REDUCTION OF CALCIUM INACTIVATION OF SARCOPLASMIC-RETICULUM CALCIUM-RELEASE BY FURA-2 IN VOLTAGE-CLAMPED CUT TWITCH FIBERS FROM FROG-MUSCLE

Cut fibers from Rana temporaria and Rana pipiens (striation spacing, 3.9-4.2 mum) were mounted in a double Vaseline-gap chamber and studied at 14-degrees-C. The Ca indicator purpurate-3,3'diacetic acid (PDAA) was introduced into the end pools and allowed to diffuse into the optical recording site. When the concentration at the site exceeded 2 mM, step depolarizations to 10 mV were applied and the [Ca] transient measured with PDAA was used to estimate Ca release from the sarcoplasmic reticulum (SR) (Baylor, S. M., W. K. Chandler, and M. W. Marshall. 1983. Journal of Physiology. 344:625-666). With depolarization, the rate of SR Ca release increased to an early peak and then rapidly decreased several-fold to a quasi-steady level. The total amount of Ca released from the SR at the time of peak rate of release appeared to be independent of SR Ca content, consistent with the idea that a single activated channel might pass, on average, a fixed number of ions, independent of the magnitude of the single channel flux. A possible explanation of this property is given in terms of locally induced Ca inactivation of Ca release. The solution in the end pools was then changed to one with PDAA plus fura-2. SR Ca release was estimated from the [Cal transient, as before, and from the DELTA[Cafura-2] signal. On average, 2-3 mM fura-2 increased the quasi-steady level of the rate of SR Ca release by factors of 6.6 and 3.8, respectively, in three fibers from Rana temporaria and three fibers from Rana pipiens. The peak rate of release was increased in five of the six fibers but to a lesser extent than the quasi-steady level. In all fibers, the amplitude of the free [Ca]-transient was markedly reduced. These increases in the rate of SR Ca release are consistent with the idea that Ca inactivation of Ca release develops during a step depolarization to 10 mV and that 2-3 mM fura-2 is able to reduce this inactivation by complexing Ca and thereby reducing free [Ca]. Once the concentration of fura-2 becomes sufficiently large, a further increase reduces the rate of SR Ca release. On average, 5-6 mM fura-2 increased the quasi-steady rate of release, compared with 0 mM fura-2, by 6.5 and 2.9, respectively, in four fibers from Rana temporaria and three from Rana pipiens. The factors for 7-8 mM fura-2 were 3.6 and 2.0, respectively, in three fibers from Rana temporaria and three from Rana pipiens. This reduction of the rate of SR Ca release at large concentrations of fura-2 may be due to a reduction of Ca-induced Ca release (jacquemond, V., L. Csernoch, M. G., Klein, and M. F. Schneider. 1991. Biophysical Journal. 60:867-873) or to an effect of a large concentration of fura-2 not related to Ca buffering.