography antiport base computerized tools design disease-causing mutation experimental study flexibility innovation molecular dynamics novel protein function rational design reconstruction simulation

项目摘要

ABSTRACT The CLC channels and transporters mediate anion transport through biological membranes. Genes encoding for CLC proteins are found in nearly all organisms, from bacteria to plants and humans. Mutations altering the properties of five of the nine human genes encoding for CLC homologues result in genetic disorders of bone, kidney, brain and muscle, highlighting the fundamental role of these proteins in a wide variety of tissues and cellular compartments. Despite their pathophysiological importance, our understanding of how these proteins function lagged far behind many other classes of ion channels and exchangers. This limits our ability to interpret their function in human physiology and to design targeted pharmacological interventions that would selectively manipulate their activity. Thus, our long-term goal is to elucidate the atomic basis for CLC Cl- channel and transporter function. Our proposal is articulated in three specific aims, each of which addresses a fundamental unanswered mechanistic question on CLC function. Our innovative use of synergistic experimental and computational approaches enables the formulation of specific hypotheses and their rigorous testing. In the first Aim we will determine the bases of substrate selectivity in the CLC Cl- channels. While selectivity of cation channels is well understood, nearly nothing is known on anion selectivity. We will probe the role of the protein backbone in this process using atomic-scale mutagenesis and will determine the consequences of these manipulations through structural, electrophysiological and computational experiments. Our second aim is to elucidate the coupling mechanism in the CLC exchangers. The CLC transporters exchange 2 Cl- for 1 H+ across biological membranes. Several disease-causing mutations affect this process through unknown mechanisms. Our goal is to elucidate the basis for Cl-/H+ coupling in the CLCs. We will utilize computational tools in conjunction to conventional and atomic mutagenesis to probe the dynamic rearrangements undergone by the protein to enable the formation of a pathway for H+ that is physically distinct from the route taken by the Cl- ions. The third aim is to determine the molecular origin of the functional divergence of the CLC channels from the transporters. Despite the availability of high resolution structural information for both subtypes, the molecular origin of this functional divergence remains unknown. We will use statistical phylogenetics and evolutionary bioinformatics to identify the most likely evolutionary sequence of events leading to the functional divergence. We will then functionally characterize sequences recapitulating these key evolutionary steps and use this information to identify a subset of amino acid substitutions necessary to enact the functional switch. Ultimately, these efforts will lead to new molecular and conceptual framework for the understanding of CLC function, which will enable the design of approaches for the amelioration of the disease conditions caused by the dysfunction of these proteins.

抽象的 CLC通道和转运蛋白通过生物膜介导阴离子的传输。基因编码对于Clc蛋白，几乎在所有生物中都发现了从细菌到植物和人类。突变改变了编码CLC同源物的九种人类基因中的五个特性导致骨骼遗传疾病，肾脏，大脑和肌肉，强调了这些蛋白质在多种组织中的基本作用细胞室。尽管具有病理生理的重要性，但我们对这些蛋白质的理解功能远远落后于许多其他类别的离子通道和交换器。这限制了我们的能力解释它们在人类生理学中的功能并设计针对的药理干预措施有选择地操纵他们的活动。因此，我们的长期目标是阐明Clc cl-的原子基础通道和转运蛋白功能。我们的建议在三个特定目标中阐明，每个目标都解决了关于CLC功能的基本未解决的机械问题。我们对协同作用的创新使用实验和计算方法可实现特定假设及其严格的表述测试。在第一个目标中，我们将确定CLC CL-通道中底物选择性的碱基。尽管阳离子通道的选择性已充分了解，几乎在阴离子的选择性上尚无任何人知道。我们将探究蛋白质主链在此过程中使用原子尺度诱变的作用，并将确定这些操作通过结构，电生理和计算实验的后果。我们的第二个目标是阐明CLC交换器中的耦合机制。 CLC转运蛋白在生物膜上交换2 cl-持续1 H+。几种引起疾病的突变影响了这一过程通过未知的机制。我们的目标是阐明CLC中Cl-/H+耦合的基础。我们将利用与常规和原子诱变结合使用的计算工具，以探测动态蛋白质进行了重新排列，以使H+的途径形成物理上不同的途径从cl的路线中。第三个目的是确定功能的分子起源 CLC通道与转运蛋白的差异。尽管有高分辨率的结构可用这两种亚型的信息，该功能差异的分子起源仍然未知。我们将使用统计系统发育和进化生物信息学，以识别最可能的进化序列导致功能差异的事件。然后，我们将在功能上表征序列的序列这些关键的进化步骤，并使用此信息来确定必要的氨基酸取代子集制定功能开关。最终，这些努力将导致新的分子和概念框架对CLC功能的理解，这将使方法设计用于改善这些蛋白质功能障碍引起的疾病状况。