Understanding the Molecular Mechanisms of Gating in TREK K2P K+ Channels

Our ability to sense changes in pressure and temperature, as well as our ability to detect a wide variety of chemical agents, is not only essential for normal bodily function, but also for the perception of pain. Understanding the molecular mechanisms which control these processes represents one of the most important goals in sensory biology.

When our body comes into contact with potentially dangerous stimuli a complex series of events initiates innate protective mechanisms designed to minimize or avoid injury. For example, extreme temperatures, mechanical stress, and chemical irritants such as acid are detected by specialised receptors clustered at the ends of sensory nerve fibres which convert these stimuli into electrical signals. These signals are then rapidly transmitted from distant sites in the body to the spinal cord and to higher processing centres in the brain which interpret these signals to initiate an appropriate response.

These electrical signals are orchestrated by distinct groups of cell membrane proteins known as 'ion channels' of which there are many hundreds of different kinds in the human body. However, there is now significant evidence that one particular group known as the 'two-pore' or 'K2P' family of potassium selective channels play an important role at many different stages of this pathway, including the specific detection of both normal and painful stimuli.

Although the sensation of pain is generally beneficial for the avoidance of greater overall tissue damage, unwanted pain confers a substantial burden on individuals, employers, healthcare systems and society in general. Indeed, the personal and socioeconomic impact of chronic pain is as great as, or greater, than that of other established healthcare priorities. There is therefore a tremendous need for better and more effective drugs for the treatment of pain and K2P channels represent attractive therapeutic targets for such drugs.

In a major recent advance, we have now determined the 3D structures of two human K2P channels (TREK-1 and TREK-2) using X-ray crystallography. We were also able to determine their structures in different conformational states which has provided new insights into how these channels open and close to 'switch' electrical signals on and off.

More importantly, we were also able to solve the structure of TREK-2 in complex with an inhibitor, fluoxetine (Prozac). Although not the principal target of this drug, identification of the binding site has provided an important insight into the biophysical mechanisms of TREK-2 channel gating and regulation by small molecules, as well as some of the potential off-target effects of this commonly prescribed drug.

In this research project we aim to exploit these exciting new findings to define a structural basis for how K2P channels open and close to control electrical signals, and also to understand how other small molecules and physiologically relevant regulatory pathways control this process.

The proposed industrial partnership with Pfizer Neusentis also provides us with access to a variety of chemical tools, expertise and resources not normally available in a standard academic environment, and therefore places us in a unique position to be able to pursue these goals.

In this industrial partnership with Pfizer Neusentis, we will use an integrated, multidisciplinary approach to address the fundamental biological questions of how an ion channel can open and close, and how this 'gating' process is regulated.

The selective up-regulation of K2P channel activity by small molecules represents an attractive strategy for the treatment of pain. However, such strategies are often limited by a lack of knowledge about the 3D structures of the protein targets involved and their mechanisms of regulation.

Fortunately, we have recently determined the X-ray crystal structures of two human K2P channels (TREK-1 and TREK2) in different conformations, and also solved structures of TREK-2 in complex with an inhibitor, fluoxetine. This has provided important insights into how these channels work, and now presents an exciting experimental framework to address more complex questions about the molecular mechanisms of K2P channel gating and regulation.

We propose to combine some of the most recent advances in membrane protein structural biology (LCP and xFEL) with a detailed biophysical, computational and functional analysis of the archetypal K2P channels TREK-1 and TREK-2. This will allow us to probe the structural basis of K2P channel gating and define the molecular mechanisms which underlie their regulation by small molecules and other physiologically relevant regulatory pathways.

Importantly, we now have extensive evidence demonstrating the feasibility of this approach. Also, the support of our industrial partner provides us with access to a range of chemical probes and experimental resources not normally available in an academic research environment.

This project therefore not only represents a unique and timely opportunity to make significant advances in our fundamental understanding of ion channel function, but also establishes an unparalleled framework for the future design of therapeutic strategies which target K2P channels.

The proposed research project will be conducted in partnership with Pfizer Neusentis as well as several world-leading international collaborators. The project therefore addresses several strategic priorities by providing an example of 'collaborative research with users' and 'international partnerships' that will increase the impact of BBSRC-funded research. The project therefore has the potential to bring considerable benefit to the UK economy and society in general.

The immediate scientific objectives within this proposal are fundamental in nature. However, the long-term social and economic benefits that will arise from associated improvements in the design and development of therapeutic strategies which target K2P channels are enormous.

A key scientific priority addressed by this project is 'healthy ageing across the lifecourse'. The unwanted sensation of pain confers a substantial burden on individuals, employers, healthcare systems and society in general. Indeed, the personal and socioeconomic impact of chronic pain is as great as, or greater, than that of other established healthcare priorities. Healthy ageing therefore requires better and more effective drugs for the treatment of pain, and K2P channels represent important targets for such drugs. However, to fulfil this potential requires a fundamental understanding of the intimate relationship between the structure of a K2P channel, and its complex functional properties. Our two groups, and those of our collaborators, have a history of delivering high impact publications on these topics, and we propose to continue leading this field.

Overall, the development of new, more effective and more specific drugs to treat both chronic and acute pain will benefit significant sections of the UK population. However, there are many different types of K2P channels found throughout the body. Therefore, increasing our knowledge of how these channels function also has the potential to impact the treatment of a range of cardiovascular and neurological disorders.

Furthermore, such benefits are not just restricted to human health and well-being. The K2P superfamily is diverse and similar channels are found in many other organisms including numerous species of phytopathogenic fungi and parasitic nematodes. This study therefore also has the potential to drive improvements in animal health, veterinary medicine and plant biology.

Other potential beneficiaries include all of the research staff directly involved with this project as well as those who are exposed to it. In particular, this project will provide two postdoctoral researchers direct experience of a wide range of cutting-edge and interdisciplinary research techniques that will enable them to enhance their career opportunities and contribute more effectively to the wider economy in the future.

The general public also has a tremendous curiosity about science and so we aim to advertise this work and highlight the relevance of the underlying scientific principles in a series of public engagement and outreach events. This will have a major impact on the public perception of science as well as public trust in UK-based scientific research programmes. It will also have the added benefit of stimulating interest in STEM subjects within the next generation of potential scientific leaders.

Finally, the proposed study will also help to raise the overall profile of industrially-sponsored basic academic research in the UK, and strengthen an interdisciplinary collaboration between two leading academic research groups. This project therefore represents a strategically important investment that will help to support and maintain the high profile that the UK currently enjoys in world-class bioscience research.