Palma E, Amici M, Sobrero F, Spinelli G, Di Angelantonio S, Ragozzino D, Mascia A, Scoppetta C, Esposito V, Miledi R, Eusebi F
Istituto Pasteur, Fondazione Cenci Bolognetti and Dipartimento di Fisiologia Umana e Farmacologia, Centro di Eccellenza Biologia e Medicina Molecolare, Università di Roma La Sapienza, Piazzale A. Moro 5, 00185 Rome, Italy.
Proc Natl Acad Sci U S A. 2006 May 30;103(22):8465-8. doi: 10.1073/pnas.0602979103. Epub 2006 May 18.
The mRNA levels of NKCC1, an inwardly directed Na(+), K(+)-2Cl(-) cotransporter that facilitates the accumulation of intracellular Cl(-), and of KCC2, an outwardly directed K(+)-Cl(-) cotransporter that extrudes Cl(-), were studied in surgically resected brain specimens from drug-resistant temporal lobe (TL) epilepsy (TLE) patients. Quantitative RT-PCR analyses of the mRNAs extracted from the human TLE-associated brain regions revealed an up-regulation of NKCC1 mRNA and a down-regulation of KCC2 mRNA in the hippocampal subiculum, compared with the hippocampus proper or the TL neocortex, suggesting an abnormal transcription of Cl(-) transporters in the TLE subiculum. In parallel experiments, cell membranes isolated from the same TLE-associated brain regions were injected into Xenopus oocytes that rapidly incorporated human GABA(A) receptors into their surface membrane. The GABA currents elicited in oocytes injected with membranes from the subiculum had a more depolarized reversal potential (E(GABA)) compared with the hippocampus proper or the neocortex. The NKCC1 blocker bumetanide or a temperature decrease of 10 degrees C shifted the GABA-current E(GABA) more negative in oocytes injected with membranes from TLE hippocampal subiculum, matching the E(GABA) of TL neocortex-injected oocytes. We conclude that the anomalous expression of both Cl(-) transporters, NKCC1 and KCC2 [corrected] in TLE hippocampal subiculum probably causes altered Cl(-) transport in the "epileptic" neurons, as revealed in the microtransplanted Xenopus oocytes, and renders GABA aberrantly "exciting," a feature that may contribute to the precipitation of epileptic seizures.
研究了内向性Na(+)、K(+)-2Cl(-)协同转运蛋白NKCC1（促进细胞内Cl(-)蓄积）和外向性K(+)-Cl(-)协同转运蛋白KCC2（排出Cl(-)）的mRNA水平，这些研究来自耐药性颞叶（TL）癫痫（TLE）患者的手术切除脑标本。对从人类TLE相关脑区提取的mRNA进行定量RT-PCR分析显示，与海马本身或TL新皮质相比，海马下托中NKCC1 mRNA上调，KCC2 mRNA下调，提示TLE下托中Cl(-)转运蛋白存在异常转录。在平行实验中，将从相同TLE相关脑区分离的细胞膜注射到非洲爪蟾卵母细胞中，这些卵母细胞会迅速将人类GABA(A)受体整合到其表面膜中。与海马本身或新皮质相比，注射下托细胞膜的卵母细胞中诱发的GABA电流具有更去极化的反转电位（E(GABA)）。NKCC1阻滞剂布美他尼或温度降低10摄氏度会使注射TLE海马下托细胞膜的卵母细胞中的GABA电流E(GABA)向更负的方向移动，与注射TL新皮质细胞膜的卵母细胞的E(GABA)相匹配。我们得出结论，TLE海马下托中Cl(-)转运蛋白NKCC1和KCC2 [校正后]的异常表达可能导致“癫痫”神经元中Cl(-)转运改变，如在微移植的非洲爪蟾卵母细胞中所揭示的那样，并使GABA异常“兴奋”，这一特征可能有助于癫痫发作的诱发。
