Regulatory Mechanisms of the Calcium Transport System of Fragmented Rabbit Sarcoplasmic Reticulum : I. The effect of accumulated calcium on transport and adenosine triphosphate hydrolysis

The rate of ATP hydrolysis decreases very rapidly during the first 2 sec of calcium uptake. It changes with time in a manner similar to that described for calcium net uptake by other workers, suggesting that the two activities are coupled. The decline in both rates may be ascribed to an inhibitory effect of accumulated calcium on calcium influx and ATPase activity for the following reasons. During the steady state, Ca-Ca and Sr-Ca exchange and the rate of ATP hydrolysis are much slower than the initial rate of net calcium uptake and the associated ATP hydrolysis. If the accumulation of free calcium is prevented by calcium-oxalate precipitation the initial rate of net calcium uptake does not decay during prolonged periods of transport. Furthermore, passive preloading of vesicles with calcium inhibits the rate of hydrolysis in proportion to the extent of preloading. The inhibition of steady-state flux is alleviated by free ATP; i.e., not chelated with magnesium, but not by free ITP.     

The Effect of Calcium Ionophores on Fragmented Sarcoplasmic Reticulum

X-537 A and A 23187, two antibiotics which form liphophilic complexes with divalent cations, function as ionophores in vesicular fragments of sarcoplasmic reticulum (SR). Addition of either ionophore to SR preloaded with calcium in the presence of adenosine triphosphate (ATP), causes rapid release of calcium. Furthermore, net calcium accumulation by SR is prevented, when the ionophores are added to the reaction mixture before ATP. On the contrary, ATP-independent calcium binding to SR is not inhibited. This effect is specific for the two antibiotics and could not be reproduced, either by inactive derivatives, or by other known ionophores. Neither ionophore produces alterations of the electron microscopic appearance of SR membranes or inhibition of the calcium-dependent ATPase. In fact, the burst of ATP hydrolysis obtained on addition of calcium, is prolonged in the presence of the ionophores. Lanthanum inhibits ATP-independent calcium binding to SR, ATP-dependent calcium accumulation and calcium-dependent ATPase. However, addition of lanthanum to SR preloaded in the presence of ATP, does not cause calcium release. The reported experiments indicated that: (a) ATP-dependent calcium accumulation by SR results in primary formation of calcium ion gradients across the membrane. (b) Most of the accumulated calcium is not available for displacement by lanthanum on the outer surface of the membrane. (c) Calcium ionophores induce rapid equilibration of the gradients, by facilitating cation diffusion across the membrane.     

The Structural Basis for Phospholamban Inhibition of the Calcium Pump in Sarcoplasmic Reticulum

P-type ATPases are a large family of enzymes that actively transport ions across biological membranes by interconverting between high (E1) and low (E2) ion-affinity states; these transmembrane transporters carry out critical processes in nearly all forms of life. In striated muscle, the archetype P-type ATPase, SERCA (sarco(endo)plasmic reticulum Ca²⁺-ATPase), pumps contractile-dependent Ca²⁺ ions into the lumen of sarcoplasmic reticulum, which initiates myocyte relaxation and refills the sarcoplasmic reticulum in preparation for the next contraction. In cardiac muscle, SERCA is regulated by phospholamban (PLB), a small inhibitory phosphoprotein that decreases the Ca²⁺ affinity of SERCA and attenuates contractile strength. cAMP-dependent phosphorylation of PLB reverses Ca²⁺-ATPase inhibition with powerful contractile effects. Here we present the long sought crystal structure of the PLB-SERCA complex at 2.8-Å resolution. The structure was solved in the absence of Ca²⁺ in a novel detergent system employing alkyl mannosides. The structure shows PLB bound to a previously undescribed conformation of SERCA in which the Ca²⁺ binding sites are collapsed and devoid of divalent cations (E2-PLB). This new structure represents one of the key unsolved conformational states of SERCA and provides a structural explanation for how dephosphorylated PLB decreases Ca²⁺ affinity and depresses cardiac contractility.     

Methionine Aminopeptidase II: A Molecular Chaperone for Sarcoplasmic Reticulum Calcium ATPase

The monoclonal antibody to the β-subunit of H⁺/K⁺-ATPase (mAbHKβ) cross-reacts with a protein that acts as a molecular chaperone for the structural maturation of sarcoplasmic reticulum (SR) Ca²⁺-ATPase. We partially purified a mAbHKβ-reactive 65-kDa protein from Xenopus ovary. After in-gel digestion and peptide sequencing, the 65-kDa protein was identified as methionine aminopeptidase II (MetAP2). The effects of MetAP2 on SR Ca²⁺-ATPase expression were examined by injecting the cRNA for MetAP2 into Xenopus oocytes. Immunoprecipitation and pulse-chase experiments showed that MetAP2 was transiently associated with the nascent SR Ca²⁺-ATPase. Synthesis of functional SR Ca²⁺-ATPase was facilitated by MetAP2 and prevented by injecting an antibody specific for MetAP2. These results suggest that MetAP2 acts as a molecular chaperone for SR Ca²⁺-ATPase synthesis.     

Role of Sarcoplasmic Reticulum Calcium in Development of Secondary Calcium Rise and Early Afterdepolarizations in Long QT Syndrome Rabbit Model

Background

L-type calcium current reactivation plays an important role in development of early afterdepolarizations (EADs) and torsades de pointes (TdP). Secondary intracellular calcium (Ca_i) rise is associated with initiation of EADs.

Objective

To test whether inhibition of sarcoplasmic reticulum (SR) Ca²⁺ cycling suppresses secondary Ca_i rise and genesis of EADs.

Methods

Langendorff perfusion and dual voltage and Ca_i optical mapping were conducted in 10 rabbit hearts. Atrioventricular block (AVB) was created by radiofrequency ablation. After baseline studies, E4031, SR Ca²⁺ cycling inhibitors (ryanodine plus thapsigargin) and nifedipine were then administrated subsequently, and the protocols were repeated.

Results

At baseline, there was no spontaneous or pacing-induced TdP. After E4031 administration, action potential duration (APD) was significantly prolonged and the amplitude of secondary Ca_i rise was enhanced, and 7 (70%) rabbits developed spontaneous or pacing-induced TdP. In the presence of ryanodine plus thapsigargin, TdP inducibility was significantly reduced (2 hearts, 20%, p = 0.03). Although APD was significantly prolonged (from 298 ± 30 ms to 457 ± 75 ms at pacing cycle length of 1000 m, p = 0.007) by ryanodine plus thapsigargin, the secondary Ca_i rise was suppressed (from 8.8 ± 2.6% to 1.2 ± 0.9%, p = 0.02). Nifedipine inhibited TdP inducibility in all rabbit hearts.

Conclusion

In this AVB and long QT rabbit model, inhibition of SR Ca²⁺ cycyling reduces the inducibility of TdP. The mechanism might be suppression of secondary Ca_i rise and genesis of EADs.     

Proton Countertransport and Coupled Gating in the Sarcoplasmic Reticulum Calcium Pump

The calcium pump of the sarcoplasmic reticulum (SERCA) is an ATP-driven active transporter of Ca²⁺ ions that functions via an “alternating-access” cycle mechanism. In each cycle, SERCA transports two Ca²⁺ ions toward the lumen of the sarcoplasmic reticulum and two to three protons to the cytoplasm. How the latter conformational transition is coupled to cytoplasmic release of protons remains poorly understood. The present computational study shows how the mechanism of proton countertransport is coupled to the alternating access gating process in SERCA. Molecular dynamics simulation trajectories are generated starting from a series of configurations taken along the E2 to E1 transition pathway determined by the string method with swarms-of-trajectories. Simulations of different protonation configurations at the binding sites reveal how deprotonation events affect the opening of the cytoplasmic gate. The results show that there is a strong coupling between the chronological order of deprotonation, the entry of water molecules into the TM region, and the opening of the cytoplasmic gate. Deprotonation of E309 and E771 is sequential with E309 being the first to lose the proton. The deprotonation promotes the opening of the cytoplasmic gate but leads to a productive gating transition only if it occurs after the transmembrane domain has reached an intermediate conformation. Deprotonation of E309 and E771 is unproductive when it occurs too early because it causes the re-opening of the luminal gate.     

Mechanism of Inhibition of Calcium Uptake into Sarcoplasmic Reticulum by Notexin,a Neurotoxic and Myotoxic Polypeptide

Notexin belongs to a class of snake venom neurotoxins and myotoxins that have phospholipase A₂ activity. Previous studies have shown that these toxins affect target cells differently from phospholipases that are not neurotoxic or myotoxic. Notexin inhibited the Ca²⁺ uptake into fragmented sarcoplasmic reticulum from rabbit skeletal muscle, but it did not cause an efflux of previously accumulated Ca²⁺ or inhibit the Ca²⁺–ATPase activity. It is suggested that notexin specifically binds to and decreases the conductance for Ca²⁺ of the Ca²⁺ pump and/or the conductance of a channel for an ion that facilitates Ca²⁺ transport. The K⁺ ionophore valinomycin reversed the notexin-induced inhibition of Ca²⁺ uptake into sarcoplasmic reticulum, suggesting that the molecular target of notexin could be a K⁺ channel. Two types of reconstitution experiments make it unlikely that notexin acts by degrading a minor lipid that is resistant to hydrolysis by nontoxic phospholipases A₂. Notexininactivated sarcoplasmic reticulum vesicles were reactivated (with respect to Ca²⁺ uptake) by simple solubilization with detergent and subsequent reconstitution by detergent removal. Second, notexin was still active on sarcoplasmic reticulum vesicles after >94% of the lipids were replaced by soybean phosphoglycerides during the reconstitution procedure.     

The Phospholamban Pentamer Alters Function of the Sarcoplasmic Reticulum Calcium Pump SERCA

The interaction of phospholamban (PLN) with the sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA) pump is a major regulatory axis in cardiac muscle contractility. The prevailing model involves reversible inhibition of SERCA by monomeric PLN and storage of PLN as an inactive pentamer. However, this paradigm has been challenged by studies demonstrating that PLN remains associated with SERCA and that the PLN pentamer is required for the regulation of cardiac contractility. We have previously used two-dimensional (2D) crystallization and electron microscopy to study the interaction between SERCA and PLN. To further understand this interaction, we compared small helical crystals and large 2D crystals of SERCA in the absence and presence of PLN. In both crystal forms, SERCA molecules are organized into identical antiparallel dimer ribbons. The dimer ribbons pack together with distinct crystal contacts in the helical versus large 2D crystals, which allow PLN differential access to potential sites of interaction with SERCA. Nonetheless, we show that a PLN oligomer interacts with SERCA in a similar manner in both crystal forms. In the 2D crystals, a PLN pentamer interacts with transmembrane segments M3 of SERCA and participates in a crystal contact that bridges neighboring SERCA dimer ribbons. In the helical crystals, an oligomeric form of PLN also interacts with M3 of SERCA, though the PLN oligomer straddles a SERCA-SERCA crystal contact. We conclude that the pentameric form of PLN interacts with M3 of SERCA and that it plays a distinct structural and functional role in SERCA regulation. The interaction of the pentamer places the cytoplasmic domains of PLN at the membrane surface proximal to the calcium entry funnel of SERCA. This interaction may cause localized perturbation of the membrane bilayer as a mechanism for increasing the turnover rate of SERCA.     

Regulation of the Sarcoplasmic Reticulum Calcium Pump by Divergent Phospholamban Isoforms in Zebrafish

The sarcoplasmic reticulum calcium pump (SERCA) is regulated by the small integral membrane proteins phospholamban (PLN) and sarcolipin (SLN). These regulators have homologous transmembrane regions, yet they differ in their cytoplasmic and luminal domains. Although the sequences of PLN and SLN are practically invariant among mammals, they vary in fish. Zebrafish (zf) appear to harbor multiple PLN isoforms, one of which contains 18 sequence variations and a unique luminal extension. Characterization of this isoform (zfPLN) revealed that SERCA inhibition and reversal by phosphorylation were comparable with human PLN. To understand the sequence variations in zfPLN, chimeras were created by transferring the N terminus, linker, and C terminus of zfPLN onto human PLN. A chimera containing the N-terminal domain resulted in a mild loss of function, whereas a chimera containing the linker domain resulted in a gain of function. This latter effect was due to changes in basic residues in the linker region of PLN. Removing the unique luminal domain of zfPLN (⁵³SFHGM) resulted in loss of function, whereas adding this domain to human PLN had a minimal effect on SERCA inhibition. We conclude that the luminal extension contributes to SERCA inhibition but only in the context of zfPLN. Although this domain is distinct from the SLN luminal tail, zfPLN appears to use a hybrid PLN-SLN inhibitory mechanism. Importantly, the different zebrafish PLN isoforms raise the interesting possibility that sarcoplasmic reticulum calcium handling and cardiac contractility may be regulated by the differential expression of PLN functional variants.     

The Role of Sarcolipin and ATP in the Transport of Phosphate Ion into the Sarcoplasmic Reticulum

In a previous study, sarcolipin (SLN) was shown to form channels selective toward chloride ion when incorporated in a mercury-supported tethered bilayer lipid membrane (tBLM). Its incorporation had only a modest permeabilizing effect on phosphate ion. In this note the resistance of a tBLM membrane incorporating sarcolipin was investigated by electrochemical impedance spectroscopy in aqueous solutions of 0.05 M sodium phosphate of pH ranging from 5.3 to 8, in the presence of ATP, adenosine monophosphate, and phenylphosphonic acid. At pH 5.3, submicromolar additions of ATP increase the conductivity of the tBLM incorporating SLN up to a maximum limiting value. The dependence of the conductivity on the ATP concentration satisfies the Michaelis-Menten equation, with an association constant of 0.1 μM. Phenylphosphonium ion and adenosine monophosphate exert an inhibitory effect on membrane permeabilization to phosphate ions by ATP if they are added before ATP, but not if they are added after it. An explanation for this behavior is provided. In conclusion, SLN acts as an ATP-induced phosphate carrier exhibiting a behavior quite similar to that of the unidentified P_i transporter described previously. No ion-channel activity is exhibited by the T18A mutant of SLN.     

Transport of Ca2+ from Sarcoplasmic Reticulum to Mitochondria in Rat Ventricular Myocytes

Studies with electron microscopy have shown that sarcoplasmic reticulum (SR) andmitochondria locate close to each other in cardiac muscle cells. We investigated the hypothesis thatthis proximity results in a transient exposure of mitochondrial Ca²⁺ uniporter (CaUP) to highconcentrations of Ca²⁺ following Ca²⁺ release from the SR and thus an influx of Ca²⁺into mitochondria. Single ventricular myocytes of rat were skinned by exposing them to aphysiological solution containing saponin (0.2 mg/ml). Cytosolic Ca²⁺ concentration ([Ca²⁺]_c)and mitochondrial Ca²⁺ concentration ([Ca²⁺]_m) were measured with fura-2 and rhod2,respectively. Application of caffeine (10 mM) induced a concomitant increase in[Ca²⁺]_c and [Ca²⁺]_m.Ruthenium red, at concentrations that block CaUP but not SR release, diminished thecaffeine-induced increase in [Ca²⁺]_m but not[Ca²⁺]_c. In the presence of 1 mM BAPTA, a Ca²⁺ chelator,the caffeine-induced increase in [Ca²⁺]_m was reduced substantially less than [Ca²⁺]_c. Moreover,inhibition of SR Ca²⁺ pump with two different concentrations of thapsigargin caused anincrease in [Ca²⁺]_m, which was related to the rate of [Ca²⁺]_c increase. Finally, electronmicroscopy showed that sites of junctions between SR and T tubules from which Ca²⁺ is released,or Ca²⁺ release units, CRUs, are preferentially located in close proximity to mitochondria.The distance between individual SR Ca²⁺ release channels (feet or ryanodine receptors) isvery short, ranging between approximately 37 and 270 nm. These results are consistent withthe idea that there is a preferential coupling of Ca²⁺ transport from SR to mitochondria incardiac muscle cells, because of their structural proximity.     

Assessment of Sarcoplasmic Reticulum Ca Depletion During Spontaneous Ca Waves in Isolated Permeabilized Rabbit Ventricular Cardiomyocytes

In this study, Ca²⁺ release due to spontaneous Ca²⁺ waves was measured both from inside the sarcoplasmic reticulum (SR) and from the cytosol of rabbit cardiomyocytes. These measurements utilized Fluo5N-AM for intra-SR Ca²⁺ from intact cells and Fluo5F in the cytosol of permeabilized cells. Restricted subcellular volumes were resolved with the use of laser-scanning confocal microscopy. Local Ca²⁺ signals during spontaneous Ca²⁺ release were compared with those induced by rapid caffeine application. The free cytoplasmic [Ca²⁺] increase during a Ca²⁺ wave was 98.1% ± 0.3% of that observed during caffeine application. Conversion to total Ca²⁺ release suggested that Ca²⁺ release from a Ca²⁺ wave was not significantly different from that released during caffeine application (104% ± 6%). In contrast, the maximum decrease in intra-SR Fluo-5N fluorescence during a Ca²⁺ wave was 82.5% ± 2.6% of that observed during caffeine application. Assuming a maximum free [Ca²⁺] of 1.1 mM, this translates to a 96.2% ± 0.8% change in intra-SR free [Ca²⁺] and a 91.7% ± 1.6% depletion of the total Ca²⁺. This equates to a minimum intra-SR free Ca²⁺ of 46 ± 7 μM during a Ca²⁺ wave. Reduction of RyR2 Ca²⁺ sensitivity by tetracaine (50 μM) reduced the spontaneous Ca²⁺ release frequency while increasing the Ca²⁺ wave amplitude. This did not significantly change the total depletion of the SR (94.5% ± 1.1%). The calculated minimum [Ca²⁺] during these Ca²⁺ waves (87 ± 19 μM) was significantly higher than control (p < 0.05). A computational model incorporating this level of Ca²⁺ depletion during a Ca²⁺ wave mimicked the transient and sustained effects of tetracaine on spontaneous Ca²⁺ release. In conclusion, spontaneous Ca²⁺ release results in substantial but not complete local Ca²⁺ depletion of the SR. Furthermore, measurements suggest that Ca²⁺ release terminates when luminal [Ca²⁺] reaches ∼50 μM.     

Interaction of a Sarcolipin Pentamer and Monomer with the Sarcoplasmic Reticulum Calcium Pump,SERCA

The sequential rise and fall of cytosolic calcium underlies the contraction-relaxation cycle of muscle cells. Whereas contraction is initiated by the release of calcium from the sarcoplasmic reticulum, muscle relaxation involves the active transport of calcium back into the sarcoplasmic reticulum. This reuptake of calcium is catalyzed by the sarcoendoplasmic reticulum Ca²⁺-ATPase (SERCA), which plays a lead role in muscle contractility. The activity of SERCA is regulated by small membrane protein subunits, the most well-known being phospholamban (PLN) and sarcolipin (SLN). SLN physically interacts with SERCA and differentially regulates contractility in skeletal and atrial muscle. SLN has also been implicated in skeletal muscle thermogenesis. Despite these important roles, the structural mechanisms by which SLN modulates SERCA-dependent contractility and thermogenesis remain unclear. Here, we functionally characterized wild-type SLN and a pair of mutants, Asn⁴-Ala and Thr⁵-Ala, which yielded gain-of-function behavior comparable to what has been found for PLN. Next, we analyzed two-dimensional crystals of SERCA in the presence of wild-type SLN by electron cryomicroscopy. The fundamental units of the crystals are antiparallel dimer ribbons of SERCA, known for decades as an assembly of calcium-free SERCA molecules induced by the addition of decavanadate. A projection map of the SERCA-SLN complex was determined to a resolution of 8.5 Å, which allowed the direct visualization of an SLN pentamer. The SLN pentamer was found to interact with transmembrane segment M3 of SERCA, although the interaction appeared to be indirect and mediated by an additional density consistent with an SLN monomer. This SERCA-SLN complex correlated with the ability of SLN to decrease the maximal activity of SERCA, which is distinct from the ability of PLN to increase the maximal activity of SLN. Protein-protein docking and molecular dynamics simulations provided models for the SLN pentamer and the novel interaction between SERCA and an SLN monomer.     

Modulation of Cardiac Sarcoplasmic Reticulum Calcium Release by Aenosine: A protein Kinase C- Dependent Pathway

We have already reported that A₃ adenosine receptor stimulation reduces [³H]-ryanodine binding and sarcoplasmic reticulum Ca²⁺ release in rat heart. In the present work we have investigated the transduction pathway responsible for this effect. Isolated rat hearts were perfused for 20 min in the presence of the following substances: 100 nM N⁶-(iodobenzyl)-adenosine-5′-N-methyluronamide (IB-MECA), an A₃ adenosine agonist; 10 μM U-73122, a phospholipase C inhibitor; 2 μM chelerythrine, a protein kinase C inhibitor. At the end of perfusion, the hearts were homogenized and [³H]-ryanodine binding was assayed. IB-MECA produced a significant decrease in ryanodine binding, which was abolished in the presence of chelerythrine but not in the presence of U-73122. RT-PCR experiments showed that ryanodine receptor gene expression was not affected by IB-MECA. In Western blot experiments, ryanodine receptor phosphorylation on serine 2809 was not modified after perfusion with IB-MECA. We conclude that modulation of SR Ca²⁺ release channel by IB-MECA is dependent on protein kinase C activation. However, in this model protein kinase C activation is not due to phospholipase C activation. In addition, changes in ryanodine receptor gene expression or direct phosphorylation of the ryanodine receptor on serine 2809 residue do not appear to occur.     

Effects of ATP-sensitive Potassium Channel Regulators on Chloride Channels in the Sarcoplasmic Reticulum Vesicles from Rabbit Skeletal Muscle

The lipid bilayer technique was used to examine the effects of the ATP-sensitive K⁺ channel inhibitor (glibenclamide) and openers (diazoxide, minoxidil and cromakalim) and Cl⁻ channel activators (GABA and diazepam) on two types of chloride channels in the sarcoplasmic reticulum (SR) from rabbit skeletal muscle. Neither diazepam at 100 μm nor GABA at 150 μm had any significant effect on the conductance and kinetics of the 75 pS small chloride (SCl) channel. Unlike the 150 pS channel, the SCl channel is sensitive to cytoplasmic glibenclamide with K _i ∼ 30 μm. Glibenclamide induced reversible decline in the values of current (maximal current amplitude, I _max and average mean current, I′) and kinetic parameters (frequency of opening F _o , probability of the channel being open P _o and mean open time, T _o , of the SCl channel. Glibenclamide increased mean closed time, T _c , and was a more potent blocker from the cytoplasmic side (cis) than from the luminal side (trans) of the channel. Diazoxide increased I′, P _o , and T _o in the absence of ATP and Mg²⁺ but it had no effect on I _max and also failed to activate or remove the glibenclamide- and ATP-induced inhibition of the SCl channel. Minoxidil induced a transient increase in I′ followed by an inhibition of I _max, whereas cromakalim reduced P _o and I′ by increasing channel transitions to the closed state and reducing T _o without affecting I _max. The presence of diazoxide, minoxidil or cromakalim on the cytoplasmic side of the channel did not prevent [ATP] _cis or [glibenclamide] _cis from blocking the channel. The data suggest that the action(s) of these drugs are not due to their effects on the phosphorylation of the channel protein. The glibenclamide- and cromakalim-induced effects on the SCl channel are mediated via a ``flicker' type block mechanism. Modulation of the SCl channel by [diazoxide] _cis and [glibenclamide] _cis highlights the therapeutic potential of these drugs in regulating the Ca²⁺-counter current through this channel. Received: 2 September 1997/Revised: 20 March 1998     

Kinetic Differences in the Phospholamban-Regulated Calcium Pump When Studied in Crude and Purified Cardiac Sarcoplasmic Reticulum Vesicles

Phospholamban (PLN) phosphorylation contributes largely to the inotropic and lusitropic effects of beta-adrenergic agonists on the heart. The mechanical effects of PLN phosphorylation on the heart are generally attributed solely to an increase in the apparent affinity of the Ca pump in the sarcoplasmic reticulum (SR) membranes for Ca²⁺ with little or no effect on V _max(Ca). In the present report, we compare the kinetic properties of the cardiac SR Ca pump in commonly studied crude microsomes with those of our recently developed preparation of light SR vesicles. We demonstrate that in crude microsomes, the increase in the apparent affinity of the pump for Ca²⁺ is larger, while the increase in V _max(Ca) is smaller, than in purified vesicles. The greater phosphorylation-induced increase in apparent Ca²⁺ affinity in crude microsomes may be further enhanced by an ATP-sensitive inhibitory effect of ruthenium red on the activity of the pump at subsaturating, but not saturating, Ca²⁺ concentrations as a result of a greater inhibition in unphosphorylated microsomes. Upon increasing the ATP concentration from 1 to 5 mm, an inhibition by 10 μm ruthenium red is eliminated in phosphorylated microsomes and reduced in control microsomes. Addition of the phosphoprotein phosphatase inhibitor okadaic acid produces a considerable increase in the phosphorylation-induced effects in both crude and purified microsomes. We conclude that the use of purified cardiac SR vesicles is critical for the demonstration of a major increase in V _max(Ca) in addition to an increase in the pump's apparent affinity for Ca²⁺ in response to phosphorylation of PLN by protein kinase A. Received: 20 May 1998/Revised: 13 November 1998     

The Role of Dyadic Organization in Regulation of Sarcoplasmic Reticulum Ca Handling during Rest in Rabbit Ventricular Myocytes

The dyadic organization of ventricular myocytes ensures synchronized activation of sarcoplasmic reticulum (SR) Ca²⁺ release during systole. However, it remains obscure how the dyadic organization affects SR Ca²⁺ handling during diastole. By measuring intraluminal SR Ca²⁺ ([Ca²⁺]_SR) decline during rest in rabbit ventricular myocytes, we found that ∼76% of leaked SR Ca²⁺ is extruded from the cytosol and only ∼24% is pumped back into the SR. Thus, the majority of Ca²⁺ that leaks from the SR is removed from the cytosol before it can be sequestered back into the SR by the SR Ca²⁺-ATPase (SERCA). Detubulation decreased [Ca²⁺]_SR decline during rest, thus making the leaked SR Ca²⁺ more accessible for SERCA. These results suggest that Ca²⁺ extrusion systems are localized in T-tubules. Inhibition of Na⁺-Ca²⁺ exchanger (NCX) slowed [Ca²⁺]_SR decline during rest by threefold, however did not prevent it. Depolarization of mitochondrial membrane potential during NCX inhibition completely prevented the rest-dependent [Ca²⁺]_SR decline. Despite a significant SR Ca²⁺ leak, Ca²⁺ sparks were very rare events in control conditions. NCX inhibition or detubulation increased Ca²⁺ spark activity independent of SR Ca²⁺ load. Overall, these results indicate that during rest NCX effectively competes with SERCA for cytosolic Ca²⁺ that leaks from the SR. This can be explained if the majority of SR Ca²⁺ leak occurs through ryanodine receptors in the junctional SR that are located closely to NCX in the dyadic cleft. Such control of the dyadic [Ca²⁺] by NCX play a critical role in suppressing Ca²⁺ sparks during rest.     

Effects of Partial Sarcoplasmic Reticulum Calcium Depletion on Calcium Release in Frog Cut Muscle Fibers Equilibrated with 20 mM EGTA

Resting sarcoplasmic reticulum (SR) Ca content ([Ca_SR]_R) was varied in cut fibers equilibrated with an internal solution that contained 20 mM EGTA and 0–1.76 mM Ca. SR Ca release and [Ca_SR]_R were measured with the EGTA–phenol red method (Pape et al. 1995. J. Gen. Physiol. 106:259–336). After an action potential, the fractional amount of Ca released from the SR increased from 0.17 to 0.50 when [Ca_SR]_R was reduced from 1,200 to 140 μM. This increase was associated with a prolongation of release (final time constant, from 1–2 to 10–15 ms) and of the action potential (by 1–2 ms). Similar changes in release were observed with brief stimulations to −20 mV in voltage-clamped fibers, in which charge movement (Q _cm) could be measured. The peak values of Q _cm and the fractional rate of SR Ca release, as well as their ON time courses, were little affected by reducing [Ca_SR]_R from 1,200 to 140 μM. After repolarization, however, the OFF time courses of Q _cm and the rate of SR Ca release were slowed by factors of 1.5–1.7 and 6.5, respectively. These and other results suggest that, after action potential stimulation of fibers in normal physiological condition, the increase in myoplasmic free [Ca] that accompanies SR Ca release exerts three negative feedback effects that tend to reduce additional release: (a) the action potential is shortened by current through Ca-activated potassium channels in the surface and/or tubular membranes; (b) the OFF kinetics of Q _cm is accelerated; and (c) Ca inactivation of Ca release is increased. Some of these effects of Ca on an SR Ca channel or its voltage sensor appear to be regulated by the value of [Ca] within 22 nm of the mouth of the channel.     

Transport of bacteria in porous media: II. A model for convective Transport and growth

A model is presented for the coupled processes of bacterial growth and convective transport of bacteria has been modeled using a fractional flow approach. The various mechanisms of bacteria retention can be incorporated into the model through selection of an appropriate shape of the fractional flow curve. Permeability reduction due to pore plugging by bacteria was simulated using the effective medium theory. In porous media, the rates of transport and growth of bacteria, the generation of metabolic products, and the consumption of nutrients are strongly coupled processes. Consequently, the set of governing conservation equations form a set of coupled, nonlinear partial differential equations that were solved numerically. Reasonably good agreement between the model and experimental data has been obtained indicating that the physical processes incorporated in the model are adequate. The model has been used to predict the in situ transport and growth of bacteria, nutrient consumption, and metabolite production. It can be particularly useful in simulating laboratory experiments and in scaling microbial-enhanced oil recovery or bioremediation processes to the field. (c) 1994 John Wiley & Sons, Inc.