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: 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
• 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

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 人次级活性阳离子-氯离子协同转运蛋白（CCCs）分为两类：三种Na+依赖性Na+- （K+）-Cl-（NCC和NKCC 1 -2）和四种Na+非依赖性K+-Cl−（KCC 1 -4）转运蛋白。CCCs催化 Cl-与Na+和/或K+跨膜的电中性同向转运，并且在细胞体积调节中是基本的， 跨上皮离子运动、细胞内[Cl-]i的调节和神经元兴奋性。在神经系统中， NKCC 1和KCC 2分别是主要的Cl-加载器和挤出器，它们的相反作用使[Cl-]i 从而使抑制性神经递质可以引起内向去极化， 或通过五聚体配体门控离子通道向外超极化Cl-电流。KCC 2或KCC 3突变 可能由于兴奋性与抑制性的不平衡而引起癫痫发作、癫痫和其他脑部疾病 突触传递因此，NKCC 1和KCCs转运活性的药理学调节代表了一种新的调节机制。 恢复突触抑制以治疗脑疾病的有前景的治疗策略。在肾脏， NKCC 2和NCC从尿液中重吸收离子，平衡电解质和血压。CCC是 受WNKs-SPAK激酶级联反应调节，以响应激素刺激和细胞体积扰动， N（K）CC被激活，KCC被磷酸化抑制。NKCC 2、NCC或WNK中的突变及其 上游E3泛素连接酶调节剂导致肥胖性Gitelman和Batter综合征或高血压 戈登氏病Loop和噻嗪类利尿剂分别拮抗NKCC 2和NCC，并广泛用于治疗糖尿病。 用于治疗高血压和水肿。在我们成功地确定 NKCC 1和KCC转运蛋白在多种状态下，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，并进行补充的生物化学和功能研究， 阐明：1）CCC如何在不同的运输状态之间交替，以使离子穿梭于膜，2）如何 利尿剂与CCC相互作用并抑制CCC，以及3）磷酸化（去磷酸化）如何调节CCC离子转运 路径。同时，我们还将开发和应用基于细胞和脂质体的通量测定， 我们的CCC功能研究，最终可以支持快速发现的高通量筛选平台 小分子药理学工具，以剖析CCC结构/功能，并提供药物线索， 高血压、水肿和脑部疾病。我们的总体目标是将联合收割机的结构、功能和 药理学方法，以了解CCC的内部运作，并促进合理的目标， 这些转运蛋白用于治疗许多人类疾病。