Structures and Pharmacology of Cation-Chloride Cotransporters

项目摘要 人次级活性阳离子-氯转运蛋白(CCCS)分为两类：三种钠离子依赖的钠离子转运体 (K+)-Cl-(NCC和NKCC1-2)和4个不依赖Na+的K+-Cl−(KCC1-4)转运体。CCCS催化 Cl-与Na+和/或K+跨膜的电子中和共生是细胞体积调节的基础， 跨上皮细胞离子移动，细胞内[Cl-]i的调节和神经元的兴奋性。在神经系统中， NKCC1和KCC2分别是主要的氯离子加载剂和挤出剂，它们的相反作用使[Cl-]i 远离电化学平衡，因此抑制性神经递质可以引起内向去极化 或通过五聚体配基门控离子通道向外超极化氯离子电流。KCC2或KCC3基因突变 引起癫痫、癫痫和其他脑部疾病，可能是由于兴奋性和抑制性失衡所致 突触传递。因此，NKCC1和KCCS转运活动的药理学调节代表着一种 恢复突触抑制治疗脑部疾病的有希望的治疗策略。在肾脏里， NKCC2和NCC从形成的尿液中重新吸收离子，平衡电解质和血压。CCS是 在激素刺激和细胞体积扰动的反应下，受WNKS-Spak激酶级联反应的调节， N(K)CC被激活，KCCs被磷酸化抑制。NKCC2、NCC或WNKS基因突变及其意义 上游E3泛素连接酶调节因子导致低血压Gitelman‘s和Batter’s综合征或高血压 戈登氏病。环状和噻嗪类利尿剂分别拮抗NKCC2和NCC，并被广泛 用于治疗高血压和水肿症的处方。在我们成功确定结构的基础上 NKCC1和KCC转运体都处于多个状态，我们现在建议确定一系列新的CCC 使用单粒子低温EM的结构并进行补充的生化和功能研究 阐明：1)CCCs如何在不同的传输状态之间切换以使离子穿透膜；2)如何 利尿剂药物与CCCS相互作用并抑制CCCS，以及3)去磷酸化如何调节CCC离子转运 小路。同时，我们还将开发和应用基于细胞和脂质体的通量分析，这将加速 我们的CCC功能研究，并最终可以支持快速发现的高通量筛查平台 利用小分子药理工具剖析CCC结构/功能并为治疗提供药物先导 高血压、浮肿和脑部疾病。我们的总体目标是将结构性、功能性和 了解CCCS内部工作机制和促进合理靶向的药理学方法 这些转运蛋白用于治疗多种人类疾病。

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.