Mechanisms of stimulated 45Ca efflux in skinned skeletal muscle fibers

Excitation–contraction (E–C) coupling in skeletal muscle can be studied in skinned fibers by direct assay of 45Ca efflux and simultaneous isometric force, under controlled conditions. Recent work provides evidence that such studies can address major current questions about the mechanisms of signal transmission between transverse tubules and sarcoplasmic reticulum and sarcoplasmic reticulum calcium release, as well as operation of the sarcoplasmic reticulum active Ca transport system in situ. Stimulation by imposed ion gradients at constant [K+][Cl−] product results in 45Ca release with two components: a large Ca2+-dependent efflux, responsible for contractile activation, and a small Ca2+-insensitive efflux. The Ca2+-insensitive stimulation is sustained, consistent with sustained depolarization, and appears to gradate the Ca2+-dependent stimulation; this component is likely to reflect intermediate steps in E–C coupling. Several lines of evidence suggest that the depolarizing stimulus acts on the transverse tubules. It is inhibited by the impermeant glycoside ouabain applied before skinning, which should specifically inhibit polarization of subsequently sealed transverse tubules. Sealed polarized transverse tubules also are the only plausible target for stimulation of 45Ca release by monensin and gramicidin D, which can rapidly dissipate Na+ and K+ gradients; a protonophore and the K+-specific ionophore valinomycin are ineffective, lonophore stimulation is prevented by the permeant glycoside digitoxin; it is also highly Ca2+ dependent. Stimulation of 45Ca release by imposed ion gradients is potentiated by perchlorate, which potentiates charge movements and activation in intact fibers, and is inhibited selectively in highly stretched fibers, presumably by transverse tubule – sarcoplasmic reticulum uncoupling. These results relate the Ca2+-dependent sarcoplasmic reticulum efflux channel to the physiological transverse tubule – sarcoplasmic reticulum coupling pathway, which also could involve Ca2+.

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Ca2+ dependence of transverse tubule-mediated calcium release in skinned skeletal muscle fibers.

The Journal of General Physiology ◽

10.1085/jgp.87.2.271 ◽

1986 ◽

Vol 87 (2) ◽

pp. 271-288 ◽

Cited By ~ 19

Author(s):

P Volpe ◽

E W Stephenson

Keyword(s):

Skeletal Muscle ◽

Sarcoplasmic Reticulum ◽

Calcium Release ◽

Muscle Fibers ◽

Transverse Tubule ◽

Force Development ◽

Skeletal Muscle Fibers ◽

Gramicidin D ◽

45Ca Efflux ◽

Stimulation Of

Isometric force and 45Ca efflux from the sarcoplasmic reticulum were measured at 19 degrees C in frog skeletal muscle fibers skinned by microdissection. After Ca2+ loading, application of the ionophores monensin, an Na+(K+)/H+ exchanger, or gramicidin D, an H+ greater than K+ greater than Na+ channel-former, evoked rapid force development and stimulated release of approximately 30% of the accumulated 45Ca within 1 min, whereas CCCP (carbonyl cyanide pyruvate p-trichloromethoxyphenylhydrazone), a protonophore, and valinomycin, a neutral, K+-specific ionophore, did not. When monensin was present in all bathing solutions, i.e., before and during Ca2+ loading, subsequent application failed to elicit force development and to stimulate 45Ca efflux. 5 min pretreatment of the skinned fibers with 50 microM digitoxin, a permeant glycoside that specifically inhibits the Na+,K+ pump, inhibited monensin and gramicidin D stimulation of 45Ca efflux; similar pretreatment with 100 microM ouabain, an impermeant glycoside, was ineffective. Monensin stimulation of 45Ca efflux was abolished by brief pretreatment with 5 mM EGTA, which chelates myofilament-space calcium. These results suggest that: monensin and gramicidin D stimulate Ca2+ release from the sarcoplasmic reticulum that is mediated by depolarization of the transverse tubules, which seal off after sarcolemma removal and form closed compartments; a transverse tubule membrane potential (myofilament space-negative) is maintained and/or established by the operation of the Na+,K+ pump in the transverse tubule membranes and is sensitive to the permeant inhibitor digitoxin; the transverse tubule-mediated stimulation of 45Ca efflux appears to be entirely Ca2+ dependent.

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Ca(2+)- and pH-dependent halothane stimulation of Ca2+ release in sarcoplasmic reticulum from frog muscle

AJP Cell Physiology ◽

10.1152/ajpcell.1996.271.2.c540 ◽

1996 ◽

Vol 271 (2) ◽

pp. C540-C546 ◽

Cited By ~ 6

Author(s):

M. Beltran ◽

R. Bull ◽

P. Donoso ◽

C. Hidalgo

Keyword(s):

Skeletal Muscle ◽

Sarcoplasmic Reticulum ◽

Calcium Release ◽

Release Kinetics ◽

Frog Muscle ◽

Time Range ◽

Frog Skeletal Muscle ◽

Open Probability ◽

Filtration System ◽

Stimulation Of

The effect of halothane on calcium release kinetics was studied in triad-enriched sarcoplasmic reticulum vesicles from frog skeletal muscle. Release from vesicles passively equilibrated with 3 mM 45CaCl2 was measured in the millisecond time range by use of a fast-filtration system. Halothane (400 microM) increased release rate constants at pH 7.1 and 7.4 as a function of extravesicular pCa. In contrast, halothane at pH 6.8 produced the same stimulation of release from pCa 7.0 to 3.0; no release took place in these conditions in the absence of halothane. Halothane shifted the calcium activation curve at pH 7.1, but not at pH 7.4, to the left and increased channel open probability at pH 7.1 in the cis pCa range of 7.0 to 5.0. These results indicate that cytosolic pCa and pH modulate the stimulatory effects of halothane on calcium release. Furthermore, halothane stimulated release in frog skeletal muscle at low pH and resting calcium concentration, indicating that in frog muscle halothane can override the closing of the release channels produced by these conditions, as it does in malignant hyperthermia-susceptible porcine muscle.

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Quercetin stimulation of calcium release from rabbit skeletal muscle sarcoplasmic reticulum

Life Sciences ◽

10.1016/0024-3205(83)90033-4 ◽

1983 ◽

Vol 32 (3) ◽

pp. 213-219 ◽

Cited By ~ 14

Author(s):

James Watras ◽

Sharon Glezen ◽

Christina Seifert ◽

Arnold M. Katz

Keyword(s):

Skeletal Muscle ◽

Sarcoplasmic Reticulum ◽

Calcium Release ◽

Rabbit Skeletal Muscle ◽

Stimulation Of

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Effect of the calcium buffer EGTA on the "hump" component of charge movement in skeletal muscle.

The Journal of General Physiology ◽

10.1085/jgp.97.5.885 ◽

1991 ◽

Vol 97 (5) ◽

pp. 885-896 ◽

Cited By ~ 16

Author(s):

J García ◽

G Pizarro ◽

E Ríos ◽

E Stefani

Keyword(s):

Skeletal Muscle ◽

Sarcoplasmic Reticulum ◽

Calcium Release ◽

Variable Interval ◽

Kinetic Properties ◽

Charge Movement ◽

Voltage Sensors ◽

Calcium Buffer ◽

Delayed Onset ◽

Charge Movements

Three manifestations of excitation-contraction (E-C) coupling were measured in cut skeletal muscle fibers of the frog, voltage clamped in a double Vaseline gap: intramembrane charge movements, myoplasmic Ca2+ transients, and changes in optical transparency. Pulsing patterns in the presence of high [EGTA] intracellularly, shown by García et al. (1989. J. Gen. Physiol. 94:973-986) to deplete Ca2+ in the sarcoplasmic reticulum, were found to change the above manifestations. With an intracellular solution containing 15 mM EGTA and 0 Ca, 10-15 pulses (100 ms) to -20 mV at a frequency of 2 min-1 reduced the "hump" component of charge movement current. This effect was reversible by 5 min of rest. The same effect was obtained in 62.5 mM EGTA and 0 Ca by pulsing at 0.2 min-1. This effect was reversible by adding calcium to the EGTA solution, for a nominal [Ca2+]i of 200 nM, and was prevented by adding calcium to the EGTA solution before pulsing. The suppression of the hump was accompanied by elimination of the optical manifestations of E-C coupling. The current suppressed was found by subtraction and had the following properties: delayed onset, a peak at a variable interval (10-20 ms) into the pulse, a negative phase (inward current) after the peak, and a variable OFF transient that could be multi-phasic and carried less charge than the ON transient. In the previous paper (Csernoch et al., 1991. J. Gen. Physiol. 97:845-884) it was shown that several interventions suppress a similar component of charge movement current, identified with the "hump" or Q gamma current (I gamma). Based on the similarity to that component, the charge movement suppressed by the depletion protocols can also be identified with I gamma. The fact that I gamma is suppressed by Ca2+ depletion and the kinetic properties of the charge suppressed is inconsistent with the existence of separate sets of voltage sensors underlying the two components of charge movement, Q beta and Q gamma. This is explicable if Q gamma is a consequence of calcium release from the sarcoplasmic reticulum.

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