FURA-2 CALCIUM SIGNALS IN SKELETAL-MUSCLE FIBERS LOADED WITH HIGH-CONCENTRATIONS OF EGTA

Fura-2 is one of the most frequently used fluorescent Ca indicator dye s; yet it has limitations in tracking large intracellular Ca transient s due to its high affinity for Ca. Since high affinity is of advantage when small Ca changes are to be detected, we tried the application of Fura-2 in skeletal muscle fibres which had been loaded with 15 mM int ernal EGTA to eliminate contractile artifacts. Under these conditions, the free Ca transients are considerably reduced in amplitude and stro ng saturation of Fura-2 is avoided. Cut segments of isolated muscle fi bres were voltage-clamped in a double vaseline gap set-up. In the pres ence of high internal EGTA, free Ca (as measured with the rapid metall ochromic indicator antipyrylazo III) drops rapidly from one value to a lower quasi steady-state value at the end of a depolarizing voltage p ulse. This property allowed inspection of the dissociation kinetics of Ca from Fura-2 in the myoplasmic environment. The dissociation rate c onstant k(off) in the fibre was determined from the time constant of t he exponential decay of the Fura-2 signal as a function of the final l evel of free Ca. We obtained a value of 26 s(-1) at the experimental t emperature of 12 degrees C. Knowledge of k(off) in the cell is essenti al for reconstructing the time course of free Ca from indicator bound Ca and for estimating the time course of the rate of release from the sarcoplasmic reticulum. The described combination of high EGTA bufferi ng with Fura-2 fluorescence recording may be particularly useful for t he determination of Ca release in small muscle cells.