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

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.

The aim of this project is to determine the structure of acid-sensing ion channels, or ASICs. ASICs are widely expressed in the central and peripheral nervous systems, and are involved in various sensory modalities such as nociception, mechanotransduction in the peripheral nervous system, and learning and memory. There are several types of ASIC subunit - ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3 and ASIC4. We will first determine the subunit stoichiometry of the homomeric ASIC1a channel complex. The channel subunit will be tagged with His6 to facilitate its isolation from a heterologous expression system. ASIC1a channels will be imaged by atomic force microscopy (AFM). The molecular volume of the channel will be determined and compared with the volume expected for a single subunit, to reveal the channel stoichiometry. We will also incubate the isolated channel with anti-His6 antibodies. The resulting channel-antibody complexes will then be imaged by AFM, and the channel stoichiometry will be determined using the geometry of the antibody-tagged channels. We will then study ASIC1a/3 and ASIC1a/2a heteromers, after producing subunit constructs tagged with different epitopes. ASIC1a will have a His6 tag, whereas the other subunit (e.g. ASIC2a or ASIC3) will have a different tag, such as Myc. Isolated channels will be decorated with antibodies against either tag, and separate liganding profiles will be constructed. The profiles for channel decoration by the two antibodies will reveal whether the hetero-oligomers have a unique stoichiometry and a unique subunit arrangement. We will image the channels after integration into a lipid bilayer in an attempt to visualise the channel pore. We will study the distribution of ASICs in domain-forming bilayers consisting of DOPC and sphingomyelin. The distribution of the ASIC between the two lipid domains will be determined, and the effect of manipulation of the cholesterol content of the bilayer on this distribution will be examined.