Simultaneous recording of calcium transients in skeletal muscle using high- and low-affinity calcium indicators.

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.