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

利用新型混合离子通道来了解钠离子选择性的机制

• 批准号: BB/F013035/1
• 负责人: Stephen Tucker
• 金额: $ 72.96万
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2008
• 资助国家: 英国
• 起止时间: 2008 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FF013035%2F1
• 关键词: Exploiting; novel; hybrid; ion; channel

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.

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，从我们的心跳方式，肌肉运动方式，到我们的思维方式。这些电信号是由一组被称为“离子通道”的蛋白质产生和控制的，这些蛋白质存在于每个活细胞的膜上，充当“电子开关”，控制钠离子（Na+）进入细胞的选择性运动，并允许钾离子（K+）离开细胞。因此，至关重要的是，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乱，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，类似于食盐溶液在变干时结晶的方式。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，我们已经知道了很多，2003年诺贝尔化学奖授予了罗德·麦金农教授（洛克菲勒大学），因为他确定了细菌K+通道中的这一机制。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。我们已经确定了这个问题的可能解决方案，因为我们最近克隆了一种新的细菌Na+通道基因，它结构简单，与已经结晶的其他几个相关的K+通道非常相似。我们已经进行了广泛的初步调查，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，可以以完整的功能形式纯化。更重要的是，我们还证明了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，我们寻求资助两位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，并进一步表征其功能特性。这将使我们能够在这些结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生物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+通道中发生提供一个框架。这一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。

项目成果

Structure of a KirBac potassium channel with an open bundle crossing indicates a mechanism of channel gating.

• DOI: 10.1038/nsmb.2208
• 发表时间: 2012-01-08
• 期刊: NATURE STRUCTURAL & MOLECULAR BIOLOGY
• 影响因子: 16.8
• 作者: Bavro, Vassiliy N.;De Zorzi, Rita;Schmidt, Matthias R.;Muniz, Joao R. C.;Zubcevic, Lejla;Sansom, Mark S. P.;Venien-Bryan, Catherine;Tucker, Stephen J.
• 通讯作者: Tucker, Stephen J.

Modular Design of the Selectivity Filter Pore Loop in a Novel Family of Prokaryotic 'Inward Rectifier' (NirBac) channels.

• DOI: 10.1038/srep15305
• 发表时间: 2015-10-16
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Zubcevic L;Wang S;Bavro VN;Lee SJ;Nichols CG;Tucker SJ
• 通讯作者: Tucker SJ

Conformational changes during the gating of a potassium channel revealed by structural mass spectrometry.

• DOI: 10.1016/j.str.2010.04.012
• 发表时间: 2010-07-14
• 期刊: Structure (London, England : 1993)
• 作者: Gupta S;Bavro VN;D'Mello R;Tucker SJ;Vénien-Bryan C;Chance MR
• 通讯作者: Chance MR

Functional complementation and genetic deletion studies of KirBac channels: activatory mutations highlight gating-sensitive domains.

• DOI: 10.1074/jbc.m110.175687
• 发表时间: 2010-12-24
• 期刊: The Journal of biological chemistry
• 作者: Paynter JJ;Andres-Enguix I;Fowler PW;Tottey S;Cheng W;Enkvetchakul D;Bavro VN;Kusakabe Y;Sansom MS;Robinson NJ;Nichols CG;Tucker SJ
• 通讯作者: Tucker SJ

Control of KirBac3.1 potassium channel gating at the interface between cytoplasmic domains.

• DOI: 10.1074/jbc.m113.501833
• 发表时间: 2014-01-03
• 期刊: The Journal of biological chemistry
• 作者: Zubcevic L;Bavro VN;Muniz JR;Schmidt MR;Wang S;De Zorzi R;Venien-Bryan C;Sansom MS;Nichols CG;Tucker SJ
• 通讯作者: Tucker SJ