Rate Of Calcium Release Research Articles

Modulation of type-1 Ins(1,4,5)P3 receptor channels by the FK506-binding protein, FKBP12.

FK506-binding protein (FKBP12) is highly expressed in neuronal tissue, where it is proposed to localize calcineurin to intracellular calcium-release channels, ryanodine receptors and Ins(1,4,5)P(3) receptors (InsP(3)Rs). The effects of FKBP12 on ryanodine receptors have been well characterized but the nature and function of binding of FKBP12 to InsP(3)R is more controversial, with evidence for and against a tight interaction between these two proteins. To investigate this, we incorporated purified type-1 InsP(3)R from rat cerebellum into planar lipid bilayers to monitor the effects of exogenous recombinant FKBP12 on single-channel activity, using K(+) as the current carrier. Here we report for the first time that FKBP12 causes a substantial change in single-channel properties of the type-1 InsP(3)R, specifically to increase the amount of time the channel spends in a fully open state. In the presence of ATP, FKBP12 can also induce co-ordinated gating with neighbouring receptors. The effects of FKBP12 were reversed by FK506. We also present data showing that rapamycin, at sub-optimal concentrations of Ins(2,4,5)P(3), decreases the rate of calcium release from cerebellar microsomes. These results provide evidence for a direct functional interaction between FKBP12 and the type-1 InsP(3)R.

A stable calcium gradient near the plasma membrane

AbstractThe influx of calcium ions from extracellular space plays a central role in regulation of function of living cells. It has been hypothesized that calcium influx causes a rise in intracellular calcium concentration ([Ca2+]i) near the plasma membrane, thereby facilitating regulation of membrane associated processes without flooding the cell with Ca2+. Such a localization is extremely important, since high [Ca2+]i is toxic to cells. However, existence of sustained areas of different Ca2+ concentrations within a continuous aquatic medium is not a trivial phenomenon. The mathematical model presented here evaluates the cytosolic concentration of Ca2+ as a function of time and distance from the plasma membrane. The model has verified quantitatively the feasibility of a stable Ca2+ gradient in the cytosol with high values of Ca2+ concentration near the plasma membrane and evaluated its properties as a function of different cellular parameters. The formation of the gradient does not require special distribution of the intracellular contents, channels and pumps. However, it requires buffering of the cytosolic calcium by the intracellular stores and that the rate of calcium release from the stores near the plasma membrane be higher than in other parts of the cell. We suggest that this model can provide an adequate description of the elevated calcium plateau generally observed in electrically non‐excitable cells. Copyright © 2001 John Wiley & Sons, Ltd.

Subcellular Mechanisms of Presenilin-Mediated Enhancement of Calcium Signaling

Mutations in presenilin-1 (PS1), the leading cause of early-onset, autosomal-dominant familial Alzheimer's disease (FAD), enhance calcium signaling mediated by inositol 1,4,5-trisphosphate (IP3). To elucidate the subcellular mechanisms underlying this enhancement, we used high resolution line-scanning confocal microscopy to image elementary calcium release events (“puffs”) in Xenopus oocytes expressing wild-type or mutant PS1. Here we report that mutant PS1-rendered puffs more sensitive to IP3 and increased both the magnitude and the rate of calcium release during each event. These effects were not attributable to quantitative changes in the levels of IP3 receptors or their distribution on the ER, but were instead associated with an abnormal elevation of ER calcium stores. Together, our results suggest that the effects of mutant PS1 on calcium signaling are manifested predominantly at the level of the regulation of calcium stores rather than via perturbations in the numbers or activity of IP3-activated calcium release channels.

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.

Feasibility of a Sustained Steep Ca2+Gradient in the Cytosol of Electrically Non-excitable Cells

In electrically non-excitable cells the predominant mode of calcium signaling is a biphasic rise in cytosolic calcium concentration. It results from Ca2+release from intracellular stores, followed by Ca2+influx across the plasma membrane. It has been hypothesized that prolonged calcium influx may result in a sustained local elevation of the cytosolic calcium concentration near the plasma membrane. The mathematical model presented here evaluates the cytosolic concentration of Ca2+as a function of time and distance from the plasma membrane. It consists of cytoplasmic calcium stores and a plasma membrane, both equipped with calcium channels and pumps, and an immobile cytoplasmic calcium buffer. The model has verified quantitatively the feasibility of a stable Ca2+gradient in the cytosol with high values of Ca2+concentration near the plasma membrane and evaluated its properties as a function of different cellular parameters. The formation of the gradient does not require special distribution of the intracellular contents, channels and pumps. However, it requires buffering of the cytosolic calcium by the intracellular stores and that the rate of calcium release from the stores near the plasma membrane be higher than in other parts of the cell. We suggest that this model can provide an adequate description of the elevated calcium plateau generally observed in electrically non-excitable cells.

Effects of Capacitative Calcium Entry on Agonist-Induced Calcium Transients in A7r5 Vascular Smooth Muscle Cells

Objective: The purpose of this study was to evaluate the contribution of capacitative calcium influx to intracellular calcium levels during agonist-induced stimulation of vascular smooth muscle cells. Methods: Aortic vascular smooth muscle cells (A7r5) were loaded with Indo-1 and intracellular calcium transients were measured. Cells were challenged with either arginine vasopressin (0.5 μM) or thapsigargin (1 μM). Lanthanum (1 mM) was used to block capacitative calcium influx through store-operated channels. Calcium traces were analyzed for basal, peak and plateau responses. Recordings were derivatized and integrated to gain additional information. Nonlinear regression provided a time constant that describes restoration of ionic equilibrium involving both sequestration and extrusion pathways. Results: Stimulation of cells with thapsigargin produced a non-L-type calcium influx that was attenuated by lanthanum. Cells excited with vasopressin exhibited a rapid calcium increase followed by a gradual decrease to a plateau level. Lanthanum pretreatment prior to stimulation caused no significant change in baseline, peak or plateau calcium levels as compared to control. Lanthanum caused no significant change in maximal calcium release rate, calcium integrals or time constant as compared to control. Conclusions: Capacitative calcium entry can occur in vascular smooth muscle cells, but does not appear to contribute significantly to the vasopressin response.

Effects of capacitative calcium entry on agonist-induced calcium transients in A7r5 vascular smooth muscle cells.

The purpose of this study was to evaluate the contribution of capacitative calcium influx to intracellular calcium levels during agonist-induced stimulation of vascular smooth muscle cells. Aortic vascular smooth muscle cells (A7r5) were loaded with Indo-1 and intracellular calcium transients were measured. Cells were challenged with either arginine vasopressin (0. 5 microM) or thapsigargin (1 microM). Lanthanum (1 mM) was used to block capacitative calcium influx through store-operated channels. Calcium traces were analyzed for basal, peak and plateau responses. Recordings were derivatized and integrated to gain additional information. Nonlinear regression provided a time constant that describes restoration of ionic equilibrium involving both sequestration and extrusion pathways. Stimulation of cells with thapsigargin produced a non-L-type calcium influx that was attenuated by lanthanum. Cells excited with vasopressin exhibited a rapid calcium increase followed by a gradual decrease to a plateau level. Lanthanum pretreatment prior to stimulation caused no significant change in baseline, peak or plateau calcium levels as compared to control. Lanthanum caused no significant change in maximal calcium release rate, calcium integrals or time constant as compared to control. Capacitative calcium entry can occur in vascular smooth muscle cells, but does not appear to contribute significantly to the vasopressin response.

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.

Calcium Release in Frog Cut Twitch Fibers Exposed to Different Ionic Environments under Voltage Clamp

Calcium release was measured in highly stretched frog cut twitch fibers mounted in a double Vaseline-gap voltage clamp chamber, with the internal solution containing 20 mM EGTA plus 0.4 or 1.8 mM added calcium. Rise in myoplasmic [Ca 2+] was monitored with antipyrylazo III as the indicator at a temperature of 13 to 14°C. The waveform of calcium release rate (Rel) computed from the absorbance change showed an early peak (Rel p) followed by a maintained phase (Rel m). Each Rel p-versus-V plot was fitted with a Boltzmann distribution function. The maximum value of Rel p (Rel p,max) was compared in various calcium-containing external solutions. The average value in a Cl − solution was about one-third larger than those in a CH 3SO 3 − or gluconate solution, whereas the values in the CH 3SO 3 − and gluconate solutions had no statistically significant difference. In external solutions containing CH 3SO 3 − or gluconate, a replacement of the Ca 2+ with Mg 2+ reduced Rel p,max by 30 to 50%, on average. The values of Rel p,max also had no statistically significant difference among calcium-free external solutions containing different impermeant anions. An increase of the nominal free [Ca 2+] in the end-pool solution from a reduced to the normal physiological level increased the value of Rel p,max, and also slowed the decay of the maintained phase of the Rel waveform. The Rel waveforms in the Cl − and CH 3SO 3 − solutions were compared in the same fiber at a fixed potential. CH 3SO 3 − increased the time to peak, reduced Rel p, and increased Rel m, and the effects were partially reversible. Under the hypothesis that the decay of the peak was due to calcium inactivation of calcium release, the inactivation was larger in Cl − than in CH 3SO 3 −, in qualitative agreement with the ratio of Rel p in the two solutions. Under the alternative hypothesis that the peak and the maintained phase were separately gated by calcium and depolarization, respectively, then CH 3SO 3 − appeared to decrease the calcium-gated component and increase the voltage-gated component.

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.

Voltage-Dependent Calcium Release in Human Malignant Hyperthermia Muscle Fibers

Malignant hyperthermia (MH) results from a defect of calcium release control in skeletal muscle that is often caused by point mutations in the ryanodine receptor gene (RYR1). In malignant hyperthermia-susceptible (MHS) muscle, calcium release responds more sensitively to drugs such as halothane and caffeine. In addition, experiments on the porcine homolog of malignant hyperthermia (mutation Arg615Cys in RYR1) indicated a higher sensitivity to membrane depolarization. Here, we investigated depolarization-dependent calcium release under voltage clamp conditions in human MHS muscle. Segments of muscle fibers dissected from biopsies of the vastus lateralis muscle of MHN (malignant hyperthermia negative) and MHS subjects were voltage-clamped in a double vaseline gap system. Free calcium was determined with the fluorescent indicator fura-2 and converted to an estimate of the rate of SR calcium release. Both MHN and MHS fibers showed an initial peak of the release rate, a subsequent decline, and rapid turn-off after repolarization. Neither the kinetics nor the voltage dependence of calcium release showed significant deviations from controls, but the average maximal peak rate of release was about threefold larger in MHS fibers.

Effects of varied fatigue protocols on sarcoplasmic reticulum calcium uptake and release rates

The purpose of this investigation was to examine changes in sarcoplasmic reticulum (SR) function in muscles subjected to different patterns of muscle activity. Frog sartorius muscles were stimulated with tetanic trains (100 ms, 100 Hz) delivered at rates of 2.0, 0.5, and 0.2 trains/s. In one set of experiments, stimulation was continued until force had declined to approximately 17% of initial (constant fatigue), whereas in the other set, stimulation was continued for 1 min (constant duration). In the constant-fatigue experiments, Ca2+ uptake (1 mM MgATP) and release rates (25 microM AgNO3, 5 mM 4-chloro-m-cresol) were depressed by similar extents following each protocol. This occurred despite 1, 4, and 17 min of stimulation, respectively, used to induce fatigue. In the constant-duration experiments, larger reductions in SR function occurred following the highest frequency stimulation protocol. These data suggest that when muscles are fatigued to similar extents, depressions in SR function are independent of the activity protocol. On the other hand, when a constant duration of activity is imposed, changes in SR function are closely linked to the extent of force reduction.

Numerical Methods to Determine Calcium Release Flux from Calcium Transients in Muscle Cells

Several methods are currently in use to estimate the rate of depolarization-induced calcium release in muscle cells from measured calcium transients. One approach first characterizes calcium removal of the cell. This is done by determining parameters of a reaction scheme from a fit to the decay of elevated calcium after the depolarizing stimulus. In a second step, the release rate during depolarization is estimated based on the fitted model. Using simulated calcium transients with known underlying release rates, we tested the fidelity of this analysis in determining the time course of calcium release under different conditions. The analysis reproduced in a satisfactory way the characteristics of the input release rate, even when the assumption that release had ended before the start of the fitting interval was severely violated. Equally good reconstructions of the release rate time course could be obtained when the model used for the analysis differed in structure from the one used for simulating the data. We tested the application of a new strategy (multiple shooting) for fitting parameters in nonlinear differential equation systems. This procedure rendered the analysis less sensitive to ill-chosen initial guesses of the parameters and to noise. A locally adaptive kernel estimator for calculating numerical derivatives allowed good reconstructions of the original release rate time course from noisy calcium transients when other methods failed.

Cortical granule exocytosis is triggered by different thresholds of calcium during fertilisation in sea urchin eggs.

In sea urchin eggs, fertilisation is followed by a calcium wave, cortical granule exocytosis and fertilisation envelope elevation. Both the calcium wave and cortical granule exocytosis sweep across the egg in a wave initiated at the point of sperm entry. Using differential interference contrast (DIC) microscopy combined with laser scanning confocal microscopy, populations of cortical granules undergoing calcium-induced exocytosis were observed in living urchin eggs. Calcium imaging using the indicator Calcium Green-dextran was combined with an image subtraction technique for visual isolation of individual exocytotic events. Relative fluorescence levels of the calcium indicator during the fertilisation wave were compared with cortical fusion events. In localised regions of the egg, there is a 6s delay between the detection of calcium release and fusion of cortical granules. The rate of calcium accumulation was altered experimentally to ask whether this delay was necessary to achieve a threshold concentration of calcium to trigger fusion, or was a time-dependent activation of the cortical granule fusion apparatus after the 'triggering' event. Calcium release rate was attenuated by blocking inositol 1,4,5-triphospate (InsP3)-gated channels with heparin. Heparin extended the time necessary to achieve a minimum concentration of calcium at the sites of cortical granule exocytosis. The data are consistent with the conclusion that much of the delay observed normally is necessary to reach threshold concentration of calcium. Cortical granules then fuse with the plasma membrane. Further, once the minimum threshold calcium concentration is reached, cortical granule fusion with the plasma membrane occurs in a pattern suggesting that cortical granules are non-uniform in their calcium sensitivity threshold.

The Role of Disseminated Calcite in Rates of Calcium Release and CO2 Consumption During the Weathering of Granitoid Rocks

Although Ca and Na reside predominately in plagioclase feldspar in granitoid rocks, recent studies have demonstrated that excess Ca often exits in surface and ground waters relative to plagioclase stochiometry. Explanations advanced to explain the Ca excess have included selective loss of Ca in soils due to watershed acidification (Hyman et al., 1998), selective weathering of the anorthite component of plagioclase (Clayton 1986), and the rapid weathering of trace mineral phases such as calcite (Mast and Drever 1990). Chemical hydrolysis of Ca-silicate rocks is recognized as impacting long term atmospheric CO2 consumption and global climate through silicate hydrolysis reactions (Bemer and Caldeira 1997). In contrast, carbonate mineral dissolution does not have a corresponding impact because the CO2 consumed in weathering is reintroduced back into the atmosphere by precipitation of carbonates in the ocean. Thus, the solute Ca/Na ratio in watersheds and rivers, reflecting proportions of silicate and carbonate weathering, is important in understanding long term global CO2 balances.

Dissolution Rate of Fluor-Apatite Crystals Inserted in Acid Soils of a Forested Catchment (Vosges Mountains, NE France)

In the Strengbach catchment (Vosges Mountain, NE France), the Ca and Mg nutrition of spruce and beech stands is very poor. Water and mineral fluxes through beech and spruce stands have been monitored at these sites since 1987. Ca supply is limited because its amount in the granite bedrock (CaO = 0.15-0.30 wt.%), in the bulk soil (CaO = 0.01-0.30 wt.%) and on the cation exchange capacity (CaO = 0.01-0.3 creole kg -1) is very low. Total and exchangeable Ca in the fine earth (< 2mm) of the layer soil (0-120 cm) is respectively about 8.4-9.6 t.ha -1 and 130-140 kg ha l . The rate of calcium release by weathering of soil primary minerals has been computed from detailed quantitative mineralogical data and mineral fluxes monitored at these two sites using steady state soil chemistry model PROFILE (Sverdrup and Warfvinge; 1993). Obtained values were extremely low (0.1-1.2 kg ha i yr l ) (Fichter et al., 1998a). Ca-bearing minerals in soils are albite and apatite. Although, apatite is the trace mineral in soil, Ca is principally located in this mineral because Ca content in albite is very low (CAO:0.45-0.53 wt.%) (Fichter et al., 1998b). Only few data of fluorapatite dissolution rate of fluor-apatite were published. In the aim to determinate the dissolution rate of fluor-apatite in these soils, the mineral test technique was used.

Base Cation Supply in Spruce and Beech Ecosystems of the Strengbach Catchment (Vosges Mountains, N-E France)

In spruce and beech stands, mineral budgets for a rotation period were calculated from measured element fluxes. The release of base cations by mineral weathering was calculated with the steady state soil chemistry model PROFILE. The calcium release rate by weathering of the mineral fine earth was extremely low. For the period of one rotation, mineral weathering cannot provide enough Ca to compensate timber harvesting and leaching. Forest sustainability depends strongly on the amounts of Ca gained from deposition and lost by biomass removal. Magnesium was supplied by atmospheric deposition and mineral weathering. Calculated weathering rates were close to present soil losses. However, as the model assumes that all dissolution reactions are congruent, the computed release rate of Mg from illite might be too high. Main inputs of K to the soil solutions were primarily attributed to canopy leaching and litterfall in upper horizons and to mineral weathering in deeper horizons. The cation budget of the beech stand was much more equilibrated than that of the spruce stand. Given possible changes in silviculture and deposition chemistry, the sustainability of the present stands is rather improbable with respect to their mineral supply.

Effect of taurine on excitation-contraction coupling of extensor digitorum longus muscle of dystrophic mdx mouse.

Dystrophin is a cytoskeletal protein associated with the sarcolemma. Genetic depletion of the protein leads to Duchenne muscular dystrophy (DMD) and dystrophic conditions in animals such as the mdx mouse9. Mechanical weakness of the sarcolemma during the stress of contraction can account for an abnormal calcium entry in the cell that in turn triggers proteolysis-dependent dystrophic muscle necrosis5,7,9. This mechanism can also account, in a positive feedback loop, for the increased activity of calcium-selective leak ion channels recorded in dystrophinless muscle fibers15. The increased cytosolic calcium is also responsible for longer calcium transients in the dystrophinless fibers7,14, which may affect muscle contractile properties. In skeletal muscle, contraction is triggered mainly by the calcium ions released from the sarcoplasmic reticulum due to depolarization of the sarcolemma and transverse tubules11. Nevertheless, higher cytosolic calcium levels can affect the rate of calcium release and reuptake1,14, thus modifying the excitation-contraction coupling mechanism.

Changes in Luminal pH Caused by Calcium Release in Sarcoplasmic Reticulum Vesicles

Fast (milliseconds) Ca 2+ release from sarcoplasmic reticulum is an essential step in muscle contraction. To electrically compensate the charge deficit generated by calcium release, concomitant fluxes of other ions are required. In this study we investigated the possible participation of protons as counterions during calcium release. Triad-enriched sarcoplasmic reticulum vesicles, isolated from rabbit fast skeletal muscle, were passively loaded with 1 mM CaCl 2 and release was induced at pCa = 5.0 and pH = 7.0 in a stopped-flow fluorimeter. Accompanying changes in vesicular lumen pH were measured with a trapped fluorescent pH indicator (pyranin). Significant acidification (∼0.2 pH units) of the lumen occurred within the same time scale ( t 1/2 = 0.75 s) as calcium release. Enhancing calcium release with ATP or the ATP analog 5′-adenylylimidodiphosphate (AMPPNP) produced >20-fold faster acidification rates. In contrast, when calcium release induced with calcium with or without AMPPNP was blocked by Mg 2+, no acidification of the lumen was observed. In all cases, rate constants of luminal acidification corresponded with reported values of calcium release rate constants. We conclude that proton fluxes account for part (5–10%) of the necessary charge compensation during calcium release. The possible relevance of these findings to the physiology of muscle cells is discussed.

Intramembrane charge movement and sarcoplasmic calcium release in enzymatically isolated mammalian skeletal muscle fibres

1. Single muscle fibres were dissociated enzymatically from the extensor digitorum longus and communis muscles of rats and guinea-pigs. The fibres were mounted into a double Vaseline gap experimental chamber and the events in excitation-contraction coupling were studied under voltage clamp conditions. 2. The voltage dependence of intramembrane charge movement followed a two-state Boltzmann distribution with maximal available charge of 26.1 +/- 1.5 and 26.1 +/- 1.3 nC microF-1, mid-point voltage of -35.1 +/- 5.0 and -42.2 +/- 1.2 mV and steepness of 16.7 +/- 2.2 and 17.0 +/- 1.9 mV (means +/- S.E.M., n = 7 and 4) in rats and guinea-pigs, respectively. 3. Intracellular calcium concentration ([Ca2+]i) was monitored using the calcium-sensitive dyes antipyrylazo III, fura-2 and mag-fura-5. Resting [Ca2+]i was similar in rats and guinea-pigs with 125 +/- 18 and 115 +/- 8 nM (n = 10 and 9), respectively, while the maximal increase for a 100 ms depolarization to 0 mV was larger in rats (6.3 +/- 1.0 microM; n = 7), than in guinea-pigs (2.8 +/- 0.3; n = 4). 4. The rate of calcium release (Rrel) from the sarcoplasmic reticulum (SR) displayed an early peak followed by a fast and a slow decline to a quasi maintained steady level. After normalizing Rrel to the estimated SR calcium content (1.2 +/- 0.1 and 0.9 +/- 0.1 mM in rats and guinea-pigs, respectively) and correcting for depletion of calcium in the SR the peak and steady levels at 0 mV, respectively, were found to be 2.50 +/- 0.08 and 0.81 +/- 0.06% ms-1 in rats and 2.43 +/- 0.25 and 0.88 +/- 0.01% ms-1 in guinea-pigs. The voltage dependence was essentially the same in both species, but different from that in amphibians. 5. These experiments show that enzymatic isolation yields functionally intact mammalian skeletal muscle fibres for Vaseline gap experiments. The data also suggest a close connection in the regulation of the different kinetic components of SR calcium release in mammalian skeletal muscle.