Molecular Cloning of Epithelial K Channels

项目描述（由申请人提供）：本项目基于禽类Kir2.2和细菌Kir1.1通道的结构知识，探索ROMK （Kir1.1）通道门控的分子细节。这延续了最初的特定目标，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)；在控制系统钾和水分平衡中起重要作用。这项研究的结果不仅与像产前巴特氏综合征这样的肾脏疾病有关，而且如果ROMK功能的部分降低可以在不显著破坏血清电解质的情况下降低血压，则可能对高血压也很重要。从鸟类Kir2.2和原核KirBac闭态的晶体学模型开始，提出的实验将检查与Kir1.1通道门控（打开和关闭）相关的构象变化，使用在体外蛋白脂质体系统中使用稀土共振能量转移（LRET）光学技术直接测量状态依赖的分子距离。AIM 1描述了使用新的单cys二聚体构建的稳态LRET测定kir11 b的尺寸，标记为单个供体和单个受体。为此，我们还将进行电生理测量，以验证这些单- cys二聚体作为ROMK门控的模型。AIM 2提出了状态相关的LRET测量，以评估c端结构域启动通道打开的其他假设；AIM 3研究了内部跨膜螺旋的ROMK束交叉时初级疏水门的不同运动。这将有助于解决一直存在争议的基尔开态构象。最后，在AIM 4中，我们将评估ROMK打开所需的PIP2结合通过缩短c端结构域和界面滑动螺旋之间的连接物来设置预打开条件的假设。提出的专家依赖于各种创新：(1)体外无细胞真核蛋白表达；(2)通过直接注射到非洲爪蟾卵母细胞中评估无细胞Kr蛋白，然后进行全细胞和切除的贴片记录；(3)使用单cys ROMK二聚体进行明确的LRET状态依赖的分子距离测量。

DESCRIPTION (provided by applicant): The proposed project relies on previous structural knowledge of avian Kir2.2 and bacterial Kir channels to probe the molecular details of ROMK (Kir1.1) channel gating. This continues the original specific goal of understanding potassium (K) permeation and gating through the renal, inward rectifying, K channel ROMK (Kir1.1); which plays an important role in control of systemic K and water balance. Results of this study would not only be relevant for renal diseases like antenatal Bartter's syndrome but might also be important for hypertension if a partial reduction of ROMK function lowers blood pressure without significantly disrupting serum electrolytes. Starting with crystallographic models of the avian Kir2.2 and prokaryotic KirBac closed-states, the proposed experiments would examine conformational changes associated with Kir1.1 channel gating (opening & closing), using direct measurement of state-dependent molecular distances with lanthanide resonance energy transfer (LRET) optical techniques in an in vitro proteoliposome system. AIM 1 describes steady-state LRET determinations of Kir1.1b dimensions using novel single-Cys dimeric constructs, labeled with a single donor and a single acceptor. In this aim we would also conduct electrophysiological measurements to validate these single-Cys dimers as models for ROMK gating. AIM 2 proposes state-dependent LRET measurements to evaluate alternative hypotheses for initiation of channel opening by the C-terminal domain; and AIM 3 examines alternative motions of the primary hydrophobic gate at the ROMK bundle-crossing of inner transmembrane helices. This would help resolve the Kir open-state conformation, which has been controversial. Finally, in AIM 4 we would evaluate the hypothesis that PIP2 binding, required for ROMK opening, sets a pre-open condition by shortening the linker between the C-terminal domain and the interfacial slide helix. The proposed expts rely on a variety of innovations: (1) cell- free, in vitro, eukaryotic protein expression, (2) evaluation of cell-free Kr protein by direct injection into Xenopus oocytes, followed by whole-cell and excised patch recording (3) unambiguous LRET state-dependent molecular distance measurements using single-Cys ROMK dimers.