Evolution and modulation of intracellular calcium release during long-lasting, depleting depolarization in mouse...

Intracellular calcium signals regulate multiple cellular functions. They depend on release of Ca(2+) from cellular stores into the cytosol, a process that in many types of cells appears to be tightly controlled by changes in [Ca(2+)] within the store. In contrast with cardiac muscle, where depletion of Ca(2+) in the sarcoplasmic reticulum is a crucial determinant of termination of Ca(2+) release, in skeletal muscle there is no agreement regarding the sign, or even the existence of an effect of SR Ca(2+) level on Ca(2+) release. To address this issue we measured Ca(2+) transients in mouse flexor digitorum brevis (FDB) skeletal muscle fibres under voltage clamp, using confocal microscopy and the Ca(2+) monitor rhod-2. The evolution of Ca(2+) release flux was quantified during long-lasting depolarizations that reduced severely the Ca(2+) content of the SR. As in all previous determinations in mammals and non-mammals, release flux consisted of an early peak, relaxing to a lower level from which it continued to decay more slowly. Decay of flux in this second stage, which has been attributed largely to depletion of SR Ca(2+), was studied in detail. A simple depletion mechanism without change in release permeability predicts an exponential decay with time. In contrast, flux decreased non-exponentially, to a finite, measurable level that could be maintained for the longest pulses applied (1.8 s). An algorithm on the flux record allowed us to define a quantitative index, the normalized flux rate of change (NFRC), which was shown to be proportional to the ratio of release permeability P and inversely proportional to Ca(2+) buffering power B of the SR, thus quantifying the 'evacuability' or ability of the SR to empty its content. When P and B were constant, flux then decayed exponentially, and NFRC was equal to the exponential rate constant. Instead, in most cases NFRC increased during the pulse, from a minimum reached immediately after the early peak in flux, to a time between 200 and 250 ms, when the index was no longer defined. NFRC increased by 111% on average (in 27 images from 18 cells), reaching 300% in some cases. The increase may reflect an increase in P, a decrease in B, or both. On experimental and theoretical grounds, both changes are to be expected upon SR depletion. A variable evacuability helps maintain a constant Ca(2+) output under conditions of diminishing store Ca(2+) load.