Activation mechanisms of voltage-gated K+ and Na+ channels

电压门控离子通道是非常重要的膜蛋白，其通过跨膜电位的变化而被激活和失活。电压门控离子通道的激活机制仍然是难以捉摸的，部分原因是缺乏高分辨率的结构信息，在静息状态下的膜电位的存在下的通道。 了解静息状态下电压门控离子通道的结构将有助于理解其激活机制。在这里，我建议使用膜蛋白生物化学，生物物理学和subtomogram平均的组合，以获得在高分辨率下的受控膜电位封闭的脂质囊泡中的电压门控离子通道的结构信息。具体而言，我们的目标是确定三种不同的电压门控离子通道在静息状态下的静息状态结构：（1）细菌电压门控钠通道NaChBac，（2）传统的哺乳动物电压门控钾通道Kv1.2和（3）具有非交换结构域的非传统哺乳动物电压门控通道。实验方法包括蛋白质的异源表达、其纯化、重组成脂质体、生物物理表征和结构分析。为了在生物解释中取得成功，关键是要实现高分辨率，从低温电子断层扫描图，这是原位结构分析的方法的选择亚断层平均。对于这种方法，将进行优化图像处理工作流程的开发。我们将使用计算机视觉领域的现代方法，以便对具有各向异性分辨率的噪声3D体积进行更精确的配准。我们将进一步考虑几何和电子光学畸变典型的处理subtomogram平均数据。两者合计的方法的发展将允许达到α-螺旋，甚至近原子resolutions.The开发的方法subtomograms平均将是一般使用，他们将被释放和使用的其他研究人员和我们确定的膜电位下封闭的脂质囊泡中的离子通道的结构。了解静息状态下电压门控离子通道的结构将大大加深我们对电压门控的分子机制的理解，这将为开发针对多种心脏和神经系统疾病的更具选择性的药物奠定基础。

Voltage-gated ion channels are critically important membrane proteins that are activated and deactivated by changes in electric potential across membranes. The activation mechanism of voltage-gated ion channels remains elusive, in part due to lack of high-resolution structural information on the channels in resting state in the presence of membrane potential. Knowledge of the structure of voltage-gated ion channels in resting state will allow understanding their activation mechanisms. Here, I propose to use a combination of membrane protein biochemistry, biophysics, and subtomogram averaging to obtain structural information on voltage-gated ion channels in closed lipid vesicles under a controlled membrane potential at high resolution. Specifically, we aim to determine the resting state structures of three different voltage-gated ion channels in the resting states: (1) a bacterial voltage-gated sodium channel NaChBac, (2) a conventional mammalian voltage-gated potassium channel Kv1.2 and (3) a non-conventional mammalian voltage-gated channel with non-swapped domains. Experimental approach involves heterologous expression of the the proteins, their purification, reconstitution into liposomes, biophysical characterization and structural analysis. In order to succeed in biological interpretation, it is critical to achieve high resolution with subtomogram averaging from cryo electron tomograms which is the method of choice for structural analysis in situ. For this method developments optimizing the image processing workflows will be performed. We will use modern approaches from the computer vision field in order to perform more precise registration of noisy 3D volumes with anisotropic resolution. We will further account for the geometrical and electron optical distortions typical to processing of subtomogram averaging data. Taken together the method developments will allow reaching alpha-helical or even near-atomic resolutions.The developed methods for subtomogram averaging will be of general use; they will be publically released and used by other researchers and by us for determining the structures of ion channels in closed lipid vesicles under the membrane potential. Knowledge of the structures of the voltage-gated ion channels in their resting states will significantly further our understanding of the molecular mechanisms of voltage gating which will serve as a basis for the development of more selective drugs against multiple cardiac and neurological disorders.