Sung K W, Kirby M, McDonald M P, Lovinger D M, Delpire E
Departments of Anesthesiology, Center for Molecular Neuroscience, Vanderbilt University Medical Center, Nashville, Tennesse, USA.
J Neurosci. 2000 Oct 15;20(20):7531-8. doi: 10.1523/JNEUROSCI.20-20-07531.2000.
We have recently disrupted Slc12a2, the gene encoding the secretory Na-K-2Cl cotransporter in mice (NKCC1) (Delpire et al., 1999). Gramicidin perforated-patch and whole-cell recordings were performed to study GABA-induced currents in dorsal root ganglion (DRG) neurons isolated from wild-type and homozygote NKCC1 knock-out mice. In wild-type DRG neurons, strong GABA-evoked inward current was observed at the resting membrane potential, suggesting active accumulation of Cl(-) in these cells. This GABA-induced current was blocked by picrotoxin, a GABA(A) receptor blocker. The strong Cl(-) accumulation that gives rise to depolarizing GABA responses is caused by Na-K-2Cl cotransport because reduction of external Cl(-) or application of bumetanide induced a decrease in Cl(-), whereas an increase in external K(+) caused an apparent Cl(-) accumulation. In contrast to control neurons, little or no net current was observed at the resting membrane potential in homozygote NKCC1 mutant DRG neurons. E(GABA) was significantly more negative, demonstrating the absence of Cl(-) accumulation in these cells. Application of bumetanide induced a positive shift of E(GABA), suggesting the presence of an outward Cl(-) transport mechanism. In agreement with an absence of GABA depolarization in DRG neurons, behavioral analysis revealed significant alterations in locomotion and pain perception in the knock-out mouse. Our results clearly demonstrate that the Na-K-2Cl cotransporter is responsible for Cl(-) accumulation in DRG neurons and that via regulation of intracellular Cl(-), the Na-K-2Cl cotransporter participates in the modulation of GABA neurotransmission and sensory perception.
我们最近破坏了Slc12a2基因，该基因编码小鼠中的分泌型钠-钾-2氯协同转运蛋白（NKCC1）（德尔皮尔等人，1999年）。进行了短杆菌肽穿孔膜片钳和全细胞记录，以研究从野生型和纯合子NKCC1基因敲除小鼠分离的背根神经节（DRG）神经元中γ-氨基丁酸（GABA）诱导的电流。在野生型DRG神经元中，在静息膜电位下观察到强烈的GABA诱发内向电流，表明这些细胞中氯离子（Cl⁻）的主动积累。这种GABA诱导的电流被GABA（A）受体阻断剂印防己毒素阻断。导致去极化GABA反应的强烈Cl⁻积累是由钠-钾-2氯协同转运引起的，因为降低细胞外Cl⁻或应用布美他尼会导致细胞内Cl⁻浓度（[Cl⁻]i）降低，而增加细胞外钾离子会导致[Cl⁻]i明显积累。与对照神经元相反，在纯合子NKCC1突变体DRG神经元的静息膜电位下几乎没有观察到净电流。GABA的反转电位（E（GABA））明显更负，表明这些细胞中不存在Cl⁻积累。应用布美他尼会导致E（GABA）正向偏移，表明存在外向Cl⁻转运机制。与DRG神经元中不存在GABA去极化一致，行为分析显示基因敲除小鼠的运动和疼痛感知有显著改变。我们的结果清楚地表明，钠-钾-2氯协同转运蛋白负责DRG神经元中[Cl⁻]i的积累，并且通过调节细胞内Cl⁻，钠-钾-2氯协同转运蛋白参与GABA神经传递和感觉感知的调节。
