Thermogenic activity of Ca-ATPase from skeletal muscle heavy sarcoplasmic reticulum: The role of ryanodine Ca channel

The sarcoplasmic reticulum Ca²⁺ ATPase 1 (SERCA 1) is able to handle the energy derived from ATP hydrolysis in such a way as to determine the parcel of energy that is used for Ca²⁺ transport and the fraction that is converted into heat. In this work we measured the heat production by SERCA 1 in the two sarcoplasmic reticulum (SR) fractions: the light fraction (LSR), which is enriched in SERCA and the heavy fraction (HSR), which contains both the SERCA and the ryanodine Ca²⁺ channel. We verified that although HSR cleaved ATP at faster rate than LSR, the amount of heat released during ATP hydrolysis by HSR was smaller than that measured by LSR. Consequently, the amount of heat released per mol of ATP cleaved (ΔH^cal) by HSR was lower compared to LSR. In HSR, the addition of 5 mM Mg²⁺ or ruthenium red, conditions that close the ryanodine Ca²⁺ channel, promoted a decrease in the ATPase activity, but the amount of heat released during ATP hydrolysis remained practically the same. In this condition, the ΔH^cal values of ATP hydrolysis increased significantly. Neither Mg²⁺ nor ruthenium red had effect on LSR. Thus, we conclude that heat production by SERCA 1 depends on the region of SR in which the enzyme is inserted and that in HSR, the ΔH^cal of ATP hydrolysis by SERCA 1 depends on whether the ryanodine Ca²⁺ channel is opened or closed.     

Ca-transport in sea urchin unfertilized eggs: Regulation by endogenous sulfated polysaccharides and K

Previous data from our laboratory showed that the reticulum of the sea cucumber smooth muscle body wall retains both a sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) and a sulfated polysaccharide. In this invertebrate, the transport of Ca²⁺ by the SERCA is naturally inhibited by these endogenous sulfated polysaccharides. The inhibition is reverted by K⁺ leading to an enhancement of the Ca²⁺ transport rate. We now show that vesicles derived from the endoplasmic reticulum of unfertilized eggs from the sea urchin Arbacia lixula retain a SERCA that is able to transport Ca²⁺ at the expense of ATP hydrolysis. As described for the sea cucumber SERCA isoform, the enzyme from the sea urchin is activated by K⁺ but not by Li⁺ and is inhibited by thapsigargin, a specific inhibitor of SERCA. A new sulfated polysaccharide was identified in the sea urchin eggs reticulum composed mainly by galactose, glucose, hexosamine and manose. After extraction and purification, this sulfated polysaccharide was able to inhibit the mammal SERCA isoform found in rabbit skeletal muscle and the inhibition is reversed by K⁺. These data suggest that the regulation of the SERCA pump by K⁺ and sulfated polysaccharides is not restricted to few marine invertebrates but is widespread.     

Thyroid hormones differentially regulate the distribution of rabbit skeletal muscle Ca2 + -ATPase (SERCA) isoforms in light and heavy sarcoplasmic reticulum

The sarcoplasmic reticulum (SR) is composed of two fractions, the heavy fraction that contains proteins involved in Ca^2?+? release, and the light fraction enriched in Ca^2?+?-ATPase (SERCA), an enzyme responsible for Ca^2?+? transport from the cytosol to the lumen of SR. It is known that in red muscle thyroid hormones regulate the expression of SERCA 1 and SERCA 2 isoforms. Here we show the effects of thyroid hormone on SERCA expression and distribution in light and heavy SR fractions from rabbit white and red muscles. In hyperthyroid red muscle there is an increase of SERCA 1 and a decrease of SERCA 2 expression. This is far more pronounced in the heavy than in the light SR fraction. As a result, the rates of Ca^2?+?- ATPase activity and Ca^2?+?-uptake by the heavy vesicles are increased. In hypothyroidism we observed a decrease in SERCA 1 and no changes in the amount of SERCA 2 expressed. This promoted a decrease of both Ca^2?+?-uptake and Ca^2?+?-ATPase activity. While the major differences in hyperthyroidism were found in the heavy SR fraction, the effects of hypothyroidism were restricted to light SR fraction. In white muscle we did not observe any significant changes in either hypo- or hyperthyroidism in both SR fractions. Thus, the regulation of SERCA isoforms by thyroid hormones is not only muscle specific but also varies depending on the subcellular compartment analyzed. These changes might correspond to the molecular basis of the altered contraction and relaxation rates detected in thyroid dysfunction.     

Reconstitution and regulation of cation-selective channels from cardiac sarcoplasmic reticulum

In order to study the conductances of the Sarcoplasmic Reticulum (SR) membrane, microsomal fractions from cardiac SR were isolated by differential and sucrose gradient centrifugations and fused into planar lipid bilayers (PLB) made of phospholipids. Using either KCl or K-gluconate solutions, a large conducting K+ selective channel was characterized by its ohmic conductance (152 pS in 150 mM K⁺), and the presence of short and long lasting subconducting states. Its open probability P_o increased with depolarizing voltages, thus supporting the idea that this channel might allow counter-charge movements of monovalent cations during rapid SR Ca²⁺ release. An heterogeneity in the kinetic behavior of this channel would suggest that the cardiac SR K⁺ channels might be regulated by cytoplasmic, luminal, or intra SR membrane biochemical mechanisms. Since the behavior was not modified by variations of [Ca²⁺] nor by the addition of soluble metabolites such as ATP, GTP, cAMP, cGMP, nor by phosphorylation conditions on both sides of the PLB, a specific interaction with a SR membrane component is postulated. Another cation selective channel was studied in asymmetric Ca²⁺, Ba²⁺ or Mg²⁺-HEPES buffers. This channel displayed large conductance values for the above divalent cations 90, 100, and 40 pS, respectively. This channel was activated by µM Ca²⁺ while its Ca²⁺ sensitivity was potentiated by millimolar ATP. However Mg²⁺ and calmodulin modulated its gating behavior. Ca²⁺ releasing drugs such as caffeine and ryanodine increased its P_o. All these features are characteristics of the SR Ca²⁺ release channel. The ryanodine receptor which has been purified and reconstituted into PLB, may form a cation selective pathway. This channel displays all the regulatory sites of the native cardiac SR Ca²⁺ release channel. However, when NA was used as charge carrier, multiple subconducting states were observed. In conclusion, the reconstitution experiments have yield a great deal of informations about the biochemical and biophysical events that may regulated the ionic flux across the SR membrane.     

Effects of monovalent cations on Ca uptake by skeletal and cardiac muscle sarcoplasmic reticulum

Ca²⁺ transport by the sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase (SERCA) is sensitive to monovalent cations. Possible K⁺ binding sites have been identified in both the cytoplasmic P-domain and the transmembrane transport-domain of the protein. We measured Ca²⁺ transport into SR vesicles and SERCA ATPase activity in the presence of different monovalent cations. We found that the effects of monovalent cations on Ca²⁺ transport correlated in most cases with their direct effects on SERCA. Choline⁺, however, inhibited uptake to a greater extent than could be accounted for by its direct effect on SERCA suggesting a possible effect of choline on compensatory charge movement during Ca²⁺ transport. Of the monovalent cations tested, only Cs⁺ significantly affected the Hill coefficient of Ca²⁺ transport (n_H). An increase in n_H from ∼2 in K⁺ to ∼3 in Cs⁺ was seen in all of the forms of SERCA examined. The effects of Cs⁺ on the maximum velocity of Ca²⁺ uptake were also different for different forms of SERCA but these differences could not be attributed to differences in the putative K⁺ binding sites of the different forms of the protein.     

Vanadate inhibition of sarcoplasmic reticulum Ca2+-ATPase and other ATPases.

Vanadate is a potent inhibitor of the Ca²⁺-ATPase activity of sarcoplasmic reticulum in the presence of A-23187. The purified enzyme is sensitive to vanadate even in the absence of the ionophore. Ca²⁺ and norepinephrine protect the enzyme against inhibition of vanadate. The nonspecificity of vanadate is emphasized by the finding of inhibition of several other ATPases including the Ca²⁺Mg²⁺-ATPases of the ascites and human red cell plasma membranes, Mg²⁺-ATPase of the ascites plasma membrane, and the K⁺-ATPases of $\text{E.}\text{coli}$ and hog gastric mucosal cell membranes. The ascites plasma membrane Ca²⁺-ATPase (an ecto ATPase) and mitochondrial ATPase are not inhibited by vanadate.     

Channel Gating Dependence on Pore Lining Helix Glycine Residues in Skeletal Muscle Ryanodine Receptor

Type 1 ryanodine receptors (RyR1s) release Ca²⁺ from the sarcoplasmic reticulum to initiate skeletal muscle contraction. The role of RyR1-G4934 and -G4941 in the pore-lining helix in channel gating and ion permeation was probed by replacing them with amino acid residues of increasing side chain volume. RyR1-G4934A, -G4941A, and -G4941V mutant channels exhibited a caffeine-induced Ca²⁺ release response in HEK293 cells and bound the RyR-specific ligand [³H]ryanodine. In single channel recordings, significant differences in the number of channel events and mean open and close times were observed between WT and RyR1-G4934A and -G4941A. RyR1-G4934A had reduced K⁺ conductance and ion selectivity compared with WT. Mutations further increasing the side chain volume at these positions (G4934V and G4941I) resulted in reduced caffeine-induced Ca²⁺ release in HEK293 cells, low [³H]ryanodine binding levels, and channels that were not regulated by Ca²⁺ and did not conduct Ca²⁺ in single channel measurements. Computational predictions of the thermodynamic impact of mutations on protein stability indicated that although the G4934A mutation was tolerated, the G4934V mutation decreased protein stability by introducing clashes with neighboring amino acid residues. In similar fashion, the G4941A mutation did not introduce clashes, whereas the G4941I mutation resulted in intersubunit clashes among the mutated isoleucines. Co-expression of RyR1-WT with RyR1-G4934V or -G4941I partially restored the WT phenotype, which suggested lessening of amino acid clashes in heterotetrameric channel complexes. The results indicate that both glycines are important for RyR1 channel function by providing flexibility and minimizing amino acid clashes.     

Dissection of the functional domains of the sarcoplasmic reticulum Ca2+-ATPase by site-directed mutagenesis

The results of site-directed mutagenesis studies of the sarcoplasmic reticulum Ca²⁺-ATPase are reviewed. More than 250 different point mutants have been expressed in cell culture and analysed by a panel of functional assays. Thereby, 40–50 important amino acid residues have been pinpointed, and the mutants have been assigned to functional classes: the Ca²⁺-affinity mutants, the phosphorylation-negative mutants, the ATP-affinity mutants, the E₁P mutants, the E₂P mutants, and the uncoupled mutants. Moreover, regions important to the specific inhibition by thapsigargin have been identified by analysis of Ca²⁺-ATPase/Na⁺, K⁺-ATPase chimeric constructs.     

The action of carboxyl modifying reagents on the ryanodine receptor/Ca2+ release channel of skeletal muscle sarcoplasmic reticulum

Summary

In this work we show that ryanodine binding to junctional sarcoplasmic reticulum (SR) membranes or purified ryanodine receptor (RyR) is inhibited in a time — and concentration-dependent fashion by prior treatment with the carboxyl reagent dicyclohexylcarbodiimide (DCCD). Exposure of the membrane-bound RyR to the water soluble carboxyl reagents 1-ethyl-3 (3-(dimethylamino) propyl carbodiimide (EDC) or N-ethyl-pheny-lisoxazolium-3 -sulfonate (WRK) only slightly affects their ryanodine binding capacity. The amphipathic reagent N-ethoxy cabonyl-2-ethoxy-1, 2-dihydroquinaline (EEDQ) inhibited ryanodine binding at relatively high concentrations. DCCD-modifica-tion of the SR decreased the binding affinities of the RyR for ryanodine and Ca²⁺ by about 3- and 18-fold, respectively.

The single channel activity of SR membranes modified with DCCD and then incorporated into planar lipid bilayers is very low (5–8%) in comparison to control membranes. Application of DCCD to either the myoplasmic (c/s) or luminal (trans) side of the reconstituted unmodified channels resulted in complete inhibition of their single channel activities. Similar results were obtained with the water soluble reagent WRK applied to the myoplasmic, but not to the luminal side. The DCCD-modified non-active channel is re-activated by addition of ryanodine in the presence of 250_üM Ca²⁺ and is stabilized in a sub-conductance state. With caffeine, ryanodine re-activated the channel in the presence of 100_üM of Ca^2+. The results suggest that a carboxyl residue(s) in the RyR is involved either in the binding of Ca²⁺, or in conformational changes that are produced by Ca²⁺ binding, and are required for the binding of ryanodine and the opening of the Ca²⁺ release channel.     

Mg2+ and ATP effects on K+ activation of the Ca2+-transport ATpase of cardiac sarcoplasmic reticulum

ATP and the divalent cations Mg²⁺ and Ca²⁺ regulated K⁺ stimulation of the Ca²⁺-transport ATPase of cardiac sarcoplasmic reticulum vesicles. Millimolar concentrations of total ATP increased the K⁺-stimulated ATPase activity of the Ca²⁺ pump by two mechanisms. First, ATP chelated free Mg²⁺ and, at low ionized Mg²⁺ concentrations, K⁺ was shown to be a potent activator of ATP hydrolysis. In the absence of K⁺ ionized Mg²⁺ activated the enzyme half-maximally at approximately 1 mM, whereas in the presence of K⁺ the concentration of ionized Mg²⁺ required for half-maximal activation was reduced at least 20-fold. Second MgATP apparently interacted directly with the enzyme at a low affinity nucleotide site to facilitate K⁺-stimulation. With a saturating concentration of ionized Mg²⁺, stimulation by K⁺ was 2-fold, but only when the MgATP concentration was greater than 2 mM. Hill plots showed that K⁺ increased the concentration of MgATP required for half-maximal enzymic activation approx. 3-fold.Activation of K⁺-stimulated ATPase activity by Ca²⁺ was maximal at anionized Ca²⁺ concentration of approx. 1 μM. At very high concentrations of either Ca²⁺ or Mg²⁺, basal Ca²⁺-dependent ATPase activity persisted, but the enzymic response to K⁺ was completely inhibited. The results provide further evidence that the Ca²⁺-transport ATPase of cardiac sarcoplasmic reticulum has distinct sites for monovalent cations, which in turn interact allosterically with other regulatory sites on the enzyme.     

The potassium channel opener NS1619 modulates calcium homeostasis in muscle cells by inhibiting SERCA

NS1619 (1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazole-2-one) is widely used as a large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel opener. It was previously reported that activation of BK_Ca channels by NS1619 could protect the cardiac muscle against ischaemia and reperfusion injury. This study reports the effects of NS1619 on intracellular Ca²⁺ homeostasis in H9C2 and C2C12 cells as well as its molecular mechanism of action. The effects of NS1619 on Ca²⁺ homeostasis in C2C12 and H9C2 cells were assessed using the Fura-2 fluorescence method. Ca²⁺ uptake by sarcoplasmic reticulum (SR) vesicles isolated from rat skeletal muscles and sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) activity were measured. The effect of NS1619 on the isometric force of papillary muscle contraction in the guinea pig heart was also examined. H9C2 and C2C12 cells treated with NS1619 released Ca²⁺ from internal stores in a concentration-dependent manner. Ca²⁺ accumulation by the SR vesicles was inhibited by NS1619 treatment. NS1619 also decreased the activity of SERCA derived from rat skeletal muscle. The calcium release from cell internal stores and inhibition of SERCA by NS1619 are pH dependent. Finally, NS1619 had a profound effect on the isometric force of papillary muscle contraction in the guinea pig heart. These results indicate that NS1619 is a potent modulator of the intracellular Ca²⁺ concentration in H9C2 and C1C12 cells due to its interaction with SRs. The primary target of NS1619 is SERCA, which is located in SR vesicles. The effect of NS1619-mediated SERCA inhibition on cytoprotective processes should be considered.     

Chloride-induced release of actively loaded calcium from light and heavy sarcoplasmic reticulum vesicles

Summary Light and heavy sarcoplasmic reticulum vesicles (LSR, HSR) isolated from rabbit leg muscle have been used in a study of chloride-induced Ca²⁺ release. The biochemical and morphological data indicate that LSR is derived from the longitudinal reticulum and HSR is derived from the terminal cisternae of the sarcoplasmic reticulum. LSR and HSR were both able to accumulate Ca²⁺ in the presence of ATP to amounts greater than 100 nmol Ca²⁺/mg of protein in less than 1 min. LSR and HSR each had a biphasic time course of Ca²⁺ uptake. The initial uptake was followed by a rapid release, after approximately 1 min, of 30–40% of the accumulated Ca²⁺, which was then followed by a slower phase of Ca²⁺ accumulation. Ca²⁺ taken up by the SR vesicles could be released from both the LSR and HSR by changing the anion outside the vesicles from methanesulfonate to chloride. Due to the difference in permeability between methanesulfonate and chloride, this change should result in a decreased positivity inside the vesicles with respect to the exterior. It could also result in osmotic swelling of the vesicles. Changing the ionic medium from chloride to methanesulfonate caused no release of Ca²⁺. The amount of accumulated Ca²⁺ released in 6 sec by changing the anion outside the vesicles from methanesulfonate to chloride was 30–35 nmol/mg membrane protein for LSR and HSR, respectively. Osmotic buffering with 200mm sucrose caused a slight inhibition of chloride-induced Ca²⁺ release from HSR (17%15%) but it greatly reduced the release of Ca²⁺ from LSR (32%15%). The specificity of Ca²⁺ release was measured using SR vesicles which were passively loaded with 10mm ²²Na⁺. LSR released five times more²²Na⁺ than HSR under same conditions as chloride-induced Ca²⁺ release occurred. Na dantrolene (20 m) had no effect on the release of Ca²⁺ from LSR but it inhibited the chloride-induced Ca²⁺ release from HSR by more than 50%. Na dantrolene also increased the Ca²⁺ uptake in the HSR by 20% while not affecting LSR Ca²⁺ uptake. Our results indicate the presence of a chloride-induced, Na dantrolene inhibited, Ca²⁺ release from HSR, which is not due to osmotic swelling.     

Thermogenic activity of Ca2+-ATPase from skeletal muscle heavy sarcoplasmic reticulum: the role of ryanodine Ca2+ channel

The sarcoplasmic reticulum Ca(2+) ATPase 1 (SERCA 1) is able to handle the energy derived from ATP hydrolysis in such a way as to determine the parcel of energy that is used for Ca(2+) transport and the fraction that is converted into heat. In this work we measured the heat production by SERCA 1 in the two sarcoplasmic reticulum (SR) fractions: the light fraction (LSR), which is enriched in SERCA and the heavy fraction (HSR), which contains both the SERCA and the ryanodine Ca(2+) channel. We verified that although HSR cleaved ATP at faster rate than LSR, the amount of heat released during ATP hydrolysis by HSR was smaller than that measured by LSR. Consequently, the amount of heat released per mol of ATP cleaved (DeltaH(cal)) by HSR was lower compared to LSR. In HSR, the addition of 5 mM Mg(2+) or ruthenium red, conditions that close the ryanodine Ca(2+) channel, promoted a decrease in the ATPase activity, but the amount of heat released during ATP hydrolysis remained practically the same. In this condition, the DeltaH(cal) values of ATP hydrolysis increased significantly. Neither Mg(2+) nor ruthenium red had effect on LSR. Thus, we conclude that heat production by SERCA 1 depends on the region of SR in which the enzyme is inserted and that in HSR, the DeltaH(cal) of ATP hydrolysis by SERCA 1 depends on whether the ryanodine Ca(2+) channel is opened or closed.     

Roles of transmembrane segment M1 of Na+,K+-ATPase and Ca2+-ATPase, the gatekeeper and the pivot

In this review we summarize mutagenesis work on the structure–function relationship of transmembrane segment M1 in the Na⁺,K⁺-ATPase and the sarco(endo)plasmic reticulum Ca²⁺-ATPase. The original hypothesis that charged residues in the N-terminal part of M1 interact with the transported cations can be rejected. On the other hand hydrophobic residues in the middle part of M1 turned out to play crucial roles in Ca²⁺ interaction/occlusion in Ca²⁺-ATPase and K⁺ interaction/occlusion in Na⁺,K⁺-ATPase. Leu⁶⁵ of the Ca²⁺-ATPase and Leu⁹⁹ of the Na⁺,K⁺-ATPase, located at homologous positions in M1, function as gate-locking residues that restrict the mobility of the side chain of the cation binding/gating residue of transmembrane segment M4, Glu³⁰⁹/Glu³²⁹. A pivot formed between a pair of a glycine and a bulky residue in M1 and M3 seems critical to the opening of the extracytoplasmic gate in both the Ca²⁺-ATPase and the Na⁺,K⁺-ATPase. All numbering of Na⁺,K⁺-ATPase amino acid residues in this article refers to the sequence of the rat α₁-isoform.     

Cytoplasmic Ca2+ inhibits the ryanodine receptor from cardiac muscle

Ca²⁺-dependent inhibition of native and isolated ryanodine receptor (RyR) calcium release channels from sheep heart and rabbit skeletal muscle was investigated using the lipid bilayer technique. We found that cytoplasmic Ca²⁺ inhibited cardiac RyRs with an average K _m = 15 mm, skeletal RyRs with K _m = 0.7 mm and with Hill coefficients of 2 in both isoforms. This is consistent with measurements of Ca²⁺ release from the sarcoplasmic reticulum (SR) in skinned fibers and with [³H]-ryanodine binding to SR vesicles, but is contrary to previous bilayer studies which were unable to demonstrate Ca²⁺-inhibition in cardiac RyRs (Chu, Fill, Stefani &; Entman (1993) J. Membrane Biol. 135, 49–59). Ryanodine prevented Ca²⁺ from inhibiting either cardiac or skeletal RyRs. Ca²⁺-inhibition in cardiac RyRs appeared to be the most fragile characteristic of channel function, being irreversibly disrupted by 500 mm Cs⁺, but not by 500 mm K⁺, in the cis bath or by solublization with the detergent CHAPS. These treatments had no effect on channel regulation by AMP-PNP, caffeine, ryanodine, ruthenium red, or Ca²⁺-activation. Ca²⁺-inhibition in skeletal RyRs was retained in the presence of 500 mm Cs⁺. Our results provide an explanation for previous findings in which cardiac RyRs in bilayers with 250 mm Cs⁺ in the solutions fail to demonstrate Ca²⁺-inhibition, while Ca²⁺-inhibition of Ca²⁺ release is observed in vesicle studies where K⁺ is the major cation. A comparison of open and closed probability distributions from individual RyRs suggested that the same gating mechanism mediates Ca²⁺-inhibition in skeletal RyRs and cardiac RyRs, with different Ca²⁺ affinities for inhibition. We conclude that differences in the Ca²⁺-inhibition in cardiac and skeletal channels depends on their Ca²⁺ binding properties.     

Methamphetamine directly accelerates beating rate in cardiomyocytes by increasing Ca entry via L-type Ca channel

Methamphetamine induces several cardiac dysfunctions, which leads to arrhythmia, cardiac failure and sudden cardiac death. Although these cardiac alterations elicited by methamphetamine were thought to be due to an indirect action of methamphetamine, namely, an excessive catecholamine release from synaptic terminals, while it seems likely that methamphetamine directly modulates the functioning of cardiomyocytes independent of neurotransmitters. However, the direct effects of methamphetamine on cardiomyocytes are still not clear. We show that methamphetamine directly accelerates the beating rate and alters Ca²⁺ oscillation pattern in cultured neonatal rat cardiomyocytes. Adrenergic receptor antagonists did not block the methamphetamine-induced alterations in cardiomyocytes. Treatment with a ryanodine receptor type 2 inhibitor and a sarcoplasmic reticulum Ca²⁺-ATPase inhibitor did not affect these responses, either. In contrast, the L-type Ca²⁺ channel inhibitor nifedipine eradicated these responses. Furthermore, methamphetamine elevated the internal free Ca²⁺ concentration in HEK-293T cells stably transfected with the L-type Ca²⁺ channel α1C subunit. In neonatal rat cardiomyocytes, methamphetamine accelerates beating rate and alters Ca²⁺ oscillation pattern by increasing Ca²⁺ entry via the L-type Ca²⁺ channels independent of any neurotransmitters.     

Modification of heart sarcolemmal Na+/K+-ATPase activity during development of the calcium paradox

This study examined the status of sarcolemmal Na⁺/K⁺-ATPase activity in rat heart under conditions of Ca²⁺-paradox to explore the existence of a relationship between changes in Na⁺/K⁺-pump function and myocardial Na⁺ as well as K⁺ content. One min of reperfusion with Ca²⁺ after 5 min of Ca²⁺-free perfusion reduced Na⁺/K⁺-ATPase activity in the isolated heart by 53% while Mg²⁺-ATPase, another sarcolemmal bound enzyme, retained 74% of its control activity. These changes in sarcolemmal ATPase activities were dependent on the duration and Ca²⁺ concentration of the initial perfusion and subsequent reperfusion periods; however, the Na⁺/K⁺-ATPase activity was consistently more depressed than Mg²⁺-ATPase activity under all conditions. The depression in both enzyme activities was associated with a reduction in V_max without any changes in K_m values. Low Na⁺ perfusion and hypothermia, which protect the isolated heart from the Ca²⁺-paradox, also prevented reperfusion-induced enzyme alterations. A significant relationship emerged upon comparison of the changes in myocardial Na⁺ and K⁺ content to Na⁺/K⁺-ATPase activity under identical conditions. At least 60% of the control enzyme activity was necessary to maintain normal cation gradients. Depression of the Na⁺/K⁺-ATPase activity by 60-65% resulted in a marked increase and decrease in intracellular Na⁺ and K⁺ content, respectively. These results suggest that changes in myocardial Na⁺ and K⁺ content during Ca²⁺-paradox are related to activity of the Na⁺/K⁺-pump; the impaired Na⁺/K⁺-ATPase activity may lead to augmentation of Ca²⁺-overload via an enhancement of the Na⁺/Ca²⁺-exchange system.     

Effects of extremely low frequency electromagnetic fields on intracellular calcium transients in cardiomyocytes

Abstract

Calcium transients play an essential role in cardiomyocytes and electromagnetic fields (EMF) and affect intracellular calcium levels in many types of cells. Effects of EMF on intracellular calcium transients in cardiomyocytes are not well studied. The aim of this study was to assess whether extremely low frequency electromagnetic fields (ELF-EMF) could affect intracellular calcium transients in cardiomyocytes. Cardiomyocytes isolated from neonatal Sprague-Dawley rats were exposed to rectangular-wave pulsed ELF-EMF at four different frequencies (15?Hz, 50?Hz, 75?Hz and 100?Hz) and at a flux density of 2?mT. Intracellular calcium concentration ([Ca²⁺]_i) was measured using Fura-2/AM and spectrofluorometry. Perfusion of cardiomyocytes with a high concentration of caffeine (10?mM) was carried out to verify the function of the cardiac Na⁺/Ca²⁺ exchanger (NCX) and the activity of sarco(endo)-plasmic reticulum Ca²⁺-ATPase (SERCA2a). The results showed that ELF-EMF enhanced the activities of NCX and SERCA2a, increased [Ca²⁺]_i baseline level and frequency of calcium transients in cardiomyocytes and decreased the amplitude of calcium transients and calcium level in sarcoplasmic reticulum. These results indicated that ELF-EMF can regulate calcium-associated activities in cardiomyocytes.     

Uncoupling of Ca2+ transport from ATP hydrolysis activity of sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase

Summary In reconstituted rabbit skeletal muscle (Ca²⁺ + Mg²⁺)-ATPase proteoliposomes, Ca²⁺-uptake is decreased by more than 90% with T₂ cleavage (Arg-198). However, no difference in the ATP dependence of hydrolysis activity is seen between SR and trypsin-treated SR. A large decrease in E-P formation and hydrolysis activity of the enzyme appear only at T₃ cleavage, which represents the cleavage of A₁ fragment to A_1a + A_1b forms. The disappearance of hydrolysis activity due to digestion is prior to the disappearance of E-P formation. No significant difference is found in the passive Ca²⁺ efflux between control SR and tryptically digested SR in the absence of Mg⁺ ruthenium red or in the presence of ATP. However, the passive Ca²⁺ efflux rate for tryptically digested SR is much larger than control SR in the presence of Mg²⁺ + ruthenium red. These results show that the Ca²⁺ channel cannot be closed after trypsin digestion of SR membranes by the presence of the Ca²⁺ channel inhibitors, Mg²⁺ and ruthenium red. In the reconstituted ATPase proteoliposomes, the Ca²⁺ efflux rates are the same regardless of digestion (T₂); also, efflux is not affected by the presence or absence of Mg²⁺ + ruthenium red. These results indicate that T₂ cleavage causes uncoupling of the Ca²⁺-pump from ATP hydrolytic activity.A theoretical model is developed in order to fit the extent of tryptic digestion of the A fragment of the (Ca²⁺ + Mg²⁺)-ATPase polypeptide with the loss of Ca²⁺-transport. Fits of the theoretical equations to the data are consistent with that Ca²⁺-transport system appears to require a dimer of the polypeptide (Ca²⁺ + Mg²⁺)-ATPase.     

Ion transport and energy transduction of P-type ATPases: Implications from electrostatic calculations

This paper summarizes our present electrostatic calculations on P-type ATPases and their contribution to understand the molecular details of the reaction mechanisms. One focus was set on analyzing the proton countertransport of the sarcoplasmic reticulum Ca²⁺-ATPase (SERCA1a). Protonation of acidic residues was calculated in dependence of pH for different enzyme states in the reaction cycle of the Ca²⁺-ATPase. We proposed that the acidic Ca²⁺ ligands Glu 771, Asp 800 and Glu 908 participate in the proton countertransport whereas Glu 309 is more likely to serve as a proton shuttle between binding site I and the cytoplasm. Complementary to infrared measurements, we assigned infrared bands to specific Ca²⁺ ligands that are hydrogen bonded. Ion pathways were proposed based on the calculations and structural data. Another focus was set on analyzing the energy transduction mechanism of P-type ATPases. In accordance to electrophysiological experiments, we simulated an electric field across the membrane. The impact of the electric field was studied by an accumulated number of residue conformational and ionization changes on specific transmembrane helices. Our calculations on the Ca²⁺-ATPase and the Na⁺/K⁺-ATPase indicated that the highly conserved transmembrane helix M5 is one structural element that is likely to act as energy transduction element in P-type ATPases. Perspectives and limitations of the electrostatic calculations for future computational studies are pointed out.