Mechanisms of clustering of GABA-A receptors

Lead Research Organisation: School of Pharmacy
Department Name: Pharmacology

Abstract

The central nervous system is an intricate network of nerve cells (neurones) whose primary function is to transmit and receive messages. This communication occurs at specialised sites of contact known as synapses. At these sites, an arriving nerve impulse causes the release of a chemical neurotransmitter which then binds to receptor molecules embedded in the cell membrane of a neighbouring neurone. Some types of these receptors (e.g. GABA-A receptors) possess specific chloride-permeable channels, formed from combinations of different protein subunits (e.g. alpha1+beta2+gamma2). The opening of these channels in response to the neurotransmitter GABA alters the electrical state of the cell, subtly altering the incoming nerve impulse. The mechanisms that regulate synaptic transmission and nerve impulse activity are important in understanding normal and diseased states of the brain. Dysfunction of nerve cells releasing GABA, or harbouring GABA receptors, have been implicated in a diverse range of human disorders including epilepsy, schizophrenia and anxiety. In support of these findings, many clinically important drugs including benzodiazepines, barbiturates and anaesthetics, act primarily via GABA-A receptors. The therapeutic nature of these agents provides a compelling reason for further understanding the molecular details of this receptor class.

This proposal will benefit research in this area by examining the mechanisms involved in locating GABA-A receptors at synapses. This process is of fundamental importance, since when disrupted in humans, a severe drug-resistant epilepsy develops. We know that a key protein, called gephyrin, is vital for GABA-A receptor clustering, but to date no-one has been able to demonstrate either a direct or indirect interaction between GABA-A receptor subunits and gephyrin. I have found that the GABA-A receptor beta2, beta3 and gamma2 subunits bind to artificially truncated forms of gephyrin. This suggests that either: i) these GABA-A receptor subunits co-operate to bind one or more gephyrin variants; ii) binding of another protein to gephyrin might unlock the GABA-A receptor binding site, or iii) cleavage or modification (e.g. addition of phosphate groups by protein kinases) might alter gephyrin or GABA-A receptors so that they can link. I will explore these possibilities using a range of molecular and proteomics techniques including the yeast three-hybrid system, tandem affinity purification and molecular targeting studies in cell culture models of GABA-A receptor clustering. My studies will uncover fundamental knowledge concerning the localisation of this important receptor class, and reveal possible additional candidate genes for epilepsy and other neurological disorders involving GABA-A receptors.

Technical Summary

Despite compelling evidence that gephyrin is responsible for the clustering of many major GABA-A receptor subtypes, as yet no-one has been able to demonstrate a direct or indirect interaction between GABA-A receptor subunits and any of the multiple gephyrin splice isoforms at synapses. My preliminary studies have revealed an interaction of GABA-A receptor beta and gamma subunits with two different artificially truncated forms of gephyrin. This suggests that either: i) the gephyrin binding site is located at the interface between GABA-A receptor beta and gamma subunits; ii) splice cassettes may alter the conformation of gephyrin, facilitating GABA-A receptor interactions; iii) binding of an accessory protein to gephyrin reveals the GABA-A receptor binding site, or iv) proteolysis or other post-translational modifications (e.g. phosphorylation, palmitoylation) of endogenous gephyrin is necessary for GABA-A receptor interactions. I will explore these possibilities using a range of molecular and proteomics techniques including the yeast three-hybrid system, tandem affinity purification and molecular targeting studies in cell culture models of GABA-A receptor clustering. Since defects in neuronal gephyrin and GABA-A receptor synaptic targeting result in a severe drug resistant form of epilepsy, understanding the molecular mechanisms involved in clustering may provide important new leads for pharmacological intervention and genetic analysis.

Publications

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Harvey RJ (2008) The genetics of hyperekplexia: more than startle! in Trends in genetics : TIG

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Harvey RJ (2008) A critical role for glycine transporters in hyperexcitability disorders. in Frontiers in molecular neuroscience

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Mukherjee J (2011) The residence time of GABA(A)Rs at inhibitory synapses is determined by direct binding of the receptor a1 subunit to gephyrin. in The Journal of neuroscience : the official journal of the Society for Neuroscience

 
Description Bloomsbury Colleges PhD Studentship
Amount £45,000 (GBP)
Organisation University College London 
Department School of Pharmacy
Sector Academic/University
Country United Kingdom
Start 10/2010 
End 09/2013
 
Description MRC DTG 4 Year PhD Studentship G0800111
Amount £81,750 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2008 
End 09/2012
 
Description MRC Project Grant
Amount £494,207 (GBP)
Funding ID MR/J004049/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 05/2012 
End 04/2015
 
Description MRC Project Grant G0800498
Amount £1,015,020 (GBP)
Funding ID G0800498 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 01/2009 
End 05/2012
 
Title Collybistin knockout mouse 
Description A knockout mouse line for the RhoGEF collybistin (gene symbol ARHGEF9) lacking exon 5. Collybistin deficient mice display a region specific loss of postsynaptic gephyrin and GABA-A receptor clusters (e.g. in the hippocampus and the basolateral amygdala). Collybistin deficiency is accompanied by significant changes in hippocampal synaptic plasticity, due to reduced dendritic GABAergic inhibition. Long-term potentiation is enhanced, and long-term depression reduced, in collybistin-deficient hippocampal slices. The animals show increased levels of anxiety and impaired spatial learning. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2007 
Provided To Others? Yes  
Impact PMID: 17690689. Together, these data indicate that collybistin is essential for gephyrin-dependent clustering of a specific set of GABA-A receptors, but not required for glycine receptor postsynaptic clustering. This finding has had a significant impact on research directions in the field of receptor clustering. 
URL http://europepmc.org/abstract/MED/17690689
 
Description Collybistin interactions with neuroligin 2 
Organisation Max Planck Society
Department Max Planck Institute for Experimental Medicine
Country Germany 
Sector Public 
PI Contribution Provision of experimental data, expertise and reagents. For this study we provided data showing that the human ARHGEF9 mutation G55A disrupts neuroligin 2 induction of collybistin-mediated gephyrin clustering.
Collaborator Contribution Discovery that neuroligin 2 binds to gephyrin and functions as a specific activator of the RhoGEF collybistin.
Impact This study contributed (together with PMIDs: 17690689 and 18615734) to the discovery of a pivotal role of collybistin in clustering of gephyrin at selected GABAergic synapses via interactions with the adhesion molecule neuroligin 2 (PMID: 19755106).
Start Year 2007
 
Description Collybistin knockout mouse 
Organisation Max Planck Society
Department Max Planck Institute for Brain Research
Country Germany 
Sector Academic/University 
PI Contribution Provision of experimental data, expertise and reagents. For this study we generated a targeting construct for the mouse collybistin gene (arhgef9).
Collaborator Contribution Generation and characterisation of a knockout mouse for the RhoGEF collybistin.
Impact This study contributed (together with PMID: 19755106 and 18615734) to the discovery of a pivotal role of collybistin in clustering of gephyrin and selected GABAAR subtypes (PMID: 17690689).
 
Description GABA-AR subunit interactions with gephyrin 
Organisation Medical University of Vienna
Department Department of Biochemistry and Molecular Biology
Country Austria 
Sector Academic/University 
PI Contribution Provision of experimental data, expertise and reagents. For these studies, we provided data showing that the GABA-AR alpha1 and alpha3 subunits interact directly with gephyrin.
Collaborator Contribution Contribution to the characterisation of the GABA-AR gephyrin interaction.Contribution to the characterisation of the GABA-AR gephyrin interaction.
Impact These studies contributed together with PMID: 20622020 to defining the complex interaction between GABA-AR alpha subunits and gephyrin important for GABA-AR clustering at inhibitory synapses (PMIDs: 21880742 and 21994384).
Start Year 2008
 
Description GABA-AR subunit interactions with gephyrin 
Organisation Tufts University
Department Department of Neuroscience
Country United States 
Sector Academic/University 
PI Contribution Provision of experimental data, expertise and reagents. For these studies, we provided data showing that the GABA-AR alpha1 and alpha3 subunits interact directly with gephyrin.
Collaborator Contribution Contribution to the characterisation of the GABA-AR gephyrin interaction.Contribution to the characterisation of the GABA-AR gephyrin interaction.
Impact These studies contributed together with PMID: 20622020 to defining the complex interaction between GABA-AR alpha subunits and gephyrin important for GABA-AR clustering at inhibitory synapses (PMIDs: 21880742 and 21994384).
Start Year 2008
 
Description Role of collybistin in X-linked intellectual disability 
Organisation Max Planck Society
Department Max Planck Institute for Molecular Cell Biology and Genetics
Country Germany 
Sector Public 
PI Contribution Provision of experimental data, expertise and reagents. For this study, we characterised mis-spliced collybistin mRNAs in a patient cell line that resulted in disruption of gephyrin and GABA-A receptor clustering in cellular and neuronal models.
Collaborator Contribution Characterisation of a balanced chromosomal translocation affecting the human collybistin gene.
Impact This study (PMID: 18615734) revealed that mutations in the collybistin gene (ARHGEF9) are unlikely to represent a significant risk factor for hyperekplexia, but rather produce complex phenotypes in humans, encompassing epilepsy, anxiety, aggression and intellectual disability.
Start Year 2006
 
Description Highlighting MRC-funded research on the UCL School of Pharmacy website 
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 MRC-funded research resulting in notable publications or achievements is routinely highlighted on the home page of the UCL School of Pharmacy website (http://www.ucl.ac.uk/pharmacy/research) in a Cluster specific 'News' sections e.g. http://www.ucl.ac.uk/pharmacy/research/disease-models-and-clinical-pharmacology/news. Previously website (http://www.pharmacy.ac.uk/latest_news.html) and the following exmples are no longer available.
Examples: http://www.pharmacy.ac.uk/2276.html
http://www.pharmacy.ac.uk/1520.html

Raising awareness of MRC-funded research with the general public, staff, other stakeholders and M.Pharm. Students.
Year(s) Of Engagement Activity 2008,2009,2010,2011,2012
URL http://www.ucl.ac.uk/pharmacy/research/disease-models-and-clinical-pharmacology