Skeletal Sarcoplasmic Reticulum Ca2 Research Articles

Adenoviral SERCA1a gene transfer to adult rat ventricular myocytes induces physiological changes in calcium handling.

We examined the functional consequences of expressing adult rabbit fast skeletal sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA1a) in isolated adult rat ventricular myocytes. Myocytes were infected with a recombinant adenovirus harboring SERCA1a. Then 2 days after myocyte infection, protein expression was estimated using Western blot and SDS-PAGE analysis. We also measured the ATP-dependent oxalate-facilitated Ca(2+) uptake of myocyte homogenates and monitored Ca(2+) transient in myocytes loaded with the Ca(2+) dye, indo-1. SERCA1a gene expression resulted in a 36% increase in the total SERCA protein level in infected myocytes compared to controls (P<0.01), while SERCA2 and phospholamban levels did not change. This increase was associated with a 42% rise in SR Ca(2+) uptake (P<0.01), while tau (the time constant of Ca(2+) transient decay), and the time to peak fell by 32% (P<0.01) and 38% (P<0.001), respectively. Increasing the frequency of stimulation from 0.2 to 2 Hz decreased tau in both cell types (P<0.01). However, the decrease was much smaller in infected (P<0.01) than in uninfected cells (P<0.001). Isoproterenol (1 microM) further decreased tau in infected myocytes by 23% (P<0.05). In these cells, the diastolic [Ca(2+)](i) decreased by 50% (P<0.05) while the systolic [Ca(2+)](i) increased by 19% (P<0.05). No difference was found in the speed of SR Ca(2+) reloading after caffeine washout between the two cell types. Adenovirus-mediated SERCA1a gene transfer to adult rat ventricular myocytes enhances SR Ca(2+) handling to a degree similar to that observed following physiological stimulation.

Clomipramine and related structures as inhibitors of the skeletal sarcoplasmic reticulum Ca2+ pump.

The Ca2+-pumping activity of skeletal sarcoplasmic reticulum vesicles is half-maximally inhibited by 120 microM clomipramine, 250 microM desipramine, and 500 microM imipramine or trimipramine. The inhibition is attributed to the dihydrodibenzazepine moiety, since 3-(dimethylamino)propionitrile, reproducing the aliphatic amine chain, has no inhibitory action. The inhibition is shown as a marked decrease of Ca2+ binding at equilibrium in the absence of ATP and as a reduction of phosphorylation of the Ca2+-free conformation by inorganic phosphate. Therefore, the drug effect is consistent with preferential interaction of tricyclic antidepressants with the Ca2+-free conformation of the nonphosphorylated enzyme. An additional decrease in the apparent rate constant of enzyme dephosphorylation, i.e., in the release of phosphate from ATP during enzyme cycling was also noticed.

Inhibition of skeletal sarcoplasmic reticulum Ca2+-ATPase activity by deferoxamine nitroxide free radical.

Deferoxamine is an inhibitor of iron-dependent free radical reactions. Despite the antioxidant roles, prolonged clinical use of the chelator is far from benign, and paradoxically, deferoxamine has been shown to promote lipid peroxidation. The possible toxicity of the drug's metabolites, such as deferoxamine nitroxide free radical, deserves attention. We, therefore, tested the hypothesis that deferoxamine nitroxide radicals produced as a result of enzymatic one-electron oxidation of deferoxamine by horseradish peroxidase in the presence of H2O2 are capable of inactivating Ca2+-ATPase of skeletal sarcoplasmic reticulum microsomes as a model system with which to explore the effect of the radical on a biological membrane. Ca2+-ATPase activity of sarcoplasmic reticulum was depressed by exposure to Fenton's reagent (H2O2/FeSO4); the observed effect was significantly enhanced by deferoxamine. We found that the Fenton reaction produced hydroxyl radical, as determined by electron spin resonance spectroscopy. The formation of hydroxyl radical was completely inhibited by deferoxamine; instead, under the same experimental conditions (in the presence of sarcoplasmic reticulum vesicles with or without FeSO4 but without spin trap 5, 5-dimethyl-1-pyrroline N-oxide), the spectral shape and hyperfine coupling constants of electron spin resonance signals confirmed to be long-lived deferoxamine radical were obtained. Furthermore, exposure of sarcoplasmic reticulum vesicles to deferoxamine radical formed by horseradish peroxidase via reaction with H2O2 caused an inhibition of the Ca2+-ATPase activity. The findings show that the sarcoplasmic reticulum vesicles can act as peroxidases and suggest that deferoxamine enhances the decreased Ca2+-ATPase activity afforded by H2O2/FeSO4 due to formation of its metabolites, possibly deferoxamine nitroxide free radical.

Polylysine-induced rapid Ca2+ release from cardiac sarcoplasmic reticulum.

The rapid kinetics of polylysine-induced Ca2+ release from cardiac sarcoplasmic reticulum (SR) was assessed in combination with its effect on ryanodine binding. SR vesicles were isolated from canine cardiac SR. The time course of SR Ca2+ release was continuously monitored by a stopped-flow apparatus, and [3H]ryanodine binding was done by using the filtration method. The initial rate of polylysine-induced Ca2+ release from cardiac SR revealed different concentration dependence from those observed in skeletal SR. The initial rate peaked at 0.11 microM, followed by a decrease at higher concentrations in skeletal SR, whereas it increased to 3.7 microM in cardiac SR. The [3H]ryanodine binding was also stimulated by polylysine with an identical parallelism with Ca2+ release in terms of polylysine concentration dependence. Thus we demonstrated that the cardiac SR Ca2+ release channel is sensitive to activation by polylysine and that there is a difference in the concentration dependence of polylysine-induced activation of cardiac and skeletal SR Ca2+ release channels.

Hypochlorous acid inhibits Ca(2+)-ATPase from skeletal muscle sarcoplasmic reticulum.

Hypochlorous acid (HOCl) is produced by polymorphonuclear leukocytes that migrate and adhere to endothelial cells as part of the inflammatory response to tissue injury. HOCl is an extremely toxic oxidant that can react with a variety of cellular components, and concentrations reaching 200 microM have been reported in some tissues. In this study, we show that HOCl interacts with the skeletal sarcoplasmic reticulum Ca(2+)-adenosinetriphosphatase (ATPase), inhibiting transport function, HOCl inhibits sarcoplasmic reticulum Ca(2+)-ATPase activity in a concentration-dependent manner with a concentration required to inhibit ATPase activity by 50% of 170 microM and with complete inhibition of activity at 3 mM. A concomitant reduction in free sulfhydryl groups after HOCl treatment was observed, paralleling the inhibition of ATPase activity. It was also observed that HOCl inhibited the binding of the fluorescent probe fluorescein isothiocyanate to the ATPase protein, indicating some structural damage may have occurred. These findings suggest that the reactive oxygen species HOCl inhibits ATPase activity via a modification of sulfhydryl groups on the protein, supporting the contention that reactive oxygen species disrupt the normal Ca(2+)-handling kinetics in muscle cells.

A carboxy-terminal peptide of the alpha 1-subunit of the dihydropyridine receptor inhibits Ca(2+)-release channels.

Excitation-contraction coupling in skeletal muscle is thought to involve a physical interaction between the alpha 1-subunit of the dihydropyridine receptor (DHPR) and the sarcoplasmic reticulum (SR) Ca(2+)-release channel (also known as the ryanodine receptor). Considerable evidence has accumulated to suggest that the cytoplasmic loop between domains II and III of the DHPR alpha 1-subunit is at least partially responsible for this interaction. Other parts of this subunit or other subunits may, however, contribute to the functional and/or structural coupling between these two proteins. A synthetic peptide corresponding to a conserved sequence located between amino acids 1487 and 1506 in the carboxy terminus of the alpha 1-subunit inhibits both [3H]ryanodine binding to skeletal and cardiac SR membranes and the activity of skeletal SR Ca(2+)-release channels reconstituted into planar lipid bilayers. A second, multiantigenic peptide synthesized to correspond to the same sequence inhibits both binding and channel activity at lower concentrations than the linear peptide. These peptides slow the rate at which [3H]ryanodine binds to its high-affinity binding site and decrease the rate at which [3H]ryanodine dissociates from this site. A third polypeptide synthesized in Escherichia coli and corresponding to amino acids 1381-1627 and encompassing the above sequence has similar effects. This portion of the alpha 1-subunit of the transverse tubule DHPR is therefore a candidate for contributing to the interaction of this protein with the Ca(2+)-release channel.

Comparison of 2,2′-azobis(2-amidinopropane) hydrochloride (AAPH) and 2,2′-azobis(2,4-dimethylvaleronitrile)(AMVN) as free radical initiators: a spin-trapping study

Spin trapping with 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) and its hydrophobic analogue 2,2-dimethyl-4-phenyl-2H-imidazole 1-oxide (DMPIO) was used to identify and to monitor the concentration of participating radical species in oxidation reactions initiated by azo compounds: water-soluble 2,2′-azobis(2-amidinopropane) hydrochloride (AAPH) and lipophilic 2,2′-azobis(2,4-dimethylvaleronitrile)(AMVN). Incubation of AAPH with spin traps in aqueous media produced alkoxyl radical spin adducts with hyperfine splitting constants being aN= 14.62 G, βaH= 15.29 G, γaH= 0.72 G and aN= 13.46 G, βaH= 12.53 G, for DMPO and DMPIO, respectively. In contrast, formation of AMVN-derived peroxyl radicals was detected by both direct EPR and spin trapping in DMSO (dimethylsulfoxide) solutions. In the presence of either rabbit skeletal sarcoplasmic reticulum (SR) membranes (10 g dm–3 of SR protein) or egg phosphatidylcholine liposomes (10 g dm–3 of lipid) preloaded with AMVN no spin adduct formation was observed, for both DMPO and DMPIO spin traps, indicating that AMVN-derived radical species do not escape the lipid environment. Only a small portion of AAPH-derived alkoxyl radicals was trapped by DMPIO in the presence of SR membranes. Spectral characteristics of the DMPIO spin adduct indicate its location at the lipid–water interface. At the same time, there was virtually no effect of SR on the rate of formation and steady-state level of the DMPO-spin adduct formed in the aqueous phase. From these data we suggest that the bulky cytosolic domains of the SR Ca2+-ATPase protect the membrane surface from radicals generated in the bulk (aqueous) solvent. Other evidence also demonstrates different mechanisms for free radical formation by AAPH and AMVN azo-initiators.

The gating of the sheep skeletal sarcoplasmic reticulum Ca(2+)-release channel is regulated by luminal Ca2+.

The effects of changes in luminal [Ca2+] have been investigated in sheep skeletal sarcoplasmic reticulum (SR) Ca(2+)-release channels after activation of the channels by different ligands from the cytosolic side of the channel. Native heavy SR membrane vesicles were incorporated into planar phospholipid bilayers under voltage-clamp conditions. Experiments were carried out in symmetrical 250 mM Cs+. Lifetime analysis indicates that channels activated solely by cytosolic Ca2+ exhibit at least two open and five closed states. The open events are very brief and are close to the minimum resolvable duration. When channels are activated solely by cytosolic Ca2+, luminal Ca2+ does not appear to exert any regulatory effect. The Po and duration of the open and closed lifetimes are unchanged. However, if channels are activated by ATP alone or by ATP plus cytosolic Ca2+, increases in luminal [Ca2+] produce marked increases in Po and in the duration of the open lifetimes. Our results demonstrate that maximum activation of the skeletal SR Ca(2+)-release channel by ATP cannot be obtained in the absence of millimolar luminal [Ca2+].

Cyclic ADP-ribose and related compounds activate sheep skeletal sarcoplasmic reticulum Ca2+ release channel

It has been suggested that adenosine 5'-cyclic-diphosphoribose (cADPR) can activate only nonskeletal isoforms of the ryanodine-sensitive Ca2+ release channel. We now demonstrate that cADPR is an effective activator of sheep skeletal sarcoplasmic reticulum (SR) Ca2+ release channels incorporated into planar phospholipid bilayers in the presence of activating levels of cytosolic Ca2+. In addition, the precursor of cADPR, beta-NAD+, and the metabolite, adenosine diphosphoribose (ADP-ribose), also increase the open probability (Po) of skeletal SR Ca2+ release channels in micromolar concentrations. At low concentrations of cADPR (1 microM), the mechanism for the increase in Po is an increase in the frequency of channel openings with no increase in the duration of the open events. We also show that the effect of cADPR is dependent on luminal [Ca2+]. cADPR has no effect on Po when the luminal [Ca2+] is < 40 microM. However, at millimolar concentrations of luminal Ca2+, cADPR 1 and 10 microM) increases Po in the presence of activating cytosolic Ca2+.

Effects of palmitoyl carnitine and related metabolites on the avian Ca(2+)‐ATPase and Ca2+ release channel.

1. In birds, prolonged cold exposure induces the development of a non-shivering thermogenesis (NST) of muscular origin that may result from an increase in ATP-dependent cycling of Ca2+ between the sarcoplasmic reticulum (SR) and the cytosol. 2. Because fatty acids are thought to play a significant role in NST, we investigated the effects of palmitic acid and related metabolites on skeletal SR Ca2+ uptake and release in ducklings. 3. Ca(2+)-ATPase activity, 45Ca2+ release and [3H]ryanodine-binding measurements indicated that palmitic acid was without effect on the Ca(2+)-ATPase and Ca2+ release channel. Palmitoyl carnitine and palmitoyl coenzyme A inhibited the Ca(2+)-ATPase at concentrations > 20 microM whereas both activated the Ca2+ release channel at concentrations < or = 20 microM in a dose-dependent manner. 4. Palmitoyl carnitine stimulated [3H]ryanodine binding to skeletal but not cardiac SR vesicles. Induction of 45Ca2+ release was observed with long-chain (C > or = 14) but not with short-chain acyl carnitines (C < or = 12). 5. Long-chain acyl carnitines accumulated significantly in duckling skeletal muscle during cold acclimation. Accordingly, these results suggest that long-chain acyl metabolites may modulate SR Ca2+ cycling and its associated thermogenesis in vivo.

3‘,3“,5‘,5”-Tetraiodophenolsulfonephthalein is a selective inhibitor of Ca(2+)-pumping ATPase in intracellular Ca2+ store

3',3",5',5"-Tetraiodophenolsulfonephthalein (I4PSP) stimulated both Ca2+ transient levels and aggregation in response to thrombin in platelets. Ca2+ uptake into skeletal sarcoplasmic reticulum (SR) was inhibited by I4PSP (IC50, 1.8 microM) whereas that of red blood cell ghosts was not affected by it. Furthermore, I4PSP inhibited the SR Ca(2+)-ATPase activity (IC50, 1.1 microM). Kinetic analysis of the inhibitory effects of I4PSP reveals that I4PSP shows an inhibition of noncompetitive and competitive type with respect to low and high concentrations of ATP, respectively. The mode of inhibition by I4PSP is an uncompetitive type with respect to Ca2+. I4PSP decreased the decomposition rate of the phosphorylated enzyme intermediate (EP). The change in the tryptophan fluorescence of SR Ca(2+)-ATPase induced by ATP was reduced by I4PSP indicating inhibition of the EP transition from Ca2E1P to E2P. The concentration-inhibitory response curves for I4PSP in Ca(2+)-ATPase activities and fluorescence changes were closely correlated. These results suggest that I4PSP slows down the structure transition from Ca2E1P to E2P, resulting in inhibition of the Ca(2+)-ATPase activity. On the basis of these results, it is suggested that the stimulation of Ca2+ transient levels in platelets and their aggregation in response to thrombin are due to the inhibition of Ca2+ pump in intracellular Ca2+ stores by I4PSP.

Activation of single cardiac and skeletal ryanodine receptor channels by flash photolysis of caged Ca2+

Single ryanodine-sensitive sarcoplasmic reticulum (SR) Ca2+ release channels isolated from rabbit skeletal and canine cardiac muscle were reconstituted in planar lipid bilayers. Single channel activity was measured in simple solutions (no ATP or Mg2+) with 250 mM symmetrical Cs+ as charge carrier. A laser flash was used to photolyze caged-Ca2+ (DM-nitrophen) in a small volume directly in front of the bilayer. The free [Ca2+] in this small volume and in the bulk solution was monitored with Ca2+ electrodes. This setup allowed fast, calibrated free [Ca2+] stimuli to be applied repetitively to single SR Ca2+ release channels. A standard photolytically induced free [Ca2+] step (pCa 7-->6) was applied to both the cardiac and skeletal release channels. The rate of channel activation was determined by fitting a single exponential to ensemble currents generated from at least 50 single channel sweeps. The time constants of activation were 1.43 +/- 0.65 ms (mean +/- SD; n = 5) and 1.28 +/- 0.61 ms (n = 5) for cardiac and skeletal channels, respectively. This study presents a method for defining the fast Ca2+ regulation kinetics of single SR Ca2+ release channels and shows that the activation rate of skeletal SR Ca2+ release channels is consistent with a role for CICR in skeletal muscle excitation-contraction coupling.

Digoxin activates sarcoplasmic reticulum Ca(2+)-release channels: a possible role in cardiac inotropy.

1. The effect of digoxin on rapid 45Ca2+ efflux from cardiac and skeletal sarcoplasmic reticulum (SR) vesicles was investigated. Additionally the interaction of digoxin with single cardiac and skeletal muscle SR Ca(2+)-release channels incorporated into planar phospholipid bilayers and held under voltage clamp was determined. 2. Digoxin (1 nM) increased the initial rate and amount of Ca(2+)-induced release of 45Ca2+ from cardiac SR vesicles, passively loaded with 45CaCl2, at an extravesicular [Ca2+] of 0.1 microM. The efflux in the presence and absence of digoxin was inhibited at pM extravesicular Ca2+ and blocked by 5 mM Mg2+. 3. To elucidate the mechanism of action of digoxin, single-channel recording was used. Digoxin (1-20 nM) increased single-channel open probability (Po) when added to the cytosolic but not the luminal face of the cardiac channel in the presence of sub-maximally activating Ca2+ (0.1 microM-10 microM) with an EC50 of 0.91 nM at 10 microM Ca2+. The mechanisms underlying the action of digoxin appear to be concentration-dependent. The activation observed at 1 nM digoxin appears to be consistent with the sensitization of the channel to the effects of Ca2+. At higher concentrations the drug appears to interact synergistically with Ca2+ to produce values of Po considerably greater than those seen with Ca2+ as the sole activating ligand. 4. Digoxin had no effect on single-channel conductance or the Ca2+/Tris permeability ratio. In channels activated by digoxin the Po was decreased by Mg2+. Single-channels were characteristically modified to along lasting open, but reduced, conductance state when 100 nM ryanodine was added to the cytosolic side of the channel.5. Activation of the cardiac SR Ca2+-release channel was observed with similar concentrations of digitoxin, however, higher concentrations of ouabain were required to increase PO. In contrast, a steroid which is not positively inotropic, chlormadinone acetate, had no effect on either cardiac or skeletal SR Ca2+-release channel activity.6. At concentrations up to 1 microM, digoxin had no effect on Ca2+-induced 45Ca2+ efflux from skeletal muscle SR vesicles nor did it affect skeletal SR Ca2+-release channel Po, reflecting a difference between the cardiac and skeletal isoforms of the Ca2+-release channel.7. Since activation of the cardiac SR Ca2+-release channel occurs within the range of concentrations of digoxin encountered therapeutically, it is possible that activation of this channel contributes to the positive inotropic effect observed with this drug. Further, activation of the channel by higher concentrations of digoxin may contribute to the toxic effects seen clinically.

Ryanodine-affinity chromatography purifies 106 kD Ca 2+ release channels from skeletal and cardiac sarcoplasmic reticulum

A 106 kD protein was isolated from skeletal sarcoplasmic reticulum (SR) vesicles and shown to have the properties of SR Ca 2+ release channels, including blockade by 5 nM ryanodine. In view of extensive reports that the ryanodine-receptor complex consists of four 565 kD junctional feet proteins (JFPs) and is the ‘physiological’ Ca 2+ release channel, we prepared ryanodine-affinity columns to isolate its receptor site(s). Conditions known to maximize the association and dissociation of ryanodine to SR proteins were respectively used to link, then elute, the receptor(s) from ryanodine-affinity columns. The method purified a protein at about 100 kD from both rabbit skeletal and canine cardiac SR vesicles. The skeletal and cardiac proteins isolated by ryanodine-affinity chromatography were identified as the low molecular weight Ca 2+ release channel through their antigenic reaction with an anti-106 kD monoclonal antibody. Upon reconstitution in planar bilayers, both skeletal and cardiac proteins revealed the presence of functional SR Ca 2+ release channels. Surprisingly, ryanodine-affinity columns did not retain JFPs but purified 106 kD Ca 2+ release channels which are a minor component (0.1–0.3%) of SR proteins.

Regulation of the skeletal sarcoplasmic reticulum Ca(2+)-ATPase by phospholamban and negatively charged phospholipids in reconstituted phospholipid vesicles.

The Ca(2+)-ATPase of skeletal sarcoplasmic reticulum was purified and reconstituted in proteoliposomes containing phosphatidylcholine (PC). When reconstitution occurred in the presence of PC and the acidic phospholipids, phosphatidylserine (PS) or phosphatidylinositol phosphate (PIP), the Ca(2+)-uptake and Ca(2+)-ATPase activities were significantly increased (2-3 fold). The highest activation was obtained at a 50:50 molar ratio of PS:PC and at a 10:90 molar ratio of PIP:PC. The skeletal SR Ca(2+)-ATPase, reconstituted into either PC or PC:PS proteoliposomes, was also found to be regulated by exogenous phospholamban (PLB), which is a regulatory protein specific for cardiac, slow-twitch skeletal, and smooth muscles. Inclusion of PLB into the proteoliposomes was associated with significant inhibition of the initial rates of Ca(2+)-uptake, while phosphorylation of PLB by the catalytic subunit of cAMP-dependent protein kinase reversed the inhibitory effects. The effects of PLB on the reconstituted Ca(2+)-ATPase were similar in either PC or PC:PS proteoliposomes, indicating that inclusion of negatively charged phospholipid may not affect the interaction of PLB with the skeletal SR Ca(2+)-ATPase. Regulation of the Ca(2+)-ATPase appeared to involve binding with the hydrophilic portion of phospholamban, as evidenced by crosslinking experiments, using a synthetic peptide which corresponded to amino acids 1-25 of phospholamban. These findings suggest that the fast-twitch isoform of the SR Ca(2+)-ATPase may be also regulated by phospholamban although this regulator is not expressed in fast-twitch skeletal muscles.

Involvement of sarcoplasmic reticulum 'Ca2+ release channels' in excitation-contraction coupling in vertebrate skeletal muscle.

1. Pharmacological blockers of calcium-induced calcium release from isolated skeletal sarcoplasmic reticulum (SR) vesicles have been introduced into frog skeletal muscle fibres to determine their effects on excitation-contraction coupling. 2. Among the blockers tested, Ruthenium Red, neomycin, gentamicin and 9-aminoacridine inhibited the SR Ca2+ release associated with excitation-contraction (E-C) coupling as much as they inhibited caffeine potentiation of that release. Protamine, certain of its derivatives, and spermine were ineffective in both in situ tests. 3. Alternative sites of polyamine action on the contractile proteins, SR Ca2+ uptake or charge movements were ruled out. 4. All polyamines tested required considerably higher concentrations to inhibit excitation-contraction coupling than to block Ca2+ release from isolated SR vesicles. 5. The quantitative pharmacological difference in sensitivity between isolated and intact systems serves as a reminder that results on isolated systems cannot generally be used to predict results of the same substances on more physiological systems. 6. Since caffeine is known to open the SR 'Ca2+ release channels' (the ryanodine receptors that mediate Ca(2+)-induced Ca2+ release), the equal effectiveness of these blockers at inhibiting excitation-contraction (E-C) coupling and its potentiation by caffeine suggests that the SR 'Ca2+ release channels' are indeed involved in excitation-concentration coupling in skeletal muscle, although the results do not indicate how the channel is gated open during E-C coupling.

Properties of immunoaffinity purified 106-kDa Ca 2+ release channels from the skeletal sarcoplasmic reticulum

The sulfhydryl-gated 106-kDa Ca 2+-release channel (SG-106) was purified by biotin-avidin chromatography from skeletal sarcoplasmic reticulum (SR) vesicles and used as an antigen to raise polyclonal anti-bodies. Western blots showed that the antisera crossreacted with the antigenic SG-106 and not with SR Ca 2+, Mg 2+-ATPase or with junctional foot proteins (JFPs) (Zaidi et al., 1989, J. Biol. Chem. 264(36), 21,725–21,736; 21,737–21,747). Polyclonal anti-body-affinity columns were used to selectively purify SG-106-kDa proteins which, upon incorporation in planar bilayers, revealed the presence of a cationic channels with properties similar to “native” Ca 2+-release channels obtained through the fusion of SR vesicles with planar bilayers. In agreement with measurements of Ca 2+ release from SR vesicles, sulfhydryl oxidizing and reducing agents (i.e., 2,2′-dithiodipyridine and dithiothreitol) respectively increased and decreased the open-time probability of 106-kDa Ca 2+-release channels. In contrast with reports on JFPs, ryanodine at 0.5–1 n m increased the open-time probability and at 2–10 n m locked 106-kDa Ca 2+-release channels in a closed state rather than an open subconductance state. The SG-106 was activated by millimolar ATP, inhibited by millimolar Mg 2+, and blocked by micromolar ruthenium red. Adriamycin (2–10 μ m) caused a transient activation of SG-106 Ca 2+-release channels, followed by closure in about 5 min, and intermittent activation to a subconductance state. Polyclonal antibodies used to purify the SG-106 also activated the channel when added to the cis side but not the trans side of the bilayer. Thus, SG-106 channels possess features that are similar to “native” SR Ca 2+-release channels, are immunologically distinct from JFPs, and interact in seconds with nanomolar ryanodine in planar bilayers.

The heavy metal ions Ag + and Hg 2+ trigger calcium release from cardiac sarcoplasmic reticulum

Heavy metal ions have been shown to induce Ca 2+ release from skeletal sarcoplasmic reticulum (SR) by binding to free sulfhydryl groups on a Ca 2+ channel protein and are now examined in cardiac SR. Ag + and Hg 2+ (at 10–25 μ m) induced Ca 2+ release from isolated canine cardiac SR vesicles whereas Ni 2+, Cd 2+, and Cu 2+ had no effect at up to 200 μ m. Ag 2+-induced Ca 2+ release was measured in the presence of modulators of SR Ca 2+ release was compared to Ca 2+-induced Ca 2+ release and was found to have the following characteristics, (i) Ag +-induced Ca 2+ release was dependent on free [Mg 2+], such that rates of efflux from actively loaded SR vesicles increased by 40% in 0.2 to 1.0 m m Mg 2+ and decreased by 50% from 1.0 to 10.0 m m Mg 2+. (ii) Ruthenium red (2–20 μ m) and tetracaine (0.2–1.0 m m), known inhibitors of SR Ca 2+ release, inhibited Ag +-induced Ca 2+ release, (iii) Adenine nucleotides such as cAMP (0.25–2.0 m m) enhanced Ca 2+-induced Ca 2+ release, and stimulated Ag +-induced Ca 2+ release. (iv) Low Ag + to SR protein ratios (5–50 nmol Ag +/mg protein) stimulated Ca 2+-dependent ATPase activity in Triton X-100-uncoupled SR vesicles. (v) At higher ratios of Ag + to SR proteins (50–250 nmol Ag +/mg protein), the rate of Ca 2+ efflux declined and Ca 2+-dependent ATPase activity decreased gradually, up to a maximum of 50% inhibition, (vi) Ag + stimulated Ca 2+ efflux from passively loaded SR vesicles (i.e., in the absence of ATP and functional Ca 2+ pumps), indicating a site of action distinct from the SR Ca 2+ pump. Thus, at low Ag 2+ to SR protein ratios, Ag 2+ is very selective for the Ca 2+ release channel. At higher ratios, this selectivity declines as Ag + also inhibits the activity of Ca 2+,Mg 2+-ATPase pumps. Ag + most likely binds to one or more sulfhydryl sites “on” or “adjacent” to the physiological Ca 2+ release channel in cardiac SR to induce Ca 2+ release.

Structural and Functional Correlation of the Trypsin-digested Ca2+ Release Channel of Skeletal Muscle Sarcoplasmic Reticulum

The effect of trypsin digestion on the (i) fragmentation pattern, (ii) activity, (iii) [3H]ryanodine binding, and (iv) sedimentation behavior of the skeletal sarcoplasmic reticulum (SR) ryanodine receptor-Ca2+ release channel complex has been examined. Mild tryptic digestion of heavy, junctional-derived SR vesicles resulted in the rapid disappearance of the high molecular weight (Mr approximately 400,000) Ca2+ release channel protein on sodium dodecyl sulfate gels and appearance of bands of lower Mr upon immunoblot analysis, without an appreciable effect on [3H]ryanodine binding or the apparent S value (30 S) of the 3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps)-solubilized channel complex. Further degradation to bands of Mr greater than 70,000 on immunoblots correlated with a reduction of channel size from 30 S to 10-15 S and loss of high affinity [3H]ryanodine binding to the trypsinized receptor, while low affinity [3H]ryanodine binding and [3H]ryanodine bound prior to digestion were retained. Parallel 45Ca2+ efflux measurements also indicated retention of the Ca2+, Mg2+, and ATP regulatory sites, although Ca2+-induced 45Ca2+ release rates were changed. In planar lipid bilayer-single channel measurements, addition of trypsin to the cytoplasmic side of the high conductance (100 pS in 50 mM Ca2+), Ca2+-activated SR Ca2+ channel initially increased the fraction of channel open time and was followed by a complete and irreversible loss of channel activity. Trypsin did not change the unitary conductance, and was without effect on single channel activity when added to the lumenal side of the channel.

Evidence that platelet and skeletal sarcoplasmic reticulum Ca2+-ATPase are structurally distinct.

Proteolytic digestion and indirect immunostaining were used to compare the platelet and sarcoplasmic reticulum Ca2+-ATPase proteins. When the platelet and sarcoplasmic reticulum Ca2+-ATPase proteins were digested in the native state with trypsin, the platelet Ca2+-ATPase, which had an apparent undigested molecular mass of 103 kDa, yielded 78-kDa and 25-kDa fragments. Calcium transport activity depended on the integrity of the 103-kDa protein, while the digested protein had residual ATPase activity. Tryptic digestion of the sarcoplasmic reticulum pump protein, which also had an undigested molecular mass of 103 kDa, yielded products with apparent molecular masses of 55 kDa, 36 kDa, and 26 kDa. Distinct patterns were also observed when the platelet and sarcoplasmic reticulum calcium pump proteins were digested with chymotrypsin and Staphylococcus aureus protease in the presence of sodium dodecyl sulfate. Chymotrypsin digestion of the platelet protein resulted in the appearance of products with apparent molecular masses of 70 kDa, 39 kDa, and 31 kDa, while a similar digestion of the sarcoplasmic reticulum calcium pump protein yielded 54-kDa, 52.5-kDa, 46-kDa, 41-kDa, and 36-kDa fragments. Exposure of the sarcoplasmic reticulum and platelet Ca2+-ATPase proteins to S. aureus protease also yielded dissimilar fragmentation patterns. These results indicate that the Ca2+-ATPases from platelets and sarcoplasmic reticulum are distinct proteins.