Engblom A C, Akerman K E
Department of Biochemistry and Pharmacy, Abo Akademi University, Finland.
Biochim Biophys Acta. 1993 Dec 12;1153(2):262-6. doi: 10.1016/0005-2736(93)90414-u.
The chloride-sensitive fluorescent indicator MQAE (N-(6-methoxyquinolyl) acetoacetyl ester) has been used for determination of the intracellular free chloride concentration in rat brain synaptoneurosomes. Loading of the synaptoneurosomes with MQAE occurs by transmembrane diffusion. Calibration of the intracellular MQAE was done by determining the correlation between fluorescence intensity and intrasynaptoneurosomal Cl- concentration in the presence of the Cl-/OH- exchanger tributyltin and the K+/H+ exchanger nigericin, starting from zero Cl- concentration. The total quenchable signal of MQAE was determined by adding KSCN in the presence of the K+ ionophore valinomycin. The correlation between the reciprocal of the fluorescence intensity and the chloride concentration was linear at least up to 50 mM Cl-. The fluorescence of freshly prepared synaptoneurosomes was then measured and the obtained value was plotted into the calibration curve and the corresponding Cl- was read. The mean intrasynaptoneurosomal chloride concentration was 14 +/- 4 mM. We also quantitatively estimated the Cl- flux after addition of the barbiturate, pentobarbitone that opens GABAA receptor-Cl(-)-channels, to the synaptoneurosomes. An addition of 1 mM pentobarbitone corresponded to an approx. 0.59 mM change in the intrasynaptoneurosomal free chloride concentration. The results show that the chloride-sensitive fluorescent indicator MQAE is a useful tool when determining intracellular chloride activity, and in quantitative determination of chloride fluxes in living cells and subcellular preparations.
氯离子敏感荧光指示剂MQAE（N-(6-甲氧基喹啉基)乙酰乙酸酯）已被用于测定大鼠脑突触神经小体中的细胞内游离氯离子浓度。MQAE通过跨膜扩散进入突触神经小体。细胞内MQAE的校准是通过在存在Cl⁻/OH⁻交换剂三丁基锡和K⁺/H⁺交换剂尼日利亚菌素的情况下，从零Cl⁻浓度开始，测定荧光强度与突触神经小体内Cl⁻浓度之间的相关性来完成的。在存在K⁺离子载体缬氨霉素的情况下加入KSCN来测定MQAE的总可淬灭信号。荧光强度的倒数与氯离子浓度之间的相关性至少在高达50 mM Cl⁻时呈线性。然后测量新鲜制备的突触神经小体的荧光，并将获得的值绘制在校准曲线上，读取相应的Cl⁻值。突触神经小体内氯离子的平均浓度为14±4 mM。我们还定量估计了在向突触神经小体中加入打开GABAA受体-Cl⁻通道的巴比妥酸盐戊巴比妥后Cl⁻的通量。加入1 mM戊巴比妥相当于突触神经小体内游离氯离子浓度约有0.59 mM的变化。结果表明，氯离子敏感荧光指示剂MQAE在测定细胞内氯离子活性以及定量测定活细胞和亚细胞制剂中的氯离子通量时是一种有用的工具。
