USE OF FURA RED AS AN INTRACELLULAR CALCIUM INDICATOR IN FROG SKELETAL-MUSCLE FIBERS

Fura red, a fluorescent Ca2+ indicator with absorbance bands at visibl e wavelengths, was injected into intact single muscle fibers that had been stretched to a long sarcomere length (approximately 3.8 mum) and bathed in a 'high-Ca2+' Ringer ([Ca2+] = 11.8 mM). From fura red's slo w diffusion coefficient in myoplasm, 0.16 (+/-0.01, SEM) X 10(-6) cm2 s-1 (N = 5; 16-degrees-C), it is estimated that approximately 85% of t he indicator molecules are bound to muscle constituents of large molec ular weight. Binding appears to elevate, by 3- to 4-fold, the indicato r's apparent dissociation constant for Ca2+ (K(D)), which is estimated to be 1.1-1.6 muM in myoplasm. Fura red's myoplasmic absorbance spect rum was used to estimate f(r), the fraction of fura red molecules in t he Ca2+-bound form at rest. In 3 fibers thought to be minimally damage d by the micro-injection, f(r) was estimated to be 0.15 (+/-0.01). Thu s, resting myoplasmic free [Ca2+] ([Ca2+]r) is estimated to be 0.19-0. 28 muM. For fibers in normal Ringer solution ([Ca2+] = 1.8 mM), at sho rter sarcomere length (approximately 2.7 mum), and containing a nonper turbing concentration of indicator (less-than-or-equal-to 0.2 mM), [Ca 2+]r is estimated to be 0.18-0.27 muM. This range is higher than estim ated previously in frog fibers with other techniques. In 6 fibers, R, the indicator's fluorescence ratio signal (equal to the emission inten sity measured with 420 nm excitation divided by that measured with 480 nm excitation), was measured at rest and following electrical stimula tion and compared with absorbance measurements made from the same fibe r region. The analysis implies that R(MIN) and R(MAX) (the values of R that would be measured if all indicator molecules were in the Ca2+-fr ee and Ca2+-bound states, respectively) were substantially smaller in myoplasm than in calibration solutions lacking muscle proteins. Severa l methods for estimation of [Ca2+]r from R are analyzed and discussed.