Parallel electrophysiological characterization of sodium channels

离子通道蛋白在多种生理过程和（慢性）疾病中起着关键作用，因此对制药工业具有相当大的兴趣。电生理学是研究通道蛋白功能及其药物调节的金标准，特别是在单通道水平，并且是唯一能够表征电压门控通道的方法。它涉及将电极放置在膜的两侧，并测量通过膜嵌入通道的电流，通常每个通道在1到150 pA之间。关键的挑战是获得“Gigaseal”配置，其中两个水性隔室通过稳定的细胞膜或脂质双层彼此电绝缘。这很难实现，使得传统的电生理学成为一个费力的过程，而且通量极低。电压门控钠通道负责所有真核生物和一系列细菌中钠离子跨细胞膜的运输，是一个离子通道家族，电生理学对此非常感兴趣。在人类以及低等真核生物中，这些通道对于正常功能是必不可少的;在不同的组织中发现了各种亚型，从心脏到大脑，在健康生物体中具有不同的功能作用。在人类中，钠通道突变会引起许多疾病，并与衰老和疼痛有关;因此，它们是许多药物的靶点，包括用于治疗癫痫、慢性疼痛和心血管疾病的药物。该提议旨在开发一种用于并行芯片上电生理学的新平台，该平台由南安普顿大学开发，用于电压门控钠通道家族的功能表征，包括人/细菌嵌合体，伯克贝克学院正在开发其表达、纯化和重组为脂质体。具体而言，该项目将使用这个高通量平台来识别调节钠通道电导特性的新型配体/药物。由于该项目代表了两个实验室之间的合作，这两个实验室具有与微电子和微流体技术开发相关的非常不同但互补的专业知识，以及重要通道系统的生化纯化和表征，因此它福尔斯跨学科主题“电子与生物学的接口”。它将战略研究和应用研究结合起来，并将培养跨学科科学和技术的研究生研究人员，这项研究的潜在公共和经济影响是多方面的。例如，电压门控钠通道是人类健康和农业的重要组成部分。提高对这些渠道的结构/功能/药物绑定的认识将对公共、第三和私营产业部门的受益者产生影响。此外，除了基础研究之外，新技术平台还将在药物发现和测试行业中有许多应用。当该项目的成果被领先的电生理公司采用时，可以实现具有医疗影响的商业产品。我们将通过各种途径与这些公司和其他利益相关者积极合作，包括针对高通量电生理学关键利益相关者的研讨会。为了最大限度地发挥其经济和社会影响，该项目开发的新平台及其在评估钠通道药物疗效方面的直接应用将通过专业出版物、行业参与的重大会议以及新闻稿和媒体积极参与进行传播。

Ion channel proteins play a pivotal role in a wide variety of physiological processes and (chronic) diseases and are consequently of considerable interest to the pharmaceutical industry. Electrophysiology is the gold standard for investigating the function of channel proteins and their modulation by pharmaceutical drugs, particularly at single-channel level, and is the only method that enables the characterization of voltage-gated channels. It involves placing an electrode on either side of a membrane and measuring the current flow through the membrane-embedded channels, which is typically between 1 and 150 pA per channel. The key challenge is to obtain a 'Gigaseal' configuration where the two aqueous compartments are electrically insulated from each other by a stable cell membrane or lipid bilayer. This is difficult to achieve, rendering conventional electrophysiology a laborious process with a notoriously low throughput.Voltage-gated sodium channels, responsible for the transport of sodium ions across cell membranes in all eukaryotic organisms and in a range of bacteria, represent an ion channel family for which the electrophysiology is of great interest. In humans as well as lower eukaryotes these channels are essential for normal functioning; various isoforms are found in different tissues, ranging from heart to brain, with different functional roles in the healthy organism. In humans, sodium channel mutations give rise to a number of disease states, as well as being associated with ageing and pain; as a result they are the targets of many pharmaceutical drugs, including ones for treatment of epilepsy, chronic pain, and cardiovascular diseases.This proposal aims to exploit a novel platform for parallel on-chip electrophysiology, developed at the University of Southampton, for the functional characterization of a family of voltage-gated sodium channels, including human/bacterial chimeras, for which the expression, purification and reconstitution into liposomes is being developed at Birkbeck College. Specifically, the project will use this high-throughput platform to identify novel ligands/drugs that modulate the conductance properties of the sodium channels. As this project represents a collaboration between two labs with the very different but complementary expertise associated with microelectronic and microfluidic technique development & biochemical purification and characterisation of an important channel system, it falls within the cross-disciplinary theme 'Interfacing Electronics to Biology'. It combines strategic and applied research and it will train postgraduate researchers in cross-disciplinary science and technology.The potential public and economic impacts of this research are manifold. For example, voltage-gated sodium channels are essential components in human health and in agriculture. Improved knowledge of the structure/function/drug binding of these channels would impact on beneficiaries in the public, third and private industry sectors. Furthermore, the new technology platform will have many applications in industries for drug discovery and testing in addition to those in fundamental research. Commercial products with medical impact can be realized when the outcomes of this project are taken up by the leading electrophysiological companies. We will actively engage with these companies and other stakeholders by various routes, including a workshop targeted to key stakeholders in high-throughput electrophysiology. To maximize its economic and societal impact, the novel platform developed in this project -and its direct application to evaluate sodium channel drug efficacy- will be disseminated through professional publications, at major conferences with industry participation, and through press releases and active media engagement.