Fluctuations in Ionic Current Through Membrane Channels

通过膜通道的离子电流的波动

• 批准号: 6994480
• 负责人: FREDERICK J SIGWORTH
• 金额: $ 58.34万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-01-01 至 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6994480
• 关键词: Xenopus oocyte; action potentials; biological transport; conformation; cryoelectron microscopy; electrophysiology; fluorescence resonance energy transfer; gene expression; inositol phosphates; ion transport; membrane channels; membrane potentials; membrane transport proteins; muscle cells; potassium channel; protein structure; site directed mutagenesis; voltage /patch clamp; voltage gated channel

DESCRIPTION (provided by applicant): About 30% of the human genome codes for membrane proteins, but membrane protein structures account for far fewer than 1 % of the entries in the Protein Data Bank. Of particular interest to neuroscience would be structures of ion channels and membrane receptors, even at low resolution but in multiple functional states so that the molecular motions that underlie their actions are visualized. Toward this goal we are working on a general and flexible method for the imaging of membrane proteins, in a membrane environment, using electron microscopy of cryogenically-cooled specimens (cryo-EM) and computer image processing. The application of this method will first be to the IPS receptor, an intracellular calcium release channel that is responsible for calcium signaling in brain and in many other cell types. We plan to observe this channel's structure in its closed and open states, in membrane vesicles. The large-conductance, calcium and voltage-activated potassium channel (BK channel) has roles ranging from the provision of local feedback in presynaptic nerve terminals to the control of blood pressure. Its characteristics also make it particularly suited to structural studies of the S4 voltage-sensor of voltage gated ion channels. Our goal is to observe the structure of its voltage sensor in both the activated and deactivated states, while the channel is in a membrane environment. To complement the structural work on the BK channel we seek to measure the time course of its conformational changes using fluorescence techniques. Making use of a library of fluorescent BK channel fusion proteins, we will employ fluorescence energy transfer to monitor changes in inter-domain distances in BK channels in a patch-clamped membrane.

描述（申请人提供）：大约 30% 的人类基因组编码膜蛋白，但膜蛋白结构仅占蛋白质数据库条目的不到 1%。神经科学特别感兴趣的是离子通道和膜受体的结构，即使在低分辨率下但处于多种功能状态，以便使它们行为背后的分子运动可视化。为了实现这一目标，我们正在研究一种通用且灵活的方法，在膜环境中使用低温冷却标本的电子显微镜 (cryo-EM) 和计算机图像处理来成像膜蛋白。该方法首先应用于 IPS 受体，这是一种细胞内钙释放通道，负责大脑和许多其他细胞类型中的钙信号传导。我们计划观察膜囊泡中该通道的关闭和开放状态的结构。 大电导钙和电压激活钾通道（BK 通道）的作用范围广泛，从提供突触前神经末梢的局部反馈到控制血压。其特性还使其特别适合电压门控离子通道的 S4 电压传感器的结构研究。我们的目标是观察其电压传感器在激活和停用状态下的结构，同时通道处于膜环境中。 为了补充 BK 通道的结构工作，我们寻求使用荧光技术测量其构象变化的时间过程。利用荧光 BK 通道融合蛋白库，我们将利用荧光能量转移来监测膜片钳膜中 BK 通道的域间距离的变化。