THE REMOVAL OF MYOPLASMIC FREE CALCIUM FOLLOWING CALCIUM RELEASE IN FROG SKELETAL-MUSCLE

Transient changes in intracellular free calcium concentration (.DELTA.[Ca2+]) in response to pulse depolarizations were monitored in isolated segments of single frog skeletal muscle fibres cut at both ends and voltage clamped at a holding potential of -90 mV in a double-Vaseline-gap chamber. Calcium transients were monitored optically using the metallochromic indicator dye Antipyrylazo III (APIII), which entered the fibre by diffusion from the solution applied to the cut ends. Optical artifacts due to fibre movement were minimized or eliminated by stretching the fibres to sarcomere lengths at which there was little or no overlap of thick and thin contractile filaments. Remaining movement-independent optical changes intrinsic to the fibre and unrelated to the dye were monitored at 850 nm, where free and dye-bound APIII have no absorbance. These 850 nm signals scaled by .lambda.-1.2 were used to remove intrinsic components from the signals at 700 or 720 nm, wave-lengths at which the APIII absorbance increases when calcium is bound. The corrected 700 or 720 nm signals were used to calculate .DELTA.[Ca2+]. The decay of .DELTA.[Ca2+] following fibre repolarization at the termination of a depolarizing pulse was well described by a single exponential plus a constant. The exponential rate constant for the decay of .DELTA.[Ca2+] decreased and the final ''steady'' level that .DELTA.[Ca2+] appeared to be approaching increased with increasing amplitude and/or duration of the depolarizing pulse. Both the decreasing decay rate and the build up of the ''steady'' level can be accounted for using a two-component model for the removal of free calcium from the myoplasm. One component consists of a set number of a single type of saturable calcium binding site in the myoplasm. The second component is a non-saturable, first-order uptake mechanism operating in parallel with the saturable binding sites. The removal model parameter values were adjusted to fit simultaneously the decay of .DELTA.[Ca2+] after pulses of various amplitudes and durations in a given fibre. The basic procedure was to track .DELTA.[Ca2+] during each pulse when an undetermined calcium release was occurring, but to calculate the decay of .DELTA.[Ca2+] starting 14 ms after repolarization when release was assumed to be negligible. After appropriate selection of parameter values, the model reproduced most aspects of the decay of .DELTA.[Ca2+]. The relatively small rate constant (0.29 .+-. 0.15 s-1) for the dissociation of calcium from the sites obtained by fitting the model to the decay of .DELTA.[Ca2+] after a variety of pulses was confirmed by a similarly slow recovery of the maximum rate of decay of .DELTA.[Ca2+] in a test pulse applied at various times after a relatively large conditioning pulse that decreased the .DELTA.[Ca2+] decay rate. The decay of .DELTA.[Ca2+] following fibre repolarization after any given pulse became slower with increasing dye concentration. This reflects the rapid calcium-buffering action of APIII and could be used to estimate the rapid calcium-buffering activity intrinsic to the fibre. The components of the removal model and the intrinsic rapid calcium buffer are discussed in terms of the biochemical constituents known to be present in skeletal muscle fibres.