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Development of novel tools and techniques for the study of the structure and dynamics of GABAergic inhibitory synapses

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Resolving synaptic inhibition

Gamma-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the vertebrate central nervous system. Scientists exploited advanced imaging and genetic techniques to study structural and functional relationships regarding GABA receptors.

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Binding of GABA to type A GABA receptors (GABA-A) results in opening of chloride-specific (Cl-) ion channels. Opening ion channels allows ions to diffuse down their concentration gradients. Since Cl- is in higher concentration in the extracellular fluid, Cl- ions flow in and inhibit initiation of electrical signals to other cells. Although clustering of GABA-A receptors is important in health and disease, the mechanisms of assembly and modulation are not well understood. Scientists initiated the EU-funded project GABAAR to study GABA-A receptors at synapses and to investigate the role of the scaffolding protein gephyrin. 'Gephyra' is the Greek word for bridge. Gephyrin forms a bridge anchoring inhibitory neurotransmitter receptors to the postsynaptic cytoskeleton. Scientists tagged GABA-A receptor subunits and gephyrin with photoconvertible fluorescent proteins and expressed them in cultured neurons from the central nervous system. They used two types of photo-activated localisation microscopy (PALM) to confirm that the subunit constructs form functional receptors correctly localised at the plasma membrane surface at synapses. Researchers then focused on gephyrin. They went on to show important relationships between gephyrin and another Cl- ion channel-linked receptor (for glycine). They also demonstrated competition between the glycine receptors and GABAA receptors for the gephyrin binding sites. GABAAR investigators described the changes in the morphology and stability of synaptic gephyrin clusters and the level of interaction between proteins, information previously not available. Results are very important to any field of science studying dynamic clustered structures, and the PALM techniques promise to be quite useful in such research. The team exploited genetic methods to minimally disrupt the protein structure of GABA-A receptors. Unnatural amino acids were encoded in both model proteins and functional neuronal receptors. The studies could make it possible to tag individual receptors with a single fluorophore, enabling single-particle imaging with combined electrophysiology to study protein structure, dynamics and function. GABAAR developed powerful techniques for visualising structural and functional changes at inhibitory neurotransmitter synapses in the vertebrate central nervous system. Application of these techniques has already shed important light on previously poorly understood topics and promises to advance understanding of many other biological systems.

Keywords

Synaptic inhibition, GABA, neurotransmitter, GABA receptors, GABA-A, gephyrin

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