An improved method for the isolation of rat cardiac sarcoplasmic reticulum

Summary Preparations of cardiac sarcoplasmic reticulum (CSR) isolated from the rat by differential centrifugation have been widely used for measuring alterations in intracellular calcium flux in response to metabolic and pharmacologic disruptions. However, the purity of these SR fractions has not been firmly established.Using a combination of differential and linear sucrose gradient centrifugation, we have isolated rat CSR with high specific activity and purity. By SDS-PAGE analysis, the preparation is enriched in a protein (110 kD) of similar size to the Ca²⁺-ATPase of SR from other sources. Gels stained with the dye Stains All reveal a blue colored 55 kD band, confirming the presence of calsequestrin, the intraluminal low-affinity calcium binding protein of SR. The presence of the transmembrane 53 kD glycoprotein of SR was confirmed by endoglycosidase-H treatment followed by SDS-PAGE and also by a modified Western blotting technique. The rate of calcium uptake in this preparation averages 130 nmol/mg over the first minute of accumulation, approximately 4 times that previously reported for rat CSR. Calcium uptake in our preparation was essentially complete within 5 minutes. Preparations isolated by this method should be of value in future studies measuring alterations in rat CSR function.     

Quantitative determination of the calcium involved in the regulation of the Ca2+-ATPase activity in sarcoplasmic reticulum vesicles

The dependence of the Ca²⁺-ATPase activity of sarcoplasmic reticulum vesicles upon the intravesicular concentration of calcium accumulated after active uptake was studied. The internal calcium concentration was modified by addition of the ionophore A23187 at the steady state of accumulation. About half of the calcium accumulated could be released at low ionophore concentration without any concomitant activation of the Ca²⁺-ATPase. This population of calcium might consist of calcium free in the lumen of the vesicles or bound to the bilayer at sites which do not interact with the ATPase activity. At higher concentrations of ionophore (above 1.75 nmol A23187/mg protein) the release of calcium activated this enzyme. This phenomenon was independent of the extravesicular calcium concentration and might be explained by assuming second species of calcium ions bound to the inner side of the membrane and in close functional interaction with the Ca²⁺-ATPase.     

Ligand-gated channel of the sarcoplasmic reticulum Ca2+ transport ATPase

In resting muscle, cytoplasmic Ca²⁺ concentration is maintained at a low level by active Ca²⁺ transport mediated by the Ca²⁺ ATPase from sarcoplasmic reticulum. The region of the protein that contains the catalytic site faces the cytoplasmic side of the membrane, while the transmembrane helices form a channel-like structure that allows Ca²⁺ translocation across the membrane. When the coupling between the catalytic and transport domains is lost, the ATPase mediates Ca²⁺ efflux as a Ca²⁺ channel. The Ca²⁺ efflux through the ATPase channel is activated by different hydrophobic drugs and is arrested by ligands and substrates of the ATPase at physiological pH. At acid pH, the inhibitory effect of cations is no longer observed. It is concluded that the Ca²⁺ efflux through the ATPase may be sufficiently fast to support physiological Ca²⁺ oscillations in skeletal muscle, that occur mainly in conditions of intracellular acidosis.     

Calcium uptake and Ca2+-ATPase activity in goat spermatozoa membrane vesicles do not require Mg2+

Microsomal membrane vesicles isolated from goat spermatozoa contain Ca²⁺-ATPase, and exhibit Ca²⁺ transport activities that do not require exogenous Mg²⁺ .The enzyme activity is inhibited by calcium-channel inhibitors,e.g. verapamil and diltiazem, like the well known Ca²⁺ , Mg²⁺-ATPase. The uptake of calcium is ATP (energy)-dependent and the accumulated Ca²⁺ can be completely released by the Ca²⁺ ionophore A23187, suggesting that a significant fraction of the vesicles are oriented inside out     

Comparison of the effects of the membraneassociated Ca2+/calmodulin-dependent protein kinase on Ca2+-ATPase function in cardiac and slow-twitch skeletal muscle sarcoplasmic reticulum

In both cardiac and slow-twitch skeletal muscle sarcoplasmic reticulum (SR) there are several systems involved in the regulation of Ca²⁺-ATPase function. These include substrate level regulation, covalent modification via phosphorylation-dephosphorylation of phospholamban by both cAMP-dependent protein kinase (PKA) and Ca²⁺/calmodulin-dependent protein kinase (CaM kinase) as well as direct CaM kinase phosphorylation of the Ca²⁺-ATPase. Studies comparing, the effects of PKA and CaM kinase on cardiac Ca²⁺-ATPase function have yielded differing results; similar studies have not been performed in slow-twitch skeletal muscle. It has been suggested recently, however, that phospholamban is not tightly coupled to the Ca²⁺-ATPase in SR vesicles from slow-twitch skeletal muscle. Our results indicate that assay conditions strongly influence the extent of CaM kinase-dependent Ca²⁺-ATPase stimulation seen in both cardiac and slow-twitch skeletal muscle. Addition of calmodulin (0.2 M) directly to the Ca²⁺ transport assay medium results in minimal ( 112–130% of control) stimulation of Ca²⁺ uptake activity when the Ca²⁺ uptake reaction is initiated by the addition of either ATP or Ca²⁺/EGTA. On the other hand, prephosphorylation of the SR by the endogenous CaM kinase and subsequent transfer of the membranes to the Ca²⁺ transport assay medium results in stimulation of Ca²⁺ uptake activity (202% of control). These effects are observable in both cardiac and slow-twitch skeletal muscle SR. PKA stimulates Ca²⁺ uptake markedly (215% of control) when the Ca²⁺ uptake reaction is initiated by the addition of prephosphorylated SR membranes or by Ca²⁺/EGTA but minimally (130% of control) when the Ca²⁺ uptake reaction is initiated by the addition of ATP. These findings imply that (a) phospholamban is coupled to the Ca²⁺-ATPase in slow-twitch skeletal muscle SR (as in cardiac SR), and (b) the amount of Ca²⁺ uptake stimulation seen upon the addition of calmodulin or PKA depends strongly on the assay conditions employed. Our observations help to explain the wide range of effects of calmodulin or PKA addition reported in previous studies. It should be noted that, since CaM kinase is now known to phosphorylate the Ca²⁺-ATPase in addition to phospholamban, further studies are required to determine the relative contributions of phospholambanversus Ca²⁺-ATPase phosphorylation in the stimulation of Ca²⁺-ATPase function by CaM kinase. Also, earlier studies attributing all of the effects of CaM kinase stimulation of Ca²⁺ uptake and Ca²⁺-ATPase activity to phospholamban phosphorylation need to be re-examined.     

Ca2+-overload inhibits the cardiac SR Ca2+-calmodulin protein kinase activity

There is increasing evidence to suggest that Ca2+-calmodulin dependent protein kinase (CaMK) regulates the sarcoplasmic reticulum (SR) function and thus plays an important role in modulating the cardiac performance. Because intracellular Ca2+-overload is an important factor underlying cardiac dysfunction in a heart disease, its effect on SR CaMK was examined in the isolated rat heart preparations. Ca2+-depletion for 5 min followed by Ca2+-repletion for 30 min, which is known to produce intracellular Ca2+-overload, was observed to attenuate cardiac function as well as SR Ca2+-uptake and Ca2+-release activities. Attenuated SR function in the heart was associated with reduced CaMK phosphorylation of the SR Ca2+-cycling proteins such as Ca2+-release channel, Ca2+-pump ATPase, and phospholamban, decreased CaMK activity, and depressed levels of SR Ca2+-cycling proteins. These results indicate that alterations in cardiac performance and SR function following the occurrence of intracellular Ca2+-overload may partly be due to changes in the SR CaMK activity.     

Ca transport and heat production in vesicles derived from the sarcoplasmic reticulum terminal cisternae: Regulation by K

In this work, we compared the effect of K⁺ on vesicles derived from the longitudinal (LSR) and terminal cisternae (HSR) of rabbit white muscle. In HSR, K⁺ was found to inhibit both the Ca²⁺ accumulation and the heat released during ATP hydrolysis by the Ca²⁺-ATPase (SERCA1). This was not observed in LSR. Valinomycin abolished the HSR Ca²⁺-uptake inhibition promoted by physiological K⁺ concentrations, but it did not modify the thermogenic activity of the Ca²⁺ pump. The results with HSR are difficult to interpret, assuming that a single K⁺ is binding to either the ryanodine channel or to the Ca²⁺-ATPase. It is suggested that an increase of K⁺ in the assay medium alters the interactions among the various proteins found in HSR, thus modifying the properties of both the ryanodine channel and SERCA1.     

Failure of short term stimulation to reduce sarcoplasmic reticulum Ca2+-ATPase function in homogenates of rat gastrocnemius

To examine the effect of short term intense activity on sarcoplasmic reticulum (SR) Ca²⁺ sequestering function, the gastrocnemius (G) muscles of 11 anaesthetized male rats (weight, 411±8 g,X±SE) were activated using supramaximal, intermittent stimulation (one train of 0.2 msec impulses per sec of 100 msec at 100 Hz). Homogenates were obtained from stimulated white (WG-S) and red (RG-S) tissues, assayed for Ca²⁺ uptake and maximal Ca²⁺ ATPase activity and compared to contralateral controls (WG-C, RG-C). Calcium uptake (nmoles/mg protein/min) determined using Indo-l and at [Ca²⁺]_f concentrations between 300–400 nM was unaffected (p>0.05) by activity in both WG (6.14+0.43 vs 5.37+0.43) and RG (3.21+0.18 vs 3.07+0.20). Similarly, no effect (p>0.05) of contractile activity was found for maximal Ca²⁺ ATPase activity (mole/mg protein/min) determined spectrophotometrically in RG (0.276+0.03 vs 0.278+0.02). In WG, Ca²⁺ ATPase activity was 15% higher in WG-S compared to WG-C (0.412+0.03 vs 0.385+0.04). Repetitive stimulation resulted in a reduction in tetanic tension of 74% (p<0.05) by 2 min in the G muscle. By the end of the stimulation period, ATP concentration was reduced (p<0.05) by 57% in the WG and by 47% in the RG. These results indicate that the repeated generation of maximal tetanic force, at least for short term periods, need not adversely affectin vitro homogenate determination of Ca²⁺ sequestering function in spite of severe alterations in energy potential and that some other mechanism must be involved to explain the depression in Ca²⁺ uptake and Ca²⁺ ATPase activity previously noted with short term intense exercise.     

Interdependence of H+ and K+ fluxes during the Ca2+-pumping activity of sarcoplasmic reticulum vesicles

The release of H⁺ during the oxalate-supported Ca²⁺ uptake in sarcoplasmic reticulum vesicles is kinetically coincident with the initial phase of Ca²⁺ accumulation. The Ca²⁺ uptake is increased and the H⁺ release is decreased in the presence of KCl and other monovalent chloride salts as expected for a H⁺-monovalent cation exchange. The functioning of the Ca²⁺-pump is disturbed by the presence of potassium gluconate and, to a lesser extent, of choline chloride. These salts do not inhibit the ATPase activity of Ca²⁺-permeable vesicles, suggesting a charge imbalance inhibition which is specially relevant in the case of gluconate. Therefore, K⁺, and also Cl^–, appear to be involved in secondary fluxes during the active accumulation of Ca²⁺. The microsomal preparation seems homogeneous with respect to the K⁺-channel, showing an apparent rate constant for K⁺ release of approximately 25 s^–1 measured with the aid of⁸⁶Rb⁺ tracer under equilibrium conditions. A Rb⁺ efflux, sensitive to Ca²⁺-ionophore, can be also detected during the active accumulation of Ca²⁺. The experimental data suggest that both monovalent cations and anions are involved in a charge compensation during the Ca²⁺ uptake and H⁺ release. Fluxes of these highly permeable ions would contribute to cancel the formation of a resting membrane potential through the sarcoplasmic reticulum membrane.     

Impaired calcium uptake by cardiac sarcoplasmic reticulum and its underlying mechanism in endotoxin shock

Effects of endotoxin administration on the ATP-dependent Ca²⁺ uptake by canine cardiac sarcoplasmic reticulum (SR) were investigated. Results obtained 4 h after endotoxin administration show that ATP-dependent Ca²⁺ uptake by cardiac SR was decreased by 27–43% (p < 0.05). Kinetic analysis indicates that the Vmax values for Ca²⁺ and for ATP were significantly decreased while the S_0.5 and the Hill coefficient values were not affected during endotoxin shock. Magnesium (1–5 mM) stimulated while vanadate (25–50 M) inhibited the ATP-dependent Ca²⁺ uptake, but the Mg²⁺-stimulated and the vanadate-inhibited activities remained significantly lower in the endotoxin-treated animals. Phosphorylation of SR by the exogenously added catalytic subunit of the cAMP-dependent protein kinase or by the addition of calmodulin stimulated the ATP-dependent Ca²⁺ uptake activities both in the control and endotoxin-injected dogs. However, the phosphorylation-stimulated activities remained significantly lower in the endotoxin-injected dogs. Dephosphorylation of SR decreased the ATP-dependent Ca²⁺ uptake, but the half-time required for the maximal dephosphorylation was reduced by 31% (p < 0.05) 4 h post-endotoxin. These data indicate that endotoxin administration impairs the ATP-dependent Ca²⁺ uptake in canine cardiac SR and the endotoxininduced impairment in the SR calcium transport is associated with a mechanism involving a defective phosphorylation and an accelerated dephosphorylation of SR membrane protein. Since ATP-dependent Ca²⁺ uptake by cardiac SR plays an important role in the regulation of the homeostatic levels of the contractile calcium, our findings may provide a biochemical explanation for myocardial dysfunction that occurs during endotoxin shock.     

Biochemical characterization of the Ca2+ release channel of skeletal and cardiac sarcoplasmic reticulum

Summary Rapid mixing-vesicle ion flux and planar lipid bilayer-single channel measurements have shown that a high-conductance, ligand-gated Ca²⁺ release channel is present in heavy, junctional-derived membrane fractions of skeletal and cardiac muscle sarcoplasmic reticulum. Using the release channel-specific probe, ryanodine, a 30S protein complex composed of polypeptides of M_r 400 000 has been isolated from cardiac and skeletal muscle. Reconstitution of the complex into planar lipid bilayers has revealed a Ca²⁺ conductance with properties characteristic of the native Ca²⁺ release channel.     

Sulfhydryl oxidation and Ca2+ release from sarcoplasmic reticulum

Summary Our interest in the role of sulfhydryl groups (SH) in regulating or altering transport across biological membranes has focused on the significance of a critical SH group associated with the Ca²⁺-release protein from skeletal muscle sarcoplasmic reticulum (SR). We have shown that binding of heavy metals to this group or oxidation of this sulfhydryl to a disulfide induces rapid Ca²⁺ release from SR vesicles [1, 2] and induces contraction in skinned muscle fibers [3]. Several models are described in which oxidation and reduction might control the state of the Ca²⁺-release channel from SR.Abbreviations DTT Dithiothreitol, redox. - oxidation-reduction - SDS Sodium Dodecyl Sulfate - SH Sulfhydryl - SR Sarcoplasmic Reticulum - T-tubule Transverse tubule     

Fluorescence study on transmembrane Ca2+ gradient-mediated conformation changes of sarcoplasmic reticulum Ca2+-ATPase

The conformational states of Ca²⁺-ATPase in sarcoplasmic reticulum (SR) vesicles with or without a thousand-fold transmembrane Ca²⁺ gradient have been studied by fluorescence spectroscopy and fluorescence quenching. In consequence of the establishment of the transmembrane Ca²⁺ gradient, the steady-state fluorescence results revealed a reproducible 8% decrease in the intrinsic fluorescence while time-resolved fluorescence measurements showed that 13 tryptophan residues in SR · Ca²⁺-ATPase could be divided into three groups. The fluorescence lifetime of one of these groups increased from 5.5 ns to 5.95 ns in the presence of a Ca²⁺ gradient. Using KI and hypocrellin B (a photosensitive pigment obtained from a parasitic fungus, growing in Yunnan, China), the fluorescence quenching further indicated that the dynamic change of this tryptophan group, located at the protein-lipid interface, is a characteristic of transmembrane Ca²⁺ gradient-mediated conformational changes in SR · Ca²⁺-ATPase.Abbreviations SR sarcoplasmic reticulum - HB hypocrellin B - Trp tryptophan - DMSO dimethysulfoxide - Hepes N-2-hydroxyethyl piperazine-N-ethanesulfonic acad - SR(50005) SR vesicles with 1000-fold transmembrane Ca²⁺ gradient - SR(5050) SR vesicles without Ca²⁺ gradient - K_sv(app) apparent Stern-Volmer constant - K_svi Stern-Volmer constant of component i for dynamic quenching     

Purification of the microsomal Ca2+-ATPase from rat liver

Summary The Ca²⁺-ATPase from rat liver microsomes has been solubilized in Triton X-100 and purified to homogeneity by ficollsucrose treatment, column chromatography with agarose-hexane adenosine 5-triphosphate Type 2, and high pressure liquid chromatography (HPLC). The purified enzyme obtained by this sequential procedure exhibited a 183-fold increase in specific activity. After ficoll-sucrose treatment, the activity of the Ca²⁺-ATPase was stable for at least two weeks when stored at –70°C. In SDS-polyacrylamide gels, several fractions from HPLC chromatography showed a single band at a position corresponding to a molecular weight of about 107 kDa. This value is consistent with the molecular weight of the phosphoenzyme intermediate of endoplasmic reticulum (ER) Ca²⁺-ATPase. Further characterization of the ER Ca²⁺-ATPase was performed by western immunoblots. Antiserum raised against the 100-kDa sarcoplasmic reticulum (SR) Ca²⁺-ATPase cross-reacted with the purified Ca²⁺-ATPase from rat liver ER membranes.     

Effect of ATP/ADP/phosphate potential on the maximal steady-state uptake of Ca2+ by skeletal sarcoplasmic reticulum

The ability of the Ca²⁺-Mg²⁺ ATPase pump of skeletal SR to produce and maintain a Ca²⁺ gradient was studied as a function of the ATP/ADP/P_i ratio. The internal free Ca²⁺ concentration [Ca²⁺]_i was monitored by changes in fluorescence of CTC. Increasing ADP concentrations in the medium reduce the maximal [Ca²⁺]_i concentration achieved. The inclusion or the omission of 4×10^–4 M P_i or doubling the absolute ATP and ADP concentrations at a constant ATP/ADP ratio does not affect the level obtained. The level depends primarily on the ATP/ADP ratio. The [Ca²⁺] concentration shows a 1.5 power dependence on the ATP/ADP ratio. Further, [Ca²⁺]_i achieved at steady state does not depend on whether the pump had been working in the forward or the reverse direction prior to testing. Analysis shows that the levels of Ca²⁺ achieved are much lower than the levels predicted thermodynamically under the assumption of ideal coupling between Ca²⁺ transport and ATP hydrolysis with a stoichiometry of 2:1. Under this condition the osmotic energy of the [Ca²⁺]_i/[Ca²⁺]_o ratio was shown to be 48% as large as the free energy of hydrolysis of ATP, giving an overall thermodynamic efficiency of 48%. Analysis shows that maximal steady-state uptake is determined by the balance between the rates of uptake by the pump and rates of leak processes (intrinsic or extrinsic to the pump). Comparison with other studies shows that the [Ca²⁺]_i achieved results in trans-inhibition of the pump by tying up the Ca²⁺ translocator in the inwardly oriented phosphorylated form. The absence of an effect of P_i can be taken as evidence that the dissociation of Ca²⁺ from the inwardly oriented translocator on the phosphoylated enzyme must precede the dephosphorylation of the enzyme.     

Partial ischemia reduces the efficiency of sarcoplasmic reticulum Ca2+ transport in rat EDL

To investigate the hypothesis that prolonged partial ischemia would result in a depression in homogenate sarcoplasmic reticulum (SR) Ca²⁺-sequestering and mechanical properties in muscle, a cuff was placed around the hindlimb of 8 adult Sprague–Dawley rats (267 ± 5.8 g; × ± S.E.) and partially inflated (315 mm Hg) for 2 h. Following occlusion, the EDL was sampled both from the ischemic (I) and contralateral control (C) leg and SR properties compared with the EDL muscles extracted from rats (n = 8) immediately following anaesthetization (CC). Ischemia was indicated by a lower (p < 0.05) concentration (mmol.kg dry wt^–1) of ATP (19.0 ± 0.7 vs. 16.7 ± 0.7) and phosphocreatine (58.1 ± 5.7 vs. 35.0 ± 4.6) in I compared to C. Although Ca²⁺-ATPase activity (mol·g protein^–1.sec^–1 ), both maximal and submaximal, was not different between C and I (19.7 ± 0.4 vs. 18.5 ± 1.3), reductions (p < 0.05) in Ca²⁺-uptake (mmol·g protein^–1.sec^–1 ) of between 18.2 and 24.7% across a range of submaximal free Ca²⁺-levels were observed in I compared to C. Lower submaximal Ca²⁺-ATPase activity and Ca²⁺-uptake were also observed in the EDL in C compared to CC animals. Time dependent reductions (p < 0.05) were found in peak twitch and maximal tetanic tension in EDL from I but not C. It is concluded that partial ischemia, resulting in modest reductions in energy state in EDL, induces a reduction in Ca²⁺-uptake independent of changes in Ca²⁺-ATPase activity. These changes reduce the coupling ratio and the efficiency of Ca²⁺-transport by SR.     

Docosahexaenoate-containing phospholipids in sarcoplasmic reticulum and retinal photoreceptors. A proposal for a role in Ca2+-ATPase calcium transport

A critical review of the experimental literature concerning the metabolism of all-cis-4, 7, 10, 13, 16, 19-docosahexaenoate-containing phospholipids in muscle and retina suggests that it plays an essential role in maximizing the Ca²⁺/ATP stoichiometry of the Ca²⁺-ATPase of sarcoplasmic reticulum and retinal photoreceptor disks. Docosahexaenoate-phosphatidylcholine is proposed to participate in oligomerization of Ca²⁺-ATPase necessary for the establishment of a high Ca²⁺/ATP coupling ratio of the Ca²⁺ pump in these tissues. Possible tests of this hypothesis are presented.     

Functional Approach to the Catalytic Site of the Sarcoplasmic Reticulum Ca2+-ATPase: Binding and Hydrolysis of ATP in the Absence of Ca2+

Isolated sarcoplasmic reticulum vesicles in the presence of Mg(2+) and absence of Ca(2+) retain significant ATP hydrolytic activity that can be attributed to the Ca(2+)-ATPase protein. At neutral pH and the presence of 5 mM Mg(2+), the dependence of the hydrolysis rate on a linear ATP concentration scale can be fitted by a single hyperbolic function. MgATP hydrolysis is inhibited by either free Mg(2+) or free ATP. The rate of ATP hydrolysis is not perturbed by vanadate, whereas the rate of p-nitrophenyl phosphate hydrolysis is not altered by a nonhydrolyzable ATP analog. ATP binding affinity at neutral pH and in a Ca(2+)-free medium is increased by Mg(2+) but decreased by vanadate when Mg(2+) is present. It is suggested that MgATP hydrolysis in the absence of Ca(2+) requires some optimal adjustment of the enzyme cytoplasmic domains. The Ca(2+)-independent activity is operative at basal levels of cytoplasmic Ca(2+) or when the Ca(2+) binding transition is impeded.     

Activation of Ca2+ release in isolated sarcoplasmic reticulum

Summary The relationship between Ca²⁺ release from sarcoplasmic reticulum, induced by elevated pH, tetraphenylboron (TPB^–) or chemical modification, and the change in the surface charge of the membranes as measured by the fluorescence intensity of anilinonaphthalene sulfonate (ANS) is examined. The stimulated Ca²⁺ release is inhibited by dicyclohexylcarbodiimide and external Ca²⁺. TPB^–, but not tetraphenylarsonium (TPA⁺), causes a decrease in ANS^– fluorescence, with 50% decrease occurring at about 5 m TPB^–. The decrease in ANS^– fluorescence as well as the inhibition of Ca²⁺ accumulation induced by TPB^– are prevented by TPA⁺. A linear relationship between the decrease in membrane surface potential and the extent of the Ca²⁺ released by TPB^– is obtained. Similar levels of [³H]TPB^– bound to sarcoplasmic reticulum membranes were obtained regardless of whether or not the vesicles have taken up Ca²⁺. The inhibition of Ca²⁺ accumulation and the [³H]TPB^– incorporation into the membranes were correlated. Ca²⁺ release from sarcoplasmic reticulum, by pH elevation, chemical modification or by addition of NaSCN (0.2 to 0.5m) or the Ca²⁺ ionophore ionomycin, is also accompanied by a decrease in ANS^– fluorescence intensity. However, chemical modification and elevated pH affects the surface potential much less than SCN^– or TPB^– do. These results suggest that the enhancement of Ca²⁺ release by these treatments is not due to a general effect on the membrane surface potential, but rather through the modification of a specific protein. They also suggest that membrane surface charges might play an important role in the control mechanism of Ca²⁺ release.