Ca2+-induced Ca2+ release from fragmented sarcoplasmic reticulum: a comparison with skinned muscle fiber studies

Uptake and release of Ca2+ in heavy and light fractions of fragmented sarcoplasmic reticulum (FSR) isolated from frog and rabbit skeletal muscle was studied under conditions similar to those employed in skinned muscle fiber experiments, where ATP and Mg2+ concentrations were considered to be physiological and free Ca2+ concentration was kept constant during the Ca2+ uptake and release. Ca2+ level in FSR monotonously approached a steady state level which depended only on the final experimental conditions. Heavy fractions, but not light fractions, exhibited characteristics similar to those of Ca2+-induced Ca2+ release reported in skinned fiber studies: i) the rate and steady state level of Ca2+ uptake increased with increase in free Ca2+ concentration in the reaction medium up to 10(-6) M. With further increase in free Ca2+ concentration, the steady state level of Ca2+ taken up decreased while the Ca2+ uptake rate increased. ii) The steady state Ca2+ level was decreased by caffeine but increased by procaine or ruthenium red. Parallel measurement of Ca2+-ATPase activity clearly showed that these drugs modify the Ca2+ efflux but hardly affect the Ca2+-pump activity. It was concluded that the Ca2+-induced Ca2+ release mechanism was in operation at as low as 10(-6) M free Ca2+ concentration. Treatment of FSR with 0.6 M KCl did not have any significant effect.     

Ca2+-induced Ca2+ release from fragmented sarcoplasmic reticulum: Ca2+-dependent passive Ca2+ efflux

Characterization of the putative Ca2+-gated Ca2+ channel of sarcoplasmic reticulum, which is thought to mediate Ca2+-induced Ca2+ release, was carried out in order to elucidate the mechanism of Ca2+-induced Ca2+ release. Heavy and light fractions of fragmented sarcoplasmic reticulum isolated from rabbit skeletal muscle were loaded passively with Ca2+, and then passive Ca2+ efflux was measured under various conditions. The fast phase of the Ca2+ efflux depended on the extravesicular free Ca2+ concentration and was assigned to the Ca2+ efflux through the Ca2+-gated Ca2+ channel. Vesicles with the Ca2+-gated Ca2+ channels comprised about 85% of the heavy fraction and about 40% of the light fraction. The amount of Ca2+ loaded in FSR was found to be much larger than that estimated on the basis of vesicle inner volume and the equilibration of intravesicular with extravesicular Ca2+, indicating Ca2+ binding inside FSR. Taking this fact into account, the Ca2+ efflux curve was quantitatively analyzed and the dependence of the Ca2+ efflux rate constant on the extravesicular free Ca2+ concentration was determined. The Ca2+ efflux was maximal, with the rate constant of 0.75 s-1, when the extravesicular free Ca2+ was at 3 microM. Caffeine increased the affinity for Ca2+ of Ca2+-binding sites for opening the channel with only a slight change in the maximum rate of Ca2+ efflux. Mg2+ inhibited the Ca2+ binding to the sites for opening the channel while procaine seemed to inhibit the Ca2+ efflux by blocking the ionophore moiety of the channel.     

Single chloride-selective channel from cardiac sarcoplasmic reticulum studied in planar lipid bilayers

Summary The behavior of single Cl^– channel was studied by fusing isolated canine cardiac sarcoplasmic reticulum (SR) vesicles into planar lipid bilayers. The channel exhibited unitary conductance of 55 pS (in 260mm Cl^–) and steady-state activation. Subconductance states were observed. Open probability was dependent on holding potentials (–60 to +60 mV) and displayed a bell-shaped relationship, with probability values ranging from 0.2 to 0.8 with a maximum at –10 mV. Channel activity was irreversibly inhibited by DIDS, a stilbene derivative. Time analysis revealed the presence of one time constant for the full open state and three time constants for the closed states. The open and the longer closed time constants were found to be voltage dependent. The behavior of the channel was not affected by changing Ca²⁺ and Mg²⁺ concentrations in both chambers, nor by adding millimolar adenosine triphosphate, or by changing the pH from 7.4 to 6.8. The presence of sulfate anions decreased the unit current amplitude, but did not affect the open probability. These results reveal that at the unitary level the cardiac SR anion-selective channel has distinctive as well as similar electrical properties characteristic of other types of Cl^– channels.     

Spin-labelling studies of fragmented sarcoplasmic reticulum

Vesicular fragments of sarcoplasmic reticulum (SR) were spin labelled with 2,2,6,6-tetramethyl, 4-isothiocyanate piperidine-1-oxyl (probe A) and 2,2,6,6-tetramethyl, 4-amino (N-iodoacetamide) piperidine-1-oxyl (probe B). Two to five moles of probe A or B were covalently bound to 10⁶g of membrane protein, with minimal loss of activity (ATPase, Ca²⁺, uptake). The EPR spectra of labelled SR were then studied in various experimental conditions.Strongly acid or alkaline pH, protein denaturation with ura, and membrane solubilization with deoxycholate produced marked alterations of the EPR spectra of spin-labelled SR, indicating changes in the local environment surrounding the probes, and the occurrence of conformational changes.A reversible modification of the EPR spectra of probe A and an accelerated efflux of accumulated Ca²⁺ were produced by increasing the temperature of SR suspensions from 30° to 40° C. Such a parallel behavior indicates that reversible structural transitions may control membrane permeability and Ca²⁺ efflux.ATP modifies the EPR spectra of probe B, suggesting that ATP binding to the membrane induces a structural change involving the local environment of certain sulfhydryl groups. The ATP concentration required for this effect is comparable to that requied for activation of ATPase. ADP and ITP are also effective, while pyrophosphate, AMP, and cyclic AMP are not. The effect of ATP is reversible.In other experiments, 2,2,6,6-tetramethylpiperidine-1-oxyl (probeC) was equilibrated with concentrated suspensions of SR. The EPR spectra obtained thereafter indicate that probe C binds to the membrane fragments, still maintaining a high degree of motional freedom. These spectra were markedly changed by deoxycholate solubilization of the membrane fragments, while they were little affected by protein denaturation with guanidine. These results confirm the hypothesis that the region of distribution of probe C into SR, is prevalently constituted by low-viscosity lipids.Supported by research grants from USPHS (HE 09878), the American Heart Association (66742), and the Muscular Distrophy Association of America.     

Efflux of Ca2+ from fragmented sarcoplasmic reticulum during AMP deamination

Deamination of AMP in skeletal muscle sarcoplasmic reticulum followed by an increase in pH from 6,5 up to 8,0 leads in a liberation of part of Ca2+ from the SR vesicles. This effect is enhanced by K+, which activate the deamination, and is suppressed by Mg2+, which inhibit the reaction. The activating effect of AMP on Ca2+ efflux from the vesicles markedly decreases after AMP deaminase dissociation from the vesicles and is restored after reconstitution of their deaminase activity. Substitution of IMP for AMP causes a decrease of Ca2+ efflux from the vesicles. The data obtained are in good agreement with the assumption that the ammonium formation from AMP can favour the release of Ca2+ from some vesicles of SR.     

Influence of Ca++ and Mg++ on the vanadate inhibition of the Ca++- ATPase from pig heart sarcoplasmic reticulum

Vanadate inhibits the Ca⁺⁺-ATPase of sarcoplasmic reticulum from pig heart half maximally at about 10^?5 M. Mg⁺⁺ promotes this inhibition by vanadate whereas increasing Ca⁺⁺-concentrations protect the enzyme against vanadate inhibition. Keeping the ratio $\text{Mg}{}^{\mathrm{++}}\text{ATP}$ constant there was no influence of ATP on the vanadate inhibition at concentrations up to 5 × 10^?3 M ATP. Whenever the ratio $\text{Mg}{}^{\mathrm{++}}\text{ATP}$ was higher than 1:1 the inhibitory effect of vanadate on the Ca⁺⁺-ATPase was increased.     

Ca2+-induced sarcoplasmic reticulum Ca2+ release in myotubularin-deficient muscle fibers

Skeletal muscle deficiency in the 3-phosphoinositide (PtdInsP) phosphatase myotubularin (MTM1) causes myotubular myopathy which is associated with severe depression of voltage-activated sarcoplasmic reticulum Ca²⁺ release through ryanodine receptors. In the present study we aimed at further understanding how Ca²⁺ release is altered in MTM1-deficient muscle fibers, at rest and during activation. While in wild-type muscle fibers, SR Ca²⁺ release exhibits fast stereotyped kinetics of activation and decay throughout the voltage range of activation, Ca²⁺ release in MTM1-deficient muscle fibers exhibits slow and unconventional kinetics at intermediate voltages, suggestive of partial loss of the normal control of ryanodine receptor Ca²⁺ channel activity. In addition, the diseased muscle fibers at rest exhibit spontaneous elementary Ca²⁺ release events at a frequency 30 times greater than that of control fibers. Eighty percent of the events have spatiotemporal properties of archetypal Ca²⁺ sparks while the rest take either the form of lower amplitude, longer duration Ca²⁺ release events or of a combination thereof. The events occur at preferred locations in the fibers, indicating spatially uneven distribution of the parameters determining spontaneous ryanodine receptor 1 opening. Spatially large Ca²⁺ release sources were obviously involved in some of these events, suggesting that opening of ryanodine receptors in one cluster can activate opening of ryanodine receptors in a neighboring one. Overall results demonstrate that opening of Ca²⁺-activated ryanodine receptors is promoted both at rest and during excitation-contraction coupling in MTM1-deficient muscle fibers. Because access to this activation mode is denied to ryanodine receptors in healthy skeletal muscle, this may play an important role in the associated disease situation.     

Role of cholesterol in the Ca uptake and ATPase activity of fragmented sarcoplasmic reticulum

With the use of continuous sucrose density gradient a highly purified preparation of vesicles of fragmented sarcoplasmic reticulum was obtained and its lipid content was determined.     

Adenine nucleotide stimulation of Ca2+-induced Ca2+ release in sarcoplasmic reticulum

Rabbit skeletal muscle sarcoplasmic reticulum was fractionated into a "Ca2+-release" and "control" fraction by differential and sucrose gradient centrifugation. External Ca2+ (2-20 microM) caused the release of 40 nmol of 45Ca2+/mg of protein/s from Ca2+-release vesicles passively loaded at pH 6.8 with an internal half-saturation Ca2+ concentration of 10-20 mM. Ca2+-induced Ca2+ release had an approximate pK value of 6.6 and was half-maximally inhibited at an external Ca2+ concentration of 2 X 10(-4) M and Mg2+ concentration of 7 X 10(-5) M. 45Ca2+ efflux from control vesicles was slightly inhibited at external Ca2+ concentrations that stimulated the rapid release of Ca2+ from Ca2+-release vesicles. Adenine, adenosine, and derived nucleotides caused stimulation of Ca2+-induced Ca2+ release in media containing a "physiological" free Mg2+ concentration of 0.6 mM. At a concentration of 1 mM, the order of effectiveness was AMP-PCP greater than cAMP approximately AMP approximately ADP greater than adenine greater than adenosine. Other nucleoside triphosphates and caffeine were minimally effective in increasing 45Ca2+ efflux from passively loaded Ca2+-release vesicles. La3+, ruthenium red, and procaine inhibited Ca2+-induced Ca2+ release. Ca2+ flux studies with actively loaded vesicles also indicated that a subpopulation of sarcoplasmic reticulum vesicles contains a Ca2+ permeation system that is activated by adenine nucleotides.     

Calcium-induced calcium release from fragmented sarcoplasmic reticulum.

A new method is introduced which allows the study of calcium-induced calcium release from fragmented sarcoplasmic reticulum. Results obtained with this method are in agreement with those obtained by previous investigators using skinned muscle fiber. It was also found that anesthetic drugs and alcohol increased the calcium- and caffeine-induced calcium release from the sarcoplasmic reticulum.     

Oxalate transfer across the membranes of sarcoplasmic reticulum during the uptake of Ca++

The kinetic profile of Ca++ uptake in the presence of oxalate is biphasic. An initial phase independent on oxalate is followed by an oxalate-dependent phase delayed in time. The ionophore X-537A only abolishes the net Ca++ uptake if added before the onset of the oxalate phase. However, during this phase, X-537A suddenly releases an amount of Ca++ similar in quantity to that released in the initial phase. The delay of the oxalate-dependent phase is a function of pH. At pH of about 5.5, the oxalate phase and simultaneous calcium oxalate precipitation would theoretically start at the beginning, with no delay. Ejection of protons during Ca++ uptake is strongly depressed by oxalate, but not by other organic anions which do not trap Ca++. It is suggested that oxalate is transferred to the inside of the vesicles as a monoprotonated species at expense of protons ejected by the Ca++-pump during the uptake of Ca++.     

Evidence for the influence of the protein-phospholipid interface on sarcoplasmic reticulum Ca++ Mg++ ATPase activity.

Sarcoplasmic reticulum from the white hind leg muscle of the rabbit was examined with 31P nuclear magnetic resonance as a nonperturbing probe of phospholipid-protein interactions in the intact membrane. The phospholipids of the sarcoplasmic reticulum appear to inhabit two distinct environments: one very similar in behavior to pure phospholipid lamellar dispersions and the other immobilized by the protein in the membrane. Measurement of the population of the latter environment suggests that it is dependent on salt concentration and probably not due to the Ca++ Mg++ ATPase of the sarcoplasmic reticulum. This immobilization can be removed completely by papain proteolysis of the membrane protein, but only partially by trypsin treatment. The phospholipid composition of recombinants with the Ca++ Mg++ ATPase was varied in order to look for effects of the phospholipid-protein interface on enzymatic activity of the Ca++ Mg++ ATPase. Both transphosphatidylated phosphatidylethanolamine (from egg phosphatidylcholine) and bovine brain phosphatidylserine readily partitioned into the putative boundary layer, whereas under the same conditions soybean phosphatidylethanolamine was excluded. Only phosphatidylserine affected the activity of the enzyme, causing an inhibition that was proportional to the phosphatidylserine content, relative to phosphatidylcholine.     

Ca++-induced fusion of proteoliposomes: Dependence on transmembrane osmotic gradient

Summary The fusion of cytochrome oxidase liposomes with liposomes reconstituted with mitochondrial hydrophobic protein is dependent on the presence of an acidic phospholipid in the liposomes and on the addition of Ca⁺⁺ ions. Liposomes which have grown, by fusion, to diameters in excess of 1000 Å lose the ability to fuse further, unless an osmotic gradient across the liposome membrane is established, with the internal osmotic pressure higher than the external. At a given Ca⁺⁺ concentration, the extent to which this second fusion step takes place is determined by the ratio of internal to external osmolarity. Single-walled liposomes with diameters exceeding 1 m have been produced by this technique. The data suggest that the thermodynamic driving force for the Ca⁺⁺-induced fusion is an excess surface free energy which can be supplied by membrane curvature or transmembrane osmotic gradients.