From Ion Channel Structure to Function: Better Tools to Annotate Membrane Protein Structures

The way we think and move, as well as the way we interact with and perceive our surroundings are just a few of the important processes controlled by ion channels and membrane transport proteins. In effect, almost every process in the body requires the movement of charged ions (like salts such as sodium chloride), or other molecules (like glucose), into and out of our cells. Without the tiny pores created by these membrane proteins a cell would just be an impermeable plastic bag incapable of interacting with its environment.

These membrane proteins are therefore not only essential for all forms of 'bioelectrical' and cellular signalling, but also for life itself. It is therefore perhaps not surprising that 50% of current drug targets are thought to reside within cell membranes. The importance of these proteins has also driven many recent advances that now allow us to visualise their 3-D structure in exquisite detail. However, these images represent only the beginning of a long journey to understand how an ion channel works.

Central to this understanding is not only the ability to visualise the transmembrane pathways which ions take through these proteins, but also to identify any barriers which exist within these pores. This is important because controlling these barriers enables cellular electrical signals to be switched on or off.

Ions generally move through channels in their 'hydrated' state i.e. dissolved in water, and so previous methods for identification of barriers have mostly focussed on comparing the relative size of a dissolved ion to the size of the pathway through which it moves. This approach has proven extremely useful in the past. However, studies by us and others, now clearly demonstrate that the relative greasiness or 'hydrophobicity' of these narrow pathways also has a profound influence on the movement of ions, i.e. it is not just a matter of how wide the holes are. This is because water behaves very differently in a narrow greasy pore compared to one where the surface is easily wetted or 'hydrophilic'. As a result, if a section of a pore does not easily fill with water then ions cannot pass through, and a barrier is created. The scientific principles underlying this process also explain why oil and water do not mix, how cell membranes and biomolecules assemble, and why water is so essential for nearly all forms of life.

Unfortunately, it is not possible to directly visualise the behaviour of water in such nanometre-sized pores. However, we have recently shown that powerful computational methods known as 'Molecular Dynamics Simulations' can accurately model these processes and therefore act as a 'computational microscope' to visualise this behaviour. Using this approach we have predicted the existence of hydrophobic barriers in several novel channel structures. Importantly, we have validated these predictions with a variety of experimental approaches, including direct changes to the 'wettability' of the channel itself. These results have had a major impact on our understanding of how these ion channels function.

In this project we will develop new tools to simulate and predict the behaviour of water in membrane pores and transport pathways. This will enable a wide range of scientists, from the casual user to expert structural biologist, to accurately predict these hydrophobic barriers in any new or existing protein structure. To underpin this we will improve our mechanistic understanding of these processes by further refining and experimentally validating our computational methods in several model channel systems. These predictive tools will be designed with the requirements of the end-user in mind and for easy implementation on a number of different platforms, ranging from desktop PCs to supercomputers. The toolkit aims to meet a rapidly increasing demand for functional annotation and will have a far reaching impact on understanding how ion channels and membrane transport proteins work.

The regulation of ion channel and transporter function requires modulation of barriers or 'gates' within their transmembrane pathways. However, despite the ever-expanding number of available crystal structures, our current understanding of these barriers is often only determined from calculating the physical dimensions of the pore.

This approach has worked well in the past, but we now have evidence that the unusual behaviour of water within narrow hydrophobic spaces (similar to those found within a channel pore) can create a major barrier to ion flow. Measurement of pore radius alone is therefore unable to identify such barriers. However, we have now shown that molecular dynamics (MD) simulations of water behaviour can be used as a proxy to accurately predict hydrophobic gates in novel channel structures, thereby providing a major new insight into how they are regulated.

We propose to build upon these findings to develop an improved mechanistic understanding of the principles of 'hydrophobic gating' in ion channels, and further define the important role that water plays within pores of certain channels and membrane transport proteins.

Firstly, we aim to study these mechanisms in detail in a number of ion channels by using a variety of computational techniques. Importantly, this will also be combined with functional (electrophysiological) analysis to further refine and validate our understanding of this process.

Secondly, the results of this improved mechanistic understanding will be used to develop a range of analytical and visualisation tools capable of predicting such barriers within novel (and existing) channels, and other transport proteins. These tools will be fast, easy to use, freely available and capable of implementation on a wide-range of computational platforms (running e.g. Linux, Mac, PC). Their adoption will enable a wide range of casual and expert users to maximise their understanding of a wide range of membrane transport processes.

The long-term socio-economic benefits of this project will primarily arise from associated improvements in the design and development of rational therapeutic strategies that target ion channels and membrane transporters. It is estimated that approximately 50% of drug-targets reside within the membrane, and so the principal beneficiaries will be the pharmaceutical and agrochemical industries. The ability to understand how ion channels and other membrane transport proteins function at the molecular level will have a major impact upon rational based drug design and other computational approaches to drug delivery across membranes. This will impact the early stages of drug design and development, thus having the potential to reduce both the overall costs involved as well as the time taken to move drugs to market.

Overall, the development of new, more effective and more specific drugs to treat neurological and cardiovascular diseases will benefit significant sections of the ageing UK population. Furthermore, such benefits are not just restricted to human health and well-being. The ability to improve our understanding of the fundamental mechanisms of membrane transport will also drive improvements in animal health, veterinary medicine and plant biology.

Other potential beneficiaries include those within the synthetic biology sector involved in the design of biomimetic nanopores, as well as the bionanotechnology sectors, especially those working towards energy efficient water purification which is one of the grand challenges of the 21st century. The project will also expose two postdoctoral researchers to a wide range of cutting-edge and interdisciplinary research techniques that will enable them to contribute more effectively to the wider economy in the future.

Finally, the general public also has a tremendous curiosity about science and so the relevance of the underlying scientific principles will also be highlighted in an extensive programme of public engagement and outreach in schools. 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.