Regulation of the gating of the sheep cardiac sarcoplasmic reticulum ca2+-release channel by luminal Ca2+

We investigated the effects of changes in luminal [Ca²⁺] on the gating of native andpurified sheep cardiac sarcoplasmic reticulum (SR) Ca²⁺-release channels reconstituted intoplanar phospholipid bilayers. The open probability (P _o )of channels activated solely by cytosolic Ca²⁺ was greater at positive than negative holding potentials. Channels activatedsolely by 10 m cytosolic Ca²⁺ exhibited no change in steady-stateP _o or in the relationship betweenP _o and voltage when the luminal[Ca²⁺] was increased from nanomolar to millimolar concentrations. In the absence of activating concentrationsof cytosolic Ca²⁺, the channel can be activated by the phosphodiesterase inhibitor sulmazole (AR-L 115BS). However, cytosolicCa²⁺-independent activation of the channel by sulmazole requires luminal Ca²⁺. In the presence ofsulmazole, at picomolar luminal [Ca²⁺] the channel remains completely closed. Increasing the luminal [Ca²⁺]to millimolar levels markedly increases the P _o via an increase in theduration of open events. The P _o and duration of the sulmazole-activated, luminalCa²⁺-dependent channel openings are voltage dependent. In the presence of micromolar luminal Ca²⁺, theP _o and duration of sulmazole-activated openings are greater atnegative voltages. However, at millimolar luminal [Ca²⁺], long openings are also observed at positive voltages and theP _o appears to be similar at positive and negative voltages. Our findings indicate thatthe regulation of channel gating by luminal Ca²⁺ depends on the mechanism of channel activation.We would like to thank Dr Allan Lindsay for the preparation of the purified SR Ca²⁺-release channels. This work was supported by the British Heart Foundation.     

Block of the sheep cardiac sarcoplasmic reticulum Ca2+-release channel by tetra-alkyl ammonium cations

Summary The purified ryanodine receptor channel of the sheep cardiac muscle sarcoplasmic reticulum (SR) membrane functions as a calcium-activated cation-selective channel under voltage-clamp conditions following reconstitution into planar phospholipid bilayers. We have investigated the effects of the tetra-alkyl ammonium (TAA) cations, (C _n H_2n+1)₄N⁺ and the trimethyl ammonium cations, ethyltrimethyl ammonium and propyltrimethyl ammonium, on potassium conductance through the receptor channel. Small TAA cations (n = 1–3) and the trimethyl ammonium derivatives act as asymmetric, voltage-dependent blockers of potassium current. Quantitative analysis of the voltage dependence of block indicates that the conduction pathway of the sheep cardiac SR ryanodine receptor channel contains two distinct sites for the interaction of these small organic cations. Sites are located at approximately 50% for tetramethyl ammonium (TMA ⁺) and 90% for tetraethyl ammonium (TEA⁺) and tetrapropyl ammonium (TPrA⁺) of the voltage drop across the channel from the cytosolic face of the protein. The chemical substitution of an ethyl or propyl group for one of the methyl groups in TMA⁺ increases the voltage dependence of block to a level similar to that of TEA ⁺ and TPrA⁺. The zero-voltage dissociation constant (K _b(0)) falls with the increasing number of methyl and methylene groups for those blockers acting 90% of the way across the voltage drop. This is interpreted as suggesting a hydrophobic binding site at this point in the conduction pathway. The degree of block increases as the concentration of small TAA cations is raised. The concentration dependence of tetraethyl ammonium block indicates that the cation interacts with a single site within the conduction pathway with a K _m of 9.8±1.7 mm (mean±sd) at 40 mV. Larger TAA cations (n = 4–5) do not induce voltage-dependent block of potassium current of the form seen with the smaller TAA cations. These data support the contention that the sheep cardiac SR ryanodine receptor channel may be occupied by at most one ion at a time and suggest that a large proportion of the voltage drop falls over a relatively wide region of the conduction pathway.This work was supported by funds from the Medical Research Council and the British Heart Foundation. We would like to thank Richard Montgomery for his considerable help with the chemical synthesis. We are grateful to Drs. John Chambers, Nick Price and staff for showing us the intricacies of NMR spectroscopy.     

Muscle-specific GSTM2-2 on the luminal side of the sarcoplasmic reticulum modifies RyR ion channel activity

We show that a glutathione transferase (GST) protein, which is recognised by an antibody against the muscle-specific human GSTM2-2 (hGSTM2-2), is associated with the lumen of the sarcoplasmic reticulum (SR) of cardiac muscle, but not skeletal muscle. We further show that hGSTM2-2 modifies both cardiac and skeletal ryanodine receptor (RyR) activity when it binds to the luminal domain of the RyR channel complex. The properties of hGSTM2-2 were compared with those of the calsequestrin (CSQ), a Ca²⁺ binding protein also present in the lumen of the SR which, like GSTM2-2, contains a thioredoxin-fold structure and modifies RyR activity (Wei, L., Varsanyi, M., Dulhunty, A. F., Beard, N. A. (2006). The Biophysical Journal, 91, 1288–1301). The glutathione transferase activity of hGSTM2-2 is strong, while CSQ is essentially inactive. Conversely CSQ is a strong Ca²⁺ binder, but hGSTM2-2 is not. The effects of luminal hGSTM2-2 on RyR activity differ from those of CSQ in that hGSTM2-2 activates RyRs by increasing their open probability and conductance and the effects are independent of luminal Ca²⁺ concentration. The results suggest that GSTM2-2 can interact with specific luminal sites on the RyR complex and that the interaction is likely to be within the pore of the RyR channel. The differences between the effects of CSQ and hGSTM2-2 suggest that the thioredoxin fold is not a major determinant of the luminal actions of either protein. The results indicate that GSTM2-2 is a novel luminal regulator of the RyR channels in the heart.     

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.     

The concentration of free Ca(2+) in the sarcoplasmic reticulum of frog cut twitch skeletal muscle fibers estimated with tetramethylmurexide

One aim of this article was to determine the resting concentration of free Ca²⁺ in the sarcoplasmic reticulum (SR) of frog cut skeletal muscle fibers ([Ca²⁺]_SR,R) using the calcium absorbance indicator dye tetramethylmurexide (TMX). Another was to determine the ratio of [Ca²⁺]_SR,R to TMX's apparent dissociation constant for Ca²⁺ (K_app) in order to establish the capability of monitoring [Ca²⁺]_SR(t) during SR Ca²⁺ release – a signal needed to determine the Ca²⁺ permeability of the SR. To reveal the properties of TMX in the SR, the surface membrane was rapidly permeabilized with saponin to rapidly dissipate myoplasmic TMX. Results indicated that the concentration of Ca-free TMX in the SR was 2.8-fold greater than that in the myoplasm apparently due to binding of TMX to sites in the SR. Taking into account that such binding might influence K_app as well as a dependence of K_app on TMX concentration, the results indicate an average [Ca²⁺]_SR,R ranging from 0.43 to 1.70 mM. The ratio [Ca²⁺]_SR,R/K_app averaged 0.256, a relatively low value which should not depend on factors influencing K_app. As a result, the time course of [Ca²⁺]_SR(t) in response to electrical stimulation is well determined by, and approximately linearly related to, the active TMX absorbance signal.     

Differentiation between Ca2+ transport and ATP-induced Ca2+ binding by sarcoplasmic reticulum

The Ca²⁺ actively accumulated by sarcoplasmic reticulum isolated from skeletal muscle is composed of two fractions; one represented by intravesicular free Ca²⁺ and another represented by Ca²⁺ selectively bound to the membranes. Both of these Ca²⁺ fractions depend on ATP, although it is not clear whether ATP hydrolysis is essential for accumulation of the second Ca²⁺ fraction. The existence of the membrane-bound Ca²⁺ induced by ATP is clearly shown in experiments in which the Ca²⁺ retention by sarcoplasmic reticulum is measured in the presence and in the absence of X-537A, a Ca²⁺ ionophore, which makes the membrane permeable to Ca²⁺. Thus, in the presence of X-537A all Ca²⁺ accumulated due to ATP is bound to the membranes. This membrane-bound Ca²⁺ represents about 30 nmol/mg protein in the range of external $\text{p}\text{Ca}$ values of 7 to 3.5. The magnitude of this Ca²⁺ fraction is slightly higher whether or not the experiments are performed in the presence of oxalate, which greatly increased the intravesicular Ca²⁺ accumulation. Furthermore, taking advantage of the impermeability of sarcoplasmic reticulum to EGTA, it is possible to show the existence of the membrane-bound Ca²⁺ as a distinct fraction from that which exists intravesicularly.     

Glycogen synthase binds to sarcoplasmic reticulum and is phosphorylated by CaMKII in fast-twitch skeletal muscle

We investigated the subcellular localization of glycogen synthase (GS) in the adductor muscle of anesthetized rabbits injected intravenously with propranolol. Under these experimental conditions, glycogen content was about 10 mmol/kg of fresh tissue. Immunofluorescent and fractionation studies showed that GS associated with sarcoplasmic reticulum (SR) membranes. Glycogen and GS always co-sedimented, suggesting a predominant role of glycogen in targeting of GS to SR. SR-associated GS was phosphorylated in vitro by SR-bound Ca2+-calmodulin dependent protein kinase (CaMKII) and dephosphorylated by endogenous protein phosphatase 1 (PP1c). Based on measurements of GS activity ratio, in vitro phosphorylation of GS by CaMKII did not significantly affect GS activity per se. However, GS activity ratio was slightly reduced, when SR membranes were further incubated with ATP after prior phosphorylation by CaMKII, suggesting that CaMKII might act sinergistically with other protein kinases. We propose that SR-bound CaMKII plays a role in regulation of glycogen metabolism in skeletal muscle, when intracellular Ca2+ is raised.     

Vanilloid receptor expressed in the sarcoplasmic reticulum of rat skeletal muscle

Vanilloid receptor subtype 1 (VR1) was cloned as a capsaicin receptor from neuronal cells of dorsal root ganglia. VR1 was subsequently found in a few non-neuronal tissues, including skeletal muscle [Onozawa et al., Tissue distribution of capsaicin receptor in the various organs of rats, Proc. Jpn. Acad. Ser. B 76 (2000) 68-72]. We confirmed the expression of VR1 in muscle cells using the RT-PCR method and Western blot analysis. Immunostaining studies with a confocal microscope and an electron microscope indicated that VR1 was present in the sarcoplasmic reticulum (SR), a store of Ca2+. The SR releases Ca2+ to cause a contraction when a muscle is excited. However, SR still releases a small amount of Ca2+ under relaxed conditions. We found that this leakage was enhanced by capsaicin and was antagonized by capsazepine, a capsaicin blocker, indicating that leakage of Ca2+ occurs through a channel composed of VR1.     

Activation of the sheep cardiac Ca2+ release channel by simple heteroaromatics

The broad range of ligands known to modulate ryanodine receptor activity includes a class of heteroaromatic compounds displaying relatively poor efficacy. Greater understanding of the physicochemical properties that predispose these molecules to interaction with the channel should facilitate the rational design of more potent analogues. To this end we are examining the structure-activity relationship for simple heteroaromatic compounds. Efficacy is assessed by the ability to stimulate [3H]ryanodine binding to heavy sarcoplasmic reticulum vesicles. The propensity to activate the channel requires notably little chemical functionality and is associated with the capacity for charge-transfer complex formation in conjunction with steric bulk.     

Increased Ca2+ storage capacity in the sarcoplasmic reticulum by overexpression of HRC (histidine-rich Ca2+ binding protein)

The histidine-rich Ca(2+) binding protein (HRC) is a high capacity Ca(2+) binding protein in the sarcoplasmic reticulum (SR). Because HRC appears to interact directly with triadin, HRC may play a role in the regulation of Ca(2+) release during excitation-contraction coupling. In this study, we examined the physiological effects of HRC overexpression in rat neonatal cardiomyocytes. Both caffeine-induced and depolarization-induced Ca(2+) release from the SR were increased significantly in the HRC overexpressing cardiomyocytes. Consistently, the Ca(2+) content, normally depleted from the SR in the presence of cyclopiazonic acid (CPA), remained elevated in these cells. In contrast, the density and the ryanodine-binding kinetics of the ryanodine receptor (RyR)/Ca(2+) release channel were slightly reduced or not significantly altered in the HRC overexpressing cardiomyocytes. We suggest that HRC is involved in the regulation of releasable Ca(2+) content into the SR.     

Cytoplasmic Ca2+ does not inhibit the cardiac muscle sarcoplasmic reticulum ryanodine receptor Ca2+ channel,although Ca2+-induced Ca2+ inactivation of Ca2+ release is observed in native vesicles

Single channel properties of cardiac and fast-twitch skeletal muscle sarcoplasmic reticulum (SR) release channels were compared in a planar bilayer by fusing SR membranes in a Cs⁺-conducting medium. We found that the pharmacology, Cs⁺ conductance and selectivity to monovalent and divalent cations of the two channels were similar. The cardiac SR channel exhibited multiple kinetic states. The open and closed lifetimes were not altered from a range of 10^–7 to 10^–3 M Ca²⁺, but the proportion of closed and open states shifted to shorter closings and openings, respectively.However, while the single channel activity of the skeletal SR channel was activated and inactivated by micromolar and millimolar Ca²⁺, respectively, the cardiac SR channel remained activated in the presence of high [Ca²⁺]. In correlation to these studies, [³H]ryanodine binding by the receptors of the two channel receptors was inhibited by high [Ca²⁺] in skeletal but not in cardiac membranes in the presence of adenine nucleotides. There is, however, a minor inhibition of [³H]ryanodine binding of cardiac SR at millimolar Ca²⁺ in the absence of adenine nucleotides.When Ca²⁺-induced Ca²⁺ release was examined from preloaded native SR vesicles, the release rates followed a normal biphasic curve, with Ca²⁺-induced inactivation at high [Ca²⁺] for both cardiac and skeletal SR. Our data suggest that the molecular basis of regulation of the SR Ca²⁺ release channel in cardiac and skeletal muscle is different, and that the cardiac SR channel isoform lacks a Ca²⁺-inactivated site.This work was supported by research grants from the National Institutes of Health HL13870 and AR38970, and the Texas Affiliate of the American Heart Association, 91A-188. M. Fill was the recipient of an NIH fellowship AR01834.     

Systemic ablation of RyR3 alters Ca2+ spark signaling in adult skeletal muscle

Ca2+ sparks are localized intracellular Ca2+ release events from the sarcoplasmic reticulum in muscle cells that result from synchronized opening of ryanodine receptors (RyR). In mammalian skeletal muscle, RyR1 is the predominant isoform present in adult skeletal fibers, while some RyR3 is expressed during development. Functional studies have revealed a differential role for RyR1 and RyR3 in the overall Ca2+ signaling in skeletal muscle, but the contribution of these two isoforms to Ca2+ sparks in adult mammalian skeletal muscle has not been fully examined. When enzyme-disassociated, individual adult skeletal muscle fibers are exposed to an osmotic shock, the resting fiber converts from a quiescent to a highly active Ca2+ release state where Ca2+ sparks appear proximal to the sarcolemmal membrane. These osmotic shock-induced Ca2+ sparks occur in ryr3(-/-) muscle with a spatial distribution similar to that seen in wild type muscle. Kinetic analysis reveals that systemic ablation of RyR3 results in significant changes to the initiation, duration and amplitude of individual Ca2+ sparks in muscle fibers. These changes may reflect the adaptation of the muscle Ca2+ signaling or contractile machinery due to the loss of RyR3 expression in distal tissues, as biochemical assays identify significant changes in expression of myosin heavy chain protein in ryr3(-/-) muscle.     

Distribution of the endoplasmic reticulum and its relationship with the sarcoplasmic reticulum in skeletal myofibers

We have analyzed the distribution of the endoplasmic reticulum (ER) within isolated rat skeletal muscle flexor digitorum brevis myofibers. Studies with confocal microscopy indicated that the resident ER proteins displayed a perinuclear and cross-striated distribution that extended over the I band areas. Interestingly, two discrete distribution patterns were observed when different receptor or viral marker proteins were blocked in the ER. Accordingly, the vesicular stomatitis virus G protein that lost its efficient export through the Golgi apparatus during myogenesis preferentially marked the A-I junctional areas. The proteins that retained their Golgi processing after myogenesis, on the contrary, concentrated around the myonuclei and over the Z lines. Furthermore, the ER exit site marker sec23 located to Z lines but not to A-I junctions. To analyze the ultrastructural organization of the ER, we infected myofibers with recombinant virus expressing KDEL-tagged peroxidase that is translocated into the ER. With transmission electron microscopy, peroxidase activity was found in perinuclear and Z line-flanking tubular structures, but also within the terminal cisternae of the sarcoplasmic reticulum. The translocon-associated protein exhibited a similar localization. Taken together, the terminal cisternae contained unevenly distributed rough ER structures apparently lacking the export function. The exporting ER comprised perinuclear and Z line-flanking structures.     

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.     

Molecular regulation of cardiac ryanodine receptor ion channel

The release of Ca(2+) ions from intracellular stores is a key step in a wide variety of cellular functions. In striated muscle, the release of Ca(2+) from the sarcoplasmic reticulum (SR) leads to muscle contraction. Ca(2+) release occurs through large, high-conductance Ca(2+) release channels, also known as ryanodine receptors (RyRs) because they bind the plant alkaloid ryanodine with high affinity and specificity. The RyRs are isolated as 30S protein complexes comprised of four 560 kDa RyR2 subunits and four 12 kDa FK506 binding protein (FKBP12) subunits. Multiple endogenous effector molecules and posttranslational modifications regulate the RyRs. This review focuses on current research toward understanding the control of the isolated cardiac Ca(2+) release channel/ryanodine receptor (RyR2) by Ca(2+), calmodulin, thiol oxidation/reduction and nitrosylation, and protein phosphorylation.     

Cyclic ADP-ribose induced Ca release in rabbit skeletal muscle sarcoplasmic reticulum

The Ca²⁺-mobilizing metabolite cyclic ADP-ribose (cADPR) has been shown to release Ca²⁺ from ryanodine-sensitive stores in many cells. We show that this metabolite at a concentration of 17μM, but not its precursor β-NAD⁺ nor non-cyclic ADPR at the same concentration, is active in releasing Ca²⁺ from rabbit skeletal muscle sarcoplasmic reticulum. The release was not sensitive to Ruthenium red (1μM) nor to the ryanodine receptor-specific scorpion toxin Buthotus₁-1 (10 μM). In planar bilayer single channel recordings, concentrations up to 50μM cADPR did not increase the open probability of Ruthenium red and toxin-sensitive Ca²⁺ release channels. Thus Ca²⁺ release induced by cADPR in skeletal muscle sarcoplasmic reticulum may not involve opening of ryanodine receptors.     

State of translocated Ca2+ by sarcoplasmic reticulum inferred from kinetic analysis of calcium oxalate precipitation

Uptake of Ca²⁺ by sarcoplasmic reticulum in the presence of oxalate displays biphasic kinetics. An initial phase of normal uptake is followed by a second phase coincident with precipitation of calcium oxalate inside the vesicles. The precipitation rate induced by actively transported Ca²⁺ is depressed by increasing the added Ca²⁺ concentration. This correlates linearly with the reciprocal of precipitation rate. Therefore, a maximal limit rate could be extrapolated at zero Ca²⁺ ($\text{V}{}_0$). The rate of precipitation, also a function of added amount protein, gives a linear correlation in a double reciprocal plot. Thus, it was possible to estimate the maximal precipitation rate occurring at infinite protein concentration ($\text{V}{}^{\mathrm{\infty }}$). With the combined extrapolated values a maximal expected precipitation rate could be calculated ($\text{V}{}_0{}^{\mathrm{\infty }}$). Kinetics of calcium oxalate precipitation was studied in the absence of calcium uptake and empirical equations relating the rate of precipitation with the added Ca²⁺ were established. Entering $\text{V}{}_0{}^{\mathrm{\infty }}$ in the equations, an internal free Ca²⁺ concentration of approx. 2.5 mM was estimated. Additionally, it is shown that the ionophore X-537A does not supress the Ca²⁺ uptake, if added during the oxalate-dependent phase, albeit the uptake proceeds at a slower rate after the release of approx. 70 nmol Ca²⁺/mg protein. This amount presumably equals the internal free Ca²⁺ not sequestered by oxalate, producing a maximal concentration approx. 14 mM. Taking into account low affinity binding of internal binding sites and the transmembrane Ca²⁺ gradients built up during the uptake of Ca²⁺, values of free Ca²⁺ ranging from 3 to 6 mM, approaching those estimated by the precipitation analysis, could be estimated.