Permeability Of Sarcoplasmic Reticulum Research Articles

New insights on sarcoplasmic reticulum calcium regulation in muscle fatigue

a consistent observation with fatigue in skeletal muscle is a decline in the amplitude of the myoplasmic Ca2+ transient, which is thought to result primarily from a reduced Ca2+ flux through the ryanodine receptor (RyR1) of the sarcoplasmic reticulum (SR) ([Fig. 1][1]). This in turn is thought to

Voltage‐controlled Ca2+ release and entry flux in isolated adult muscle fibres of the mouse

The voltage-activated fluxes of Ca(2+) from the sarcoplasmic reticulum (SR) and from the extracellular space were studied in skeletal muscle fibres of adult mice. Single fibres of the interosseus muscle were enzymatically isolated and voltage clamped using a two-electrode technique. The fibres were perfused from the current-passing micropipette with a solution containing 15 mm EGTA and 0.2 mm of either fura-2 or the faster, lower affinity indicator fura-FF. Electrical recordings in parallel with the fluorescence measurements allowed the estimation of intramembrane gating charge movements and transmembrane Ca(2+) inward current exhibiting half-maximal activation at -7.60 +/- 1.29 and 3.0 +/- 1.44 mV, respectively. The rate of Ca(2+) release from the SR was calculated after fitting the relaxation phases of fluorescence ratio signals with a kinetic model to quantify overall Ca(2+) removal. Results obtained with the two indicators were similar. Ca(2+) release was 2-3 orders of magnitude larger than the flux carried by the L-type Ca(2+) current. At maximal depolarization (+50 mV), release flux peaked at about 3 ms after the onset of the voltage pulse and then decayed in two distinct phases. The slower phase, most likely resulting from SR depletion, indicated a decrease in lumenal Ca(2+) content by about 80% within 100 ms. Unlike in frog fibres, the kinetics of the rapid phase of decay showed no dependence on the filling state of the SR and the results provide little evidence for a substantial increase of SR permeability on depletion. The approach described here promises insight into excitation-contraction coupling in future studies of genetically altered mice.

Deterministic inactivation of calcium release channels in mammalian skeletal muscle.

Enzymatically dissociated fibres from the extensor digitorum communis muscle of rats were mounted into a double Vaseline gap chamber. The rate of calcium release (R(rel)) from the sarcoplasmic reticulum (SR) and changes in SR permeability to Ca2+ (PSR) were calculated from measured changes in intracellular calcium concentration. Calcium release during a prepulse attenuated the inactivating component of PSR of the subsequent test pulse. The suppression was graded, larger release causing greater suppression, as expected from a calcium-dependent inactivation process. However, if the dissociation constant of the putative inhibitory calcium binding site (Kd) was estimated using different test pulses different affinities were obtained: a smaller test pulse yielded a smaller Kd. Comparing the suppression of the inactivatable component of PSR during the test pulse (suppression) with the inactivatable component during the prepulse (pre-inactivation) revealed a linear relationship with a regression coefficient close to unity. Lowering intracellular magnesium by decreasing its concentration to 25 microM in the internal solution altered the time course of PSR. The maximal peak-to-steady-level ratio was increased to 6.3 +/- 0.4 (n = 10, mean +/- s.e.m.) from a control value of 3.0 +/- 0.2 (n = 19). Despite the apparent change in steady-state inactivation, suppression remained equal to that pre-inactivation. Our results support the view that a depolarizing pulse always recruits the same set of calcium release channels and a portion of these channels undergoes a deterministic inactivation process.

Differential effects of caffeine and perchlorate on excitation-contraction coupling in mammalian skeletal muscle.

1. Enzymatically dissociated single muscle fibres of the rat were studied under voltage clamp conditions in a double Vaseline gap experimental chamber. Intramembrane charge movement and changes in intracellular calcium concentration ([Ca2+]i) were measured and the rate of calcium release (Rrel) from the sarcoplasmic reticulum (SR) was calculated. This enabled the determination of SR permeability and thus the estimation of the transfer function between intramembrane charge movement and SR permeability. 2. Perchlorate (3 mM) shifted the membrane potential dependence of intramembrane charge movement to more negative voltages without any effect on the steepness or on the maximal available charge. The drug increased SR permeability at every membrane potential but did not alter the peak-to-steady level ratio. It also increased the slope of the transfer function, indicating a more efficient coupling between the voltage sensors and the ryanodine receptors. 3. Caffeine (1 mM), on the other hand, increased SR permeability without altering the voltage dependence of intramembrane charge movement. It neither prolonged the depolarization-induced increase in [Ca2+]i at short pulse durations nor altered the time to peak of Rrel. The augmentation of SR permeability by the drug was more pronounced during the peak caffeine response than during its steady level. This was manifested in a leftward shift of the transfer function rather than an increase in its slope. 4. These observations indicate that perchlorate and caffeine alter the coupling between the voltage sensors and SR calcium release channels in mammalian skeletal muscle. They do not, however, share a common mechanism for enhancing the depolarization-induced release of calcium from the SR.

Effects of tetracaine on sarcoplasmic calcium release in mammalian skeletal muscle fibres

1. Single muscle fibres were dissociated enzymatically from the extensor digitorum communis muscle of rats. The fibres were mounted into a double Vaseline gap experimental chamber and the events in excitation-contraction coupling were studied under voltage clamp conditions in the presence and absence of the local anaesthetic tetracaine. 2. Changes in intracellular calcium concentration ([Ca2+]i) were monitored using the calcium sensitive dyes antipyrylazo III and fura-2 and the rate of calcium release (Rrel) from the sarcoplasmic reticulum (SR) was calculated. Tetracaine decreased the maximal attained [Ca2+]i and suppressed, in a dose-dependent manner, both the early peak and the steady level of Rrel in the voltage range examined. 3. The concentration dependence of the effects on the two kinetic components of Rrel were almost identical with a half-effective concentration (K50) of 70 and 71 microM and a Hill coefficient (nH) of 2.7 and 2.3 for the peak and the steady level, respectively. Furthermore, the drug did not alter the peak to steady level ratio up to a concentration (50 microM) that caused a 35 +/- 5 % reduction in calcium release. Higher concentrations did suppress the ratio but the degree of suppression was voltage independent. 4. Tetracaine (50 microM) neither influenced the total available intramembrane charge nor altered its membrane potential dependence. It shifted the transfer function, the normalized SR permeability versus normalized charge to the right, indicating that similar charge transfer caused a smaller increase in SR permeability. 5. To explore the site of action of tetracaine further the ryanodine receptor (RyR) calcium release channel of the SR was purified and reconstituted into planar lipid bilayers. The reconstituted channel had a conductance of 511 +/- 14 pS (n = 8) in symmetric 250 mM KCl that was not affected by tetracaine. Tetracaine decreased the open probability of the channel in a concentration-dependent manner with K50 = 68 microM and nH = 1.5. 6. These experiments show that tetracaine suppresses SR calcium release in enzymatic isolated mammalian skeletal muscle fibres. This effect is due, presumably, to the decreased open probability of the RyR in the presence of the drug. Since both the inactivating peak and the steady level of Rrel were equally affected by tetracaine, our observations suggest that there is a tight coupling between these kinetic components of SR calcium release in mammalian skeletal muscle.

Myocardial Ca-sequestration failure and compensatory increase in Ca-ATPase with congestive cardiomyopathy: kinetic characterization by a homogenate microassay using real-time ratiometric indo-1 spectrofluorometry

A novel, simple, rapid and reproducible microassay is used for kinetic analysis of Ca-sequestration by homogenates of myocardium of turkeys with furazolidone-induced congestive cardiomyopathy. The assay monitors Ca in real-time using dual-emission ratiometric spectrofluorometry and the Ca-indicator dye indo-1. Using this assay and isolated SR studies we make several novel findings regarding the mechanism of SR failure in furazolidone cardiomyopathy. Qualitative differences in Ca-sequestration were not detected between groups. However, compared to controls the furazolidone treatment resulted in: 1) 50% depression in maximal activities (1.54 +/- 0.36 vs 0.73 +/- 0.12 microM/sec); 2) 2-fold increases in post-sequestration concentrations of ionized Ca (79 +/- 23 vs 141 +/- 13 nmol Ca/L homogenate); 3) 2-fold increases in Ca half-life (415 vs 790 msec); and 4) 25% increased passive Ca-binding capacity of homogenates. The Ca-ATPase specific activity of isolated sarcoplasmic reticulum was 60% increased in congestive cardiomyopathy (543 +/- 140 vs 873 +/- 108 nmol ATP hydrolyzed/min/mg membrane protein) although membrane yield was 20% decreased (0.79 +/- 0.09 vs 0.63 +/- 0.03 mg/g heart). The increased ATPase and decreased Ca-uptake activities in combination with the occurrence of 36% cardiac hypertrophy and 19% decreased body weights resulted in estimates of the relative energy cost to the animal for myocardial Ca transport being 5.5-fold increased with cardiomyopathy (20.5 vs 111 nmol ATP hydrolyzed per microM decrease of sarcoplasmic free Ca/kg body weight). These data indicate that congestive cardiomyopathy is associated with markedly increased permeability of sarcoplasmic reticulum to Ca and compensatorily increased Ca-ATPase activity. Accelerated energy consumption due to the increased energy cost of Ca transport and increased time of myocyte activation are predicted to predispose the myocardium to fatigue and irreversible failure.

33] Permeability of sarcoplasmic reticulum

This chapter describes the methodology to measure ion permeability of isolated SR vesicles. Sarcoplasmic reticulum (SR) regulates the cytoplasmic Ca 2+ concentration of muscle cells and—thereby, controls muscular contraction and relaxation. The cytoplasmic Ca 2+ concentration is controlled by Ca 2+ release through the Ca channel and Ca 2+ uptake by the Ca 2+ -pump. In order to elucidate these functions on a molecular level, studies using isolated SR vesicles are essential. It has been established that Ca 2+ uptake is performed by Ca 2+ -ATPase molecules. As the SR vesicle is a spherical-shaped entity with a diameter of about 0.1μm, the method described here is applicable to vesicles of different origin and different sizes including small vesicles, organelles, or cells. Further, the method to measure Ca 2+ permeability is applicable to Ca 2+ uptake or release. When the radioactive tracer is placed on one side of the membrane and movement of radioactive tracer to the other side is followed, one can measure the net flux or equilibrium exchange rate depending on the constitution of the solution in the other side.

Calcium efflux from sarcoplasmic reticulum microsomes due to oxidation and sulfhydryl-binding agents

Calcium permeability of sarcoplasmic reticulum (SR) microsomes was measured after aging or after exposure to peroxydisulfate or to sulfhydryl-binding agents. Under conditions where the Ca 2+-ATPase was active, the maximum net release of Ca 2+ was not significantly different between control and oxidized SR. However, when calcium uptake was prevented by EGTA or apyrase, the Ca 2+ permeability of oxidized microsomes was 2 to 3 times greater than control of low (10 −9, 10 −7 M) but not high (10 −6 M) levels of external calcium. The observation that vesicles preincubated with 5 mM dithiothreitol loaded up to 3 times as much calcium and had a slightly lower calcium permeability coefficient than control vesicles suggested that suflhydryl oxidation might modulate calcium flux. This hypothesis was tested by exposing to sulfydryl-binding agents:silver, arsenite, and p-chloromercuri-phenylsulfonic acid. Sulfhydryl-binding agents initiated a rapid release of calcium from microsomes, and release was halted by dithiothreitol. Inhibition of calcium transport could not entirely account for the apparent increase in permeability because the calcium permeability of SR treated with sulfhydryl-binding agents was 5 times greater than that of SR exposed to Ca 2+-ATPase inhibitors. These results suggest that oxidation may increase the calcium permeability of SR. by allowing calcium loss through a channel that can be gated by sulfhydryl oxidation.

Chronic alcohol ingestion alters the calcium permeability of sarcoplasmic reticulum of rat skeletal muscle.

Heavy sarcoplasmic reticulum (SR) was prepared from skeletal muscle of control and chronic alcoholic rats, and the effect of in vitro addition of ethanol on the passive Ca2+ permeability was studied. The SR was loaded with Ca2+ in the absence of ATP. Then efflux was initiated by adding an EGTA solution to decrease the extravesicular Ca2+ concentration. The decrease of Ca2+ content of the SR was measured by an optical method using an encapsulated metallochromic indicator (calcein). The Ca2+ permeability of alcoholic rat SR was higher than that of control rats, especially at low external Ca2+ concentrations (below 1 microM). An in vitro (acute) exposure of SR to ethanol increased the Ca2+ permeability of the SR. However, the degree of increase in alcoholic rat SR was smaller than that in control rat SR.