Silayeva Liliya, Deeb Tarek Z, Hines Rochelle M, Kelley Matt R, Munoz Michaelanne B, Lee Henry H C, Brandon Nicholas J, Dunlop John, Maguire Jaime, Davies Paul A, Moss Stephen J
AstraZeneca-Tufts Laboratory for Basic and Translational Neuroscience, Boston, MA 02111; Department of Neuroscience, Tufts University School of Medicine, Boston, MA 02111;
Department of Neuroscience, Tufts University School of Medicine, Boston, MA 02111;
Proc Natl Acad Sci U S A. 2015 Mar 17;112(11):3523-8. doi: 10.1073/pnas.1415126112. Epub 2015 Mar 2.
The K(+)/Cl(-) cotransporter (KCC2) allows adult neurons to maintain low intracellular Cl(-) levels, which are a prerequisite for efficient synaptic inhibition upon activation of γ-aminobutyric acid receptors. Deficits in KCC2 activity are implicated in epileptogenesis, but how increased neuronal activity leads to transporter inactivation is ill defined. In vitro, the activity of KCC2 is potentiated via phosphorylation of serine 940 (S940). Here we have examined the role this putative regulatory process plays in determining KCC2 activity during status epilepticus (SE) using knockin mice in which S940 is mutated to an alanine (S940A). In wild-type mice, SE induced by kainate resulted in dephosphorylation of S940 and KCC2 internalization. S940A homozygotes were viable and exhibited comparable basal levels of KCC2 expression and activity relative to WT mice. However, exposure of S940A mice to kainate induced lethality within 30 min of kainate injection and subsequent entrance into SE. We assessed the effect of the S940A mutation in cultured hippocampal neurons to explore the mechanisms underlying this phenotype. Under basal conditions, the mutation had no effect on neuronal Cl(-) extrusion. However, a selective deficit in KCC2 activity was seen in S940A neurons upon transient exposure to glutamate. Significantly, whereas the effects of glutamate on KCC2 function could be ameliorated in WT neurons with agents that enhance S940 phosphorylation, this positive modulation was lost in S940A neurons. Collectively our results suggest that phosphorylation of S940 plays a critical role in potentiating KCC2 activity to limit the development of SE.
钾离子/氯离子共转运体（KCC2）使成年神经元能够维持较低的细胞内氯离子水平，这是γ-氨基丁酸受体激活后有效突触抑制的前提条件。KCC2活性缺陷与癫痫发生有关，但神经元活动增加如何导致转运体失活尚不清楚。在体外，KCC2的活性通过丝氨酸940（S940）的磷酸化而增强。在这里，我们使用S940突变为丙氨酸（S940A）的敲入小鼠，研究了这一假定的调节过程在癫痫持续状态（SE）期间决定KCC2活性中所起的作用。在野生型小鼠中，海藻酸诱导的SE导致S940去磷酸化和KCC2内化。S940A纯合子存活，并且相对于野生型小鼠表现出相当的KCC2表达和活性基础水平。然而，将S940A小鼠暴露于海藻酸会在注射海藻酸后30分钟内导致死亡，并随后进入SE。我们评估了S940A突变在培养的海马神经元中的作用，以探索这种表型的潜在机制。在基础条件下，该突变对神经元氯离子外流没有影响。然而，短暂暴露于谷氨酸后，S940A神经元中观察到KCC2活性存在选择性缺陷。值得注意的是，虽然谷氨酸对KCC2功能的影响可以在野生型神经元中通过增强S940磷酸化的试剂得到改善，但这种正向调节在S940A神经元中丧失。我们的结果共同表明，S940的磷酸化在增强KCC2活性以限制SE的发展中起关键作用。
