Fluctuations in Ionic Current Through Membrane Channels

描述（由申请人提供）：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的条目中所占的比例远远不到1%。神经科学特别感兴趣的是离子通道和膜受体的结构，即使是在低分辨率下，但在多种功能状态下，这样就可以可视化其行为背后的分子运动。为了实现这一目标，我们正在研究一种通用而灵活的膜蛋白成像方法，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。该方法将首先应用于IPS受体，这是一种细胞内钙释放通道，负责大脑和许多其他细胞类型的钙信号传导。我们计划在膜囊中观察这个通道在封闭和开放状态下的结构。

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.