Gating mechanism of a potassium channel (KirBac3.1) studied using high resolution cryo electron crystallography

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 ion channels which act as electrical 'nano-switches' to control the selective movement of charged ions like potassium (K+) and sodium (Na+) into and out of the cell. As a consequence of this fundamental importance, a large number of genetically inherited diseases or 'channelopathies' are the result of defective ion channel function. These diseases include cystic fibrosis, diabetes, epilepsy and many other rarer diseases of the heart, brain, nerves and kidneys. Ion channels also have considerable potential to be exploited as molecular switches in nanoscale electrical devices and so understanding how they open and close, and how this process is controlled is of considerable interest. Inwardly-rectifying potassium (Kir) channels are expressed in almost every cell of the body and underlie the basic electrical activity which is found in these cells. Structural studies of these human channels is extremely difficult. However, related 'KirBac' channels exist in bacteria and these are much more amenable to biochemical and structural studies. In our preliminary investigations we have expressed and purified KirBac3.1 and shown we are able to obtain images of this channel in both the open and the closed state using a powerful electron microscope. In this proposal we aim to use a new state-of-the-art microscope and mutant KirBac3.1 proteins to improve the resolution of our images we are able to achieve. This will allow us to obtain high-resolution images of the KirBac3.1 channel in both the closed and open states and to address the structural changes which occur as the channel changes shape between the open and closed states. Improving our understanding of how this process occurs at the molecular level will provide a major insight into how related K+ channels open and close and how this process becomes defective in the disease state.

Inwardly-rectifying potassium (Kir) channels control many important electrical processes in the body. As these dynamic proteins open and close to control the flow of K+ ions across the membrane they undergo large conformational changes. Yet, despite detailed information about the closed-state structure of these channels, we still know very little about their open state structure. This represents a major problem to understanding their molecular mechanism of gating. We can produce 2D crystals of a prokaryotic Kir homolog (KirBac3.1) trapped in either the open state or the closed state, and have obtained low resolution projection images of these 2D crystals. In this proposal we aim to harness state-of-the-art electron crystallographic approaches to determine a high resolution 3D structure of the KirBac3.1 open state. Our extensive preliminary studies show that we can produce high quality 2D crystals suitable for high-tilt angle imaging and 3D image reconstruction. To increase the resolution of our images we have access to a powerful Titan Krios FEI Microscope which is unique in Europe and is ideal for advanced, high resolution, 2D electron crystallography. We have also developed novel approaches to improve the quality of our open state crystals by generating a number of gating mutations in KirBac3.1 which stabilise the channel in the open state. These mutant channels will improve the stability and ordering of our 2D open state crystals and further increase the resolution we are able to achieve. The use of this high-powered microscope and well-ordered crystals will enable us to obtain high resolution 3D images of the channel in the open state, and allow us to begin addressing the dynamic conformational rearrangements which occur during channel gating.

Inwardly rectifying potassium channels are found in almost every cell type in the body where they control membrane electrical excitability and many K+ transport processes. This physiological importance is highlighted by the fact that genetically inherited defects in Kir channels are responsible for a number of human diseases (channelopathies) including Bartter's syndrome, Andersen's Syndrome, and certain forms of neonatal diabetes and other insulin secretory disorders. In this basic science proposal, we aim to study the structure of a prokaryotic Kir homolog, KirBac3.1 stabilised in the open state at high resolution in order to address the fundamental biophysical principles which underlie the gating mechanism. It will allow us a greater understanding of the mechanism of gating of both bacterial KirBac and mammalian Kir channels and provide a huge impetus to both academic and applied research in the field. Although the proposed study is unashamedly fundamental in its experimental approach, its objectives are firmly rooted in furthering our understanding of a group of proteins which pay a major role in health and disease and the long-term potential implications of determining such an open state structure are enormous. The technological developments made during the course of this study will also be important for the field of membrane protein structural biology and potentially expand the range of targets which can be examined with this technique. The potential application of ion channels as electrical nano-devices in bionanotechnology also requires a greater understanding of the mechanism by which they open and close. This study will ultimately assist with that objective. The principal beneficiaries of the work outlined in this proposal will therefore include those working in both pure and applied ion channel research here in the UK and internationally. The study will provide a foundation upon which more specific applied research can be based to address the role of these channels in health and disease. Furthermore, the rational design of novel therapeutic strategies which target these channels also relies upon a basic understanding of the intimate relationship between the ion channel structure and function. This study will therefore help to achieve that goal by providing a fundamental insight into the structural mechanism of Kir channel gating and regulation, and how this process becomes defective in the disease state. Achievement of the objectives outlined in this proposal would also place UK ion channel research at the forefront of international work in this field. Finally, the professional development of the PDRA employed on this project will also be carefully considered . They will be exposed to many different experimental and computational techniques and have opportunity to develop a wide range of transferable skills, including working with and training graduate research students working alongside them. They will also have considerable opportunity to interact with our collaborative partners, both in Oxford and in Switzerland. Their ability to engage in such international cooperation is important for generating a greater awareness of how scientific research is conducted in different laboratories and in different countries. They will also be encouraged to present their findings at both national and international conferences and to engage with existing programmes within the University of Oxford for the public dissemination of their work.