Fluctuations in Ionic Current Through Membrane Channels

描述(申请人提供)：大约30%的人类基因组编码膜蛋白，但膜蛋白结构在蛋白质数据库中的条目中所占比例远远不到1%。神经科学特别感兴趣的是离子通道和膜受体的结构，即使在低分辨率但处于多种功能状态下也是如此，这样就可以直观地看到支撑其作用的分子运动。为了实现这一目标，我们正在研究一种通用而灵活的方法，在膜环境中使用低温冷却样品的电子显微镜(Cryo-EM)和计算机图像处理来对膜蛋白进行成像。这种方法将首先应用于IPS受体，这是一种细胞内钙释放通道，负责大脑和许多其他类型细胞中的钙信号。我们计划在膜小泡中观察这个通道在闭合和开放状态下的结构。 大电导、钙和电压激活的钾通道(BK通道)具有多种作用，从提供突触前神经末梢的局部反馈到控制血压。它的特性也使其特别适合于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失活状态下的结构，而通道处于膜环境中。 为了补充BK通道的结构工作，我们试图使用荧光技术测量其构象变化的时间进程。利用荧光BK通道融合蛋白文库，我们将利用荧光能量转移来监测膜片钳膜中BK通道结构域间距离的变化。

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.