CHEMICAL & MOLECULAR PROPERTIES OF ION CHANNEL PROTEINS

化学

• 批准号: 2332923
• 负责人: TED B BEGENISICH
• 金额: $ 19.25万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-12-01 至 1999-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2332923
• 关键词: Xenopus; Xenopus oocyte; chemical kinetics; chimeric proteins; conformation; cysteine; divalent cations; histidine; lysine; mathematical model; molecular cloning; potassium channel; protein structure function; site directed mutagenesis; voltage /patch clamp; voltage gated channel; zinc

Voltage-dependent, potassium ion selective, membrane proteins exist in virtually all animal cells. The physiological roles of these K channels are diverse and include the maintenance of cell resting potentials, repolarization of cell action potentials, and regulation of rhythmic electrical activity. As a consequence, these channels are involved in various pathologies and are the targets of diverse therapeutic agents including those directed toward diseases of the pancreas and the cardiovascular system. Molecular level knowledge of the structure and function of these channels will be valuable in understanding the pathologies and will lead to the design of improved therapeutic measures. The long-term goal of this research is to provide information for a molecular level view of the conformational change process in voltage- gated K channels. A combination of biochemical, molecular biological, and electrophysiological methods will be used to examine certain specific features of this process in channels of known primary structure. Previous studies of the channel conformational change process have concentrated on control by membrane voltage-ignoring weakly voltage dependent conformational changes. Our past work has implicated a histidine group in some K channels that is involved in a weakly voltage dependent step in channel opening. One specific focus of the research proposed here is to determine the location of this amino acid and confirm its role in channel gating. Another specific goal of this proposal is to identify the region of the channel protein that contains the site for modulation by external divalent cations like Ca2+. Drawing on work in the past grant period, we hypothesize two possible loci which will be tested with site specific mutagenesis and electrophysiological methods. Channel gating is substantially perturbed by divalent cations interacting with an important internal region of these proteins. Another study in this application is designed to provide information on the chemical nature of this region. In all these studies, cloned ion channels will be expressed by RNA injection in Xenopus oocytes. Voltage clamp electrophysiology techniques will be used to assay channel function and will include measurement of macroscopic and single channel currents. The functional properties that are the focus of this research are common to many K channel types in cells as diverse as lymphocytes and heart and brain cells. Thus, the knowledge gained in these studies will be of general utility in revealing the molecular properties of the physiology , pathology, and pharmacology of ion channels.

电压依赖性、钾离子选择性、膜蛋白存在于 几乎所有动物细胞。 这些 K 通道的生理作用 多种多样，包括维持细胞静息电位， 细胞动作电位的复极化和节律的调节 电活动。因此，这些渠道参与 多种病理学，是多种治疗药物的靶标 包括那些针对胰腺疾病和 心血管系统。分子水平的结构知识 这些渠道的功能对于理解 病理学并将导致改进治疗措施的设计。 这项研究的长期目标是为 电压构象变化过程的分子水平视图 门控 K 通道。生物化学、分子生物学和 电生理学方法将用于检查某些特定的 该过程在已知一级结构的通道中的特征。 先前对通道构象变化过程的研究有 集中于膜电压控制-忽略弱电压 依赖性构象变化。我们过去的工作涉及到 某些 K 通道中的组氨酸基团参与弱电压 通道打开的相关步骤。研究的一个具体重点 这里提出的是确定该氨基酸的位置并确认 它在通道门控中的作用。该提案的另一个具体目标是 识别通道蛋白的区域，其中包含以下位点： 通过外部二价阳离子如 Ca2+ 进行调节。借鉴工作中 过去的资助期，我们假设两个可能的基因座将被测试 采用位点特异性诱变和电生理学方法。渠道 门控受到与相互作用的二价阳离子的显着干扰 这些蛋白质的重要​​内部区域。这方面的另一项研究 应用程序旨在提供有关化学性质的信息 这个地区。在所有这些研究中，克隆的离子通道将被表达 通过将 RNA 注射到非洲爪蟾卵母细胞中。电压钳电生理学 技术将用于分析通道功能，包括 宏观和单通道电流的测量。功能性的 本研究重点关注的属性对于许多 K 来说都是共同的 淋巴细胞、心脏和大脑等多种细胞中的通道类型 细胞。 因此，在这些研究中获得的知识将具有一般性 在揭示生理学分子特性方面的实用性， 离子通道的病理学和药理学。