Plásek J, Dale R E, Sigler K, Laskay G
Institute of Physics of Charles University, Prague, Czech Republic.
Biochim Biophys Acta. 1994 Dec 30;1196(2):181-90. doi: 10.1016/0005-2736(94)00209-6.
The mechanism by which the fluorescent cationic dye diS-C3(3) reports on cellular transmembrane potential has been investigated in murine haemopoietic cells. Due to the large molar absorbance of diS-C3(3) and its high quantum yield of fluorescence in cells, this dye can be used at very low labelling concentrations (5 x 10(-8) to 2 x 10(-7) M). In contrast to the quenching of fluorescence observed for the most commonly used voltage-sensitive dyes of the carbocyanine class, the fluorescence intensity of diS-C3(3) increases when the dye accumulates in the cells. The method of synchronous emission spectroscopy was used to resolve the intracellular and extracellular components of the diS-C3(3) fluorescence of suspensions of labelled cells. In comparing changes in these signals consequent on changes in transmembrane potential induced by varying the extracellular concentration of potassium ions in the presence of valinomycin, the logarithm of the ratio of intensities of these two components, as predicted theoretically, was found to be a good linear measure of transmembrane potential under these conditions. The dye was also demonstrated to be suitable for flow-cytofluorimetric analysis, the logarithm of the mean population signal similarly being found to provide a good linear measure of the transmembrane potential. The conditions under which such linearity may be expected with respect to possible effects due to changes in the capacity for binding of the dye to proteins and various cytosolic structures are delineated and their validity with respect to the possibly contentious role of mitochondria in such measurements examined in particular. The use of the method in indicating changes in the transmembrane potential and/or changes in the transport numbers of the major ions determining transmembrane potential between different physiological states, the possible extension to determinations of absolute differences in potential between different cell states without calibration or comparison with potassium-ion potentials, and the conditions for validity and limitations of these partially complementary measurements, are discussed.
已在小鼠造血细胞中研究了荧光阳离子染料二硫代氰基三碳菁（diS-C3(3)）报告细胞跨膜电位的机制。由于diS-C3(3)的摩尔吸光度大及其在细胞中的荧光量子产率高，该染料可在非常低的标记浓度（5×10⁻⁸至2×10⁻⁷ M）下使用。与最常用的碳菁类电压敏感染料观察到的荧光猝灭相反，当染料在细胞中积累时，diS-C3(3)的荧光强度会增加。采用同步发射光谱法解析标记细胞悬液中diS-C3(3)荧光的细胞内和细胞外成分。在比较缬氨霉素存在下通过改变细胞外钾离子浓度诱导跨膜电位变化后这些信号的变化时，发现这两个成分强度比的对数，如理论预测的那样，是这些条件下跨膜电位的良好线性度量。该染料也被证明适用于流式细胞荧光分析，同样发现平均群体信号的对数可提供跨膜电位的良好线性度量。描述了在染料与蛋白质和各种胞质结构结合能力变化可能产生的影响方面预期这种线性关系的条件，并特别研究了它们在这种测量中线粒体可能存在争议的作用方面的有效性。讨论了该方法在指示不同生理状态之间跨膜电位变化和/或决定跨膜电位的主要离子转运数变化方面的应用、无需校准或与钾离子电位比较即可确定不同细胞状态之间电位绝对差异的可能扩展，以及这些部分互补测量的有效性条件和局限性。
