Regulating synaptic and extrasynaptic GABA-A receptors in health and disease

For the brain to function coherently, it is vital that individual nerve cells exert some control over their innate excitability. The main mechanism by which this is achieved involves the process of synaptic inhibition. Principally, this requires nerve cells to express GABA-A receptors at selected locations on their cell surface which are located opposite presynaptic nerve terminals releasing the neurotransmitter GABA. These structures are known as synapses. It is the release of GABA that activates the receptors causing a rapid but brief flow of chloride ions across the cell membrane through ion channels contained within the receptor structure. This flow either causes the cell to hyperpolarize, or massively increases the membrane conductance of the cell membrane, thereby reducing neuronal excitation. These receptors are known targets for numerous drugs, including, benzodiazepines, barbiturates, general anaesthetics and neurosteroids. A malfunction in synaptic inhibition, which could occur as a result of, a receptor mutation; a reduction in receptor numbers near GABA-releasing terminals; or because of dysregulation by naturally-occurring agents in the brain, can easily result in uncontrolled excitability with devastating consequences for humans, e.g., epilepsy. This research programme will focus on understanding the molecular mechanisms by which GABA-A receptors are regulated at or near synapses, and specifically what controls their movement (trafficking) into and out of synapses in the cell surface membrane. We will also concentrate on identifying which endogenous regulatory processes are important in modulating GABA-A receptor function and how they influence receptor trafficking. To this end, we have recently solved where a group of endogenous compounds in the brain, known as neurosteroids, bind to and modulate GABA-A receptors. With this knowledge we can use a genetic approach to dissect the importance of particular GABA-A receptor subtypes in health and disease processes. We plan to integrate our new knowledge into achieving a better understanding of how GABA-A receptors are regulated at synapses by studying synaptic plasticity: a phenomenon whereby synaptic transmission between neurones can be manipulated over time; and also by using animal models of disease, principally epilepsy. These aims will be achieved using cellular, genetic, molecular and pharmacological approaches in conjunction with novel techniques to explain how the most important inhibitory receptor in the brain controls nerve cell excitability. This study will be performed at UCL which is a recognised centre of excellence in neuroscience. The results of this research will be disseminated to the public via lectures and open publications

GABA-A receptors are vitally important for mediating synaptic inhibition in the CNS. This is emphasised not only by the consequences of their dysregulation, which is associated with neurological diseases, but also because they are targets for therapeutic agents. It is therefore important to understand those mechanisms that control the function and trafficking of GABA-A receptors at inhibitory synapses and extrasynaptic domains and how they ultimately shape synaptic inhibition. This work will build upon the foundations laid by our previous programme grant on the construction of inhibitory synapses. The main objectives will be addressed in four closely integrated sections:

We will ascertain the important factors that determine the level and extent of lateral mobility for GABA-A receptors and why some receptor subtypes appear to be relatively static. We will study how new accessory proteins and their binding sites on receptor subunits influence receptor movement in addition to using electrophysiological methods to measure, in real time, GABA-A receptor movements between synapses and extrasynaptic domains. How endogenous modulators in the brain affect lateral receptor mobility will also be addressed.

The second section will build on our discovery of the neurosteroid binding sites on GABA-A receptors. We will establish the importance of these sites in other GABA-A receptor isoforms and deduce whether one or both binding sites needs to be occupied for modulation of GABA-A receptors. Lenti viral constructs will help us to decipher the role of receptor subtypes in mediating neurosteroid modulation and overall, their importance in health and disease will be addressed using two a subunit knock-ins generated by homologous recombination.

In the third section, the mechanisms underlying inhibitory synaptic plasticity will be further investigated by studying the role of GABA-A receptor phosphorylation. We will establish how important surface trafficking mechanisms are for affecting long-term synaptic inhibition by changing receptor numbers at synaptic and extrasynaptic sites. New knock-in animals in which we have ablated selected phosphorylation sites will also be used to assess their impact on receptor trafficking and function.

The fourth aim will concentrate on understanding the importance of surface stability, subunit composition and mutations of GABA-A receptors in epilepsy using established animal models. This will include the association of GABA-A receptors with protein kinases and how this affects synaptic inhibition as well as the trafficking of GABA-A receptors.

Overall, this programme will provide insight into how neurones regulate their synaptic and extrasynaptic GABA-A receptors under physiological and pathophysiological conditions.