Fura-2 calcium signals in skeletal muscle fibres loaded with high concentrations of EGTA.

Fura-2 is one of the most frequently used fluorescent Ca indicator dyes; yet it has limitations in tracking large intracellular Ca transients 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 internal EGTA to eliminate contractile artifacts. Under these conditions, the free Ca transients are considerably reduced in amplitude and strong saturation of Fura-2 is avoided. Cut segments of isolated muscle fibres were voltage-clamped in a double vaseline gap set-up. In the presence of high internal EGTA, free Ca (as measured with the rapid metallochromic indicator antipyrylazo III) drops rapidly from one value to a lower quasi steady-state value at the end of a depolarizing voltage pulse. This property allowed inspection of the dissociation kinetics of Ca from Fura-2 in the myoplasmic environment. The dissociation rate constant koff in the fibre was determined from the time constant of the exponential decay of the Fura-2 signal as a function of the final level of free Ca. We obtained a value of 26 s-1 at the experimental temperature of 12 degrees C. Knowledge of koff in the cell is essential 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 buffering with Fura-2 fluorescence recording may be particularly useful for the determination of Ca release in small muscle cells.