Molecular Cloning of Epithelial K Channels

上皮 K 通道的分子克隆

• 批准号: 7623692
• 负责人: HENRY SACKIN
• 金额: $ 23.08万
• 依托单位: ROSALIND FRANKLIN UNIV OF MEDICINE & SCI
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7623692
• 关键词: Acid-Base Equilibrium; Address; Brain; Buffers; C-terminal; Closure; Collection; Computers; Congenital Abnormality; Crystallography; Cysteine; Cytoplasmic Tail; Data; Decompression Sickness; Dependence; Diabetes Mellitus; Disease; Energy Transfer; Epithelial; Equilibrium; Excision; Family; Glycine; Goals; Helix (Snails); Homology Modeling; Human; Hypoglycemia; Institution; Ion Channel; Ions; KCNJ1 gene; Kidney; Label; Lanthanoid Series Elements; Ligands; Link; Lysine; Measures; Mechanics; Methods; Modeling; Molecular; Molecular Cloning; Molecular Conformation; Molecular Structure; Motion; Movement; Neuroglia; Neurons; Patients; Physiological; Play; Potassium; Potassium Channel; Process; Publishing; Regulation; Relative (related person); Renal function; Role; Rotation; Series; Side; Signal Transduction; Site-Directed Mutagenesis; Slide; Sodium Chloride; Structure; Structure of beta Cell of islet; Techniques; Variant; Work; abstracting; base; design; driving force; ear helix; heart cell; insight; inward rectifier potassium channel; molecular modeling; research study; sensor

ABSTRACT The proposed project continues the original specific goal of understanding potassium (K) permeation and gating through the renal, inward rectifying, K channel: ROMK (Kir1.1). However, the project now encompasses a more general theme of understanding the structural mechanics of gating (opening & closing) in the inward rectifier K channel family (Kir). Recent crystallographic data on the closed and partially open structures of the bacterial channels: KirBac1.1 and KirBac3.1 have allowed us to construct detailed homology models of ROMK in the closed state and partial open-state. The ROMK channel is uniquely suited for combined structure and function studies to elucidate gating in a mammalian channel because we already have a large collection of physiological data on both ligand (pH) gating and permeant-ion gating in ROMK. This, together with our homology modeling, allows us to design new experiments that should hopefully clarify the molecular processes of ROMK gating as well as provide new insight into the gating dynamics of other inward rectifier channels. Our experiments would address 5 aspects of conformational change during gating. (1) Is the pH sensor formed by C-terminal salt bridging? (2) How is the ligand (pH) signal transmitted from the C-terminus to the transmembrane helices and to the principal gate at the inner helix bundle crossing? (3) Is gating produced by bending all along the inner (TM2) helix or only at 2 conserved glycines? (4) Are there other gates in the permeation path besides the principal ligand gate at the bundle crossing? If so, how are these two gates linked together at a structural level? Is external K gating of ROMK dependent on the molecular structure of the pH gate at the bundle crossing? Do changes in selectivity filter conformation constitute a second (C-type inactivation) gate in series with the bundle-crossing gate? (5) We also propose to directly measure conformational changes during gating, using lanthanide resonance energy transfer (LRET) methods. This would specifically address two hypotheses. Do the Kir C-termini move toward each other during opening of the bundle-crossing gate? Do the slide, outer and inner helices rotate relative to each other during gating? We plan to use a variety of techniques to answer these questions: (1) computer based molecular modeling, (2) lanthanide resonance energy transfer to measure changes in molecular distance between labelled residues during channel opening and closure, and (3) site-directed mutagenesis to determine the locus of putative salt-bridge sensors in the cytoplasmic domain. This project would do much to further our understanding of the renal ROMK potassium channel that is essential for K balance in the human kidney. This would not only help patients with the antenatal variant of Bartter¿s disease, caused by a congenital defect in ROMK, but would also pave the way for a molecular characterization of gating in other inward rectifier potassium channels. These channels play essential roles in heart cells, pancreatic beta cells (diabetes and hypoglycemia), disorders of acid-base balance, modulation of neuronal activity as well as potassium buffering in brain glial cells. A thorough characterization of their gating is essential for understanding the molecular basis of a variety of channelopathies.

抽象的 拟议的项目继续理解钾（K）渗透和门控的最初特定目标 通过肾脏，向内纠正，k频道：romk（kir1.1）。但是，该项目现在包括更多 了解内向整流器中门控（打开和关闭）的结构机制的一般主题 K频道家庭（KIR）。有关细菌的封闭和部分开放结构的最新晶体学数据 频道：Kirbac1.1和Kirbac3.1允许我们在封闭的Romk中构建详细的同源模型 状态和部分开放状态。 ROMK渠道非常适合组合结构和功能研究 在哺乳动物通道中阐明门控，因为我们已经有大量的物理数据 罗克（Romk）的配体（pH）门控和Perseant-ion门控。这与我们的同源性建模一起允许 我们设计新实验，希望应该阐明Romk Gating的分子过程以及 提供有关其他内向整流器通道的门控动力学的新见解。我们的实验将解决 门控过程中构象变化的5个方面。 （1）pH传感器是由C端盐桥片形成的吗？ （2） 配体（pH）信号如何从C末端传输到跨膜螺旋和主螺旋 内螺旋束交叉处的门？ （3）是通过沿着内部（TM2）螺旋弯曲或 仅在2个保守的甘氨酸？ （4）除主要配体门以外的渗透路径中还有其他门 捆绑交叉？如果是这样，这两个大门如何在结构层面上连接在一起？是外部k门 romk取决于束交叉处pH门的分子结构？选择更改选择性过滤器 构象构成与捆绑门串联的第二个（C型灭活）门？ （5）我们也 使用灯笼谐振能量传递的提案，以直接测量门控变化的构象变化 （LRET）方法。这将特别提出两个假设。 Kir C-Termini互相迈向 在打开捆绑门的过程中？进行幻灯片，外部和内螺旋彼此旋转 在门控？我们计划使用各种技术回答以下问题：（1）基于计算机的分子 建模，（2）灯笼的共振能传递以测量标记的分子距离的变化 通道打开和关闭期间的残留物，以及（3）定点诱变，以确定推定的轨迹 细胞质结构域中的盐桥传感器。该项目将对我们对肾脏的理解有很大帮助 ROMK钾通道对于人类肾脏中的K平衡至关重要。这不仅会帮助患者 与先天性缺陷引起的易货疾病的产前变体有关，但也会铺平 在其他内部整流器钾通道中对门控的分子表征的方法。这些频道播放 在心脏细胞，胰腺β细胞（糖尿病和低血糖），酸碱平衡疾病中的基本作用， 神经元活性的调节以及脑神经胶质细胞中的钾缓冲。彻底的表征 他们的门控对于理解各种通道病的分子基础至关重要。