Direct estimate of 1:1 stoichiometry of K+-Cl- cotransport in rabbit erythrocytes
Direct estimate of 1:1 stoichiometry of K+-Cl- cotransport in rabbit erythrocytes
复制标题
DOI：
10.1152/ajpcell.2001.281.3.c825
复制
发表时间：
2001-09-01
影响因子：
5.5
通讯作者：
Adame, MF
中科院分区：
文献类型：
--
作者：
Jennings, ML;Adame, MF
本工作以兔红细胞为模型体系，直接测定Na~+非依赖性K~+-Cl~-共转运(KCC)的化学计量比。为了确定Rb-86(+)是否可以定量地作为KCC的示踪剂，用N-乙基马来酰亚胺(NEM)激活KCC后，平行测定了Rb-86(+)和K+外流。NEM刺激的K+外流到等渗氯化钠的速率常数比Rb-86(+)的速率常数小0.68+/-0.11倍(SD，n=5)。这个修正系数用于所有其他实验，以从测量的RB-86(+)外流计算K+外流。为了最大限度地减少阴离子交换剂的干扰，用SO42-取代了胞外的Cl-，在通量介质中加入了4，4‘-diisothiocyanothiocyanatodihydrostilbene-2，2’-disulfonic酸。用原载体2，4-二硝基苯酚将膜电位钳制在0 mV附近。在此条件下，25℃时的氯离子外流类似于未抑制的氯离子/氯离子交换通量的100,000倍，而被NEM刺激的类似2倍。正如KCC所预期的那样，NEM刺激的Cl-36(-)通量被冈田酸和花盏花素A抑制。NEM刺激的K+/Cl-外流比率为1.12+/-0.26(SD，n=5)。我们认为K+-Cl-在兔红细胞内的共转运具有1：1的化学计量比。
This work was undertaken to obtain a direct measure of the stoichiometry of Na+-independent K+-Cl- cotransport (KCC), with rabbit red blood cells as a model system. To determine whether Rb-86(+) can be used quantitatively as a tracer for KCC, Rb-86(+) and K+ effluxes were measured in parallel after activation of KCC with N-ethylmaleimide (NEM). The rate constant for NEM-stimulated K+ efflux into isosmotic NaCl was smaller than that for Rb-86(+) by a factor of 0.68 +/- 0.11 (SD, n = 5). This correction factor was used in all other experiments to calculate the K+ efflux from the measured Rb-86(+) efflux. To minimize interference from the anion exchanger, extracellular Cl- was replaced with SO42-, and 4,4'-diisothiocyanothiocyanatodihydrostilbene-2,2'-disulfonic acid was present in the flux media. The membrane potential was clamped near 0 mV with the protonophore 2,4-dinitrophenol. The Cl- efflux at 25 degreesC under these conditions is similar to 100,000-fold smaller than the uninhibited Cl-/Cl- exchange flux and is stimulated similar to2-fold by NEM. The NEM-stimulated Cl-36(-) flux is inhibited by okadaic acid and calyculin A, as expected for KCC. The ratio of the NEM-stimulated K+ to Cl- efflux is 1.12 +/- 0.26 (SD, n = 5). We conclude that K+-Cl- cotransport in rabbit red blood cells has a stoichiometry of 1:1.