Brain-type creatine kinase activates neuron-specific K+-Cl- co-transporter KCC2
Brain-type creatine kinase activates neuron-specific K+-Cl- co-transporter KCC2
复制标题
DOI：
10.1111/j.1471-4159.2005.03560.x
复制
发表时间：
2006-01-01
影响因子：
4.7
通讯作者：
Fukuda, A
中科院分区：
文献类型：
--
作者：
Inoue, K;Yamada, J;Fukuda, A
GABA是成年中枢神经系统中的一种主要抑制性神经递质，在发育早期由于细胞内氯离子浓度([Cl-](I))升高而兴奋。这种功能转换主要归因于K+-Cl-共转运蛋白KCC2，它的表达在神经元中受到发育调节。此前，我们报道了KCC2与脑型肌酸激酶(CKB)相互作用。为了阐明这种相互作用的功能意义，将KCC2和甘氨酸受体α2亚单位导入HEK293细胞，通过膜片钳记录测量甘氨酸翻转电位(E-Gly)，估计[Cl-](I)。表达KCC2的细胞在改变细胞外K+浓度([K+](O))或给予KCCs的抑制剂后，E-Gly显示出预期的变化，表明KCC2的功能得到了适当的评估。与野生型CKB相比，当加入KCC2表达细胞时，显性负性CKB诱导E-Gly发生去极化漂移，并减弱E-Gly因[K+](O)降低而出现的超极化漂移。此外，CKS的抑制剂2，4-二硝基氟苯(DNFB)使E-Gly向去极化方向移动。在表达CKB的原代皮质神经元中，DNFB也使GABA翻转电位向去极化方向移动。我们的发现表明，在细胞微环境中，CKB激活了KCC2功能。
GABA, a major inhibitory neurotransmitter in the adult CNS, is excitatory at early developmental stages as a result of the elevated intracellular Cl- concentration ([Cl-](i)). This functional switch is primarily attributable to the K+-Cl- co-transporter KCC2, the expression of which is developmentally regulated in neurons. Previously, we reported that KCC2 interacts with brain-type creatine kinase (CKB). To elucidate the functional significance of this interaction, HEK293 cells were transfected with KCC2 and glycine receptor alpha 2 subunit, and gramicidin-perforated patch-clamp recordings were performed to measure the glycine reversal potential (E-gly), giving an estimate of [Cl-](i). KCC2-expressing cells displayed the expected changes in E-gly following alterations in the extracellular K+ concentration ([K+](o)) or administration of an inhibitor of KCCs, suggesting that the KCC2 function was being properly assessed. When added into KCC2-expressing cells, dominant-negative CKB induced a depolarizing shift in E-gly and reduced the hyperpolarizing shift in E-gly seen in response to a lowering of [K+](o) compared with wild-type CKB. Moreover, 2,4-dinitrofluorobenzene (DNFB), an inhibitor of CKs, shifted E-gly in the depolarizing direction. In primary cortical neurons expressing CKB, the GABA reversal potential was also shifted in the depolarizing direction by DNFB. Our findings suggest that, in the cellular micro-environment, CKB activates the KCC2 function.