ATPase Of Sarcoplasmic Reticulum Research Articles

The effect of mercazolil and thyroid parathyroidectomy on the properties of the Ca2 + -ATPase of the sarcoplasmic skeletal muscle reticulum

The kinetic and thermodynamic properties of Ca2+-ATPase of the skeletal muscle sarcoplasmic reticulum (SR) are studied in rats subjected to thyroparathyroidectomy and in rats with thyroid hypofunction induced by mercazolyl (10 mg/kg). The studies included measurements of the blood serum calcitonin and thyroxin.
 Removal of the thyroid and parathyroid glands led to an early (in 3 days) significant decrease of Vmax and a drop of Ca by the end of experiment (30 days) and altered the thermodynamic characteristics of Ca2^-ATPase, which indicated changes in the SR membrane protein phase. Mercazolyl induced an increase of Vmax in 3 days and a decrease in Ca in 30 days and changed the thermodynamic properties of the enzyme. Thyroid hypofunction was associated with a drop of thyroxin and calcitonin levels in the serum.
 Thyroid and parathyroid dysfunctions involved changes in the structure and function of Ca2+-ATPase of SR.

Rearrangement of boundary, stand-by, and fluid lipids during the formation of two-dimensional crystals of Ca2+-ATPase

Ca2+-transport ATPase of sarcoplasmic reticulum of rabbit skeletal and carp abdominal muscle is essential for the removal of large amounts of Ca2+ ions during the relaxation of the muscle. As in the case of other lipid–protein systems, fluid lipid and motionally restricted boundary lipid sites lead to multicomponent spin label EPR spectra. Having subtracted the dominating fluid component a two-component lineshape is recovered as a subtraction endpoint, suggesting the presence of a third, motionally less restricted stand-by lipid shell as fraction of boundary lipids. On adding 5mM decavanadate the phosphate binding site is blocked and the protein dimers form a two dimensional array, at least in the case of the sarcoplasmic reticulum of rabbit. During two-dimensional crystal formation of rabbit Ca2+-ATPase an increase of lipids was found in boundary and stand-by shells. No such change was observed in the case of the sarcoplasmic reticulum of carp abdominal muscle.

Luminal dissociation of Ca2+ from the phosphorylated Ca2+-ATPase is sequential and gated by Mg2+

Transport of Ca2+ across the membrane by the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum involves the transfer of two Ca2+ ions from a pair of cytoplasmic sites to a pair of luminal sites, driven by phosphorylation of the ATPase. The ATPase is inhibited by Mg2+ at alkaline pH values. Inhibition follows from a decrease in the rate of release of Ca2+ from the phosphorylated ATPase. Phosphorylation-induced release of Ca2+ from the ATPase is biphasic at alkaline pH, which is consistent with sequential release of Ca2+ from the phosphorylated ATPase; the rates of both components decrease with increasing Mg concentration. The effect of Mg2+ on the slow phase of release follows from the binding of Mg2+ at the empty outer luminal site, vacated by the release of the first Ca2+ ion. The effect of Mg2+ on the rate of release of the first Ca2+ ion could follow from binding to a gating site also affecting the binding of Ca2+ to the cytoplasmic sites.

Luminal dissociation of Ca2+ from the phosphorylated Ca2+-ATPase is sequential and gated by Mg2+

Transport of Ca2+ across the membrane by the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum involves the transfer of two Ca2+ ions from a pair of cytoplasmic sites to a pair of luminal sites, driven by phosphorylation of the ATPase. The ATPase is inhibited by Mg2+ at alkaline pH values. Inhibition follows from a decrease in the rate of release of Ca2+ from the phosphorylated ATPase. Phosphorylation-induced release of Ca2+ from the ATPase is biphasic at alkaline pH, which is consistent with sequential release of Ca2+ from the phosphorylated ATPase; the rates of both components decrease with increasing Mg concentration. The effect of Mg2+ on the slow phase of release follows from the binding of Mg2+ at the empty outer luminal site, vacated by the release of the first Ca2+ ion. The effect of Mg2+ on the rate of release of the first Ca2+ ion could follow from binding to a gating site also affecting the binding of Ca2+ to the cytoplasmic sites.

Mechanism of nitric oxide-induced vasodilatation: refilling of intracellular stores by sarcoplasmic reticulum Ca2+ ATPase and inhibition of store-operated Ca2+ influx.

The precise mechanisms by which nitric oxide (NO) decreases free [Ca2+]i, inhibits Ca2+ influx, and relaxes vascular smooth muscle are poorly understood. In rabbit and mouse aorta, agonist-induced contractions and increases in [Ca2+]i were resistant to nifedipine, suggesting Ca2+ entry through non-L-type Ca2+ channels. Relaxations to NO were inhibited by thapsigargin (TG) or cyclopiazonic acid (CPA) indicating the involvement of sarcoplasmic reticulum ATPase (SERCA). Studies of the effect of NO on [Ca2+]i and the rate of Mn2+ influx with fura-2 fluorometry in rabbit aortic smooth muscle cells in primary culture were designed to test how SERCA is involved in mediating the response to NO. When cells were stimulated with angiotensin II (AII), NO accelerated the removal of Ca2+ from the cytoplasm, decreased [Ca2+]i, and inhibited Ca2+ and Mn2+ influx. Inhibition of SERCA abolished all the effects of NO. In contrast, inhibition of the Na+/Ca2+exchanger or the plasma membrane Ca2+ ATPase had no influence on the ability of NO to decrease [Ca2+]i. NO maximally decreased [Ca2+]i within 5 s, whereas significant inhibition of AII-induced Ca2+ and Mn2+ influx required more than 15 s. The inhibition of cation influx strictly depended on [Ca2+]o and functional SERCA, suggesting that during the delay before NO inhibits Ca2+ influx, the influx of Ca2+ and the uptake into intracellular stores are required. In the absence of [Ca2+]o, NO diminished the AII-induced [Ca2+]i transient by a SERCA-dependent mechanism and increased the amount of Ca2+ in the stores subsequently released by ionomycin. The present study indicates that the initial rapid decrease in [Ca2+]i caused by NO in vascular smooth muscle is accounted for by the uptake of Ca2+ by SERCA into intracellular stores. It is proposed that the refilling of the stores inhibits store-operated Ca2+ influx through non-L-type Ca2+ conducting ion channels and that this maintains the decrease in [Ca2+]i and NO-induced relaxation.

Effects of ivermectin and midecamycin on ryanodine receptors and the Ca2+-ATPase in sarcoplasmic reticulum of rabbit and rat skeletal muscle.

1. Ryanodine receptor (RyR) Ca2+ channels in the sarcoplasmic reticulum (SR) of skeletal muscle are regulated by the 12 kDa FK506- (or rapamycin-) binding protein (FKBP12). Rapamycin can also activate RyR channels with FKBP12 removed, suggesting that compounds with macrocyclic lactone ring structures can directly activate RyRs. Here we tested this hypothesis using two other macrocyclic lactone compounds, ivermectin and midecamycin. 2. Rabbit skeletal RyRs were examined in lipid bilayers. Ivermectin (cis, 0.66-40 microM) activated six of eight native, four of four control-incubated and eleven of eleven FKBP12-'stripped' RyR channels. Midecamycin (cis, 10-30 microM) activated three of four single native channels, six of eight control-incubated channels and six of seven FKBP12-stripped channels. Activity declined when either drug was washed out. 3. Neither ivermectin nor midecamycin removed FKBP12 from RyRs. Western blots of terminal cisternae (TC), incubated for 15 min at 37 C with 40 microM ivermectin or midecamycin, showed normal amounts of FKBP12. In contrast, no FKBP12 was detected after incubation with 40 microM rapamycin. 4. Ivermectin reduced Ca2+ uptake by the SR Ca2+-Mg2+-ATPase. Ca2+ uptake by TC fell to approximately 40% in the presence of ivermectin (10 microM), both with and without 10 microM Ruthenium Red. Ca2+ uptake by longitudinal SR also fell to approximately 40% with 10 microM ivermectin. Midecamycin (10 microM) reduced Ca2+ uptake by TC vesicles to approximately 76% without Ruthenium Red and to approximately 90 % with Ruthenium Red. 5. The rate of rise of extravesicular [Ca2+] increased approximately 2-fold when 10 microM ivermectin was added to TC vesicles that had been partially loaded with Ca2+ and then Ca2+ uptake blocked by 200 nM thapsigargin. Ivermectin also potentiated caffeine-induced Ca2+ release to approximately 140% of control. These increases in Ca2+ release were not seen with midecamycin. 6. Ivermectin, but not midecamycin, reversibly reduced Ca2+ loading in four of six skinned rat extensor digitorum longus (EDL) fibres to approximately 90%, and reversibly increased submaximal caffeine-induced contraction in five of eight fibres by approximately 110% of control. Neither ivermectin nor midecamycin altered twitch or tetanic tension in intact EDL muscle fibres within 20 min of drug addition. 7. The results confirm the hypothesis that compounds with a macrocyclic lactone ring structure can directly activate RyRs. Unexpectedly, ivermectin also reduced Ca2+ uptake into the SR. These effects of ivermectin on SR Ca2+ handling may explain some effects of the macrolide drugs on mammals.

Ca 2+ buffering action of sarcoplasmic reticulum on Bay k 8644-induced Ca 2+ influx in rat femoral arterial smooth muscle

We examined the Ca 2+ buffering action of sarcoplasmic reticulum during the stimulation of arterial smooth muscle with Bay k 8644 [methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine-5-carboxylate]. The effects of Bay k 8644 on tension and cellular Ca 2+ level were first determined in endothelium-denuded strips of rat femoral artery. The Ca 2+ buffering action was examined by using cyclopiazonic acid and thapsigargin to inhibit Ca 2+-ATPase of sarcoplasmic reticulum and ryanodine to deplete Ca 2+ stored in sarcoplasmic reticulum. The addition of Bay k 8644 (0.3–300 nM) to the resting strips almost failed to cause a contraction. When the strips were preincubated with 10 μM cyclopiazonic acid, Bay k 8644 induced a concentration-dependent contraction that is antagonized by nifedipine. The maximum contraction induced by Bay k 8644 in the presence of cyclopiazonic acid was comparable to the maximum contraction induced by 65.9 mM K +-depolarization and the ED 50 value for Bay k 8644 was around 5 nM. Similar results were obtained when the strips were preincubated with 30 nM thapsigargin or 10 μM ryanodine. Bay k 8644 also induced a strong contraction when the extracellular K + concentration was elevated. During the stimulation with 100 nM Bay k 8644, the Ca 2+ influx was increased. We conclude that in rat femoral arterial smooth muscle, (1) the Ca 2+ influx induced by Bay k 8644 is completely buffered by Ca 2+ uptake into the sarcoplasmic reticulum, and (2) this sarcoplasmic reticulum can buffer a large amount of Ca 2+ that induces a maximum contraction.

Volume Changes of the Myosin Lattice Resulting from Repetitive Stimulation of Single Muscle Fibers

Single muscle fibers at 1°C were subjected to brief tetani (20 Hz) at intervals of between 20 s and 300 s over a period of up to 2 h. A band lattice spacing increased during this period at a rate inversely dependent on the rest interval between tetani. Spacing increased rapidly during the first 10 tetani at a rate equivalent to the production of 0.04 mOsmol·liter −1 of osmolyte per contraction, then continued to expand at a much slower rate. For short rest intervals, where lattice expansion was largest, spacing increased to a limiting value between 46 and 47 nm (sarcomere length 2.2 μm), corresponding to accumulation of 30 mOsmol·liter −1 of osmolytes, where it remained constant until repetitive stimulation was terminated. At this limiting spacing, force was reduced by up to 30%. The effect of lattice swelling on the lattice compression that accompanies isometric force recovery from unloaded shortening was to increase the compression, similar to that observed in hypotonic media at a similar spacing. During recovery from repetitive stimulation, spacing recompressed to its original value with a half-time of 15–30 min. These findings suggest that mechanical activity produces an increase in osmotic pressure within the cell as a result of product accumulation from cross-bridge and sarcoplasmic reticulum ATPases and glycolysis.

Phospholamban: protein structure, mechanism of action, and role in cardiac function.

A comprehensive discussion is presented of advances in understanding the structure and function of phospholamban (PLB), the principal regulator of the Ca2+-ATPase of cardiac sarcoplasmic reticulum. Extensive historical studies are reviewed to provide perspective on recent developments. Phospholamban gene structure, expression, and regulation are presented in addition to in vitro and in vivo studies of PLB protein structure and activity. Applications of breakthrough experimental technologies in identifying PLB structure-function relationships and in defining its interaction with the Ca2+-ATPase are also highlighted. The current leading viewpoint of PLB's mechanism of action emerges from a critical examination of alternative hypotheses and the most recent experimental evidence. The potential physiological relevance of PLB function in human heart failure is also covered. The interest in PLB across diverse biochemical disciplines portends its continued intense scrutiny and its potential exploitation as a therapeutic target.

Contribution of Abnormal Sarcoplasmic Reticulum ATPase Activity to Systolic and Diastolic Dysfunction in Human Heart Failure

Two of the most significant characteristics of failing human myocardium are an increased diastolic [Ca2+]i and a prolonged diastolic relaxation. These abnormalities are more pronounced at higher frequencies of stimulation and may be caused by an altered Ca2+ resequestration into the sarcoplasmic reticulum (SR). The force-frequency relationship was determined in multicellular preparations obtained from non-failing (n=6) and failing human myocardium (n=11). The active force in non-failing tissue increased as a function of the frequency of stimulation. In failing myocardium, an increase in frequency of stimulation (>1 Hz) was accompanied by a decrease in active force. Changes in the frequency of stimulation and active force were also associated with changes in intracellular calcium concentrations. The diastolic force in failing myocardium was augmented following an increase in frequency of stimulation, whereas in non-failing tissue, no increase in diastolic force was observed. Associated with the increase in diastolic force was an increase in intracellular diastolic calcium concentrations. The SR Ca2+ ATPase activity was reduced in failing compared to non-failing myocardium. SR Ca2+ ATPase was positively correlated with diastolic force in non-failing myocardium. The relationship between Ca2+ ATPase activity at 1 micromol/l [Ca2+] and active force between 0.5 and 2.0 Hz was different between failing and non-failing myocardium. The diastolic force demonstrate an inverse relationship with the SR Ca2+ ATPase activity in failing myocardium. These data suggest that a reduction in SR Ca2+ ATPase activity contributes to the impairment in both systolic and diastolic function of failing human hearts.

High-level coexpression of the canine cardiac calcium pump and phospholamban in Sf21 insect cells.

Phospholamban is a pentameric transmembrane phosphoprotein that regulates the activity of the Ca(2+)-transporting ATPase (SERCA2a) in cardiac sarcoplasmic reticulum. To better understand the structure and function of phospholamban and its mode of regulation of the ATPase, phospholamban and SERCA2a were coexpressed at high levels in Sf21 insect cells using the baculovirus expression system. SERCA2a was expressed as a functionally active Ca2+ pump, accounting for > or = 20% of the total protein in Sf21 cell microsomes. Wild-type phospholamban, as well as phospholamban with different point mutations in the transmembrane region, inhibited both Ca2+ transport and ATP hydrolysis by the recombinant Ca2+ pump. The inhibition of SERCA2a activity was reversed by an anti-phospholamban monoclonal antibody. The phospholamban molecules studied decreased the apparent Ca2+ affinity of the Ca2+ pump, but had no effect on enzyme velocity measured at saturating Ca2+ concentration. Monomeric phospholamban produced by mutations in the leucine/isoleucine zipper domain decreased the apparent Ca2+ affinity the most, giving stronger inhibition of the Ca2+ pump than even wild-type phospholamban. Thus, the baculovirus cell expression system is ideally suited for examining functional interactions between phospholamban and SERCA2a. The results obtained suggest that the phospholamban monomer may be the active species inhibiting the Ca2+ pump in the cardiac sarcoplasmic reticulum membrane.

Oxidative damage to sarcoplasmic reticulum Ca 2+-ATPase at submicromolar iron concentrations: evidence for metal-catalyzed oxidation

The sarcoplasmic reticulum (SR) calcium ATPase carries out active Ca 2+ pumping at the expense of ATP hydrolysis. We have previously described the inhibition of SR ATPase by oxidative stress induced by the Fenton reaction (Fe 2+ + H 2O 2 → HO + HO − + Fe 3+). Inhibition was not related to peroxidation of the SR membrane nor to oxidation of ATPase thiols, and involved fragmentation of the ATPase polypeptide chain. The present study aims at further characterizing the mechanism of inhibition of the Ca 2+-ATPase by oxygen reactive species at Fe 2+ concentrations possibly found in pathological conditions of iron overload. ATP hydrolysis by SR vesicles was inhibited in a dose-dependent manner by micromolar concentrations of Fe 2+, H 2O 2, and ascorbate. Measuring the rate constants of inactivation ( k inact ) at different Fe 2+ concentrations in the presence of saturating concentrations of H 2O 2 and ascorbate (100 μM each) revealed a saturation profile with half-maximal inactivation rate at ca. 2 μM Fe 2+. Inhibition was not affected by addition of 200 μM Ca 2+ to the medium, indicating that it was not related to iron binding to the high affinity Ca 2+ binding sites in the ATPase. Furthermore, inhibition was not prevented by the water-soluble hydroxyl radical scavengers mannitol or dimethylsulfoxide, nor by butylated hydroxytoluene (a lipid peroxidation blocker) or dithiothreitol (DTT). However, when Cu 2+ was used instead of Fe 2+ in the Fenton reaction, ATPase inhibition could be prevented by DTT. We propose that functional impairment of the Ca 2+-pump may be related to oxidative protein fragmentation mediated by site-specific Fe 2+ binding at submicromolar or low micromolar concentrations, which may occur in pathological conditions of iron overload.

Phencyclidine block of Ca 2+ ATPase in rat heart sarcoplasmic reticulum

Phencyclidine hydrochloride (PCP) also known as Angel Dust is a very potent psychotomimetic drug of abuse. Besides its central nervous system (CNS) effects PCP produces a number of adverse effects in a variety of tissues including the cardiovascular system. Since PCP is known to alter the cellular calcium homeostasis the present studies were initiated to determine the changes in cardiac Ca 2+ ATPase activity in rats treated with PCP. For in vitro studies the cardiac sarcoplasmic reticulum (SR) fractions prepared from normal rats were incubated with 25, 50 and 100 μM PCP and the enzyme activities were estimated. Whereas, for in vivo studies the cardiac SR fractions prepared from rats treated with PCP (10 mg/kg body wt. single dose, intra-peritoneally (i.p.)) and sacrificed at different time intervals were used. PCP reduced the Ca 2+ ATPase activity significantly both in vitro and in vivo. A 50% inhibition of the enzyme activity was obtained with 100 μM PCP in vitro. A significant reduction of SR Ca 2+ ATPase was also evident as early as 1 h after treatment of rats with PCP. The reduction of Ca 2+ ATPase activity in SR was irreversible even at 12 h after treatment. The in vitro kinetic studies revealed that PCP was found to be a competitive inhibitor of Ca 2+ ATPase with respect to the substrate, ATP, and non-competitive with respect to Ca 2+ activation. These results indicate that PCP alters the myocardial Ca 2+ homeostasis by inhibiting the Ca 2+ ATPase in cardiac SR in rats. Inhibition of SR Ca 2+ ATPase may result in the impairment of contraction and relaxation coupling processes in the myocardium.

Influence of dietary n-3 fatty acids on the membrane properties of skeletal muscle in pigs

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Calcium pool size modulates the sensitivity of the ryanodine receptor channel and calcium‐dependent ATPase of heavy sarcoplasmic reticulum to extravesicular free calcium concentration

We have examined calcium cycling and associated ATP consumption by isolated heavy sarcoplasmic reticulum (HSR) vesicles incubated in conditions believed to exist in resting muscle. Our goals were to estimate the magnitude of calcium cycling under those conditions and identify the main mechanisms involved in its regulation. The integrity of the HSR vesicles was documented by the retention of [14C]-sucrose and electron microscopy. HSR actively exchanged Ca2+ with the medium through a partially open ryanodine-binding channel (RyR), as evidenced by the rapid attainment of a steady-state gradient between HSR and medium, which was promptly increased by the closure of the channel with ruthenium red (RR) or collapsed by its opening with caffeine. The ATP dependency was evidenced by the sustained ATP consumption after the steady state was attained and by the abrogation of the gradient following inhibition of the pump with thapsigargin (Tg) or the omission of ATP. When HSR vesicles were incubated in a comparatively large pool of calcium (≈1 μmol/mg HSR protein), ATP consumption was 1–1.5 μmol × [min × mg protein]−1 at 0.1 μM free Ca2+. Under such conditions, the main regulator of the sarcoplasmic Ca2+-dependent ATPase (SERCA) was extravesicular-free Ca2+ concentration, with a four- to fivefold increase between 0.1 and 2 μM Ca2+, whereas RyR channel activity and the replenishment of the HSR vesicles had only a modest effect on ATP consumption. When calcium pool size was reduced to 0.1 μmol/mg HSR protein, a steady state was established at a lower level of HSR calcium. In spite of a slightly lower free extravesicular Ca2+ at equilibrium (≈0.07 μM following an initial concentration of 0.1 μM), both ATP consumption and the open probability of the RyR channel were increased by a factor of three to five. Compared to the large calcium pool, the sensitivity of both RyR channel and SERCA to extravesicular free Ca2+ concentration as well as to caffeine and RR was markedly enhanced. Conclusions: (1) In conditions present in resting muscle, HSR calcium is in dynamic equilibrium with the medium through a partially open RyR channel, which requires continuous ATP hydrolysis. (2) The availability of calcium is a major determinant of the sensitivity of both RyR channel and SERCA to free extravesicular Ca2+ and possibly other stimuli. (3) These observations are consistent with the concept that calcium cycling in resting muscle may account for a significant fraction of muscle energy demands and further suggest that restricting calcium availability may enhance the energetic demands of this process. J. Cell. Physiol. 175:283–294, 1998. © 1998 Wiley-Liss, Inc.

Control of heat production by the Ca2+-ATPase of rabbit and trout sarcoplasmic reticulum.

The sarcoplasmic reticulum Ca2+-ATPase of rabbit skeletal muscle can convert the energy derived from a Ca2+ gradient into heat (L. de Meis, M. L. Bianconi, and V. A. Suzano. FEBS Lett. 406: 201-204, 1997). In this report, it is shown that this conversion varies depending on the temperature and on whether rabbit (endotherm) or trout (poikilotherm) sarcoplasmic reticulum vesicles are used. The gradient doubled the yield of heat produced during ATP hydrolysis and the calorimetric enthalpy of ATP hydrolysis (DeltaHcal) value found with both rabbit and trout varied between -10 and -12 kcal/mol in leaky vesicles (no gradient) and between -20 and -22 kcal/mol with intact vesicles (gradient). For the rabbit, the difference of DeltaHcal measured with and without gradient was detected in the range of 30-35 degrees C and disappeared when the temperature was decreased below 30 degrees C. For the trout, the difference was detected between 20 and 25 degrees C and disappeared below 20 degrees C. The effect of the gradient on the DeltaHcal for ATP hydrolysis was modified by DMSO, trifluoperazine, and heparin sodium.

Calcium pool size modulates the sensitivity of the ryanodine receptor channel and calcium-dependent ATPase of heavy sarcoplasmic reticulum to extravesicular free calcium concentration.

We have examined calcium cycling and associated ATP consumption by isolated heavy sarcoplasmic reticulum (HSR) vesicles incubated in conditions believed to exist in resting muscle. Our goals were to estimate the magnitude of calcium cycling under those conditions and identify the main mechanisms involved in its regulation. The integrity of the HSR vesicles was documented by the retention of [14C]-sucrose and electron microscopy. HSR actively exchanged Ca2+ with the medium through a partially open ryanodine-binding channel (RyR), as evidenced by the rapid attainment of a steady-state gradient between HSR and medium, which was promptly increased by the closure of the channel with ruthenium red (RR) or collapsed by its opening with caffeine. The ATP dependency was evidenced by the sustained ATP consumption after the steady state was attained and by the abrogation of the gradient following inhibition of the pump with thapsigargin (Tg) or the omission of ATP. When HSR vesicles were incubated in a comparatively large pool of calcium (approximately 1 micromol/mg HSR protein), ATP consumption was 1-1.5 micromol x [min x mg protein](-1) at 0.1 microM free Ca2+. Under such conditions, the main regulator of the sarcoplasmic Ca2+-dependent ATPase (SERCA) was extravesicular-free Ca2+ concentration, with a four- to fivefold increase between 0.1 and 2 microM Ca2+, whereas RyR channel activity and the replenishment of the HSR vesicles had only a modest effect on ATP consumption. When calcium pool size was reduced to 0.1 micromol/mg HSR protein, a steady state was established at a lower level of HSR calcium. In spite of a slightly lower free extravesicular Ca2+ at equilibrium (approximately 0.07 microM following an initial concentration of 0.1 microM), both ATP consumption and the open probability of the RyR channel were increased by a factor of three to five. Compared to the large calcium pool, the sensitivity of both RyR channel and SERCA to extravesicular free Ca2+ concentration as well as to caffeine and RR was markedly enhanced. 1) In conditions present in resting muscle, HSR calcium is in dynamic equilibrium with the medium through a partially open RyR channel, which requires continuous ATP hydrolysis. 2) The availability of calcium is a major determinant of the sensitivity of both RyR channel and SERCA to free extravesicular Ca2+ and possibly other stimuli. 3) These observations are consistent with the concept that calcium cycling in resting muscle may account for a significant fraction of muscle energy demands and further suggest that restricting calcium availability may enhance the energetic demands of this process.

Phospholamban domain I/cytochrome b5 transmembrane sequence chimeras do not inhibit SERCA2a

A series of chimeras between the transmembrane domains of phospholamban (PLN) and cytochrome b 5 were coexpressed with the Ca 2+-ATPase of cardiac sarcoplasmic reticulum (SERCA2a). The chimeric molecules were not inhibitory, in line with our view that inhibitory PLN/SERCA2a interactions occur in transmembrane sequences, while cytoplasmic interactions regulate the inhibitory interactions in a four-base circuit.

Ryanodine receptors regulate arterial diameter and wall [Ca2+] in cerebral arteries of rat via Ca2+-dependent K+ channels.

1. The effects of inhibitors of ryanodine-sensitive calcium release (RyR) channels in the sarcoplasmic reticulum (SR) and Ca2+-dependent potassium (KCa) channels on the membrane potential, intracellular [Ca2+], and diameters of small pressurized (60 mmHg) cerebral arteries (100-200 micron) were studied using digital fluorescence video imaging of arterial diameter and wall [Ca2+], combined with microelectrode measurements of arterial membrane potential. 2. Ryanodine (10 microM), an inhibitor of RyR channels, depolarized by 9 mV, increased intracellular [Ca2+] by 46 nM and constricted pressurized (to 60 mmHg) arteries with myogenic tone by 44 micron (approximately 22 %). Iberiotoxin (100 nM), a blocker of KCa channels, under the same conditions, depolarized the arteries by 10 mV, increased arterial wall calcium by 51 nM, and constricted by 37 micron (approximately 19 %). The effects of ryanodine and iberiotoxin were not additive and were blocked by inhibitors of voltage-dependent Ca2+ channels. 3. Caffeine (10 mM), an activator of RyR channels, transiently increased arterial wall [Ca2+] by 136 +/- 9 nM in control arteries and by 158 +/- 12 nM in the presence of iberiotoxin. Caffeine was relatively ineffective in the presence of ryanodine, increasing [calcium] by 18 +/- 5 nM. 4. In the presence of blockers of voltage-dependent Ca2+ channels (nimodipine, diltiazem), ryanodine and inhibitors of the SR calcium ATPase (thapsigargin, cyclopiazonic acid) were without effect on arterial wall [Ca2+] and diameter. 5. These results suggest that local Ca2+ release originating from RyR channels (Ca2+ sparks) in the SR of arterial smooth muscle regulates myogenic tone in cerebral arteries solely through activation of KCa channels, which regulate membrane potential through tonic hyperpolarization, thus limiting Ca2+ entry through L-type voltage-dependent Ca2+ channels. KCa channels therefore act as a negative feedback control element regulating arterial diameter through a reduction in global intracellular free [Ca2+].

CDNA cloning and predicted primary structure of scallop sarcoplasmic reticulum Ca 2+-ATPase

Sarcoplasmic reticulum (SR) Ca 2+-ATPase of the scallop cross-striated adductor muscle was purified with deoxycholate and digested with lysyl endopeptidase for sequencing of the digested fragments. Overlapping cDNA clones of the ATPase were isolated by screening the cDNA library with an RT-PCR product as a hybridization probe, which encodes the partial amino acid sequence of the ATPase. The predicted amino acid sequence of the ATPase contained all the partial sequences determined with the proteolytic fragments and consisted of the 993 residues with ∼70% overall sequence similarity to those of the SR ATPases from rabbit fast-twitch and slow-twitch muscles. An outline of the structure of the scallop ATPase molecule is predicted to mainly consist of ten transmembrane and five ‘stalk’ domains with two large cytoplasmic regions as observed with the rabbit ATPase molecules. The sequence relationship between scallop and other sarco/endoplasmic reticulum-type Ca 2+-ATPases is discussed.