Na+, K+, 2Cl- cotransport and intracellular chloride regulation in rat primary sensory neurons:: Thermodynamic and kinetic aspects
Na+, K+, 2Cl- cotransport and intracellular chloride regulation in rat primary sensory neurons:: Thermodynamic and kinetic aspects
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DOI：
10.1152/jn.01007.2007
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发表时间：
2008-07-01
影响因子：
2.5
通讯作者：
Alvarez-Leefmans, Francisco J.
中科院分区：
文献类型：
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作者：
Rocha-Gonzalez, Hector I.;Mao, Shihong;Alvarez-Leefmans, Francisco J.
成人初级传入神经元在其整个表面，包括位于背根神经节（DRG）的胞体，都被GABA去极化。由脊髓中间神经元释放的GABA介导的初级传入去极化（PAD）决定了突触前抑制，这是躯体感觉加工的关键机制。去极化是由于通过GABA（A）通道的Cl-流出;向外的Cl-梯度是由首次在两栖动物中建立的Na+，K+，2Cl（-）协同转运蛋白（NKCC）产生的。用荧光成像显微镜，我们测量了[Cl-]（i）和细胞水体积（CWV）在分离的大鼠DRG细胞（P0-P21）负载N-（乙氧羰基甲基）-6-甲氧基喹啉溴化和钙黄绿素，分别。基础[Cl-]（i）为44.2 +/- 1.2 mM（平均值+/- SE），Cl-平衡电位（E-Cl）为-27.0 +/- 0.7 mV（n = 75）。该[Cl-]（i）比电化学平衡高约4倍。在等渗去除外部Cl-，细胞失去Cl-和萎缩。在返回到对照溶液中时，细胞重新积累Cl-并恢复CWV。Cl-再积累有Na+依赖性（SDC）和Na+非依赖性（SIC）两种成分. SIC稳定在[Cl-]（i）= 13.2 +/- 1.2 mM，表明其为被动（E-Cl = -60.5 +/- 3 mV）。布美他尼阻断CWV恢复和大多数（65%）SDC（IC 50 = 5.7 μ M），表明两者均由NKCC介导。活性Cl-吸收随[Cl-]（i）的增加而下降，当[Cl-]（i）达到基础水平时，活性Cl-吸收可忽略不计。主动Cl-摄取的动力学表明了一个负反馈系统，其中细胞内Cl-调节其自身的流入，从而保持[Cl-]（i）恒定，高于电化学平衡，但低于NKCC达到热力学平衡时将达到的值。
Adult primary afferent neurons are depolarized by GABA throughout their entire surface, including their somata located in dorsal root ganglia (DRG). Primary afferent depolarization (PAD) mediated by GABA released from spinal interneurons determines presynaptic inhibition, a key mechanism in somatosensory processing. The depolarization is due to Cl- efflux through GABA(A) channels; the outward Cl- gradient is generated by a Na+, K+, 2Cl(-) cotransporter (NKCC) as first established in amphibians. Using fluorescence imaging microscopy we measured [Cl-](i) and cell water volume (CWV) in dissociated rat DRG cells (P0-P21) loaded with N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide and calcein, respectively. Basal [Cl-](i) was 44.2 +/- 1.2 mM (mean +/- SE), Cl- equilibrium potential (E-Cl) was -27.0 +/- 0.7 mV (n = 75). This [Cl-](i) is about four times higher than electrochemical equilibrium. On isosmotic removal of external Cl-, cells lost Cl- and shrank. On returning to control solution, cells reaccumulated Cl- and recovered CWV. Cl- reaccumulation had Na+ -dependent (SDC) and Na+ -independent (SIC) components. The SIC stabilized at [Cl-](i) = 13.2 +/- 1.2 mM, suggesting that it was passive (E-Cl = -60.5 +/- 3 mV). Bumetanide blocked CWV recovery and most (65%) of the SDC (IC50 = 5.7 mu M), indicating that both were mediated by NKCC. Active Cl- uptake fell with increasing [Cl-](i) and became negligible when [Cl-](i) reached basal levels. The kinetics of active Cl- uptake suggests a negative feedback system in which intracellular Cl- regulates its own influx thereby keeping [Cl-](i) constant, above electrochemical equilibrium but below the value that would attain if NKCC reached thermodynamic equilibrium.