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

Lead Research Organisation: University of Oxford
Department Name: Structural Genomics Consortium

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description Through our collaboration with Thomas Baukrowitz we have identified a novel mechanism for activation of a range of channels, including K2P channels, but also a range of other channels.
Through our collaboration with Thomas Mueller at Bayer and Niels Decher in Marburg, we have been able to identify a novel mechanism for gating TASK channels, a subset of KeP channels. This structure, which we have called an X-gate, acreates a new type of lower gate, unrelated to gates in other channels, seen only in the TASK channels in the K2P family. This structure helps to explain trapping of compounds in the TASK channels, a feature that is important for drug design and for understanding the pharmacokinetics in a range of diseases including heart, lung and sleep disorders.
Exploitation Route This information provides extensive highly valuable data for development of novel therapeutics, both in the field of small molecule and biologics.
Sectors Healthcare

 
Description LC attended the "EMBO Practical Course: High throughput LC attended and gave a talk at the EMBL: High Throughput Practical Protein Production and Crystallisation", in Hamburg, on the 12th of September, 2018.
Geographic Reach Europe 
Policy Influence Type Influenced training of practitioners or researchers
 
Description LC gave a talk at the Hamburg Life Science Summit, in Hamburg, Germany, on the 30th of January, 2018, entitled "Challenging Targets for Structural Biology and Drug Design".
Geographic Reach Europe 
Policy Influence Type Participation in a advisory committee
 
Description Collaboration with Prof. Stephen Tucker, Physics, Oxford on structure and function of K2P potassium channels 
Organisation University of Oxford
Department Department of Physics
Country United Kingdom 
Sector Academic/University 
PI Contribution The Carpenter group has solved a series of structures of K2P ion channels
Collaborator Contribution Our collaborator, Stephen Tucker, has contributed functional data on the electrophysiology of K2P channels
Impact This work has lead to a series of publication in a range of journals, please see relevant sections. This work has lead to two research council responsive mode grants, with Liz Carpenter and PI and Stephen Tucker as CoI. It has also lead to several grants in which Stephen Tucker is the PI. Post-doctoral scientists, PhD students and technicians have all been trained on this project, as a result of this collaboration, both in the Carpenter and Tucker groups. Those that have left have gone on to positions in academia and industry.
Start Year 2012
 
Description Collaborations with Philip Stansfeld and Mark Sansom on in silico molecular dynamics simulations of human membrane proteins in membranes 
Organisation University of Oxford
Department Department of Biochemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution The Carpenter group solves structures of human membrane proteins, then shares the coordinates with Phillip Stansfield and Mark Sansom in the Biochemistry Department in Oxford, and with Stephen Tucker in the Physics Department in Oxford wrt K2P potassium channel simulations.
Collaborator Contribution Our stimulations collaborators use molecular dynamics simulations to study the movements of membrane proteins in membranes in silico. This has allowed us to answer fundamental biology questions such as (1) regulation of K2P channels by membrane stretch, (2) movement of lipid scramblases in membrane and movement of headgroups of lipids across membranes through the groove in the surface of the TMEM16K scramblase. (3) conformational stability of the unusual fold of the ZMPSTE24 membrane enzyme, which encloses a large chamber inside the lipid bilayer. (4) lipid binding to the polycystic kidney disease protein PKD2.
Impact This work has lead to a series of publications on membrane protein conformational changes, structure and function. This project has provided excellent training opportunities for several post-docs and students in a combination of lab based and in silico studies.
 
Description Collaborations with Philip Stansfeld and Mark Sansom on in silico molecular dynamics simulations of human membrane proteins in membranes 
Organisation University of Oxford
Department Department of Physics
Country United Kingdom 
Sector Academic/University 
PI Contribution The Carpenter group solves structures of human membrane proteins, then shares the coordinates with Phillip Stansfield and Mark Sansom in the Biochemistry Department in Oxford, and with Stephen Tucker in the Physics Department in Oxford wrt K2P potassium channel simulations.
Collaborator Contribution Our stimulations collaborators use molecular dynamics simulations to study the movements of membrane proteins in membranes in silico. This has allowed us to answer fundamental biology questions such as (1) regulation of K2P channels by membrane stretch, (2) movement of lipid scramblases in membrane and movement of headgroups of lipids across membranes through the groove in the surface of the TMEM16K scramblase. (3) conformational stability of the unusual fold of the ZMPSTE24 membrane enzyme, which encloses a large chamber inside the lipid bilayer. (4) lipid binding to the polycystic kidney disease protein PKD2.
Impact This work has lead to a series of publications on membrane protein conformational changes, structure and function. This project has provided excellent training opportunities for several post-docs and students in a combination of lab based and in silico studies.
 
Description Jan Steyaert lab 
Organisation Vrije Universiteit Brussel
Country Belgium 
Sector Academic/University 
PI Contribution Crystallisation and functional studies with nanobodies developed in Jan Steyaert's lab
Collaborator Contribution Jan Steyaert's lab generated a series of nanobodies to TREK1 and TREK2 which we are now studying
Impact This project has provided nanobodies for crystallisation and functional studies. There are now three PhD students working on characterising these nanobodies, as well as the post-doc who works on this grant, so the work has provided projects and training opportunities for several students, in addition to the scientific impact.
Start Year 2014
 
Description TASK-1 structural and functional studies 
Organisation Bayer
Department Bayer HealthCare
Country Germany 
Sector Private 
PI Contribution The Carpenter group at the SGC produced and solved the structure of the TASK-1 K2P potassium channel, revealing a unique and completely unexpected gating system for the TASK channels. Working with Bayer we were able to show how high affinity compounds bind and block TASK-1.
Collaborator Contribution Thomas Mueller and his colleagues at Bayer had performed a very high throughput screen of > 3million compounds, to identify molecules that bound and inhibited TASK-1. They made a series of TASK-1 binding compounds available to the SGC group with which we were able to solve structures of the complexes, thus showing how the compounds inhibit the channels. Although it is not possible to put an exact monitary value of a high thoroughput screen with 3million+ compounds, this work is of termendous value to the collaboration, a unique resource only available within a small number of large Pharma companies, so the value of this work would be more than £100,000 in in kind contributions. Niels Decher's group at the University of Marburg in Germany provided a full characterisation of the novel gate and the compound binding using electrophysiology. Stephen Tucker's group in Oxford also contributed to the understanding of the TASK-1 constructs used in these studies.
Impact We have recently had a paper accepted in the journal Nature on the role of the X-gate in K2P function: Rödström, K.E.J., Kiper, A.K., Zhang, W., Rinné, S., Pike, A.C.W., Goldstein, M., Conrad, L., Delbeck, M., Hahn, M., Meier, H., Platzk, M., Quigley, A., Speedman, D., Shrestha, L., Mukhopadhyay, S.M.M., Burgess-Brown, N.A., Tucker, S.J., Mueller, T., Decher, N., Carpenter, E.P. (July, 2019). "A unique lower X-gate in TASK channels traps inhibitors within the vestibule". BioRxiv, DOI: https://doi.org/10.1101/706168. Now accepted for publication in Nature.
Start Year 2017
 
Description TASK-1 structural and functional studies 
Organisation University of Hamburg
Country Germany 
Sector Academic/University 
PI Contribution The Carpenter group at the SGC produced and solved the structure of the TASK-1 K2P potassium channel, revealing a unique and completely unexpected gating system for the TASK channels. Working with Bayer we were able to show how high affinity compounds bind and block TASK-1.
Collaborator Contribution Thomas Mueller and his colleagues at Bayer had performed a very high throughput screen of > 3million compounds, to identify molecules that bound and inhibited TASK-1. They made a series of TASK-1 binding compounds available to the SGC group with which we were able to solve structures of the complexes, thus showing how the compounds inhibit the channels. Although it is not possible to put an exact monitary value of a high thoroughput screen with 3million+ compounds, this work is of termendous value to the collaboration, a unique resource only available within a small number of large Pharma companies, so the value of this work would be more than £100,000 in in kind contributions. Niels Decher's group at the University of Marburg in Germany provided a full characterisation of the novel gate and the compound binding using electrophysiology. Stephen Tucker's group in Oxford also contributed to the understanding of the TASK-1 constructs used in these studies.
Impact We have recently had a paper accepted in the journal Nature on the role of the X-gate in K2P function: Rödström, K.E.J., Kiper, A.K., Zhang, W., Rinné, S., Pike, A.C.W., Goldstein, M., Conrad, L., Delbeck, M., Hahn, M., Meier, H., Platzk, M., Quigley, A., Speedman, D., Shrestha, L., Mukhopadhyay, S.M.M., Burgess-Brown, N.A., Tucker, S.J., Mueller, T., Decher, N., Carpenter, E.P. (July, 2019). "A unique lower X-gate in TASK channels traps inhibitors within the vestibule". BioRxiv, DOI: https://doi.org/10.1101/706168. Now accepted for publication in Nature.
Start Year 2017
 
Description "Mechanisms of Membrane Transport" Gordon Research Conference, in New London, NH, USA - 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact LC gave a talk entitled "Structures of K2P and TRPP Channels by X-Ray, xFEL and Cryo-EM" at the "Mechanisms of Membrane Transport" Gordon Research Conference, in New London, NH, USA. The meeting was from the June 25 to June 30, 2017.
Year(s) Of Engagement Activity 2017
 
Description "Molecular Mechanisms of Transporters, Ion Channels, and G-Protein Coupled Receptors Gordon Research Conference", on the 4th - 9th of March, 2018, in Ventura Park, California, 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact LC was an invited speaker at the "Molecular Mechanisms of Transporters, Ion Channels, and G-Protein Coupled Receptors Gordon Research Conference", on the 4th - 9th of March, 2018, in Ventura Park, California, where she gave a talk entitled: "Structure and Function of Ion Channels Involved in Human Genetic Disease: PKD2 and Polycystic Kidney Disease".
Year(s) Of Engagement Activity 2018
 
Description EMBO conference "Towards Novel Therapies: Emerging Insights from Structural and Molecular Biology on the 6 and 7 of March 2017 in Groningen 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact LC gave a talk at the EMBO conference "Towards Novel Therapies: Emerging Insights from Structural and Molecular Biology on the 6 and 7 of March 2017 in Groningen, The Netherlands entitled "Structural studies of human membrane proteins and the development of 'Target Enabling Packages' at the SGC".
Year(s) Of Engagement Activity 2017
URL http://events.embo.org/17-structural-biol/
 
Description K2P talk at the Weatherall Institute 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact LC gave a talk entitled "Structure and function of K2P and PKD ion channels, targets for pain and polycystic kidney disease", this was followed by a member of LC's group obtaining a fellowship to work at the Weatherall Institute.
Year(s) Of Engagement Activity 2017
 
Description Novo Nordisk Symposium, Oxford, 21st September, 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Novo Nordisk Symposium, Oxford, 21st September, 2017, LC gave a talk entitled "Structural Studies of Human Integral Membrane Proteins Involved in Genetic and Metabolic disease at the SGC".
Year(s) Of Engagement Activity 2017
 
Description Oxfordshire Science Festival - Biophysics 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Members of the public attended an open day which highlighted research in Biophysics currently taking place in Oxford Physics. Members of my group who work on this BBSRC funded project operated a display which demonstrated some of the scientific principles underlying 'why oil and water do not mix'.
Year(s) Of Engagement Activity 2016