No abstract

The transient increase in free myoplasmic calcium concentration due to depolarization of a skeletal muscle fiber is the net result of the release of calcium from the sarcoplasmic reticulum (SR) and its simultaneous removal by binding to various sites and by reuptake into the SR. We present a procedure for empirically characterizing the calcium removal processes in voltage-clamped fibers and for using such characterization to determine the time course of SR calcium release during a depolarizing pulse. Our results reveal a decline of the SR calcium release rate during depolarization that was not anticipated from simple inspection of the calcium transients.

A general procedure for using myoplasmic calcium transients measured with a metallochromic indicator dye to calculate the time course of calcium release from the sarcoplasmic reticulum in voltage-clamped skeletal muscle fibers is described and analyzed. Explicit properties are first assigned to all relatively rapidly equilibrating calcium binding sites in the myoplasm so that the calcium content (CaF) in this pool of "fast" calcium can be calculated from the calcium transient. The overall properties of the transport systems and relatively slowly equilibrating binding sites that remove calcium from CaF are then characterized experimentally from the decay of CaF following fiber repolarization. The rate of calcium release can then be calculated as dCaF/dt plus the rate of removal of calcium from CaF. Two alternatives are assumed for the component of CaF that is due to fast binding sites intrinsic to the fiber: a linear instantaneous buffer or a set of binding sites having properties similar to thin filament troponin. Both assumptions yielded similar calcium release wave forms. Three alternative methods for characterizing the removal system are presented. The choice among these or other methods for characterizing removal can be based entirely on convenience since any method that reproduces the decay of CaF following fiber repolarization will give the same release wave form. The calculated release wave form will be accurate provided that the properties assumed for CaF are correct, that release turns off within a relatively short time after fiber repolarization, that the properties of the slow removal system are the same during and after fiber depolarization, and that possible spatial nonuniformities of free or bound calcium do not introduce major errors.

SUMMARY1. Intramembrane charge movement and myoplasmic free calcium transients (A[Ca2+]) were monitored in voltage-clamped segments of isolated frog muscle fibres cut at both ends and mounted in a double Vaseline-gap chamber. The fibres were stretched to sarcomere lengths of 3-54-6 ,tm to minimize mechanical movement and the related optical artifacts.2. The over-all calcium removal capability of each fibre was characterized by analysing the decay of A[Ca2+] following pulses of several different amplitudes and durations. The rate of sarcoplasmic reticulum (s.r.) calcium release was then calculated for each A[Ca2+] using the calcium removal properties determined for that fibre.3. The calculated calcium release wave form reached a relatively early peak and then declined appreciably during a 100-150 ms depolarizing pulse. The voltage dependence of the peak rate of calcium release was steeper and was centred at more positive membrane potentials than the steady-state voltage dependence of charge movement in the same fibres.4. A considerable fraction of the total intramembrane charge was moved at potentials at which A[Ca2+] and calcium release were only a few per cent of maximum.This 'subthreshold' charge may correspond to charge moved in preliminary transitions that precede a final charge transition that activates release. 5. A 'stepped on' pulse protocol was used to experimentally separate the subthreshold charge movement from the charge movement of the final transitions that may control calcium release. The stepped on pulse consisted of a set 50 ms pre-pulse to a potential just at or below the potential for detectable A W. MELZER AND OTHERS 6. For a wide range of test pulse amplitudes and durations in the stepped on protocol the peak rate of calcium release was linearly related to the charge movement during the test pulse. This result points to a tight control of activation of s.r. calcium release by intramembrane charge movement.7. The voltage dependence of both charge movement and of the rate of calcium release could be fitted simultaneously with a three-state, two-transition sequential model in which charge moves in both transitions but only the final transition activates s.r. calcium release. A model with three identical and independent charged gating particles per channel gave an equally good fit to the data. Both models closely fit the charge movement and release data except within about 10 mV of the voltage at which release became detectable, where release varied more steeply with membrane potential than predicted by either model. 8. Fits with the three-state model gave about equal amounts of charge movement in each transition but a 30-50 mV difference in the mid-point voltages for the two transitions. The single channel current calculated for the s.r. calcium channel based on this model was similar to values obtained for single surface membrane calcium channels in other preparations under similar transmembrane conditions.

SUMMARY1. Transient changes in intracellular free calcium concentration (A[Ca2+]) in response to pulse depolarizations were monitored in isolated segments of single frog skeletal muscle fibres cut at both ends and voltage clamped at a holding potential of -90 mV in a double-Vaseline-gap chamber. Calcium transients were monitored optically using the metallochromic indicator dye Antipyrylazo III (APIII), which entered the fibre by diffusion from the solution applied to the cut ends.2. Optical artifacts due to fibre movement were minimized or eliminated by stretching the fibres to sarcomere lengths at which there was little or no overlap of thick and thin contractile filaments. Remaining movement-independent optical changes intrinsic to the fibre and unrelated to the dye were monitored at 850 nm, where free and dye-bound APIII have no absorbance. These 850 nm signals scaled by A-1 2 were used to remove intrinsic components from the signals at 700 or 720 nm, wave-lengths at which the APIJI absorbance increases when calcium is bound. The corrected 700 or 720 nm signals were used to calculate A[Ca2+].3. The decay of A[Ca2+] following fibre repolarization at the termination of a depolarizing pulse was well described by a single exponential plus a constant. Ca2+] following fibre repolarization after any given pulse became slower with increasing dye concentration. This reflects the rapid calcium-buffering action of APIII and could be used to estimate the rapid calcium-buffering activity intrinsic to the fibre.8. The components of the removal model and the intrinsic rapid calcium buffer are discussed in terms of the biochemical constituents known to be present in skeletal muscle fibres.