Missense mutations T166M, Q242L, T336M, and Y474C in the GABA_A receptor (GABA_AR) α3 subunit gene are associated with epileptic seizures, dysmorphic features, intellectual disability, and developmental delay. When incorporated into GABA_ARs 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 GABA_AR 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 GABA_ARs with a defined stoichiometry. We first demonstrated that IPSCs mediated by α3-containing GABA_ARs (α3β3γ2) decay significantly slower than those mediated by α1-containing isoforms (α1β2γ2 or α1β3γ2). GABA_AR 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.

GABA _A受体（GABA _A R）α3亚基基因中的错义突变T166M，Q242L，T336M和Y474C与癫痫发作，畸形特征，智力残疾和发育延迟有关。当掺入到卵母细胞中表达的GABA _A Rs中时，所有突变都会降低GABA诱发的全细胞电流。但是，它们对抑制性突触电流（IPSCs）性质的影响尚不清楚，主要是因为很难建立自然神经突触中表达的GABA _A R的化学计量，而难以控制。为了解决这个问题，我们采用了HEK293细胞-神经元共培养表达系统，该系统可以记录由纯GABA _A群体介导的IPSC具有定义的化学计量比的Rs。我们首先证明了由含α3的GABA _A Rs（α3β3γ2）介导的IPSCs的衰减比由含α1的同工型（α1β2γ2或α1β3γ2）介导的IPSC的衰减要慢得多。GABA _ARα3突变不影响IPSC的峰值幅度或10-90％的上升时间，但其中三个突变影响IPSC的衰减。T336M显着加速了IPSC衰减速率，而T166M和Y474C则相反。由T366M突变引起的IPSC衰减动力学的加速通过抗癫痫药物咪达唑仑恢复为野生型。与以前的卵母细胞数据一致，HEK293细胞中的定量实验显示所有突变体的细胞表面表达均显着降低。两者合计，我们的结果表明，受损的表面表达和改变的IPSC衰减率可能都是与这些突变相关的病理基础的重要因素。