Abstract
Missense mutations T166M, Q242L, T336M, and Y474C in the GABAA receptor (GABAAR) α3 subunit gene are associated with epileptic seizures, dysmorphic features, intellectual disability, and developmental delay. When incorporated into GABAARs expressed in oocytes, all mutations are known to reduce GABA-evoked whole-cell currents. However, their impact on the properties of inhibitory synaptic currents (IPSCs) is unknown, largely because it is difficult to establish, much less control, the stoichiometry of GABAAR expressed in native neuronal synapses. To circumvent this problem, we employed a HEK293 cell-neuron co-culture expression system that permits the recording of IPSCs mediated by a pure population of GABAARs with a defined stoichiometry. We first demonstrated that IPSCs mediated by α3-containing GABAARs (α3β3γ2) decay significantly slower than those mediated by α1-containing isoforms (α1β2γ2 or α1β3γ2). GABAAR α3 mutations did not affect IPSC peak amplitudes or 10–90% rise times, but three of the mutations affected IPSC decay. T336M significantly accelerated the IPSC decay rate whereas T166M and Y474C had the opposite effect. The acceleration of IPSC decay kinetics caused by the T366M mutation was returned to wild-type-like values by the anti-epileptic medication, midazolam. Quantification experiments in HEK293 cells revealed a significant reduction in cell-surface expression for all mutants, in agreement with previous oocyte data. Taken together, our results show that impaired surface expression and altered IPSC decay rates could both be significant factors underlying the pathologies associated with these mutations.
Highlights
Type A γ-aminobutyric acid receptors (GABAARs) are pentameric ligand-gated ion channels found at a majority of inhibitory synapses in the central nervous system
We compared the kinetics of IPSCs mediated by this combination with those mediated by α1β2γ2, the most widely-expressed synaptic GABAA receptor (GABAAR) subtype in the brain (Sieghart and Sperk, 2002)
This study reveals unique kinetic properties of IPSCs mediated by α3-containing GABAARs in a co-culture system, and shows that they have slower decay kinetics than other synaptic GABAAR isoforms
Summary
Type A γ-aminobutyric acid receptors (GABAARs) are pentameric ligand-gated ion channels found at a majority of inhibitory synapses in the central nervous system. They are anion-selective channels that mediate fast synaptic inhibitory neurotransmission on a millisecond timescale and are crucial for maintaining the excitatory/inhibitory balance of activity in the brain. GABAA Receptor α3 Epilepsy Mutations of π, θ, δ and ε (McKernan and Whiting, 1996). Each of these subunits consists of a hydrophilic extracellular N-terminal domain containing a Cys-loop, followed by four α-helical transmembrane domains (TM1–4) and an extracellular Cterminus. TM2 lines the integral ion channel and the intracellular loop between TM3 and TM4 interacts with various proteins involved in receptor trafficking, phosphorylation, and clustering (Kasaragod and Schindelin, 2019)
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