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 通道和转运蛋白介导阴离子通过生物膜的转运。基因编码 CLC 蛋白几乎存在于所有生物体中，从细菌到植物和人类。突变改变了编码 CLC 同源物的九个人类基因中的五个的特性导致骨遗传疾病，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。尽管它们具有病理生理学的重要性，但我们对这些蛋白质如何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体生理学中的功能并设计有针对性的药理学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功能。我们的提案阐述了三个具体目标，每个目标都涉及一个关于 CLC 功能的基本未解答的机制问题。我们创新地利用协同效应实验和计算方法能够制定具体的假设及其严格的假设测试。在第一个目标中，我们将确定 CLC Cl- 通道中底物选择性的基础。尽管阳离子通道的选择性是众所周知的，但阴离子选择性几乎一无所知。我们将探究蛋白质主链在此过程中使用原子级诱变的作用，并将决定通过结构、电生理学和计算实验来分析这些操作的后果。我们的第二个目标是阐明 CLC 交换器中的耦合机制。 CLC转运蛋白跨生物膜将 2 个 Cl- 交换为 1 个 H+。几种致病突变会影响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。我们将利用计算工具结合常规和原子诱变来探测动态蛋白质进行重排，形成物理上不同的 H+ 通路来自Cl-离子所走的路线。第三个目标是确定功能性物质的分子起源。 CLC 通道与转运蛋白的分歧。尽管有高分辨率的结构尽管关于这两种亚型的信息，这种功能差异的分子起源仍然未知。我们将使用统计系统发育学和进化生物信息学，以确定最可能的进化序列导致功能分歧的事件。然后我们将从功能上表征序列重演这些关键的进化步骤，并使用这些信息来识别必要的氨基酸取代的子集执行功能开关。最终，这些努力将带来新的分子和概念框架对 CLC 功能的理解，这将有助于设计改善由这些蛋白质功能障碍引起的疾病。