Single molecule quantification of the activation, biophysics and pharmacology of GlyREM, a new structural model for pentameric ligand-gated channels

Ion channels are present in all our cells. The channels we work on open when a neurotransmitter or a drug binds to them, and mediate fast cell-to-cell communication at synapses, including those in the brain. Channels are also important in disease: mutations in channel genes can cause heritable human disease, such as cystic fibrosis. Also, many drugs used for common diseases or in anaesthesia act by binding to channels. For instance, the group of channels that we study, the nicotinic superfamily, are targeted by sleeping pills, drugs for epilepsy, the nicotine in tobacco and some insecticides.
The aim of our research is to understand how ion channels function as molecules. For this we need to know their 3-D structure and how this changes when the channel is activated. Structure is usually obtained by crystallography, and function by recording the channel's electrical activity. For channels, it is slow and difficult to get good crystals that can be used for structure. It is even harder to get multiple structures that show how the channels change shape as they activate. Furthermore, some of the channels that can be imaged do not give good functional data, so integrating structure and function is difficult.
Progress in a technique that does not require crystals, cryo electronmicroscopy (EM) is changing this, and is beginning to give structures for GlyR-EM, a form of the very group of channels my lab specialises on, glycine receptors. We propose to work on this form of channel, co-ordinating our work with the US group that is doing the structural work, to obtain the maximum insight.
At UCL, we perfected a technique to see and interpret the tiny currents (a billion times smaller than the current used by a kettle) produced by one channel molecule. This work is needed, because it is the only way to measure how tightly neurotransmitters and drugs bind to the channel in its different shapes, and how quickly the channel moves between these different states, with and without the drug. It is also the only technique that can measure accurately how strong (in pharmacologist's jargon, how efficacious) a drug is, because it measures how good the drug is at keeping the channel open, when it has bound. In glycine channels, analysis of these data allowed us to find out that strong drugs are strong because they are effective at producing the initial conformational change. After that, the channel opens in a similar manner for all drugs. This research started on glycine channels, but the finding is now known to apply to all nicotinic channels that have been tested. It is obviously important to understand how the channel structure moves in this initial step and for that we need to integrate our functional work with that of the structural biologists that can image the channel.
Our pilot data show that the GlyR-EM channel gives good electrical signals, and is slightly different from the various forms of human glycine receptor we have worked with. This is not a problem, on the contrary, the differences give us information on what determines the functional properties of the channel. This is particularly true for glycine receptors, where the amino-acid sequence of the different forms is very similar, making the causes of the differences in function potentially easier to identify.
Finally Cryo-EM is carried out in conditions that are different from the ones we normally would choose for electrical recording, and there is no information on channel function in these conditions (low temperature and holding potential, long drug applications). We must do these experiments so that we can identify and interpret the new structures. Importantly, the UCL functional work will allow us to indicate to our US collaborators which new structural experiments would be the most useful and informative.
This is basic research but, ultimately it can give us information on how we should modify the structure of drugs in order to make them more effective.

The functional properties of the GlyR-EM channel construct that has produced the new cryoEM structures (Du et al., 2015) are not known in any detail.
We will characterize this receptor by patch-clamp electrophysiology. We will use the whole-cell mode to describe the channel's pharmacology cf. agonists, antagonists, blockers and modulators, and fast agonist applications (ca 0.15 ms exchange) to outside-out patches to measure its macroscopic kinetics of activation/deactivation. We will also record in conditions approximating those in the structural experiments, to estimate desensitisation rates and the distribution of the channel across functional states during very long agonist exposures. Finally, low-noise single-channel recording will measure agonist efficacy as maximum open probability. Kinetic analysis of these data by global mechanism fitting with full missed event correction will establish a detailed quantitative mechanism for the glycine activation/deactivation of this channel. Extensive preliminary experiments data show that the work is feasible.

Our pilot data show also higher agonist efficacy and much slower deactivation for this channel vs its human homologue. We will investigate these properties with electrophysiology on wild type and mutant channels.

Another question arises from the structural data, namely whether the glycine/ivermectin bound channel is partially open. We will obtain precise single channel conductance measurements to clarify that.

Our research on pentameric ligand-gated channels (pLGIC) aims to improve our understanding of their molecular function, and map it to structure. pLGICs mediate fast synaptic transmission in human CNS and are the target of many therapeutic drugs and insecticides. There are many reasons why the work we plan will be useful to the future well-being and wealth of our society. Our work on GlyRs has already yielded important information about how drugs act, and why some agonists are more effective than others on the same target (it is to do with the initial conformational change in the protein). This was done without structural information. Now we can investigate agonist efficacy in a model GlyR whose structure can be investigated across its conformational range, in collaboration with one of the best structural labs in the world, co-ordinating efforts to understand structural changes and drug-receptor interactions during activation.
The main non-academic beneficiary of our work will be the pharmaceutical industry (development of new drugs, improving selectivity of existing ones) and the agro-chemical industry (control of insect and nematode pests with nicotinoids, phenylpyrazoles and avermectins). pLGICs are the target of many therapeutic drugs (sleeping pills, neuromuscular blockers, antiepileptics, ondansetron, antiparasitics such as ivermectin, drugs to facilitate smoking cessation) and of drugs of abuse, as they mediate the effects of nicotine and some of those of alcohol. Note that the two recent crystal structures of alpha3 GlyR (Huang et al, 2015 and 2017) were obtained at Amgen, a drug company.
In time our results will help in the design of new drugs, hopefully achieving greater specificity for particular receptor subtypes. Drugs are developed mostly by screening many compounds, an expensive, low yield process. Few drugs have been discovered by designing them to fit a particular protein target. For channels, the problem often is not that we don't know which channel to target, but that we don't understand channel structure-function well enough to identify which structure/state we should target. It will take a long time to get to design drugs mostly in silico, but our sort of data is precisely what is needed to make it eventually possible, because it will map function to structure and thus identify which of the structures is the state that a drug should try to stabilize.
The UK has a big tradition in drug discovery, partly because much of the basic science occurred here. The basic research we plan can 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 (pest control in agriculture).
Our project is basic research, so it is to be expected that its impact on our economy or health will take time to come to fruition. In 1996, the applicant carried out the first electrophysiological characterization of NaV 1.8, a sodium channel expressed selectively in nociceptive neurons (Akopian, Sivilotti & Wood, Nature 379, 257-262). Despite its clear relevance as a target for analgesics, it took 10 years for selective blockers of NaV 1.8 to be developed and be tested in rodents, where they do suppress various pain symptoms, validating the target. A drug suitable for humans has not emerged yet and none of the original team are involved in the development work. Obtaining selective therapeutic agents is not easy, and it is interesting that Genentech are working on this problem for another sodium channel subtype by using a combination of structural and functional approaches (Science, 2015, doi: 10.1126/science.aac5464)
Further impact lies in developing professional skills of the researchers involved. Exposure to techniques different to the ones they use will give them critical appreciation of these techniques and training in collaborating effectively across disciplines.