Molecular Cloning of Epithelial K Channels

上皮 K 通道的分子克隆

• 批准号: 8813435
• 负责人: HENRY SACKIN
• 金额: $ 35.1万
• 依托单位: ROSALIND FRANKLIN UNIV OF MEDICINE & SCI
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8813435
• 关键词: Address; Affect; Bartter Disease; Binding; Birds; C-terminal; Cations; Cell membrane; Cells; Characteristics; Charge; Chicago; Cysteine; Diabetes Mellitus; Dimensions; Drug Design; Electrolyte Balance; Electrolytes; Energy Transfer; Epithelial; Equilibrium; Evaluation; Family; Figs - dietary; Fluorescein; GTP-Binding Proteins; Goals; Heart; Hypertension; Hypoglycemia; Hypotension; In Vitro; Injection of therapeutic agent; Institution; Ion Channel Gating; Ions; KCNJ1 gene; KCNJ1 protein; Kidney; Kidney Diseases; Knowledge; Label; Lanthanoid Series Elements; Lateral; Lipids; Liposomes; Maleimides; Measurement; Measures; Messenger RNA; Modeling; Molecular; Molecular Cloning; Molecular Conformation; Molecular Probes; Motion; Nervous system structure; Oocytes; Optics; Pancreas; Phosphatidylinositol 4,5-Diphosphate; Physiological; Play; Potassium; Potassium Channel; Proteins; Relative (related person); Roentgen Rays; Role; Rotation; Serum; Slide; Structure; Structure of beta Cell of islet; System; Techniques; Water; Xenopus oocyte; base; dimer; innovation; interfacial; novel; patch clamp; protein expression; proteoliposomes; public health relevance; research study; trafficking

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.

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来探测 ROMK (Kir1.1) 通道门控的分子细节。这延续了最初的具体目标，即了解钾 (K) 渗透和门控通过肾脏、内向整流、K 通道 ROMK (Kir1.1)；它在控制系统钾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，而且如果部分降低 ROMK 功能可以降低血压而不显着扰乱血清电解质，那么这项研究的结果也可能对高血压很重要。从鸟类 Kir2.2 和原核 KirBac 闭合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共振能量转移 (LRET) 光学技术直接测量状态依赖性分子距离。 AIM 1 描述了使用新型单 Cys 二聚体构建体对 Kir1.1b 尺寸进行稳态 LRET 测定，并用单个供体和单个受体进行标记。为此，我们还将进行电生理学测量，以验证这些单 Cys 二聚体作为 ROMK 门控的模型。 AIM 2 提出了状态依赖的 LRET 测量，以评估 C 端结构域启动通道开放的替代假设； AIM 3 检查内部跨膜螺旋 ROMK 束交叉处主要疏水门的替代运动。这将有助于解决一直存在争议的基尔开放态构象。最后，在 AIM 4 中，我们将评估这样的假设：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体来设置预打开条件。所提出的实验依赖于各种创新：(1) 无细胞体外真核蛋白表达，(2) 通过直接注射到非洲爪蟾卵母细胞中评估无细胞 Kr 蛋白，然后进行全细胞和切除的斑块记录 (3) 使用单 Cys ROMK 二聚体进行明确的 LRET 状态依赖性分子距离测量。