Optical Control of Neuronal Inhibition with Genetically Engineered Light Inhibited GABAA Receptors (Li-GABARs)

GABA is the main inhibitory neurotransmitter in the brain, acting primarily at ionotropic GABAA receptors. The α subunit is critical in determining GABAA receptor functional properties and pharmacological regulation. There are six α subunit subtypes that are differentially expressed in the nervous system but few selective antagonists are available to dissect the functional role of those subtypes in neurons. We have therefore engineered α subunit specific light inhibited GABAA receptors (Li-GABARs). We generated α subunits with a cysteine mutation that allows attachment of a photoswitchable tethered ligand (PTL) consisting of a cysteine reactive maleimide group, a photoisomerizable azobenzene core and a variable GABAR ligand. Using cysteine-scanning mutagenesis we identified optimal photoswitch attachment sites near the GABA binding pocket for both α1 and α5 subunits, and tested multiple PTLs to produce maximal light dependent block of GABAR activity while minimally affecting basal receptor properties. We recorded from hippocampal slices expressing Li-GABARs and found evidence that α5, but not α1 containing receptors counteract NMDA receptor dependent synaptic depolarization. This novel interaction could provide a basis for the regulation of synaptic plasticity and memory by α5 receptors. This approach in general provides a strategy to study the role of specific GABAA receptor subunits and their importance in neuronal function and disease.