Molecular Cloning of Epithelial K Channels

描述(由申请人提供)：建议的项目依赖于以前对禽类Kir2.2和细菌KIR通道的结构知识来探索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)通过肾脏的渗透和门控，内向整流，K通道ROMK(Kir1.1)；这在控制全身K和水平衡方面发挥着重要作用。这项研究的结果不仅与产前巴特综合征等肾脏疾病有关，而且如果ROMK功能的部分降低可以降低血压，而不显著扰乱血清电解质，那么对高血压也可能是重要的。从禽类Kir2.2和原核生物KirBac闭合状态的结晶学模型出发，拟议的实验将在体外蛋白质脂质体系统中，通过使用稀土共振能量转移(LRET)光学技术直接测量依赖于状态的分子距离，来研究与Kir1.1通道门控(打开和关闭)相关的构象变化。目的1描述了用单一供体和单一受体标记的新型单半胱氨酸二聚体结构来稳定测定Kir1.1b维度的LRET。在这个目标中，我们还将进行电生理测量，以验证这些单半胱氨酸二聚体是否为ROMK门控的模型。AIM 2提出状态依赖的LRET测量来评估由C-末端结构域启动通道开放的替代假设；AIM 3研究了初级疏水门在内部跨膜螺旋的ROMK束交叉处的替代运动。这将有助于解决一直存在争议的KIR开放国家结构。最后，在AIM 4中，我们将评估这样的假设，即ROMK开放所需的PIP2结合通过缩短C-末端结构域和界面滑动螺旋之间的连接子来设置预开放条件。建议的EXPT依赖于各种创新：(1)无细胞的体外真核蛋白表达，(2)通过直接注射到非洲爪哇卵母细胞中评估无细胞Kr蛋白，随后进行全细胞和切除斑片记录(3)使用单半胱氨酸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.