Exploiting a novel hybrid ion channel to understand the mechanism of sodium ion selectivity

几乎人体的每一个过程都在某种程度上受到电信号的控制，从我们的心脏跳动的方式，我们的肌肉运动的方式，到我们思考的方式。这些电信号是由一个称为“离子通道”的蛋白质家族产生和控制的，这些蛋白质存在于每个活细胞的膜中，并充当“电开关”来控制带电离子如钠（Na+）进入细胞的选择性运动，并允许钾（K+）离开细胞。因此，绝对必要的是，这些膜孔能够对这些离子具有“选择性”，即区分Na+和K+，否则这些离子的运动将变得混合，这些电信号将无法发生。要了解控制这种“离子选择性”的基本机制，需要对这些蛋白质的三维形状和结构有详细的了解。获得高分辨率结构信息的最常见方法包括纯化蛋白质，然后将其浓缩，使其形成晶体，类似于食盐溶液在开始干燥时结晶的方式。然后将这些晶体置于X射线束中，这些X射线束被晶体内的原子散射的方式告诉我们蛋白质的3D结构。关于通道选择K+离子而不是Na+离子的方式已经知道很多，2003年诺贝尔化学奖授予了Rod MacKinnon教授（洛克菲勒大学），以确定细菌K+通道中的这种机制。然而，类似通道选择Na+而不是K+的机制仍然难以捉摸，并且仍然是离子通道生物物理学的主要挑战之一。其中一个问题是很少有Na+通道产生足够的蛋白质适合晶体学。我们已经确定了一个可能的解决方案，这个问题，因为我们最近克隆了一个新的细菌Na+通道基因，这是简单的结构和非常相似的其他几个相关的K+通道已经结晶。我们已经进行了广泛的初步研究，证明了这个项目的可行性，即我们表明，这种新的通道（KirBac9.1）表达良好，可以在一个完整的功能形式纯化。更重要的是，我们还证明，这种纯化的蛋白质能够形成蛋白质晶体，产生“衍射图案”，即确定其结构所需的数据。基于这些令人兴奋的新发现，我们寻求资助两名研究助理和相关费用，以追求这一令人兴奋的新机会，以确定这种新型Na+通道的结构，并进一步验证其功能特性。这将使我们能够在这些成果的基础上继续推进这个项目。这些目标的实现将产生国际影响，因为它不仅将解开这一最基本的生物学过程的基本分子机制，而且还将为理解这一过程如何在人类Na+通道中发生提供框架。这些知识的潜在应用不仅有利于基础科学，而且对新药的设计也有重大影响，因为Na+通道是重要的治疗靶点。

Almost every single process in the human body is controlled at some level by electrical signals, from the way our hearts beat, the way our muscles move, to the way we think. These electrical signals are generated and controlled by a family of proteins called 'ion channels' which reside in the membrane of every living cell and which act as 'electrical switches' to control the selective movement of charged ions like sodium (Na+) into the cell and and to allow potassium (K+) out of the cell. It is therefore absolutely essential that these membrane pores are able to be 'selective' for these ions i.e. to distinguish between Na+ and K+, as otherwise the movement of these ions would become mixed up and these electrical signals would not be able to happen. To understand the fundamental mechanisms which control this 'ionic selectivity' requires a detailed knowledge about the three-dimensional shape and structure of these proteins. The most common way of obtaining high-resolution structural information involves purifying the protein and then concentrating it so that it forms crystals, similar to the way a solution of table salt crystallises when it begins to dry out. These crystals are then placed in an X-ray beam and the way in which these X-ray beams are scattered by the atoms within the crystal tells us about the 3D-structure of the protein. A lot is already known about the way in which channels select for K+ ions over Na+ ions and the 2003 Nobel Prize in chemistry was awarded to Prof Rod MacKinnon (Rockefeller University) for determining this mechanism in a bacterial K+ channel. However, the mechanism by which similar channels select for Na+ over K+ remains elusive and remains one of the major challenges in ion channel biophysics. One of the problems has been that there are very few Na+ channels which produce enough protein suitable for crystallography. We have identified a possible solution to this problem as we have recently cloned a novel bacterial Na+ channel gene which is simple in structure and very similar to several other related K+ channels which have already been crystallised. We have conducted extensive preliminary investigations which demonstrate the viability of this project i.e. we show that this novel channel (KirBac9.1) expresses well and can be purified in an intact functional form. More importantly, we also demonstrate that this purified protein is capable of forming protein crystals which produce 'diffraction patterns' i.e. the data required to determine their structure. Based upon these exciting new findings we seek funding for two research assistants and associated costs to pursue this exciting new opportunity to determine the structure of this novel Na+ channel and further characterise its functional properties. This will enable us to build upon these results and move this project forwards. Achievement of these goals would have an international impact as it would not only unlock the basic molecular mechanisms of this most fundamental biological process, but would also provide a framework for understanding how this process occurs in human Na+ channels. The potential applications of this knowledge would not only benefit basic science but also have a major effect on the design of novel drugs, as Na+ channels are important therapeutic targets.