Intramembrane charge movement and calcium release in frog skeletal muscle.

Abstract

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.

Kinetic isoforms of intramembrane charge in intact amphibian striated muscle.

“…There were no obvious delayed qv components, even at relatively high display gains. In this respect, the charge movements now resembled the exponential transients previously reported in some cut fiber preparations (Melzer et al, 1986;Rakowski et al, 1985), as opposed to the complex waveforms reported in intact fiber preparations at certain voltages (Adrian and Peres, 1979;Huang, 1982). Both ON and OFF responses decayed completely to leave unambiguous DC baselines with insignificant slopes everywhere < +_ 0.0004 txA (I.LF ms) -~ (n = 5 fibers in the presence of ryanodine, and n = 4 fibers in the presence of daunorubicin, respectively) at all the test voltages examined.…”

“…Alternatively, the entire charge might still relax but with a simple time course in states that precluded such interactions. Thus, Ca 2+ release rates followed the fourth power of the charge movement Q(/)4 when qv humps were absent in charge movements from cut fiber preparations that nevertheless demonstrated sarcoplasmic reticular Ca 2+ release (Simon and Hill, 1992;Kovacs et al, 1979;Rakowski et al, 1985;Melzer et al, 1986). This suggested that Ca 2+ release-channel opening required all four of its associated but otherwise independent charge movement particles to reach their activating configurations, whatever their kinetics.…”

“…The latter returned the membrane potential to the -90-mV prepulse potential 500 ms after application of prepulses to -120 mV. These steady levels could be determined directly from the traces without the sloping baseline corrections required for both control and test responses in cut fiber preparations (Melzer et al, 1986). It was further possible to assess fiber condition and stability in the intact preparations through computations of length constants, h, internal longitudinal resistances, r~, and membrane resistances of unit fiber length, rm.…”

“…The final steady current slopes for both the ON and the OFF responses were everywhere less than 0.0007 ~A (p~F ms) 1 and did not vary significantly with test potential. Thus, corrections previously required for sloping current baselines in both control and test responses in some cut fiber preparations (Melzer et al, 1986) and in test records in intact preparations (Huang and Peachey, 1989) proved unnecessary. This expedited reproducible integrations of transients to derive steady-state charge.…”

Intramembranous charge movement in frog cut twitch fibers mounted in a double vaseline-gap chamber.

“…Melzer et al (1986) used a somewhat different approach and separated charge movement into two components, Q1 and Q2, that are linked according to a three-state two-transition model,…”