Expression of GABAA receptor subtypes varies throughout the brain. Understanding the properties of different subtypes can help us understand brain inhibition, and inform the development of new drugs targeting these receptors. The main GABAA receptor subtype in the brain is α1β2γ2, and receptors are known to differ functionally when they have different α subunits. However, differences between γ subunits could be just as important, and have not been extensively investigated.
This thesis investigated the pharmacology and physiology of GABAA receptors that contain the γ1 subunit. These receptors came to our attention because they are highly expressed in the amygdala, but it is unclear what physiological consequences this expression might have. Studying receptor subtypes in brain tissue is difficult because most neurons express mixtures of subtypes, and there are few selective drugs to differentiate them. Therefore, I set out to identify novel modulators of α2β2γ1 GABAA receptors. A high-throughput screen of 2464 natural product extracts resulted in 26 reproducible hits, and from those I tested 23 pure compounds. Further study of the most promising compounds showed that one compound was a positive modulator at α2β2γ2L receptors and an antagonist at α2β2γ1 receptors.
In order to investigate the physiological properties of γ1-containing receptors, I used co-cultures of neurons and HEK cells expressing the synaptic adhesion molecule neuroligin to make heterosynapses that have a defined receptor composition. Spontaneous currents recorded in the HEK cells transfected with α1β2γ2 resulted in synaptic currents that compared well with literature values for neuron-neuron synaptic events. I tested α2 and γ1-containing synaptic currents and found that α2 and γ1 subunits both cause slower rise and decay times when compared to α1 and γ2. I then used rapid application of GABA onto outside-out patches pulled from the HEK cells to investigate whether the slow synaptic currents were caused by slow channel gating. This was true for α2-containing receptors, which did indeed gate more slowly than α1-containing, however this pattern was not observed for γ1 channels, proving that some other mechanism was responsible for the slow γ1 synaptic currents.
γ1 and γ2 subunits are 90% homologous, however the large intracellular loop is only about 50% conserved. I used chimeras in which the intracellular loop had been switched between γ1 and γ2 to show that this part of the protein determines fast or slow synaptic kinetics. Based on other work showing that the large intracellular loop is important for synaptic localisation of GABAA receptors, I hypothesise that γ1 subunits are less clustered at the synapse than γ2 subunits.
Clearly, important differences exist between γ1 and γ2 subunits. There are still challenges to overcome before selective drugs can be used to definitively identify these receptors, however the heterosynapse model I have used represents an excellent way to identify differences that can then be tested in brain tissue. From this work, I predict that γ1 receptors are clustered differentially from γ2 receptors, and this may represent a new source of variation in synaptic inhibition in the brain.