CELL-PERMEABLE FLUORESCENT INDICATOR FOR CYTOSOLIC CHLORIDE
CELL-PERMEABLE FLUORESCENT INDICATOR FOR CYTOSOLIC CHLORIDE
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DOI：
10.1021/bi00246a001
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发表时间：
1991-08-13
期刊：
影响因子：
2.9
通讯作者：
VERKMAN, AS
中科院分区：
文献类型：
--
作者：
BIWERSI, J;VERKMAN, AS
基于喹啉的细胞溶质Cl-的荧光指示剂的主要限制是侵入性细胞加载的必要性，因为带正电荷的环氮赋予高极性和膜不渗透性。开发了一种新的方法来掩盖正氮，并评估了快速，非侵入性的指示剂加载到活细胞和有效的细胞内捕获。非极性亲脂性化合物6-甲氧基-N-乙基-1，2-二氢喹啉（diH-MEQ）对Cl-不敏感，但很容易被氧化成膜不可渗透的Cl-敏感荧光指示剂6-甲氧基-N-乙基喹啉氯化物（MEQ）。MEQ具有344 nm的吸光度和440 nm的发射最大值，0.70的量子产率和4100 M-1 cm-1的摩尔消光系数。在水性缓冲液中，MEQ的荧光通过碰撞机制被Cl-猝灭，Stern-Volmer常数（K（Cl））为145 M-1。MEQ荧光被其它阴离子（K（Br）= 275 M-1，K（I）= 360 M-1，K（SCN）= 300 M-1）猝灭，但不被NO3-，SO 4（2-），阳离子，Swiss 3 T3成纤维细胞和结肠T84细胞通过在37 ℃下与25-50 μ M diH-MEQ孵育5-10分钟，然后加入diH-MEQ-1，MEQ染色细胞明亮，均匀，是无毒的细胞生长，cAMP和Ca 2+信号，和电生理特性的研究。MEQ在60 min内漏出细胞< 10%，对胞质Cl-敏感，K（Cl）= 19 M-1。DiH-MEQ和类似的减少N-取代的二氢喹啉应该有应用在测量的Cl-运输和调节机制，在活细胞和上皮细胞。
A major limitation of quinolinium-based fluorescent indicators for cytosolic Cl- has been the necessity of invasive cell loading because the positively charged ring nitrogen confers high polarity and membrane impermeability. A novel approach to mask the positive nitrogen was developed and evaluated for rapid, noninvasive indicator loading into living cells and effective intracellular trapping. The nonpolar and lipophilic compound 6-methoxy-N-ethyl-1,2-dihydroquinoline (diH-MEQ) was Cl- insensitive but was readily oxidized to the membrane-impermeable and Cl--sensitive fluorescent indicator 6-methoxy-N-ethylquinolinium chloride (MEQ). MEQ had 344-nm absorbance and 440-nm emission maxima, 0.70 quantum yield, and 4100 M-1 cm-1 molar extinction coefficient. In aqueous buffers, the fluorescence of MEQ was quenched by Cl- by a collisional mechanism with a Stern-Volmer constant (K(Cl)) of 145 M-1. MEQ fluorescence was quenched by other anions (K(Br) = 275 M-1, K(I) = 360 M-1, K(SCN) = 300 M-1) but not by NO3-, SO4(2-), cations, and pH. Swiss 3T3 fibroblasts and colonic T84 cells were loaded with MEQ by incubation at 37-degrees-C with 25-50-mu-M diH-MEQ for 5-10 min followed by diH-MEQ-free buffer for 15 min. MEQ stained cells brightly and uniformly and was nontoxic in studies of cell growth, cAMP and Ca2+ signaling, and electrophysiological properties. MEQ leaked out of cells by < 10% in 60 min and was sensitive to cytosolic Cl- with K(Cl) = 19 M-1. DiH-MEQ and similar reduced N-substituted dihydroquinolines should have applications in measurements of Cl- transport and regulatory mechanisms in living cells and epithelia.