Cation transport by the neuronal K(+)-Cl(-) cotransporter KCC2: thermodynamics and kinetics of alternate transport modes.
Cation transport by the neuronal K(+)-Cl(-) cotransporter KCC2: thermodynamics and kinetics of alternate transport modes.
复制标题
神经元 K( )-Cl(-) 协同转运蛋白 KCC2 的阳离子转运：替代转运模式的热力学和动力学。
DOI：
10.1152/ajpcell.00005.2004
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Payne,JohnA
中科院分区：
文献类型：
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作者：
Williams,JefferyR;Payne,JohnA
Cs+和NH4+都能改变神经元Cl -稳态，但其机制尚不清楚。我们假设这两种阳离子改变了神经元K+-Cl−共转运体（KCC2）的运作。利用外源表达的KCC2蛋白，我们首先通过监测不同固定外源阳离子浓度（Na+、Li+、K+、Cs+和NH4+）下的速敏性86rb +内流作为外源Rb+浓度的函数，研究了KCC2转运位点阳离子的相互作用。Na+和Li+都不影响速尿敏感的86rb +内流，表明它们不能在KCC2的阳离子易位位点相互作用。正如预期的那样，作为一种接受Rb+和K+作为交替底物的酶，K+是KCC2转运Rb+的竞争性抑制剂。与K+一样，Cs+和NH4+都表现为KCC2转运Rb+的竞争性抑制剂，表明它们作为转运底物的潜力。使用离子色谱法测量单向Rb+和Cs+流入，我们确定尽管KCC2能够运输Cs+，但与Rb+相比，它的表观亲和力和最大速度较低。为了评估KCC2对NH4+的转运，我们在NH4+诱导的碱性负荷后，用pH敏感的荧光染料监测细胞内pH （pHi）。表达KCC2蛋白的细胞比未转染的细胞恢复phimi的速度更快，表明KCC2可以介导NH4+的净摄取。与kcc2介导的NH4+转运一致，表达kcc2的细胞中的ph恢复可以被速尿（200 μM）或去除外部[Cl−]所抑制。KCC2在不同运输模式下运行的热力学和动力学考虑可以解释Cs+和NH4+存在下神经元Cl -稳态的改变。
Both Cs+and NH4+alter neuronal Cl−homeostasis, yet the mechanisms have not been clearly elucidated. We hypothesized that these two cations altered the operation of the neuronal K+-Cl−cotransporter (KCC2). Using exogenously expressed KCC2 protein, we first examined the interaction of cations at the transport site of KCC2 by monitoring furosemide-sensitive86Rb+influx as a function of external Rb+concentration at different fixed external cation concentrations (Na+, Li+, K+, Cs+, and NH4+). Neither Na+nor Li+affected furosemide-sensitive86Rb+influx, indicating their inability to interact at the cation translocation site of KCC2. As expected for an enzyme that accepts Rb+and K+as alternate substrates, K+was a competitive inhibitor of Rb+transport by KCC2. Like K+, both Cs+and NH4+behaved as competitive inhibitors of Rb+transport by KCC2, indicating their potential as transport substrates. Using ion chromatography to measure unidirectional Rb+and Cs+influxes, we determined that although KCC2 was capable of transporting Cs+, it did so with a lower apparent affinity and maximal velocity compared with Rb+. To assess NH4+transport by KCC2, we monitored intracellular pH (pHi) with a pH-sensitive fluorescent dye after an NH4+-induced alkaline load. Cells expressing KCC2 protein recovered pHimuch more rapidly than untransfected cells, indicating that KCC2 can mediate net NH4+uptake. Consistent with KCC2-mediated NH4+transport, pHirecovery in KCC2-expressing cells could be inhibited by furosemide (200 μM) or removal of external [Cl−]. Thermodynamic and kinetic considerations of KCC2 operating in alternate transport modes can explain altered neuronal Cl−homeostasis in the presence of Cs+and NH4+.