Hartmann Anna-Maria, Blaesse Peter, Kranz Thorsten, Wenz Meike, Schindler Jens, Kaila Kai, Friauf Eckhard, Nothwang Hans Gerd
Department of Neurogenetics, Institute for Biology and Environmental Sciences, Carl von Ossietzky University, Oldenburg, Germany.
J Neurochem. 2009 Oct;111(2):321-31. doi: 10.1111/j.1471-4159.2009.06343.x. Epub 2009 Aug 17.
In the majority of neurons, the intracellular Cl(-) concentration is set by the activity of the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) and the K(+)-Cl(-) cotransporter (KCC2). Here, we investigated the cotransporters' functional dependence on membrane rafts. In the mature rat brain, NKCC1 was mainly insoluble in Brij 58 and co-distributed with the membrane raft marker flotillin-1 in sucrose density flotation experiments. In contrast, KCC2 was found in the insoluble fraction as well as in the soluble fraction, where it co-distributed with the non-raft marker transferrin receptor. Both KCC2 populations displayed a mature glycosylation pattern. Disrupting membrane rafts with methyl-beta-cyclodextrin (MbetaCD) increased the solubility of KCC2, yet had no effect on NKCC1. In human embryonic kidney-293 cells, KCC2 was strongly activated by a combined treatment with MbetaCD and sphingomyelinase, while NKCC1 was inhibited. These data indicate that membrane rafts render KCC2 inactive and NKCC1 active. In agreement with this, inactive KCC2 of the perinatal rat brainstem largely partitioned into membrane rafts. In addition, the exposure of the transporters to MbetaCD and sphingomyelinase showed that the two transporters differentially interact with the membrane rafts. Taken together, membrane raft association appears to represent a mechanism for co-ordinated regulation of chloride transporter function.
在大多数神经元中，细胞内氯离子浓度由钠钾氯共转运体（NKCC1）和钾氯共转运体（KCC2）的活性设定。在此，我们研究了这两种共转运体对膜筏的功能依赖性。在成熟大鼠脑中，NKCC1在Brij 58中主要不溶，且在蔗糖密度浮选实验中与膜筏标记物弗洛蒂林-1共分布。相比之下，KCC2在不溶部分以及可溶部分均有发现，在可溶部分它与非膜筏标记物转铁蛋白受体共分布。两种KCC2群体均呈现成熟的糖基化模式。用甲基-β-环糊精（MβCD）破坏膜筏会增加KCC2的溶解度，但对NKCC1没有影响。在人胚肾-293细胞中，KCC2经MβCD和鞘磷脂酶联合处理后被强烈激活，而NKCC1则受到抑制。这些数据表明膜筏使KCC2失活而使NKCC1激活。与此一致的是，围产期大鼠脑干中无活性的KCC2大部分分布在膜筏中。此外，将这两种共转运体暴露于MβCD和鞘磷脂酶表明它们与膜筏的相互作用存在差异。综上所述，膜筏结合似乎代表了一种协调调节氯离子转运体功能的机制。
