Understanding gating kinetics in Cys-loop receptors

Ligand-gated ion channels are proteins that are present throughout the body and mediate the fast cell-to-cell communication that occurs at the synapses. They are necessary for controlling many important processes including those fundamental to memory and learning as well as muscle control. It is therefore perhaps unsurprising that these receptors have been implicated in a range of neurological conditions including epilepsy and spasticity. In order to perform their function, these proteins must, upon binding a neurotransmitter, change conformation (shape) in order to allow ions to pass into or out of the cell. This process is a very dynamic one and the speeds of movement between the channel being in closed or open states directly underpins the behaviour of the central nervous system.

Despite huge progress, exactly how these receptors change conformation (shape) and what factors control the overall dynamic response remain very poorly understood at the molecular level. In this proposal, we will try and address this most fundamental of questions, via examination of the glycine receptor as a key example channel protein. The structure is usually obtained by crystallography and the functional behavior of the channel is usually monitored by recoding the channel's electrical activity via electrophysiological experiments. To make the link between structure and function and to elucidate key information about the underling dynamics, the most appropriate and useful tool is molecular simulation and computational modelling.

One of the key questions we wish to try and understand is exactly why do very similar agonists (compounds that open the channel) give very different functional responses? For example, the response of the glycine receptor to glycine is significantly stronger (larger overall current) compared to alanine, a molecule that differs only by the presence of a methyl group (compared to a single hydrogen atom in glycine). In order to answer this kind of question, we could wait for more structural information (via crystallography), but there is no guarantee that a high-resolution structure will be solved in the near future and regardless, we would like to understand the behaviour of several different agonists. Furthermore, our previous data suggests that even when bound to the receptor, agonists may be quite mobile and exhibit multiple binding modes (perhaps contributing to their functional complexity).

Thus, in order to address these kinds of questions we are proposing to using various molecular dynamics methodologies. These simulations can provide working hypotheses which can be tested via our on-going collaboration with colleagues at UCL. In turn, the functional experiments performed at UCL can be explored with molecular simulation in order to provide insight into results that might otherwise be difficult to rationalize. Our proposal utilizes the power of molecular simulations to provide atomic-level detail of what controls the way the binding site behaves in response to different compounds. A full understanding of this is necessary if we are to not only extend our fundamental knowledge of ion channel behavior but also to have any chance of developing compounds that target these kinds of proteins in the future as treatments for various neurological conditions.

The cys-loop family of receptors are central to mediating synaptic function and are implicated in a range of neurological conditions including epilepsy and spasticity. Despite years of study, several fundamental questions still exist regarding their functional behaviour and how we should interpret that at the molecular level. We are interested in two interrelated questions; i) Why do some agonist that appear to bind with similar affinity to full agonists only elicit a partial response and ii) Why do some receptors exhibit the phenomenon known as modal gating and can that be rationally explained in terms of structural/dynamic models?

We propose to address this via a computational approach using the glycine receptor as a model system of the cys-loop family of receptors. There are several advantages for selecting the glycine receptor. 1. Our proposed work is tightly aligned with the ongoing work of the Sivilotti lab at UCL, who are world experts in single channel electrophysiology 2. The glycine receptor currently has the most structural data associated with it (and there is likely to more appearing soon). 3. We have a proven track record in modelling the glycine receptor to the extent we were able to make key predictions that have since been confirmed experimentally.

We will use a combination of unbiased and advanced MD simulation methods to investigate the mechanisms of partial agonism and also begin to address the molecular basis of modal gating. A full and complete description of the gating transitions (say from resting to flipped or flipped to open) is not feasible. However, investigating the early events governing the transition from resting to flipped is a realistic possibility, and given that once in the flipped state, the transition to the open state is almost independent of the nature of the agonist, we believe we can shed some important insight into the early stages of activation in these receptors.

The work proposed here, which is aimed at understanding how members of the cys-loop family of receptors work is very much at the level of basic research. Consequently, the impact when it arises, will take time to come to fruition. However, as we are utilizing our simulations to interpret single channel data in a collaborative fashion, the impact is potentially high as it can provide a comprehensive and coherent understanding of function. The beauty is that this approach can not only account for other pre-existing data including crystallographic and macroscopic channel data, but can provide working hypotheses that encapsulate the dynamic nature of these proteins.

There will ultimately be many different beneficiaries of the research outside of the academic circle. In the long-term, the most obvious group will be the general public and in particular patient groups who are suffering from neurological conditions. This group would specifically include sufferers of inflammatory pain, spasticity, and neurodegeneration but by extension to other members of the cys-loop family, would include other neurological conditions. Thus, there is a direct impact on the nation's health. Our prior work has already produced important information about the dynamics of these and related (ionotropic glutamate) receptors and that understanding is critical to us designing better and improved drugs in the future.

Aside from the general public, the main beneficiary will be the pharmaceutical industry, which although currently going though a period of upheaval and rationalization, particularly for neuroscience programs, will still benefit from early basic-level research that puts any drug discovery process on a firmer footing. The drug-design process is still extremely difficult and time-consuming. Drugs are developed at vast expense, typically by screening large numbers of compounds and using large numbers of experimental animals. Only a handful of drugs have been discovered by designing them to fit a particular protein target. For channel proteins like glycine receptors, there are two key stumbling blocks: The issue of side-effects - ie making the compounds selective enough that we don't "hit" receptors that are functioning normally and secondly that actually we don't understand channel function well enough, especially with regards to their underling dynamics, regardless of disease state. It will take a long time to get to design drugs mostly in silico, but our approaches outlined here have the demonstrated power to take this forward in the right direction. The UK has a big tradition and massive knowledge in drug discovery, partly because much of the basic science discoveries occurred here.

Thus, the basic research we are proposing here will, in the long term, benefit our society through its impact both on human health and well-being (drug discovery, better understanding of physiological and pathological processes) and on economic productivity (development of novel drugs). These processes will take a long time - the drug discovery process is usually between 10 and 20 years, but the impact can be long lasting and life-changing.