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Unlocking the Potential of K2P Potassium Channels with Nanobodies

Reference	BB/T002018/1
Principal Investigator / Supervisor	Professor Stephen Tucker
Co-Investigators / Co-Supervisors	Professor Elisabeth Carpenter, Professor Ray Owens, Dr Ashley Pike
Institution	University of Oxford
Department	Structural Genomics Consortium
Funding type	Research
Value (£)	808,687
Status	Current
Type	Research Grant
Start date	01/12/2019
End date	31/05/2023
Duration	42 months

Abstract

Two-Pore Domain (K2P) K+ channels are involved in control of the resting membrane potential and represent attractive therapeutic targets for a wide range of disorders. However, a general lack of selective probes means many of their properties and functional roles are still unclear. In particular, many members remain poorly characterised, and the role of heteromeric K2P channels in cells and tissues is also still not properly understood. Fresh approaches to study their structure, function and physiology are therefore required. Single-chain nanobodies represent exciting alternatives to small molecules in a field where there is a pressing need for high affinity, selective probes. We now show that for one K2P channel, we can generate highly-selective nanobody binders, some of which also act as highly-effective activators and others as inhibitors. We have also obtained several preliminary structures of these K2P/nanobody complexes which reveal many important insights, including how to generate nanobodies against heteromeric K2P channels. Therefore, based upon the success of these initial studies, and our ability to produce K2P channel proteins for many members of this family, we aim to generate nanobodies against these other K2P channels, including their most common physiologically-relevant heteromeric combinations. We also intend to use a wide range of biophysical, biochemical, structural, and functional (electrophysiological) assays to characterise these new probes. Finally, we also aim to exploit them by examining the role of homo- and heteromeric K2P channels in native cells and tissues. The feasibility of this project is supported by extensive preliminary evidence and represents a unique and timely opportunity to make a significant advance in our understanding of these important channels.

Summary

K2P channels contribute to the basic electrical currents found in nearly all human cells. They are regulated by diverse stimuli, including acid, anaesthetics and small molecules, and represent attractive therapeutic targets for treatment of many different diseases including pain, migraine and various heart & lung disorders. However, our ability to fully understand their functional properties and role is severely hampered by a lack of selective tools that can be used analyse them in many living cells and tissues. Furthermore, a lack of selective, well validated antibodies against K2P channels means there is little convincing data on their expression profiles and localisation throughout the body. Another major complication of studying any ion channel in living tissues is that they often are made up of multiple subunits which can coassemble with related members to form novel 'heteromeric' combinations. This 'mix and match' principle of heteromultimerisation is important for generating diversity in living cells, but often complicates analysis. Better and more selective probes against these channels, especially heteromeric K2P combinations, are therefore desperately needed. Nanobodies are very small antibodies derived from an unusual type of antibody produced by camelids and sharks. Nanobodies can now be easily manipulated and produced in large quantities in bacteria. They therefore represent an exciting alternative to small molecule chemicals when searching for new methods to detect and/or manipulate K2P channels in cells and tissues. We now have extensive preliminary data demonstrating the feasibility of our proposed approach and so intend to use these new probes to unlock the structure, function and physiological role of these important ion channels.

Impact Summary

Many common diseases involving the peripheral and central nervous system as well as the pulmonary and cardiovascular systems are now associated with K2P channel dysfunction. These include various forms of pain, migraine, depression, obstructive sleep apnea, pulmonary hypertension, cardiac arrhythmias, neuroinflammation and diabetes. The long-term social and economic benefits that may eventually arise from improvements in our understanding of these channels, and the development of new therapeutic strategies that target them, are therefore significant. Such benefits are also not just restricted to human health and well-being. K2P channels are also found in many other organisms including numerous species of phytopathogenic fungi and parasitic nematodes, as well as many plants, and other organisms. This study therefore also has the potential to drive improvements in animal health, veterinary medicine and plant biology. Furthermore, in addition to the obvious scientific impact this study will have, the general public has a tremendous curiosity about science. We therefore aim to advertise this work widely and to highlight the relevance of the underlying scientific principles in public engagement and outreach events. This will have a major impact on the public perception of science. It also has the potential to enhance public trust in UK-based scientific research programmes, especially the importance of 'basic bioscience underpinning health'. Finally, the project will also help strengthen interdisciplinary collaboration between a number of different UK and international research groups, as well as a range of our industrial partners who are also interested in the outcome of this study. It therefore represents a strategically important investment that will help to support and maintain the world-class profile that UK bioscience research currently enjoys.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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