Molecular neuroscience of glycine receptors: insights into ligand-gated ion channel function

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

Abstract

When one nerve cell meets another, the nerve impulse is transmitted from the first cell to the second by release of a chemical (neurotransmitter) from the first cell. The chemical diffuses rapidly across the narrow gap between the two cells and stimulates the second cell to produce a nerve impulse. The neurotransmitter works by combining with specialized proteins (receptors) embedded in the membrane of the second cell, and this combination causes the protein to open up to form a pore that allows electrical current to flow across the membrane. This current is what stimulates the second cell.
There is a second class of receptors that, rather than stimulating the second cell, have the opposite effect; they inhibit the response of the second cell to other neurotransmitters. This inhibition is enormously important for the proper working of the brain and spinal cord. The receptor for one such inhibitory neurotransmitter, glycine, is the subject of our proposed work. Its importance can be illustrated by two examples. The glycine receptor is blocked by strychnine, poisoning by which illustrates the importance of the receptor dramatically. Less dramatically, but more commonly, consider what happens when you touch something hot. Your arm is withdrawn rapidly. To achieve this, the muscles for withdrawal contract, but at the same time the muscles for extending the arm must relax so they don t oppose withdrawal. This relaxation is coordinated by glycine receptors in the spinal cord. Human disease can be caused by genetic defects in the glycine receptor (startle disease, which resembles mild strychnine poisoning).
We have already made a good start on investigation of the glycine receptor, and this work has opened up some very interesting questions, which we how hope to pursue. We would like to know what steps lie between the binding of glycine and the opening of the pore, and how this is affected by disease-causing mutations. We will also investigate the way the receptor is regulated by other substances that are present in the brain (in particular the trace metal, zinc). This work will not only cast light on how the glycine receptor works, and therefore on the mechanisms of inhibition in the brain and spinal cord, but will also serve as an example for several other neurotransmitter receptors that are closely related in structure, but are harder to investigate than the glycine receptor.

Technical Summary

Glycine-mediated synaptic transmission is important in spinal cord and brainstem. We propose to investigate the glycine receptor, at both molecular and synaptic levels. This work is timely, because we have recently shown that the glycine receptor is more amenable to the fitting of mechanisms to single ion channel data than any other (including nicotinic receptors, on which we have worked extensively).

At the molecular level, our premise is that, if we wish to learn about the relationship between structure and function, the functional data must be fitted with a reaction scheme that is based on physical reality. Empirical modelling is not good enough. We have recently found that we can resolve what appears to be a conformation change (domain closure?) that follows binding but precedes opening. If this proves to be right, it will change the way we look at gating efficacy and will provide a crucial link with structural studies. It is, therefore, an important aim of the work to verify (or otherwise) this new way of looking at receptor function. That will set the scene for a wider look at the whole pathway between agonist binding and channel opening. We shall fit putative mechanisms to single ion channel recordings, using the maximum likelihood method that we have developed and tested. We shall test a range of agonists, and of receptor subtypes, and investigate mutations designed to cast light on mechanisms, from the binding site, through the transduction machinery to the channel itself. We shall also use linear-free energy relationships (phi-analysis), and mutant cycle analysis.

Another branch of the work will be to investigate the effects of zinc ions on the glycine receptor. Zinc is present in the CNS and it is released at synapses, but much remains to be done to elucidate its importance for physiological, and perhaps pathological, processes in man. Therefore, as well as working on the action of zinc at the molecular level (as above), we shall also investigate its action under more physiological circumstances.

Ultimately, glycine receptors matter because they mediate synaptic transmission, so we shall look at synapses too. What receptor types are involved at synapses? What determines the time course of the inhibitory currents mediated by glycine receptors? Are endogenous ions such as Zn2+ or protons important for regulation of synaptic transmission? These will be investigated by fast concentration jumps, and at real synapses in spinal cord slice and organotypic culture.

Publications

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Sivilotti Lucia (2017) Agonist efficacy in the nicotinic superfamily in JOURNAL OF NEUROCHEMISTRY

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Miller PS (2010) Binding, activation and modulation of Cys-loop receptors. in Trends in pharmacological sciences

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Moroni M (2011) Chloride ions in the pore of glycine and GABA channels shape the time course and voltage dependence of agonist currents. in The Journal of neuroscience : the official journal of the Society for Neuroscience

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Smart TG (2010) Handling accumulated internal Cl- at inhibitory synapses. in Nature neuroscience

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Pitt SJ (2008) High intracellular chloride slows the decay of glycinergic currents. in The Journal of neuroscience : the official journal of the Society for Neuroscience

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Houston CM (2009) Intracellular chloride ions regulate the time course of GABA-mediated inhibitory synaptic transmission. in The Journal of neuroscience : the official journal of the Society for Neuroscience

 
Description BBSRC project grant
Amount £395,000 (GBP)
Funding ID BB/J005312/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 02/2012 
End 02/2015
 
Description MRC Project Grant
Amount £389,771 (GBP)
Funding ID MR/R009074/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 07/2018 
End 06/2021
 
Description MRC agonist grant
Amount £538,000 (GBP)
Funding ID MR/J007110/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 11/2012 
End 10/2015
 
Description UCL Impact studentship - Marabelli
Amount £32,000 (GBP)
Organisation University College London 
Sector Academic/University
Country United Kingdom
Start 06/2010 
End 06/2013
 
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 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
 
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
 
Description blog 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact one of the PIs is a regular blogger http://www.dcscience.net/ on a variety of subjects that go from science policy to the statistical analysis of evidence. From time to time he mentions our own work

our colleague is now pretty famous and his opinion often sought by the press
Year(s) Of Engagement Activity 2006,2007,2008,2009