Mechanisms of clustering of GABA-A receptors

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.

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.