Combining structure and function in the nicotinic superfamily: the single-channel activation mechanism for the prokaryotic model channel ELIC

Lead Research Organisation: University College London
Department Name: Neuroscience Physiology and Pharmacology

Abstract

The purpose of our work is to understand how ion channels function as molecules. The key to this is to know the 3-D structure of the channel (by X-ray crystallography) and to find out how this structure moves when the channel is activated, mainly by recording its electrical activity. It is rarely possible to get both sorts of information for the same molecule. New data mean that this is now possible for the group of channels we work on.
Ion channels are proteins coded in our genome and are essential components of many cells in our bodies. For instance, they allow cells to communicate with each other at cell-to-cell junctions called synapses. This is essential not only in the brain but also to in the rest of our bodies where it allows the appropriate commands to reach muscles in our limbs and to regulate our blood pressure and heart rate. Channels also allow each neurone to process the information it receives from other neurones. Channels are important for normal human physiology and for disease. Mutations in the genes that code for channels can damage channel function and produce inherited human disease, such as cystic fibrosis. In addition to that, many drugs used for common diseases or in anaesthesia act by binding to channels. In particular, the group of channels that we study, the nicotinic superfamily, are targeted by sleeping pills, drugs for epilepsy, nicotine in tobacco and some insecticides.
There are two main sources of information about channels: the first is X-ray crystallography, which provides us with information about the shape of the protein and the second is electrophysiology, which measures the electrical signal the channel produces. In the most advanced form, which is our special expertise, this technique detects the current that passes through a single protein molecule in real time, even though it is very small (more than a billion times smaller than the current in a kettle). This technique is very useful, because it allows us to measure the speed with which molecular events in the function of the channel occur. Hence we can understand channel function precisely as a chemical reaction, quantifying each step, from the binding of the neurotransmitter to the opening of the channel. By doing this in channels in the nicotinic group, we have found how tightly neurotransmitters and drugs bind to the protein when it is active or inactive and why some drugs act more strongly than others.
Ideally we should study the structure and the function of the SAME channel. This is not easy because channels are difficult to crystallize, and so far we have good structures only for three channels in this group (GLIC, ELIC and GluCl). Of these, GLIC produces electrical signals that are too small for good electrophysiology. As for GluCl, we don't know how good its signal is (the structure has literally just been published). A potential problem with GluCl is that it does not open like all other channels in the group do, in response to a neurotransmitter-like compound, but it requires TWO different substances, binding to different places, so we don't know how good a model it will be.
Until now ELIC was thought not to be able to open. Other scientists have now discovered the right substances that activate ELIC, and it turns out to open well and to give an excellent, big signal. We want to apply the single-molecule recording that is our special skill to ELIC, so that we can understand how it functions as a molecule. Once we have that, we can push our understanding much further, because we can refer to precise structural information (available for ELIC) in interpreting the effect of drugs and the effect of mutations in the channel.
This is basic research but it is what is needed if we want to explain what bits of the molecule change and how they move when the channel is activated, where exactly drugs bind to the protein and how we should modify the structure of drugs in order to make them more effective.

Technical Summary

In the nicotinic superfamily, high resolution crystal structures are available for two prokaryotic channels, GLIC and ELIC. Neither channel has been studied by detailed functional analysis, such as single-channel kinetics, because GLIC has very low conductance, and the agonist for ELIC was unknown. Recent work by the two European groups that have solved the structure of ELIC (see Letter of Support) showed that ELIC is a non-selective cation channel that opens in response to many small-molecule agonists, including GABA. When open, ELIC has a very high conductance (80 pS) that makes it ideal for single-channel recording. We propose to apply our expertise in single channel kinetics to characterize in detail the activation mechanism of ELIC, by our established method of computational global fitting of mechanisms. This technique is the only one that can estimate microscopic association and dissociation rate constants for the binding of agonists to the channel in its resting, pre-open and open states. It recently allowed us to identify reaction intermediates in the activation of two other nicotinic channels, the glycine receptor and the ACh muscle receptor. These reaction intermediates are the key to understanding differences in agonist efficacy in this superfamily. Applying our methods to ELIC, which is amenable to structural investigation, will allow much more robust interpretation of the effect of mutations, and bring on a new era for the study of structure-function relations in the nicotinic superfamily, making ELIC an extremely attractive model system for this purpose. Once we have obtained a quantitative model for the activation by the full agonists available, we will extend our work to two specific further questions, namely the activation of ELIC by partial agonists (including GABA) and the role of the M2-M3 loop in transducing agonist binding in the extracellular domain to channel opening.

Planned Impact

Our aim is to establish by single channel analysis the quantitative activation mechanism of the prokaryotic channel ELIC, a member of the nicotinic superfamily of ligand-gated channels whose structure was solved to high resolution by X-ray crystallography. This will allow the combination of advanced structural and functional data on the same nicotinic channel, a combination that has revolutionised the field of potassium channels.
The results of our work will be useful to all biophysicists and physiologists that work on ligand-gated channels in this superfamily. At the moment we have good structures only for members of the superfamily that are either non-functional (soluble ACh binding proteins) or hard to characterize (such as GLIC). The recent discovery that ELIC can be activated to a high conductance (ideal for advanced electrophysiology) means that we can model quantitatively its behaviour, from the number and nature of the agonist binding steps to the conformational changes it must undergo to open. These results are also essential to pharmacologists. Measuring maximum open probability and agonist efficacy allows reliable interpretation of the more common macroscopic measurement (eg dose-response curves) used to characterise the effects of drugs and that of mutations. Notably, our single-channel method is the only way to estimate the agonist microscopic binding affinity for the receptor resting and activated states. This is key to understanding how agonist activity relates to the structure of the agonist molecule itself and to its sites of interaction with the protein (that may be verified by further structural work).
Having a model for ELIC activation poses the basis of structure-function work in the protein, in which mutational strategies can be designed with firm structural knowledge of the position of each residue, and can be more easily interpreted. ELIC will provide the best model system to monitor the timing of domain motion in the protein by the application of phi analysis, casting light on the dynamics of channel activation in the whole superfamily, a group of channels of great physiological and pharmacological importance.
As modelling the motions of membrane proteins by molecular dynamics becomes more feasible, researchers in this field will find that our results are the ideal experimental counterpart to verify the predictions of computational simulations, as they provide measurements of microscopic ligand affinity for the different states of the receptor, probe the conformational energy landscape of the protein and eventually build a map of the timing of domain motion.
Because of the usefulness of these results to the fields of structure, function and computational modelling, we will take special care to disseminate our results at meetings that are especially designed to be interdisciplinary and bring together scientists with different expertise.
A key aspect in maximising the benefits of our work to the nicotinic field is our collaboration with the group of Prof Dutzler (Zurich), one of the foremost labs working on the structure of prokaryotic channels in this superfamily. They have obtained structures for ELIC and GLIC and showed that ELIC can be activated by a range of agonists (see Letter of Support). Keeping each other up to date on progress on our work on this channel will enable us to identify quickly promising projects for joint work that exploit the complementary points of strength of our labs. This will become possible as soon as the first preliminary characterization of the main features of the activation mechanism become available, because this will then allow us to draw on our expertise on a range of other channels in the superfamily to design appropriate mutational strategies. Further structural characterization is also always a possibility, bearing in mind the difficulty, unpredictability and low success rate for this work in membrane proteins, even of prokaryotic origin.

Publications

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Marabelli A Probing the activation mechanism of ELIC, a model channel for the nicotinic superfamily in Gordon Research Conference on Ion channels 2012

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Marabelli A (2012) ELIC channel activation in response to agonist concentration jumps in Proceedings of the Physiological Society

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Marabelli A (2013) ELIC channel activation and block by propylamine in Proceedings of the Physiological Society

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Marabelli A (2014) Activation Mechanism of Elic by Propylamine in Biophysical Journal

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Calderhead B (2013) Bayesian approaches for mechanistic ion channel modeling. in Methods in molecular biology (Clifton, N.J.)

 
Description ELIC is a bacterial channel that has given structural information for the nicotinic superfamily.
1) By single molecule recording we have established a quantitative model of how the channel activates. We found that maximum channel activation, with a very high open probability, is reached when two-three of the five potential binding sites are occupied by the agonist propylamine. Activation requires the channel to go through at least one activation intermediate.
2) We discovered that the channel pore of ELIC is blocked by agonists with low affinity (100 mM).
3) We confirmed that ELIC has a high single channel conductance
4) We discovered that the rate with which the channel desensitises and the rate with which the agonist dissociates are similar. This poses technical difficulties to computational modelling and kinetic analysis by single channel recording. This required adding other techniques (agonist relaxations) to provide a validation of the activation model and its conclusions.
5) Findings 1-4) have been published in the Journal of General Physiology. We have extended our work to agonists other than propylamine, and we are preparing the work for publication- a new cryo-EM structure is helping us better to interpret these results
6) we have identified a mutant form of ELIC that yields itself as a better model channel for future structure-function research work and this will be useful to us and other researchers
7) we have identified a residue that controls activation and deactivation rate in parallel - we have done some bioinformatics on this- it looks like the residue does not interact with lipids; a new cryo-EM structure (Kumar... Grosman 2020, PNAS) will help us interpret these results and publish them
Exploitation Route ELIC is an important channel in which to model structure function activity for agonist drugs and for channel blockers.
This is of interest to medicinal chemists, pharmacologists in terms of basic science, and to agricultural scientists because the bacterium that makes ELIC is an important cause of food rot (cf potatoes).
Sectors Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology

 
Description We have published about half of our results in january 2015. The impact comes from the precise quantification of the activation mechanism of a channel in the nicotinic superfamily for which the structure is known. This is a first and poses the foundation for work on the structure-function relation for the two key features of agonist action, binding and gating. As such we are making sure to inform colleagues in the Pharmaceutical industry (Novartis Basel). Additionally the work is relevant to academics away from our specialty, in particular crystallographers and molecular dynamics specialists, who will have the possibility to use our experimental measurements of reaction rates with their calculations in silico.
First Year Of Impact 2012
Sector Pharmaceuticals and Medical Biotechnology
Impact Types Cultural,Economic

 
Description ARCHER EPSRC
Amount £93,069 (GBP)
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 10/2015 
End 05/2016
 
Description Leverhulme Project Grant
Amount £207,280 (GBP)
Funding ID RPG-2016-407 
Organisation The Leverhulme Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2017 
End 07/2019
 
Description UCL research software development calls
Amount £15,000 (GBP)
Organisation University College London 
Sector Academic/University
Country United Kingdom
Start 01/2013 
End 04/2013
 
Description Bayesian inference 
Organisation University College London
Department Department of Statistical Science
Country United Kingdom 
Sector Academic/University 
PI Contribution Providing data from our single channel work to Professor Girolami
Collaborator Contribution Prof Girolami is applying advanced Bayesian inference techniques to parameter estimation and model distinction in our channel problems
Impact one paper so far, we have a joint PhD student (funded by CoMPLEX)
Start Year 2012
 
Description K2P single channel analysis 
Organisation University of Kent
Country United Kingdom 
Sector Academic/University 
PI Contribution We are going to help the group of Professor Mathie implement kinetic analysis of single channel data from K2P channels.
Collaborator Contribution Mathie has the expertise on this channel and they will do the bulk of the recording
Impact we are preparing a project grant application
Start Year 2015
 
Description Unnatural mutagenesis 
Organisation University of Copenhagen
Country Denmark 
Sector Academic/University 
PI Contribution We are going to provide single channel recording and analysis for unnatural aminoacid mutations in the glycine receptor binding site.
Collaborator Contribution This technique is not in use in the UK. We are going to start the experiments in Dr Pless's lab in Copenhagen and learn how to do it.
Impact None yet
Start Year 2015
 
Description mechanisms of block and permeation in ELIC 
Organisation University of Cambridge
Department Department of Biochemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution We are going to collaborate with Professor Lummis to elucidate blocking and permeation on ELIC. We are going to carry out single channel recording at UCL with the compounds that the Cambridge lab characterized
Collaborator Contribution The Lummis lab has long worked on ELIC and has characterized the macroscopic pharmacology of many compounds. They are now interested in further dissecting the mechanism of action of these compounds with our help
Impact We are going to apply together to the BBSRC to obtain funding for this as a joint project
Start Year 2016
 
Description optical patch clamp at the Crick 
Organisation Francis Crick Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution we are trying to set up temporally resolved recording of calcium fluxes through single channel molecules, in order to combine them with our electrophysiology data
Collaborator Contribution Dr Molloy at the Crick is providing expertise and the use of a TIRF setup
Impact pilot data obtained in 2016 led to the award of a Leverhulme trust project grant
Start Year 2016
 
Title DCPROGS analysis software for single channel analysis 
Description This is a complete suite of programs for the analysis of single channel kinetics initially developed by David Colquhoun and the programmer Ms Vais. The software comprises several programs and the most important are 1) SCAN, to idealise recordings of single channels by time course fitting*; 2) EKDIST to analyse the distributions of single channel events; 3) HJCFIT to perform global mechanism fits to the idealised data with full missed event correction* (based on the theory developed by Colquhoun and Hawkes). This software is unique for the features asterisked. We use it all the time, we maintain it with the help of the UCL Research Software Development Team. We are currently redeveloping it for parallelization, so we can use it on ARCHER This is an ongoing project (and output) 
Type Of Technology Software 
Year Produced 2015 
Open Source License? Yes  
Impact We were awarded an EPSRC ARCHER award (2015) to develop the programs further. Without these programs the resolution of our analysis would be much worse, as the second best software that does the same analysis is inferior in several aspects. Use of this software made it possible for us to detect an intermediate state in the process of receptor activation and our new understanding of partial agonism would not have been possible without it. We have scientific visitors from all over the world that come to learn about the programs 
URL https://github.com/DCPROGS