RESTING MYOPLASMIC FREE CALCIUM IN FROG SKELETAL-MUSCLE FIBERS ESTIMATED WITH FLUO-3

Fluo-3 is an unusual tetracarboxylate Ca2+ indicator. For recent lots supplied by Molecular Probes Inc. (Eugene, OR), F(MAX), the fluorescence intensity of the indicator in its Ca2+-bound form, is approximately 200 times that of F(MIN), the fluorescence intensity of the indicator in its Ca2+-free form. (For earlier lots, impurities may account for the smaller reported values of (F(MAX)/(F(MIN), 36-40). We have injected fluo-3 from a high-purity lot into intact single fibers from frog muscle and measured the indicator's absorbance and fluorescence signals at rest (A and F, respectively) and changes in absorbance and fluorescence following action potential stimulation (DELTAA and DELTAF, respectively). As for other high-affinity Ca2+ indicators used previously in frog muscle, the time course of the indicator's DELTAA and DELTAF signals substantially lagged behind that of the myoplasmic free Ca2+ transient. Our analysis of fluo-3's signals from myoplasm therefore focused on information about the level of resting myoplasmic free [Ca2+] ([Ca2+]r). From A, DELTAA, and in vitro estimates of fluo-3's molar extinction coefficients, the change in the fraction of fluo-3 in the Ca2+-bound form during activity (DELTAf) was estimated. From DELTAf, DELTAF, and F, the fraction of the indicator in the Ca2+-bound form in the resting fiber (f(r)) was estimated by f(r) = (DELTAf x F/DELTAF) + (1 - F(MAX/)F(MIN))-1. Since (F(MAX)/F(MIN) is large, the contribution of the second term to the estimate of f(r) is small. At 16-degrees-C, the mean value (mean +/- S.E.) of f(r) was 0.086 +/- 0.004 (N = 15). From two estimates of the apparent dissociation constant of fluo-3 for Ca2+ in the myoplasm, 1.09 and 2.57 muM, the average value of [Ca2+]r is calculated to be 0.10 and 0.24 muM, respectively. The smaller of these estimates lies near the upper end of the range of values for [Ca2+]r in frog fibers (0.02-0.12 muM) estimated by others with aequorin and Ca2+-selective electrodes. The larger of the estimates lies within the range of values (0.2-0.3 muM) previously estimated in this laboratory with fura red. We conclude that [Ca2+]r in frog fibers is at least 0.1 muM and possibly as large as 0.3 muM.