Molecular Cloning of Epithelial K Channels

摘要 拟议的项目延续了最初的具体目标，即了解钾的渗透和门控 经肾，内向整流，K通道：ROMK(Kir1.1)。然而，该项目现在包含了更多的 了解内向整流器中闸门(开闭)结构机理的总主题 K通道家族(KIR)。细菌封闭和部分开放结构的最新结晶学数据 渠道：KirBac1.1和KirBac3.1使我们能够构建ROMK在封闭的 状态和部分开放状态。ROMK通道特别适合于结构和功能相结合的研究 为了阐明哺乳动物通道中的门控，因为我们已经收集了大量的生理数据 ROMK中既有配基(PH)门控，也有离子门控。这一点，加上我们的同源建模，允许 美国将设计新的实验，有望阐明ROMK门控的分子过程以及 为其他内向整流通道的选通动态提供新的见解。我们的实验将解决 浇注过程中构象变化的5个方面。(1)pH传感器是由C端盐桥形成的吗？(2) 配基(PH)信号是如何从C末端传递到跨膜螺旋和主链的 内螺旋束处的浇口？(3)浇注是通过沿内(TM2)螺旋弯曲产生的还是 只有2个保守的甘氨酸？(4)在渗透路径中，除了主配体门之外，还有其他门吗？ 捆绑式穿越？如果是这样的话，这两扇门是如何在结构层面上连接在一起的？外部K门控是否为 ROMK依赖于束交叉处pH门的分子结构？在选择性过滤器中进行更改 构象构成与束交叉门串联的第二个(C型失活)门？(5)我们还 提出利用稀土共振能量转移直接测量浇注过程中的构象变化 (LRET)方法。这将具体解决两个假设。Kir C-末端是否相互移动 在打开捆绑门的过程中？滑轨、外螺旋和内螺旋是否相对于彼此旋转 在门禁期间？我们计划使用各种技术来回答这些问题：(1)基于计算机的分子 模拟，(2)镧系元素的共振能量转移，以测量标记之间分子距离的变化 通道开放和关闭过程中的残留物，以及(3)定位突变以确定可能的基因座。 细胞质区域的盐桥传感器。这个项目将极大地促进我们对肾脏的了解 ROMK钾通道是人体肾脏钾平衡所必需的。这不仅有助于患者 与产前变异的巴特S病，引起的先天性缺陷的ROMK，但也将铺平 其他内向整流钾通道门控的分子表征方法。这些频道播放 在心脏细胞、胰岛β细胞(糖尿病和低血糖)、酸碱平衡紊乱、 脑神经胶质细胞中神经元活动的调节以及钾的缓冲。详尽的人物刻画 它们的门控对于理解各种通道病的分子基础是必不可少的。

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.