Division of Neuroscience, Ninewells Hospital and Medical School, University of Dundee, Dundee, UK.
Institute for Physiology and Pathophysiology, University of Heidelberg, Heidelberg, Germany.
Lancet. 2015 Feb 26;385 Suppl 1:S85. doi: 10.1016/S0140-6736(15)60400-7.
Epilepsy is characterised by disturbed neuronal activity in the brain rendering it more susceptible to seizures. An understanding of the molecular mechanisms by which the balance between excitability and inhibition in neuronal networks is controlled will help to devise better treatment options. Hyperpolarising synaptic inhibition through GABAA (γ aminobutyric acid type A) and glycine receptors depends on the presence of the neuronal cation-chloride-cotransporter protein, KCC2. Several transcriptional and post-transcriptional mechanisms have been shown to regulate KCC2 and thereby affect the polarity and efficacy of inhibitory synaptic transmission. However, it is unknown whether regulation of KCC2 enables the transporter to attain different levels of activity, thus allowing a neuron to modulate the strength of inhibitory synaptic transmission to its changing requirements. We therefore investigated whether phosphorylation can allow KCC2 to achieve distinct levels of intracellular chloride ion concentrations in neurons.
A variety of KCC2 alanine dephosphorylation mimics were created and NH4(+)-induced pHi shifts were used in cultured hippocampal neurons to quantify the rate of KCC2 transport activity exhibited by these mutants. The association between KCC2 transport strength and GABAA receptor-mediated current amplitudes was investigated by performing gramicidine perforated-patch recordings. The correlation between reversal potential of GABAergic currents (EGABA) and NH4(+)-induced pHi shifts enabled an estimate of the range of chloride extrusion possible by kinase-phosphatase regulation of KCC2. Finally, we used the Goldman-Hodgkin-Katz equation to examine how EGABA would vary with increasing concentrations of extracellular K(+) in neurons expressing KCC2 mutants with different rates of transport.
KCC2 transport strength varied considerably in magnitude (from -0·02 to -1·00 pHi shifts) depending on the combination of alanine mutations present on the protein. KCC2 transport strength determined the direction and magnitude of GABAA receptor-mediated current amplitudes and was observed to have a linear correlation with the reversal potential of GABAergic currents.
Our findings highlight the considerable potential for regulating the inhibitory tone by KCC2-mediated chloride extrusion. Transport can be enhanced to sufficiently high levels that hyperpolarising GABAA responses can be obtained even at high extracellular K(+) concentrations and in neurons with an extremely negative resting membrane potential. We conclude that cellular signalling pathways might act together to alter the state of KCC2 phosphorylation and dephosphorylation and thereby tune the strength of synaptic inhibition.
Royal Society.
癫痫的特征是大脑神经元活动紊乱，使其更容易发生癫痫发作。了解控制神经元网络兴奋与抑制平衡的分子机制将有助于设计更好的治疗方案。通过 GABA A（γ-氨基丁酸 A 型）和甘氨酸受体实现的超极化突触抑制依赖于神经元阳离子-氯离子共转运蛋白 KCC2 的存在。已经有几种转录和转录后机制被证明可以调节 KCC2，从而影响抑制性突触传递的极性和效率。然而，目前尚不清楚 KCC2 的调节是否使转运体能够达到不同的活性水平，从而使神经元能够调节抑制性突触传递的强度以适应其不断变化的需求。因此，我们研究了磷酸化是否可以使 KCC2 在神经元中达到不同的细胞内氯离子浓度水平。
创建了各种 KCC2 丙氨酸去磷酸化模拟物，并在培养的海马神经元中使用 NH4(+)诱导的 pHi 变化来量化这些突变体表现出的 KCC2 转运活性的速率。通过进行革兰氏菌素穿孔膜片钳记录来研究 KCC2 转运强度与 GABA A 受体介导的电流幅度之间的关系。GABA 能电流的反转电位（EGABA）与 NH4(+)诱导的 pHi 变化之间的相关性使我们能够估计激酶-磷酸酶对 KCC2 的调节可实现的氯离子外排范围。最后，我们使用 Goldman-Hodgkin-Katz 方程研究了在表达具有不同转运速率的 KCC2 突变体的神经元中，随着细胞外 K(+)浓度的增加，EGABA 将如何变化。
KCC2 转运强度的幅度差异很大（从 -0·02 到 -1·00 pHi 变化），这取决于蛋白上存在的丙氨酸突变的组合。KCC2 转运强度决定了 GABA A 受体介导的电流幅度的方向和幅度，并与 GABA 能电流的反转电位呈线性相关。
我们的发现突出表明，通过 KCC2 介导的氯离子外排来调节抑制性音调具有相当大的潜力。转运可以增强到足够高的水平，即使在高细胞外 K(+)浓度和具有极度负静息膜电位的神经元中，也可以获得超极化的 GABA A 反应。我们得出结论，细胞信号通路可能共同作用改变 KCC2 磷酸化和去磷酸化的状态，从而调节突触抑制的强度。
英国皇家学会。
