Exploitation of a Pharmacological 'Master Switch' to Probe the K+ Channel Selectivity Filter Gating Mechanism

Almost every living cell is electrically active because a voltage difference exists across its membrane. However, unlike the electronic currents we are so familiar with domestically , this 'biological electricity' results from the movement of charged ions (like Na+ and K+) into and out of cells through tiny pores in their membrane known as 'ion channels'.

These ion movements generate electrical currents that control or influence almost every process in the human body, from the way we move and breathe, the way our heart and kidneys work, to the way we think, and how we sense pain. Even bacteria use similar changes in their membrane potential to control important processes. So it is no overstatement to say that ion channels are essential for all forms of life on earth. As a consequence, a wide range of medicinal drugs and many poisons exert their effects by regulating the activity of these tiny molecular machines, and many human and animal diseases result from impaired ion channel function.

Potassium (K+) selective channels are one of the largest groups of ion channels; there are >80 different human K+ channel genes and K+ channels are found in almost every living organism (and even some viruses). As a result, their structural architecture is very highly conserved across these different life forms.

To operate effectively, all K+ channels need two key properties: a 'filter' so they can be selective for K+ over other ions, and a 'gate' that opens and closes to regulate the flow of ions. This opening and closing or 'gating' is regulated by many different cellular signals (including many potential drugs). This makes K+ channels attractive therapeutic targets for the treatment of disease.

The first 3D structure of a K+ channel was determined exactly 20 years ago and revealed how the K+ selective filter and gate are formed by two distinct features at either end of the channel. However, more recent studies now suggest that in some types of K+ channel the normal (lower) gate remains open, and instead all regulation of ionic flow is done via the selectivity filter which acts as both filter AND gate.

Unfortunately, the mechanism of filter gating has been difficult to study because few tools exist to manipulate this process. Some computational approaches can directly study the permeation of individual ions, but these measurements are often limited to just a few millionths of a second and even the most sensitive of electrical recordings are unable to directly measure ion channel activity on this timescale. Consequently, it is difficult to relate the movement of ions to the 3D structure of a channel and new approaches to this problem are required.

Luckily, we have now identified a new class of drugs which interact with the filter gate to directly activate the flow of K+. Interestingly, these drugs work across a range of different K+ channels and therefore provide a new set of tools to manipulate the filter gate and dissect its mechanism of action.

In this project we propose to exploit these exciting new tools to manipulate ion permeation through single ion channels. Other approaches will also allow us to compare our measurements with structural and computational models of permeation. In addition, these studies may allow us to classify (and predict) how other K+ channel drugs interact with the channel, and where they may bind. This will have important implications for the rational design of new drugs that target these channels.

Overall, this proposal is supported by extensive data that demonstrate the feasibility of our approach. It therefore offers a unique and timely opportunity to understand this most fundamental of biological signalling processes, and it will improve our ability to design new drugs that target K+ channels.

Advances in crystallography and cryo-EM have led to an explosion in the number of ion channel structures being solved, including many eukaryotic K+ channels. However, our precise understanding of how K+ channels work is often limited by our ability to relate these high-resolution snapshots to the detailed electrophysiological assays of channel activity developed over the past 50 years.

In this study we propose an integrated, multidisciplinary approach to ask the fundamental question of how do K+ channels open and close, and how is this 'gating' process is regulated?

In particular, we aim to address the mechanisms that control 'filter-gating' in K+ channels which lack a traditional lower 'bundle crossing gate' i.e. channels where the selectivity filter acts as BOTH filter AND gate.

The project is based upon exciting new unpublished work where we have identified the binding site of the TREK K+ channel agonist, BL1249. This drug appears to directly modulate the filter gate to enhance K+ permeation. Importantly, it also appears to work in a similar way in both the hERG and BK K+ channels (which also possess filter gates). Likewise, we found several hERG and BK channel activators with a common pharmacophore that also activate K2P channels. This supports ideas that a common gating mechanism may exist within the K+ channel filter and identifies a new experimental framework to address this complex process.

We propose to exploit these tools to manipulate the filter gate and dissect the underlying molecular mechanisms. Importantly, these functional studies may also allow us to begin the validation of predictions made by Molecular Dynamics simulation. They will also allow us to predict the way other novel K+ channel agonists bind and work.

The feasibility of the project is supported by extensive preliminary evidence and represents a unique and timely opportunity to make a significant advance in our understanding of K+ channel function.

The principal scientific objectives within this proposal are unashamedly fundamental in nature. However, the long-term social and economic benefits that may eventually arise from improvements in the design and development of therapeutic strategies that target K+ channels are significant.

The three classes of K+ channel that will be studied in this project represent important targets (i.e. the K2P channels, BK channel, and hERG channel) and the development of drugs/mechanisms which can selectively activate these channels have the potential to be used to treat a wide range of cardiovascular and neurological disorders including (but not limited to) pain, depression, epilepsy, stroke, migraine, diabetes, hypertension and cardiac arrhythmias. Therefore, the development of new, more effective and more specific drugs that target K+ channels has the potential to benefit significant sections of the UK population. However, to fulfil the potential of these channels as drug targets requires a deep and fundamental understanding of the intimate relationship between channel structure and their complex functional properties.

Such benefits are also not just restricted to human health and well-being. K+ channels are found in many other organisms including numerous species of pathogenic microbes, fungi, plants, and other organisms. This study therefore also has the potential to drive improvements in animal health, veterinary medicine and plant biology.

In addition to the obvious academic impact this study will have, and its possible role in the search for new and better medicines, 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 funding research in 'basic bioscience underpinning health'.

The project will also help strengthen interdisciplinary collaboration between leading UK research groups. It therefore represents a strategically important investment that will help to support and maintain the world-class profile that UK bioscience research currently enjoys.