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

• 批准号: 10491128
• 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
• 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/10491128
• 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; Hypotensives; 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; hypertensive; improved; inhibitor; 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

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.

项目摘要 人类二级活性阳离子 - 氯化物共转运蛋白（CCC）分为两个类别：三个Na+依赖性Na+ - （K+） - Cl-（NCC和NKCC1-2）和四个Na+独立的K+-Cl-（KCC1-4）转运蛋白。 CCC催化 cl-的cl-的电荷对象与Na+和/或K+在整个膜中，并且在细胞体积调节中是基本的， 反式上皮离子运动，细胞内[Cl-] I和神经元令人兴奋的调节。在神经系统中， NKCC1和KCC2分别是主要的CL载器和挤出机，它们的对立动作移动[Cl-] I 远离电化学平衡，以便抑制性神经递质可以唤起向内的分离化 或通过五聚合配体门控离子通道向外过度溶质。 KCC2或KCC3的突变 导致癫痫发作，癫痫和其他脑部疾病，因为兴奋性与抑制性不平衡 突触传输。因此，NKCC1和KCCS运输活动的药理调整代表 有前途的治疗策略，以恢复脑疾病治疗的突触抑制作用。在孩子们， NKCC2和NCC从形成尿液，平衡电解质和血压平衡的NKCC2和NCC离子。 CCC是 受wnks-spak激酶级联反应响应骑马刺激和细胞体积扰动的调节， N（K）CC激活，KCC被磷酸化抑制。 NKCC2，NCC或WNK的突变及其 上游E3泛素连接酶调节剂导致降压Gitelman和Batter综合征或高血压 戈登病。循环和噻嗪类利尿剂分别对抗NKCC2和NCC，并且广泛 规定用于治疗高血压和水肿。以我们成功确定结构的成功为基础 NKCC1和KCC转运蛋白在多个状态下，我们现在建议确定一系列新的CCC 使用单粒子冷冻EM的结构，并进行完整的生化和功能研究 阐明：1）CCC如何在不同的运输状态之间替代跨膜的离子，2） 利尿药与CCC相互作用并抑制CCC，3）（DE）磷酸化如何调节CCC离子转运 途径。同时，我们还将开发和应用基于细胞和脂质体的通量测定，以加速 我们的CCC功能研究，最终可以支持高吞吐量筛选平台以快速发现 小分子的药物工具，用于剖析CCC结构/功能并提供药物导线以治疗 高血压，水肿和脑部疾病。我们的总体目标是结合结构，功能和 了解CCC的内部工作并促进合理靶向的药理方法 这些用于治疗多种人类疾病的转运蛋白。