Structures and Pharmacology of Cation-Chloride Cotransporters

阳离子-氯化物协同转运蛋白的结构和药理学

基本信息

批准号：
10367176
负责人：
Erhu Cao
金额：
$ 33.55万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-20 至 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10367176
关键词：
Affect Agonist Antiepileptic Agents Antihypertensive Agents Architecture Binding Biochemical Biological Assay Blood Pressure Brain Brain Diseases Cations Cell Volumes Cells Chlorides Clinical Complex Cryoelectron Microscopy Development Disease Diuretics Drosophila genus Edema Electrolyte Balance Epilepsy Epithelial Equilibrium Family Family member Fluorescence Foundations Goals Homeostasis Hormones Human Hypertension Ion Channel Gating Ion Cotransport Ion Transport Ions Kidney Lead Learning Ligands Liposomes Membrane Molecular Molecular Conformation Molecular Target Movement Mus Mutation Nervous system structure Neurons Neurotransmitters Outcome Paralysed Pathway interactions Pharmaceutical Chemistry Pharmaceutical Preparations Pharmacology Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Physiological Play Proteins Recovery Regulation Resolution Role Seizures Series Sodium Chloride Specificity Spinal cord injury Structure Synapses Synaptic Transmission Syndrome Testing Therapeutic Thiazide Diuretics Urine base biophysical analysis dimer drug development falls gamma-Aminobutyric Acid high throughput screening human disease hypertension treatment improved inhibitor/antagonist innovation interdisciplinary approach man neuronal excitability novel novel therapeutic intervention particle pre-clinical protein structure reconstitution recruit response small molecule success synaptic inhibition tool trafficking ubiquitin-protein ligase wasting

项目摘要

Project Summary Human secondary active cation-chloride cotransporters (CCCs) fall into two classes: three Na+-dependent Na+- (K+)-Cl- (NCC and NKCC1-2) and four Na+-independent K+-Cl− (KCC1-4) transporters. CCCs catalyze electroneutral symport of Cl- with Na+ and/or K+ across membrane and are fundamental in cell volume regulation, trans-epithelia ion movement, regulation of intracellular [Cl-]i and neuronal excitability. In the nervous system, NKCC1 and KCC2 are the major Cl- loader and extruder, respectively, and their opposing actions move [Cl-]i away from electrochemical equilibrium so that inhibitory neurotransmitters can evoke either inward depolarizing or outward hyperpolarizing Cl- currents via pentameric ligand-gated ion channels. Mutations in KCC2 or KCC3 cause seizure, epilepsy, and other brain disorders possibly owing to an imbalance in excitatory versus inhibitory synaptic transmission. Pharmacological tuning of NKCC1 and KCCs transport activities thus represents a promising therapeutic strategy to restore synaptic inhibition for the treatment of brain disorders. In the kidneys, NKCC2 and NCC reabsorb ions from the forming urine, balancing electrolytes and blood pressure. CCCs are regulated by the WNKs-SPAK kinase cascade in response to hormone stimulation and cell volume perturbations, with N(K)CC activated and KCCs inhibited by phosphorylation. Mutations in NKCC2, NCC, or WNKs and their upstream E3 ubiquitin ligase regulators lead to hypotensive Gitelman's and Batter's syndromes or hypertensive Gordon's disease. Loop and thiazide diuretics antagonize NKCC2 and NCC, respectively, and are widely prescribed for the treatment of hypertension and edema. Building on our success in determining structures of both NKCC1 and KCC transporters in multiple states, we now propose to determine a series of new CCC structures using single-particle cryo-EM and to perform complementary biochemical and functional studies to elucidate: 1) how CCCs alternate between different transport states to shuttle ions across membranes, 2) how diuretic drugs interact with and inhibit CCCs, and 3) how (de)phosphorylation regulates CCC ion transport pathways. In parallel, we will also develop and apply cell- and liposome-based flux assays that will accelerate our CCC functional studies and, ultimately, could support high throughput screening platforms for rapid discovery of small molecule pharmacological tools to dissect CCC structures/functions and provide drug leads to treat hypertension, edema, and brain disorders. Our overarching goals are to combine structural, functional, and pharmacological approaches to understand the inner-workings of CCCs and to facilitate rational targeting of these transporters for the treatment of numerous human diseases.

项目概要人类次级活性阳离子-氯离子协同转运蛋白 (CCC) 分为两类：三种 Na+ 依赖性 Na+- (K+)-Cl-（NCC 和 NKCC1-2）和四个不依赖 Na+ 的 K+-Cl− (KCC1-4) 转运蛋白发挥催化作用。 Cl- 与 Na+ 和/或 K+ 跨膜电中性对称，是细胞体积调节的基础，跨上皮离子运动、细胞内 [Cl-]i 的调节和神经系统中的神经元兴奋性。 NKCC1和KCC2分别是主要的Cl-装载机和挤出机，它们的相反动作移动[Cl-]i 远离电化学平衡，以便抑制性神经递质可以引起内向去极化或通过 KCC2 或 KCC3 中的五聚体配体门控离子通道突变产生向外超极化 Cl- 电流。引起癫痫、癫痫和其他脑部疾病，可能是由于兴奋性与抑制性不平衡所致因此，NKCC1 和 KCC 转运活性的药理学调节代表了一种恢复突触抑制以治疗肾脏疾病的治疗策略。 NKCC2 和 NCC 从形成的尿液中重吸收离子，平衡电解质和血压。受 WNKs-SPAK 激酶级联调节，响应激素刺激和细胞体积扰动， N(K)CC 被激活，KCC 被 NKCC2、NCC 或 WNK 及其磷酸化抑制。上游 E3 泛素连接酶调节因子导致低血压 Gitelman 综合征和 Batter 综合征或高血压戈登氏病。袢利尿剂和噻嗪类利尿剂分别拮抗 NKCC2 和 NCC，并且广泛应用。建立在我们成功确定高血压和水肿结构的基础上。 NKCC1 和 KCC 转运蛋白在多个州，我们现在建议确定一系列新的 CCC 使用单粒子冷冻电镜研究结构并进行补充生化和功能研究阐明：1) CCC 如何在不同的运输状态之间交替以将离子穿梭于膜上，2) 如何利尿药与 CCC 相互作用并抑制 CCC，以及 3)（去）磷酸化如何调节 CCC 离子转运与此同时，我们还将开发和应用基于细胞和脂质体的通量测定，这将加速。我们的 CCC 功能研究最终可以支持高通量筛选平台以实现快速发现小分子药理学工具来剖析 CCC 结构/功能并提供治疗药物我们的首要目标是将结构、功能和脑部疾病结合起来。药理学方法来了解 CCC 的内部运作并促进合理靶向这些转运蛋白用于治疗多种人类疾病。