Probing the dynamics of agonist drug interaction with Cys-loop channels by single-molecule recording

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

Abstract

Ion channels are proteins that act as "nanoswitches" to translate voltage or chemical stimuli into electrical currents. They are essential to many bodily functions, including information processing in neurones and cell-to cell communication at synapses outside the brain which allow nerve impulses to move muscles and regulate blood pressure and heart rate. Unsurprisingly, inherited channel mutations can produce human disease, from cystic fibrosis to neurological conditions.

Channels are targeted by many drugs. The nicotinic-type channels we work on mediate the effects of sleeping pills, drugs for epilepsy, nicotine, alcohol, insecticides and antiparasitic drugs. If we were better at designing drugs to activate or modulate nicotinic channel function, they could be useful in other hard-to-treat conditions, from chronic pain to spasticity after stroke, especially if we could exploit the great diversity of channel subtypes to design agents selective for single subtypes.

To this day, drugs are discovered by large scale screening of chemicals to find if they are effective on a human drug receptor. The process is expensive and wasteful, and few new drugs become available every year. Instead of that, it would be ideal to be able to design chemicals to have a specific action on a particular human channel. For this we need to understand two steps: how a chemical binds to the channel and how it then changes the shape and function of the channel protein. Part of the problem is that, working as a switch, the channel itself changes shape and we don't know how this affects the binding of the drug.

This is what we want to find out. At UCL we perfected a technique to see and interpret the tiny current (more than a billion times smaller than the current in a kettle) produced by one channel protein. This analysis is the only one that can tell us how tightly the drug binds to different states of the protein and how quickly the protein moves between these different states, with and without the drug.
We will use in our work the glycine channel as a model for the nicotinic family. This channel has ideal properties for single molecule recording and has recently allowed us to see why some drugs are less effective than others in turning the receptor on, a result we found to be applicable to other nicotinic channels. We will extend our work and obtain these measurements for chemicals that activate the receptor, and differ from each other in their chemical structure in a systematic way. We will also change the protein itself, by mutating appropriate positions. Combining this information will allow us to see where the drugs "touch" the protein most closely.

Glycine channels are also the mammalian channel that is closest to a well-resolved X-ray structure (that of an invertebrate channel, GluCl, published in June 2011). This makes it possible to use the structural data in relation to channel function. At Oxford we will model the structure of the glycine channel by homology to GluCl by computer calculations and use this work to plan and interpret the experiments on channel function in terms of channel 3-D shape.

Ultimately our work should lead us to understand what features in a chemical determine its affinity and efficacy for a nicotinic channel and how the different parts of the channel move with activation. It should give us indications on how the structure of drugs should be modified, in order to make them more effective. Hence this fundamental research will be useful to lay the basis for future drug development and hopefully enable rational drug design, in the glycine receptor itself (a therapeutic orphan) and in the nicotinic superfamily as a whole. Our results will also help our drug industry colleagues interpret their data that come from the quick assay techniques used by in high-throughput screening of libraries of compounds, such as binding and macroscopic functional measurements.

Technical Summary

We will carry out ultra-low noise single channel recording of recombinant glycine channels activated by a series of agonists (with and without appropriate channel mutations). Kinetic analysis by time course idealization and global fitting of detailed activation mechanisms will be used to obtain microscopic equilibrium and rate constants, including the agonist microscopic affinity for the different states of the receptor.

We will use in silico computational modelling, both to help us choose the agonists and the mutations to be tested and to interpret our results. Our homology model will be based on the structure of GluCl, a nematode channel 34% homologous to the glycine channel. We will use molecular dynamics to explore the conformational behaviour of the receptor, particularly the binding site. We will also dock series of ligands to the receptor to examine the conformational dependence of the bound states.

Docking trials, whole-cell recordings and maximum open probability measurements will be used to select agonists suitable for full characterization, focussing on series of compounds, each series introducing small systematic structural differences in one moiety of the agonist molecule. If contaminated by glycine, agonists will be purified before experimental use.

We will also mutate the channel, choosing, from the literature and our in silico work, binding site residues likely to interact with specific parts of the agonist molecules, and repeat the agonist experiments with these mutant channels. Thermodynamic cycle analysis will be carried out on equilibrium dissociation constant values obtained by single channel recording to map the direct interactions between agonist molecule and channel at rest and after activation. Should a particularly informative agonist or mutation give rise to data beyond experimental bandwidth, we shall engineer appropriate compensating background mutations in domains distant from the binding site to allow data collection.

Planned Impact

Understanding how ion channel molecules function is basic research: this does not mean that it has no impact on our economy or health, but simply that this impact takes time to come to fruition. 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 the nicotinic group of channels has already produced 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). We plan to continue and exploit our findings, extending them to a series of chemically-related agonists, to understand channel activation and drug-protein interaction in greater detail and depth.

We expect the main non-academic beneficiary of our work to be the pharmaceutical industry (development of new drugs, improving the selectivity of existing ones) and the agro-chemical industry (control of insect and nematode pests with nicotinoids and avermectins). Nicotinic channels are the target of many therapeutic drugs (sleeping pills, neuromuscular blockers, antiepileptics, ondansetron, antiparasitic drugs 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.

In time our results will help in the design of new drugs, hopefully achieving greater specificity for particular receptor subtypes. At the moment, drugs are developed at vast expense, mostly by screening large numbers of compounds and using large numbers of experimental animals. It is getting more and more expensive and difficult. Only a handful of drugs have been discovered by designing them to fit a particular protein target. For channels, the problem is not that we don't know which target to go for, but that we don't understand channel function well enough, especially with regards to how they change in shape when they are functioning in the body, be it healthy, be it sick. It will take a long time to get to design drugs mostly in silico, but our sort of data is precisely what is needed in order to make it eventually possible. The UK has a big tradition and massive knowledge in drug discovery, partly because much of the basic science discoveries occurred here.

Because of the reasons outlined above, the basic research we plan 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 (pest control in agriculture).
Naturally, the timescale of these outcomes is long, given that the development of a drug takes ten years or more, as shown by an example from the work of one of us (LGS). In 1996 the PI participated to the discovery of the sodium channel NaV 1.8, by carrying out its first electrophysiological characterization (Akopian, Sivilotti & Wood, Nature 379, 257-262). Because of the discrete expression of this protein, in nociceptive pathways, it was obvious that blockers of this channel could be selective analgesics. It took 10-11 years for selective blockers of this channel subtype to be described. Work in rodents now shows that this channel is indeed a good target for analgesics, but a drug suitable for humans has not emerged yet.

Publications

10 25 50
 
Description ARCHER EPSRC
Amount £93,069 (GBP)
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Academic/University
Country United Kingdom
Start 10/2015 
End 05/2016
 
Description Impact PhD studentship - Hurdiss
Amount £32,000 (GBP)
Organisation University College London 
Sector Academic/University
Country United Kingdom
Start 10/2012 
End 10/2015
 
Description Leverhulme Project Grant
Amount £207,280 (GBP)
Funding ID RPG-2016-407 
Organisation The Leverhulme Trust 
Sector Academic/University
Country United Kingdom
Start 01/2017 
End 07/2019
 
Description MRC Project Grant
Amount £389,771 (GBP)
Funding ID MR/R009074/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 07/2018 
End 06/2021
 
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 Novartis 
Organisation Novartis
Country Global 
Sector Private 
PI Contribution I advised Novartis Basel on a project concerning a Cys-loop ligand-gated channel
Collaborator Contribution Interesting idea
Impact It changed the direction of the Novartis project
Start Year 2012
 
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 function for Cryo EM 
Organisation Oregon Health and Science University
Country United States 
Sector Academic/University 
PI Contribution we are recording single channel currents and agonist jumps for glycine receptors that are being characterized structurally
Collaborator Contribution Structural characterization of glycine receptors
Impact Data to identify the functional state of different structural forms
Start Year 2015
 
Description glycine receptors in spinal cord synapses 
Organisation International School for Advanced Studies
Country Italy 
Sector Multiple 
PI Contribution Professor Ballerini is investigating spinal cord development in mouse organotypic cultures. She found that glycinergic inhibition was altered in SOD1 mutant mice (a model of ALS). I suggested experiments and analysis to understand what caused this difference and was able to exclude differences in the nature of the postsynaptic glycine receptors by looking at single channel recordings.
Collaborator Contribution The project was initiated by Ballerini whose group carried out the experiments
Impact we have a paper in revision at J.Physiol. the collaboration uses several electrophysiology techniques and immunohistochemical staining
Start Year 2013
 
Description optical patch clamp at the Crick 
Organisation Francis Crick Institute
Country United Kingdom 
Sector Charity/Non Profit 
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