Molecular Cloning of Epithelial K Channels

描述（由申请人提供）：拟议项目依赖于鸟类Kir2.2和细菌Kir通道的先前结构知识，以探测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）渗透和通过肾脏内向整流钾通道ROMK（Kir1.1）的门控; ROMK在控制全身钾和水平衡中起着重要作用。这项研究的结果不仅与产前Bartter综合征等肾脏疾病有关，而且如果ROMK功能的部分降低降低血压而不显著干扰血清电解质，则对高血压也可能很重要。从鸟类Kir2.2和原核KirBac闭合状态的晶体学模型开始，拟议的实验将检查与Kir1.1通道门控（打开和关闭）相关的构象变化，在体外蛋白脂质体系统中使用镧系元素共振能量转移（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二聚体构建体（用单个供体和单个受体标记）的Kir1.1b尺寸的稳态LRET测定。在这个目标中，我们还将进行电生理学测量，以验证这些单半胱氨酸二聚体作为ROMK门控的模型。AIM 2提出了状态依赖性LRET测量，以评估C-末端结构域启动通道开放的替代假设; AIM 3检查了内部跨膜螺旋ROMK交叉处主要疏水门的替代运动。这将有助于解决Kir开放态构象，这一直是有争议的。最后，在AIM 4中，我们将评估这样的假设，即ROMK打开所需的PIP 2结合通过缩短C-末端结构域和界面滑动螺旋之间的接头来设置预打开条件。所提出的expts依赖于多种创新：（1）无细胞的体外真核蛋白表达，（2）通过直接注射到非洲爪蟾卵母细胞中评估无细胞的Kr蛋白，随后进行全细胞和切除的斑块记录（3）使用单Cys ROMK二聚体的明确的LRET状态依赖性分子距离测量。

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.