Molecular Cloning of Epithelial K Channels

上皮 K 通道的分子克隆

• 批准号: 7653298
• 负责人: HENRY SACKIN
• 金额: $ 36.96万
• 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 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7653298
• 关键词: Acid-Base Equilibrium; Address; Affect; Bartter Disease; Binding; Brain; Buffers; C-terminal; Chicago; Collaborations; Collection; Congenital Abnormality; Coupled; Data; Diabetes Mellitus; Disease; Drug Design; Energy Transfer; Epithelial; Equilibrium; Family; Fluorescein; Fluoresceins; GTP-Binding Proteins; Glycine; Goals; Heart; Homology Modeling; Human; Hypoglycemia; Ion Channel Gating; Ions; KCNJ1 gene; Kidney; Kidney Diseases; Kinetics; Lanthanoid Series Elements; Ligands; Link; Measures; Mechanics; Mediating; Membrane; Metalcaptase; Methods; Molecular; Molecular Cloning; Molecular Conformation; Molecular Structure; Motion; Movement; Nervous system structure; Neuroglia; Neurons; Oocytes; Pancreas; Patients; Physiological; Play; Potassium; Potassium Channel; Process; Relative (related person); Role; Series; Side; Slide; Sodium Chloride; Structure; Structure of beta Cell of islet; Techniques; Variant; base; heart cell; insight; inward rectifier potassium channel; novel; public health relevance; research study; sensor

DESCRIPTION (provided by applicant): 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, should allow us to 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 4 aspects of conformational change during gating. (1) Is the pH sensor formed by C-terminal salt bridging? (2) Do the 2 conserved Gly in the inner TM2 helix function as hinge points or are they more important for helix packing? (3) 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? (4) We also propose to directly measure conformational changes during gating, using lanthanide resonance energy transfer (LRET) methods. This technique, which involves genetically encoding donor and acceptor tags into the channel, would permit us to look at state-dependent changes in distance and address several important structural questions about ROMK gating: (a) Do the Kir C-termini move toward each other during opening of the bundle-crossing gate? (b) What are the helix motions at the level of the bundle crossing gate during ROMK (Kir1.1) pH gating? (c) What is the angular motion of the slide helix during ROMK (Kir1.1) pH gating? These experiments would be done in collaboration with Prof. Francisco Bezanilla, nearby at the Univ. of Chicago, using a novel technique for isolating large segments of (inside-up) oocyte membrane suitable for the proposed LRET experiments. Results of 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. PUBLIC HEALTH RELEVANCE: The proposed project would characterize the underlying molecular basis for ion channel gating (opening & closing) in the renal inward rectifier (ROMK) family of potassium (K) channels. Results of this study would not only be relevant for renal diseases like Bartter's syndrome but would also have profound implications for G-protein regulated inward rectifier channels in the heart and nervous system, as well as K channels in pancreatic beta cells that are implicated in diabetes and hypoglycemia. This could ultimately be used for targeted drug design to correct a variety of congenital ion channelopathies affecting the kidney, heart, and pancreas.

描述（由申请人提供）：拟议项目延续了最初的特定目标，即了解钾（K）通过肾脏内向整流钾通道（ROMK（Kir1.1））的渗透和门控。然而，该项目现在包括一个更一般的主题，即了解内向整流钾通道家族（Kir）中门控（打开和关闭）的结构机制。最近关于细菌通道KirBac1.1和KirBac3.1的封闭和部分开放结构的晶体学数据使我们能够构建ROMK在封闭状态和部分开放状态下的详细同源模型。ROMK通道是唯一适合于组合的结构和功能的研究，阐明门控在哺乳动物通道，因为我们已经有一个大的收集的生理数据的配体（pH值）门控和渗透离子门控在ROMK。这一点，连同我们的同源性建模，应该使我们能够澄清ROMK门控的分子过程，以及提供新的见解门控动力学的其他内向整流通道。我们的实验将解决门控过程中构象变化的4个方面。(1)pH传感器是否由C端盐桥形成？(2)2保守的甘氨酸在内部TM 2螺旋功能铰链点或他们更重要的螺旋包装？(3)除了在束交叉处的主配体门之外，在渗透路径中是否还有其他门？如果是这样的话，这两个门是如何在结构层面上联系在一起的？ROMK的外部K门控是否依赖于束交叉处pH门控的分子结构？选择性过滤器构象的变化是否构成与交叉门串联的第二个（C型灭活）门？(4)我们还建议直接测量门控过程中的构象变化，使用镧系共振能量转移（LRET）方法。这种技术，它涉及到基因编码供体和受体标签进入通道，将允许我们看看状态依赖的变化的距离和解决几个重要的结构问题ROMK门控：（a）Kir C-末端移动到对方在开放的竞争交叉门？(b)在ROMK（Kir1.1）pH门控过程中，束交叉门水平的螺旋运动是什么？(c)ROMK（Kir1.1）pH门控期间载玻片螺旋的角运动是什么？这些实验将与附近芝加哥大学的弗朗西斯科·贝萨尼拉教授合作完成，使用一种新技术分离适合于拟议的LRET实验的大片段（由内而外）卵母细胞膜。该项目的结果将大大有助于我们进一步了解肾脏ROMK钾通道，这是人类肾脏钾平衡所必需的。这不仅有助于患有由ROMK先天性缺陷引起的巴特氏病产前变异的患者，而且还为其他内向整流钾通道门控的分子表征铺平了道路。这些通道在心脏细胞、胰腺β细胞（糖尿病和低血糖症）、酸碱平衡紊乱、神经元活动的调节以及脑胶质细胞中的钾缓冲中发挥重要作用。门控的彻底表征对于理解各种通道病的分子基础是必不可少的。 公共卫生相关性：拟议的项目将表征钾（K）通道的肾内向整流（ROMK）家族中离子通道门控（打开和关闭）的潜在分子基础。这项研究的结果不仅与Bartter综合征等肾脏疾病相关，而且还对心脏和神经系统中G蛋白调节的内向整流通道以及与糖尿病和低血糖有关的胰腺β细胞中的K通道具有深远的意义。这最终可以用于靶向药物设计，以纠正影响肾脏，心脏和胰腺的各种先天性离子通道病。