9-Methyl-7-bromoeudistomin D induces Ca2+ release from cardiac sarcoplasmic reticulum

9-Methyl-7-bromoeudistomin D (MBED), the most powerful caffeine-like releaser of Ca2+ from skeletal muscle sarcoplasmic reticulum, induced Ca2+ release from the cardiac sarcoplasmic reticulum. MBED (5 microM) and caffeine (1 mM) caused rapid Ca2+ release from the fragmented cardiac sarcoplasmic reticulum in a Ca2+ electrode experiment. [3H]MBED bound to a single class of high-affinity binding sites in cardiac sarcoplasmic reticulum membranes (Kd = 150 nM). These results suggest that MBED binds to a specific binding site on cardiac sarcoplasmic reticulum membranes to induce Ca2+ release from the cardiac sarcoplasmic reticulum. Thus, MBED is a useful probe for characterizing Ca2+ release the channels not only in skeletal sarcoplasmic reticulum but also in cardiac sarcoplasmic reticulum.     

Regulation of cardiac muscle Ca2+ release channel by sarcoplasmic reticulum lumenal Ca2+

The cardiac muscle sarcoplasmic reticulum Ca2+ release channel (ryanodine receptor) is a ligand-gated channel that is activated by micromolar cytoplasmic Ca2+ concentrations and inactivated by millimolar cytoplasmic Ca2+ concentrations. The effects of sarcoplasmic reticulum lumenal Ca2+ on the purified release channel were examined in single channel measurements using the planar lipid bilayer method. In the presence of caffeine and nanomolar cytosolic Ca2+ concentrations, lumenal-to-cytosolic Ca2+ fluxes >/=0.25 pA activated the channel. At the maximally activating cytosolic Ca2+ concentration of 4 microM, lumenal Ca2+ fluxes of 8 pA and greater caused a decline in channel activity. Lumenal Ca2+ fluxes primarily increased channel activity by increasing the duration of mean open times. Addition of the fast Ca2+-complexing buffer 1,2-bis(2-aminophenoxy)ethanetetraacetic acid (BAPTA) to the cytosolic side of the bilayer increased lumenal Ca2+-activated channel activities, suggesting that it lowered Ca2+ concentrations at cytosolic Ca2+-inactivating sites. Regulation of channel activities by lumenal Ca2+ could be also observed in the absence of caffeine and in the presence of 5 mM MgATP. These results suggest that lumenal Ca2+ can regulate cardiac Ca2+ release channel activity by passing through the open channel and binding to the channel's cytosolic Ca2+ activation and inactivation sites.     

Dual mechanisms of Mg2+ block of ryanodine receptor Ca2+ release channel from cardiac sarcoplasmic reticulum

We investigated the effects of cytosolic Mg2+ on ryanodine receptor Ca2+ release channel (RyR) of bovine cardiac sarcoplasmic reticulum incorporated into planar lipid bilayers recording single channel activities. Channels were activated by > or = 0.1 microM Ca2+ in the cis solution. At constant Ca2+, application of Mg2+ (0.1-1 mM) to cis side decreased channel activity in a concentration-dependent manner. A half maximal blocking concentration (Kd) was 35 microM and a complete block was obtained at 1 mM. In the presence of 1 mM free Mg2+ in cis solution, the relation between the channel open probability (Po) and concentration of free Ca2+ in cis solution ([Ca2+]cis) shifted to the right, indicating the competition of Mg2+ and Ca2+. Blocking effects of Mg2+ on RyR were antagonized by increasing [Ca2+]cis > or = 0.1 mM. In the presence of 1 m Mg2+ and 1 mM Ca2+ in cis solution, the channel conductance was markedly depressed to approximately 400 pS (n = 7) from 603 +/- 40 pS (mean +/- S.D., n = 22) in the absence of Mg2+, indicating the flickering block. These results show that Mg2+ causes a direct inhibition of RyR in cardiac SR and this inhibition may be mediated through two different mechanisms. A competition of Mg2+ and Ca2+ at a Ca2+ sensitive site on the RyR and a flickery block of the open channel by Mg2+.     

Glutathione modulates ryanodine receptor from skeletal muscle sarcoplasmic reticulum. Evidence for redox regulation of the Ca2+ release mechanism

In this report, we demonstrate the ability of the cellular thiol glutathione to modulate the ryanodine receptor from skeletal muscle sarcoplasmic reticulum. Reduced glutathione (GSH) inhibited Ca2+-stimulated [3H]ryanodine binding to the sarcoplasmic reticulum and inhibited the single-channel gating activity of the reconstituted Ca2+ release channel. The effects of GSH on both the [3H]ryanodine binding and single-channel measurements were dose-dependent, exhibiting an IC50 of approximately 2.4 mM in binding experiments. Scatchard analysis demonstrated that GSH decreased the binding affinity of ryanodine for its receptor (increased Kd) and lowered the maximal binding occupancy (Bmax). In addition, GSH did not modify the Ca2+ dependence of [3H]ryanodine binding. In single-channel experiments, GSH (5-10 mM), added to the cis side of the bilayer lipid membrane, lowered the open probability (Po) of a Ca2+ (50 microM)-stimulated Ca2+ channel without modifying the single-channel conductance. Subsequent perfusion of the cis chamber with an identical buffer, containing 50 microM Ca2+ without GSH, re-established Ca2+-stimulated channel gating. GSH did not inhibit channel activity when added to the trans side of the bilayer lipid membrane. Similar to GSH, the thiol-reducing agents dithiothreitol and beta-mercaptoethanol also inhibited high affinity [3H]ryanodine binding to sarcoplasmic reticulum membranes. In contrast to GSH, glutathione disulfide (GSSG) was a potent stimulator of high affinity [3H]ryanodine binding and it also stimulated the activity of the reconstituted single Ca2+ release channel. These results provide direct evidence that glutathione interacts with reactive thiols associated with the Ca2+ release channel/ryanodine receptor complex, which are located on the cytoplasmic face of the SR, and support previous observations (Liu, G, Abramson, J. J., Zable, A. C., and Pessah, I. N. (1994) Mol. Pharmacol. 45, 189-200) that reactive thiols may be involved in the gating of the Ca2+ release channel.     

Ca2+ release from the sarcoplasmic reticulum compared in amphibian and mammalian skeletal muscle

This paper describes and evaluates a method for quantifying the amounts of specific plasma proteins adsorbed to biomaterial surfaces. In particular, it demonstrates that macroscopic images ('stains'), that assess the spatial distribution of albumin, IgG, fibrinogen, and HMK (high molecular weight kininogen), can be obtained over areas of at least 12 cm2 using immunospecific adhesion of dyed polystyrene beads. Stain intensities, measured with a scanner and an image analysis system, were found to quantify the amount of specific protein in the solution used to coat the surfaces. Results obtained with the proposed method produced single protein isotherms for albumin, immunoglobulin G (IgG) and fibrinogen that followed Langmuir-like adsorption behavior and were similar to previously published isotherms. The HMK isotherm also exhibited Langmuir-like adsorption behavior. The proposed method also detected the presence of an expected maximum in the adsorption of fibrinogen onto glass as a function of plasma dilution. Adsorption of fibrinogen out of 6.4% plasma onto glass from a separated flow produced results indicating the quantity as well as the location of fibrinogen at the boundary of the separated region. This result confirmed the utility of the proposed method for detecting spatial distributions of specific proteins adsorbed from plasma in practical devices.     

Uridine triphosphate-sensitive pathway of Ca2+ release from the sarcoplasmic reticulum of rat skeletal muscle fibers

The pyrimidine nucleotide, uridine triphosphate (UTP), was tested with skinned skeletal muscle fibers in order to investigate the UTP-sensitive pathway of Ca2+ release from the sarcoplasmic reticulum. The presence of ryanodine (200 microM), ruthenium red (10 microM) or heparin (2.5 mg/ml) did not affect the tension elicited in the presence of UTP, demonstrating that the UTP-induced Ca2+ release involved neither ryanodine nor inositol triphosphate-sensitive channels. Drugs such as compound 48/80 or cyclopiazonic acid used to inhibit Ca2+-ATPase in its reverse function appeared to be, respectively, non-specific or without any inhibitory effect on the tension induced by UTP. Finally, the UTP-induced tension as well as the trifluoperazine-induced tension were abolished in the presence of spermidine (50 mM), supporting the hypothesis that the UTP-sensitive pathway of the SR Ca2+ release might occur through the uncoupled calcium ATPase.     

Role of malignant hyperthermia domain in the regulation of Ca2+ release channel (ryanodine receptor) of skeletal muscle sarcoplasmic reticulum

A fusion protein encompassing Gly341 of the skeletal muscle ryanodine receptor was used to raise monoclonal antibodies; epitope mapping demonstrates that monoclonal antibody 419 (mAb419) reacts with a sequence a few residues upstream from Gly341. The mAb419 was then used to probe ryanodine receptor (RYR) functions. Our results show that upon incubation of triads vesicles with mAb419 the Ca2+-induced Ca2+ release rate at pCa 8 was increased. Equilibrium evaluation of [3H]ryanodine binding at different [Ca2+] indicates that mAb419 shifted the half-maximal [Ca2+] for stimulation of ryanodine binding to lower value (0.1 versus 1.2 microM). Such functional effects may be due to a direct action of the Ab on the Ca2+ binding domain of the RYR or to the perturbation by the Ab of the intramolecular interaction between the immunopositive region and regulatory domain of the RYR. The latter hypothesis was tested directly using the optical biosensor BIAcore (Pharmacia Biotech Inc.): we show that the immunopositive RYR polypeptide is able to interact with the native RYR complex. Ligand overlays with immunopositive digoxigenin-RYR fusion protein indicate that such an interaction might occur with a calmodulin binding domain (defined by residues 3010-3225) and with a polypeptide defined by residues 799-1172. In conclusion our results suggest that the stimulation by the mAb419 of the RYR channel activity is due to the perturbation of an intramolecular interaction between the immunopositive polypeptide and a Ca2+ regulatory site probably corresponding to a calmodulin binding domain.     

The role of sarcoplasmic reticulum and sarcoplasmic reticulum Ca2+-ATPase in the smooth muscle tone of the cat gastric fundus

Circular smooth muscle strips isolated from cat gastric fundus were studied in order to understand whether the sarcoplasmic reticulum (SR) and SR Ca2+-ATPase could play a role in the regulation of the muscle tone. Cyclopiazonic acid (CPA), a specific inhibitor of SR Ca2+-ATPase, caused a significant and sustained increase in muscle tone, depending on the presence of extracellular Ca2+. Nifedipine and cinnarizin only partially suppressed the CPA-induced tonic contraction. Bay K 8644 antagonized the relaxant effect of nifedipine in CPA-contracted fundus. Nitric-oxide-releasing agents sodium nitroprusside and 3-morpholino-sydnonimine completely suppressed the CPA-induced tonic contraction. The blockers of Ca2+-activated K+ channels, tetraethylammonium, charybdotoxin and/or apamin, decreased the contractile effect of CPA. Vanadate increased the tone but did not change significantly the effect of CPA. CPA exerted its contractile effect even when Ca2+ influx was triggered through the Na+/Ca2+ exchanger and the other Ca2+ entry pathways were blocked. Thapsigargin, another specific SR Ca2+-ATPase inhibitor, also increased the muscle tone. The effect of thapsigargin was completely suppressed by sodium nitroprusside and 3-morpholino-sydnonimine and partially by nifedipine. In conclusion, under conditions when the SR Ca2+-ATPase is inhibited, the tissue develops a strong tonic contraction and a large part of this is mediated by Ca2+ influx presumably via nifedipine-sensitive Ca2+ channels. This study suggests the important role of SR Ca2+-ATPase in the modulation of the muscle tone and the function of SR as a "buffer barrier" to Ca2+ entry in the cat gastric fundus smooth muscle.     

Calcium release from skeletal muscle sarcoplasmic reticulum: site of action of dantrolene sodium

The muscle relaxant dantrolene sodium acts directly and specifically on skeletal muscle, unlike other pharmacological agents which affect the central nervous system or act at the nueromuscular junction. Dantrolene sodium markedly suppresses the release of calcium previously sequestered by skeletal, but not cardiac, muscle sarcoplasmic reticulum. No effect in the total amount of calcium accumulated was found. In situ, the drug may reduce the amount of calcium necessary for muscle contraction.     

Fluorescence probe study of the lumenal Ca2+ of the sarcoplasmic reticulum vesicles during Ca2+ uptake and Ca2+ release

A limited amount of information is available about the lumenal Ca2+ kinetics of the sarcoplasmic reticulum (SR). Incubation of mag-fura-2AM permitted to incorporate a sufficient amount of the probe into the SR vesicles, as determined by Mn2+ quenching. Rapid changes in the lumenal [Ca2+] ([Ca2+]lum) during Ca2+ uptake and release could be monitored by following the signal derived from the lumenal probe while clamping the extra-vesicular Ca2+ ([Ca2+]ex) at various desired levels with a BAPTA/Ca buffer. Changes in the [Ca2+]lum during uptake and release show the characteristics intrinsic to the SR Ca2+ pump (the [Ca2+]ex-dependence of the activation and inhibition by thapsigargin) and the Ca2+ release channel (blocking by ruthenium red), respectively. A new feature revealed by the [Ca2+]lum measurement is that during the uptake reaction the free [Ca2+]lum showed a significant oscillation. Several pieces of evidence suggest that this is due to some interactions between the Ca2+ pump and lumenal proteins.     

Purification of the cardiac sarcoplasmic reticulum membrane protein phospholamban from recombinant Escherichia coli

Phospholamban (PLN) was expressed in Escherichia coli as a protein fusion with glutathione S-transferase (GST). GST-PLN was mostly present in the insoluble protein fraction and accounted for approximately 50% of total insoluble protein. Attempts to suppress inclusion body formation or to use GST as an affinity-purification tag failed. A successful purification method is based on preparative SDS/PAGE and electrodialysis. From 1 g cells we typically purified 13.5 mg fusion protein with a PLN content of 2.8 mg. We genetically inserted an enterokinase (EK) protease site just in front of the PLN sequence and demonstrated the proteolytical liberation of PLN from the carrier protein. The approach described represents a substantial advancement in PLN expression and purification.     

Targeted overexpression of the sarcoplasmic reticulum Ca2+-ATPase increases cardiac contractility in transgenic mouse hearts

Cardiac hypertrophy and heart failure are known to be associated with a reduction in Ca2+-ATPase pump levels of the sarcoplasmic reticulum (SR). To determine whether, and to what extent, alterations in Ca2+ pump numbers can affect contraction and relaxation parameters of the heart, we have overexpressed the cardiac SR Ca2+-ATPase specifically in the mouse heart using the alpha-myosin heavy chain promoter. Analysis of 2 independent transgenic lines demonstrated that sarco(endo)plasmic reticulum Ca2+-ATPase isoform (SERCA2a) mRNA levels were increased 3.88+/-0. 4-fold and 7.90+/-0.2-fold over those of the control mice. SERCA2a protein levels were increased by 1.31+/-0.05-fold and 1.54+/-0. 05-fold in these lines despite high levels of mRNA, suggesting that complex regulatory mechanisms may determine the SERCA2a pump levels. The maximum velocity of Ca2+ uptake (Vmax) was increased by 37%, demonstrating that increased pump levels result in increased SR Ca2+ uptake function. However, the apparent affinity of the SR Ca2+-ATPase for Ca2+ remains unchanged in transgenic hearts. To evaluate the effects of overexpression of the SR Ca2+ pump on cardiac contractility, we used the isolated perfused work-performing heart model. The transgenic hearts showed significantly higher myocardial contractile function, as indicated by increased maximal rates of pressure development for contraction (+dP/dt) and relaxation (-dP/dt), together with shortening of the normalized time to peak pressure and time to half relaxation. Measurements of intracellular free calcium concentration and contractile force in trabeculae revealed a doubling of Ca2+ transient amplitude, with a concomitant boost in contractility. The present study demonstrates that increases in SERCA2a pump levels can directly enhance contractile function of the heart by increasing SR Ca2+ transport.     

Biochemical regulation of sarcoplasmic reticulum Cl- channel from human atrial myocytes: involvement of phospholamban

Sarcoplasmic reticulum (SR) membrane vesicles derived from human atrium were characterized by specific ryanodine binding assay and fused into planar lipid bilayers. The tritiated form of the alkaloid bound to its receptor with a K(D) of 2.2 nM and a Bmax of 268 fmol/mg protein respectively. Special emphasis was placed on an anion-selective channel present in the SR membrane, which exhibited a mean conductance value of 67 pS when recorded in asymmetrical 50 mM trans/250 mM cis CsCl buffer system and a sensitivity to SITS (1 to 100 microM). Single and multiple channel activities displayed low voltage sensitivity and variability in its gating behavior which might result in spontaneous channel inactivation. However, the majority of the recordings (60%) resulted in a steady-state high open probability. The inactivated channel could be transiently reactivated with depolarizing voltage steps. This behavior is very similar, if not identical, to that observed for the SR Cl- channel in ventricular cells. The inactivation process is probably not directly related to a phosphorylation/dephosphorylation mechanism since PKA and PKG in presence of an adequate phosphorylation cocktail failed to reactivate the SR Cl- channel. In contrast, the use of a monoclonal anti-phospholamban antibody allowed the inhibition of the activity of the anionic channels. These results suggest that the regulation of the human atrial SR Cl- channel is dependent upon an interaction with phospholamban, which was clearly identified in our atrial preparations by Western blot analysis using monoclonal antibody.     

Effects of cyclopiazonic acid on Ca2+ regulation by the sarcoplasmic reticulum in saponin-permeabilized skeletal muscle fibres

Slices of lapine meniscus produced large amounts of nitric oxide after stimulation with interleukin-1, tumor necrosis factor alpha, or a mixture of lapine synovial cytokines known as chondrocyte-activating factors. Monolayer cultures of meniscal cells produced from the proteolysis of meniscal tissue contained a mixed population of chondrocytic and fibroblastic cells. These cultures also produced large amounts of nitric oxide in response to cytokines. Monolayer cultures of meniscal cells produced by the explant method, in contrast, were uniformly fibroblastic and did not produce nitric oxide in response to cytokines. We conclude that menisci contain two populations of cells, one fibroblastic and the other chondrocytic. The chondrocytic cells are responsible for generating most of the nitric oxide in response to cytokines. Endogenously generated nitric oxide suppressed the synthesis of collagen and proteoglycan by menisci but protected proteoglycan from the catabolic effects of interleukin-1. The inhibitory effect of nitric oxide on collagen synthesis occurred without greatly altering the abundance of mRNAs encoding the various collagen alpha chains. During further investigation, arginine was unexpectedly found to stimulate the synthesis of collagen and, to a lesser degree, of noncollagenous proteins but not of proteoglycans. Fragments of meniscus, but not meniscal cells in monolayer culture, increased their production of matrix metalloproteinases, lactate, and, especially, prostaglandin E2 in response to interleukin-1. Inhibition of nitric oxide production with NG-monomethyl-L-arginine enhanced production of matrix metalloproteinases but had little effect on the synthesis of lactate or prostaglandin E2.     

2,3-Butanedione monoxime (BDM) decreases sarcoplasmic reticulum Ca content by stimulating Ca release in isolated rat ventricular myocytes

The effects of 2,3-butanedione monoxime (BDM) were examined using rat ventricular myocytes loaded with Indo-1 to measure the intracellular Ca concentration ([Ca2+]i). BDM (10 mM) produced a transient increase of the systolic Ca transient with no steady-state effect on its magnitude. This transient increase was more marked when BDM was applied after having decreased the external Ca concentration from 1 to 0.1 mM. There was a transient increase of resting [Ca2+]i in both quiescent and electrically stimulated cells. Prior application of BDM decreased the rise of [Ca2+]i produced by caffeine. In voltage-clamped cells the rise of [Ca2+]i produced by BDM was accompanied by a transient inward current attributed to the electrogenic Na-Ca exchange. The amount of Ca lost from the cell upon application of 10 mM BDM could be estimated either from the integral of the BDM-evoked current or from the reduction of the integral of a caffeine-evoked current and corresponded to about 50% of the sarcoplasmic reticulum (s.r.) Ca content. The decrease of s.r. Ca content and the transient potentiation of the systolic Ca transient suggest that BDM acts by stimulating Ca-induced Ca release. These effects must be allowed for when using BDM.     

The oxidative inactivation of sarcoplasmic reticulum Ca(2+)-ATPase by peroxynitrite

The oxidative inactivation of rabbit skeletal muscle Ca(2+)-ATPase in sarcoplasmic reticulum (SR) vesicles by peroxynitrite (ONOO-) was investigated. The exposure of SR vesicles (10 mg/ml protein) to low peroxynitrite concentrations ( < or = 0.2 mM) resulted in a decrease of Ca(2+)-ATPase activity primarily through oxidation of sulfhydryl groups. Most of this deactivation (ca.70%) could be chemically reversed by subsequent reduction of the enzyme with either dithiothreitol (DTT) or sodium borohydride (NaBH4), indicating that free cysteine groups were oxidized to disulfides. The initial presence of 5 mM glutathione failed to protect the SR Ca(2+)-ATPase activity. However, as long as peroxynitrite concentrations were kept < or = 0.45 mM, the efficacy of DTT to reverse Ca(2+)-ATPase inactivation was enhanced for reaction mixtures which initially contained 5 mM glutathione. At least part of the disulfides were formed intermolecularly since gel electrophoresis revealed protein aggregation which could be reduced under reducing conditions. The application of higher peroxynitrite concentrations ( > or = 0.45 mM) resulted in Ca(2+)-ATPase inactivation which could not be restored by exposure of the modified protein to reducing agents. On the other hand, treatment of modified protein with NaBH4 recovered all SR protein thiols. This result indicates that possibly the oxidation of other amino acids contributes to enzyme inactivation, corroborated by amino acid analysis which revealed some additional targets for peroxynitrite or peroxynitrite-induced processes such as Met, Lys, Phe, Thr, Ser, Leu and Tyr. Tyr oxidation was confirmed by a significant lower sensitivity of oxidized SR proteins to the Lowry assay. However, neither bityrosine nor nitrotyrosine were formed in significant yields, as monitored by fluorescence spectroscopy and immunodetection, respectively. The Ca(2+)-ATPase of SR is involved in cellular Ca(2+)-homeostasis. Thus, peroxynitrite mediated oxidation of the Ca(2+)-ATPase might significantly contribute to the loss of Ca(2+)-homeostasis observed under biological conditions of oxidative stress.     

Lamellar stacking in three-dimensional crystals of Ca(2+)-ATPase from sarcoplasmic reticulum

Electron microscopy of multilamellar crystals of CA(2+)-ATPase currently offers the best opportunity for obtaining a high-resolution structure of this ATP-driven ion pump. Under certain conditions small, wormlike crystals are formed and provide views parallel to the lamellar plane, from which parameters of lamellar stacking can be directly measured. Assuming that molecular packing is the same, data from these views could supplement those obtained by tilting large, thin platelike crystals. However, we were surprised to discover that the lamellar spacing was variable and depended on the amount of glycerol present during crystallization (20% versus 5%). Projection maps (h,0,l) from these womklike crystals suggest different molecular contacts that give rise to the different lamellar spacings. Based on an orthogonal projection map (h,k,0) from collapsed, wormlike crystals and on x-ray powder patterns, we conclude that molecular packing within the lamellar plane is the same as that in thin, platelike crystals and is unaffected by glycerol. Finally, the orientation of molecules in the lamellar plane was characterized from freeze-dried, shadowed crystals. Comparing the profile of molecules in these multilamellar crystals with that previously observed in helical tubes induced by vanadate gives structural evidence of the conformational change that accompanies binding of calcium of Ca(2+)-ATPase.     

Ryanodine perfusion decreases cardiac mechanical function without affecting homogenate sarcoplasmic reticulum Ca2+ uptake: comparison with the stunned heart

This study tested the hypothesis that perfusion with low concentrations of ryanodine, which opens the sarcoplasmic reticulum (SR) Ca2+ channel in a sub-conducting state, could mimic the effects of stunning on both mechanical and SR activity. Perfusion of isolated rat hearts with 10-160 nM ryanodine progressively decreased left ventricular developed pressure (LVDP) and increased end-diastolic pressure (EDP), but LVDP decreased more and EDP increased less than in the stunned heart. The effect of ryanodine perfusion on LVDP and EDP is consistent with the opening of the SR Ca2+ channel by high-affinity ryanodine binding, reducing SR Ca2+ content and interfering with mechanical function. In contrast to stunning, ryanodine perfusion did not affect the homogenate Ca2+ uptake rates measured in the presence or absence of high [ryanodine]. Perfusion with 80 nM 3H-ryanodine resulted in a large decline in LVDP, but only a small degree of ryanodine binding. Thus, prolonged opening of only a few channels affects the SR in situ, whereas this is undetectable in the homogenate. Higher levels of ryanodine binding (0.3 pmol/mg) to the in vitro homogenate also did not affect the homogenate Ca(2+)-uptake rate in the presence or absence of high [ryanodine], whereas it reduced the stimulation of Ca2+ uptake by ruthenium red. High-affinity ryanodine binding to the SR Ca2+ channel, either by perfusion or by binding after homogenisation, did not duplicate the increased Ca2+ efflux observed in the stunned heart, suggesting that the SR defect in the stunned heart is not a prolonged opening of a sub-conducting state of the SR Ca2+ channel.     

Protein kinase A-activated chloride channel is inhibited by the Ca(2+)-calmodulin complex in cardiac sarcoplasmic reticulum

Cardiac sarcoplasmic reticulum (SR) has several chloride (Cl-) channels, which may neutralize the charge across the SR membrane generated by Ca2+ movement. We recently reported a novel 116-picosiemen Cl- channel that is activated by protein kinase A-dependent phosphorylation in cardiac SR. This Cl- channel may serve as a target protein in the receptor-dependent regulation of cardiac excitation-contraction coupling. To understand further regulatory mechanisms, the effects of Ca2+ on the Cl- channel were studied using the planar lipid bilayer-vesicle fusion technique. In the presence of calmodulin (CaM, 0.1 mumol/L per microgram SR vesicles), Ca2+ (3 mumol/L to 1 mmol/L) added to the cis solution reduced the channel openings in a concentration-dependent fashion, whereas Ca2+ (1 nmol/L to 1 mmol/L) alone or CaM (0.1 to 1 mumol/L per microgram SR vesicles) with 1 nmol/L Ca2+ did not affect the channel activity. This inhibitory effect of Ca2+ in the presence of CaM was prevented by CaM inhibitors N-(6 aminohexyl)-5-chloro-1-naphthalenesulfonamide and calmidazolium but not by CaM kinase II inhibitor KN62. These results suggest that the Ca(2+)-CaM complex itself, but not CaM kinase II, is involved in this channel inhibition. Thus, the cardiac SR 116-picosiemen Cl- channel is regulated not only by protein kinase A-dependent phosphorylation but also by the cytosolic Ca(2+)-CaM complex. This is a novel second messenger-mediated regulation of Cl- channels in cardiac SR membrane.     

Ca-activated chloride current and Na-Ca exchange have different timecourses during sarcoplasmic reticulum Ca release in ferret ventricular myocytes

The aim of this work was to measure membrane currents activated by Ca release from the cardiac sarcoplasmic reticulum (s.r.). Intracellular Ca concentration ([Ca2+]i) was measured using fluo-3 in patch clamped cells. Calcium release from the s.r. (whether occurring spontaneously or evoked by caffeine) produced changes of membrane current which could be separated into a Ca-activated Cl current which was inhibited by DIDS or Cl removal and a Na-Ca exchange current. Both these currents had different time courses from the measured [Ca2+]i. Furthermore the Ca-activated Cl current decayed more quickly than did Na-Ca exchange. Possible explanations for the different kinetics of these two Ca-sensitive currents are discussed.