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）：拟议项目继续了解钾（K）渗透和门控的最初特定目标，通过肾脏，内部整流，K渠道：Romk（Kir1.1）。但是，该项目现在涵盖了一个更一般的主题，即了解内向整流器K频道家族（KIR）中门控（开放和关闭）的结构机制。有关细菌通道的封闭和部分开放结构的最新晶体学数据：Kirbac1.1和Kirbac3.1使我们能够在封闭状态和部分开放状态下构建ROMK的详细同源模型。 ROMK通道非常适合组合结构和功能研究，以阐明哺乳动物通道中的门控，因为我们已经在Romk中拥有大量有关配体（pH）门控和Perseant-ion Gating的生理数据。这与我们的同源性建模一起，应该使我们能够阐明Romk门控的分子过程，并提供对其他内部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四个方面。 （1）pH传感器是由C端盐桥片形成的吗？ （2）内部TM2螺旋中的2个保守的GLY作为铰链点功能是否对螺旋填料更重要？ （3）除束交叉处的主要配体门外，渗透路径中还有其他门吗？如果是这样，这两个大门如何在结构层面上连接在一起？ ROMK的外部K座是否取决于束交叉处pH栅极的分子结构？选择性滤光片构象的变化是否构成与束交叉门串联的第二个（C型灭活）门？ （4）我们还建议使用灯笼谐振能量转移（LRET）方法直接测量门控过程中的构象变化。这项技术涉及将遗传编码的捐赠者和受体标签纳入渠道，将使我们能够查看距离状态依赖的距离变化，并解决有关ROMK门控的几个重要结构问题：（a）KIR C-termini在捆绑捆绑门的打开期间是否相互朝向彼此？ （b）在romk（Kir1.1）pH门上，束横梁级别的螺旋运动是什么？ （c）在romk（kir1.1）pH门口期间，幻灯螺旋的角运动是什么？这些实验将与大学附近的弗朗西斯科·贝扎尼拉教授合作进行。芝加哥，使用一种新型技术来隔离适合拟议的LRET实验的（内部）卵母细胞膜的大段。该项目的结果将极大地促进我们对人类肾脏K平衡至关重要的肾脏Romk钾渠道的理解。这不仅可以帮助患有Bartter病产前变体的患者，这是由Romk中的先天性缺陷引起的，而且还将为其他内部整流性钾通道中门控的分子表征铺平道路。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，神经元活性的调节以及脑神经胶质细胞中的钾缓冲中起着重要作用。对他们的门控的透彻表征对于理解各种通道病的分子基础至关重要。 公共卫生相关性：拟议的项目将表征肾脏内整流器（ROMK）钾（K）渠道家族中离子通道门控（开放和关闭）的基本分子基础。这项研究的结果不仅与Bartter综合征等肾脏疾病有关，而且对G蛋白的心脏和神经系统中调节的内向整流通道以及胰腺β细胞中与糖尿病和低血糖症有关的胰腺β细胞中的K通道具有深远的影响。这最终可用于靶向药物设计，以纠正影响肾脏，心脏和胰腺的各种先天性离子通道病。