Klein M G, Simon B J, Szucs G, Schneider M F
Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore 21201.
Biophys J. 1988 Jun;53(6):971-88. doi: 10.1016/S0006-3495(88)83178-3.
To monitor cytosolic [Ca2+] over a wide range of concentrations in functioning skeletal muscle cells, we have used simultaneously the rapid but relatively low affinity calcium indicator antipyrylazo III (AP III) and the slower but higher affinity indicator fura-2 in single frog twitch fibers cut at both ends and voltage clamped with a double vaseline gap system. When both dyes were added to the end pool solution the cytosolic fura-2 concentration reached a steady level equal to the end pool concentration within approximately 2.5 h, a time when the AP III concentration was still increasing. For depolarizing pulses of increasing amplitude, the fura-2 fluorescence signal approached saturation when the simultaneously recorded AP III absorbance change was far from saturation. Comparison of simultaneously recorded fura-2 and AP III signals indicated that the mean values of the on and off rate constants for calcium binding to fura-2 in 18 muscle fibers were 1.49 x 10(8) M-1 s-1 and 11.9 s-1, respectively (mean KD = 89 nM), if all AP III in the fiber is assumed to behave as in calibrating solution and to be in instantaneous equilibrium with [Ca2+]. [Ca2+] transients calculated from the fura-2 signals using these rate constants were consistent with the [Ca2+] transients calculated from the AP III signals. Resting [Ca2+] or small changes in [Ca2+] which could not be reliably monitored with AP III could be monitored with fura-2 with little or no interference from changes in [Mg2+] or from intrinsic signals. The fura-2 signal was also less sensitive to movement artifacts than the AP III signal. After a [Ca2+] transient the fura-2 signal demonstrated a relatively small elevation of [Ca2+] that was maintained for many seconds.
为了在功能正常的骨骼肌细胞中监测广泛浓度范围内的胞质[Ca2+]，我们在两端切断并用双凡士林间隙系统进行电压钳制的单个青蛙单收缩肌纤维中，同时使用了快速但亲和力相对较低的钙指示剂安替比拉宗III（AP III）和较慢但亲和力较高的指示剂fura-2。当将两种染料都添加到终池溶液中时，胞质fura-2浓度在大约2.5小时内达到与终池浓度相等的稳定水平，此时AP III浓度仍在增加。对于幅度不断增加的去极化脉冲，当同时记录的AP III吸光度变化远未达到饱和时，fura-2荧光信号接近饱和。同时记录的fura-2和AP III信号的比较表明，如果假设纤维中的所有AP III都表现得如同在校准溶液中一样并且与[Ca2+]处于瞬时平衡，则18条肌纤维中钙与fura-2结合的开和关速率常数的平均值分别为1.49×10(8) M-1 s-1和11.9 s-1（平均KD = 89 nM）。使用这些速率常数从fura-2信号计算出的[Ca2+]瞬变与从AP III信号计算出的[Ca2+]瞬变一致。静息[Ca2+]或无法用AP III可靠监测的[Ca2+]的微小变化，可以用fura-2进行监测，几乎不受[Mg2+]变化或内在信号的干扰。fura-2信号对运动伪影的敏感性也低于AP III信号。在[Ca2+]瞬变后，fura-2信号显示[Ca2+]有相对较小的升高，并持续了许多秒。
