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

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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.