Decline of myoplasmic calcium ion recovery of calcium release and sarcoplasmic calcium ion pump properties in frog skeletal muscle
Klein, M.G.; Kovacs, L.; Simon, B.J.; Schneider, M.F.
Journal of Physiology (Cambridge) 441: 639-672
ISSN/ISBN: 0022-3751 Accession: 007178991
The two calcium indicators Antipyrylazo III (AP III) and Fura-2 were used simultaneously to monitor free myoplasmic [Ca2+] in voltage-clamped cut segments of frog skeletal muscle fibres (8-10.degree.C). Antipyrylazo III was used for the relatively large [Ca2+] transients during 100-200 ms depolarizing pulses to -20 to 0 mV and for the rapid decline of [Ca2+] during the 200 ms after the pulses. Fura-2 was used to follow the slow decline of the small remaining elevation of [Ca2+] during the following 16 s (slow recovery period) and to monitor resting [Ca2+]. From 1 to 16 s of the slow recovery period [Ca2+] declined with two exponential components, having time constants of 1.9 .+-. 0.3 and 13.5 .+-. 1.5 s (these and all other values are means .+-. S.E.M. of eleven runs from seven fibers). At 1.2 s after the end of the pulses the amplitudes of the fast and slow exponential components of decline of [Ca2+] were 34 .+-. 7 and 31 .+-. 4 nM, respectively. The resting [Ca2+] in these runs was 40 .+-. 4 nM. The time course of calcium bound to parvalbumin ([Ca-Parv]) was calculated from the [Ca2+] records using literature values for the parvalbumin kinetic constants. From 1 to 16 s of the slow recovery period the total calcium [Ca]T outside the sarcoplasmic reticulum (SR) was assumed to equal [Ca-Parv] + [Ca-Fura]. During this period [Ca]T declined with two exponential components having time constants of 1.7 .+-. 0.2 and 14.2 .+-. 1.4 s, the same as those for [Ca2+]. Assuming the total concentration of parvalbumin cation binding sites to be 1000 .mu.M, the fast and slow components of [Ca]T had amplitudes of 117 .+-. 21 and 147 .+-. 16 .mu.M, respectively, at 1.2 s after the pulses. The rate of decline of [Ca]T, -d[Ca]T/dt, was used as a measure of the net rate of removal of calcium from the myoplasm by the SR. From 3 to 16 s of the slow recovery period and in the resting fibre -d[Ca]T/dt varied with [Ca2+] according to A[Ca2+]n-L. The term A[Ca2+]n represents the pump rate and L represents a constant rate of calcium leak from the SR. For 40 nM .ltoreq. [Ca2+] .ltoreq. 80 nM, the power n for the [Ca2+] dependence of pump rate was 3.9 .+-. 0.6. This indicates that at least four highly co-operative calcium binding sites are present in the functional unit of the SR calcium pump. Since the SR calcium ATPase binds two calcium ions per monomer, a simple interpretation is that ATPase dimers constitute the functional pump unit. The rate of release (Rrel) of calcium from the SR was calculated from the [Ca2+] records. The recovery of releasable calcium following a conditioning pulse was determined by applying test pulses at various intervals from 1 to 16 s after the conditioning pulse and determining the relative suppression of the test release compared to the conditioning release. The recovery of releasable calcium occurred with two exponential components, having time constants of 1.5 .+-. 0.2 and 31.6 .+-. 5.6 s. The slower phase of recovery of releasable calcium was significantly slower than the slower phase of decline of [Ca]T, perhaps indicative of a delay between removal of calcium from the myoplasm and its availability for release from the SR. The rate of recovery of releasable calcium varied with [Ca2+] to a power not significantly different from that obtained from the decline of -d[Ca]T/dt. Also, the magnitude of the calcium pump flux estimated from the decline of -d[Ca]T/dt was similar to the flux estimated from the recovery of releasable calcium after determining values for the SR calcium content and fibre parvalbumin concentration. Thus using either the rate of recovery of releasable calcium or the rate of decline of [Ca]T as a measure of the rate of SR calcium uptake, the results were consistent with a fourth power dependence on [Ca2+] and with pump dimers being the functional unit of the SR calcium pump in voltage-clamped skeletal muscle fibres.