Structure of acid-sensing ion channels studied using atomic force microscopy

细胞之间的信号传递涉及细胞膜中通道的运作。这些通道的打开让离子穿过细胞膜，从而改变细胞的行为;例如，激活或抑制神经元。我们打算研究的通道被称为酸敏感离子通道，或ASIC，它会响应细胞周围介质酸度的增加而打开。当打开时，通道让钠离子穿过膜进入细胞。ASIC参与重要的功能，如感知疼痛，对机械刺激的反应，以及学习和记忆。它们似乎也参与炎症等病理过程。我们希望更多地了解ASIC是如何组装的，希望我们能够设计出更好的治疗方法（例如使用新药）来治疗炎症等疾病。ASIC蛋白有几种形式，已知完整的通道由几种单独的蛋白质或亚基构成。亚单元围绕着一个中央通道构建，离子通过该通道。ASIC可以由相同亚基的多个拷贝或两个或更多个不同亚基的混合物制成。我们不知道有多少亚基组成一个通道，也不知道不同类型的亚基以何种方式组装在一起。我们已经开发了一种新的技术来观察多亚基蛋白质的结构。该技术涉及使用原子力显微镜（AFM），其工作原理是通过在样品表面上扫描尖锐的探针。当探针遇到蛋白质时，它会发生偏转，这些偏转被感知并用于构建样品的图像。AFM能够看到物体，例如光学显微镜看不到的单个蛋白质分子。AFM的另一个优点是样品可以在流体中成像，再现蛋白质存在的正常条件。ASIC将被标记一个短的蛋白质序列，使它们能够从培养的细胞中分离出来，并通过使用针对该标签的抗体进行鉴定。当通道与抗体一起孵育时，两种蛋白质之间的复合物产生，可以通过AFM可视化。从复合物的几何形状，我们将能够推断出通道内亚基的排列。例如，如果通道由四个亚基组成，那么当它被两个抗体结合时，抗体之间的角度应该是90度或180度，这取决于结合的亚基是彼此相邻还是被另一个亚基分开。我们将使用这种方法来确定由一种类型的亚基组成的通道内亚基的排列。通过在两个不同的子单元上放置不同的标签，我们也将能够回答关于从两个子单元构建的通道的相同问题。

Signalling between cells involves the operation of channels in the cell membranes. The opening of these channels lets ions pass across the membranes, which changes the behaviour of the cells; for instance, activating or inhibiting neurons. The channels that we propose to study are called acid-sensing ion channels, or ASICs, which open in response to an increase in the acidity of the medium around the cell. When opened, the channels let sodium ions pass across the membrane into the cell. ASICs are involved in important functions, such sensing pain, responding to mechanical stimuli, and learning and memory. They also seem to be involved in pathological processes such as inflammation. We would like to know more about how the ASICs are assembled in the hope that we might then be able to design better treatments (e.g. using new drugs) for conditions like inflammation. There are several forms of the ASIC protein, and it is known that complete channels are constructed from several individual proteins, or subunits. The subunits are built around a central channel, through which the ions pass. ASICs can be made either from multiple copies of the same subunit, or from mixtures of two or more different subunits. We do not know how many subunits comprise one channel or in what arrangements the different types of subunit assemble together. We have developed a new technique for looking at the structure of multi-subunit proteins. The technique involves the use of atomic force microscopy (AFM), which works by scanning a sharp probe over the surface of the sample. When the probe encounters a protein, it is deflected, and these deflections are sensed and used to construct a picture of the sample. AFM has the ability to see objects, such as individual protein molecules that are invisible by light microscopy. An additional advantage of AFM is that the sample can be imaged under fluid, reproducing the normal conditions under which the protein exists. ASICs will be tagged with a short protein sequence that allows them to be isolated from cultured cells and identified by the use of an antibody raised against the tag. When the channels are incubated with the antibody, complexes between the two proteins are produced that can be visualized by AFM. From the geometry of the complexes, we will be able to deduce the arrangement of the subunits within the channel. For example, if the channel consists of four subunits, then when it is bound by two antibodies, the angles between the antibodies should be either 90 or 180 degrees, depending on whether the bound subunits are adjacent to each other or separated by another subunit. We will use this method to determine the arrangement of subunits within a channel made from one type of subunit. By placing different tags on two different subunits, we will also be able to answer the same question about channels built from two subunits.