Molecular Cloning of Epithelial K Channels

描述（由申请人提供）：拟议的项目延续了最初的具体目标，即了解钾(K)通过肾脏向内整流的渗透和门控，钾通道：ROMK （Kir1.1）。然而，该项目现在包含了一个更普遍的主题，即理解内向整流器K通道族（Kir）中的门控（打开和关闭）的结构力学。最近关于细菌通道的封闭和部分开放结构的晶体学数据：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通道特别适合于结合结构和功能研究来阐明哺乳动物通道，因为我们已经收集了ROMK中配体（pH）门控和渗透离子门控的大量生理数据。这与我们的同源建模一起，应该使我们能够澄清ROMK门控的分子过程，并为其他内向整流通道的门控动力学提供新的见解。我们的实验将解决四个方面的构象变化在门控过程中。(1) pH传感器是由c端盐桥接形成的吗？(2)内部TM2螺旋中的2个保守的Gly是否作为铰链点，还是它们对螺旋填充更重要？(3)除了束交叉处的主配体通道外，渗透路径上是否还有其他通道？如果是这样，这两个门在结构层面上是如何连接在一起的？ROMK的外部K门是否依赖于束交叉处pH门的分子结构？选择性过滤器构象的改变是否构成与束交叉门串联的第二个（c型失活）门？(4)我们还提出使用镧系共振能量转移（LRET）方法直接测量门控过程中的构象变化。这项技术涉及将供体和受体标签遗传编码到通道中，将使我们能够观察距离的状态依赖变化，并解决有关ROMK门控的几个重要结构问题：(a)在打开束交叉门时，Kir c -末端是否相互移动？(b) ROMK (Kir1.1) pH浇注过程中束交叉栅水平的螺旋运动是什么？(c) ROMK (Kir1.1) pH浇注过程中，滑动螺旋的角运动是什么？这些实验将与芝加哥大学附近的Francisco Bezanilla教授合作完成，使用一种新技术分离适合LRET实验的大片段（内向上）卵母细胞膜。该项目的结果将有助于我们进一步了解肾脏ROMK钾通道，这是人体肾脏钾平衡所必需的。这不仅可以帮助患有由ROMK先天性缺陷引起的Bartter氏病产前变异的患者，而且还可以为其他内向整流钾通道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）、酸碱平衡紊乱、神经元活动调节以及脑胶质细胞钾缓冲中发挥重要作用。彻底表征它们的门控对于理解各种通道病变的分子基础是必不可少的。

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.