Characterization of human cardiac calsequestrin and its deleterious mutants

Mutations of conserved residues of human cardiac calsequestrin (hCSQ2), a high-capacity, low-affinity Ca²⁺-binding protein in the sarcoplasmic reticulum, have been associated with catecholamine-induced polymorphic ventricular tachycardia (CPVT). In order to understand the molecular mechanism and pathophysiological link between these CPVT-related missense mutations of hCSQ2 and the resulting arrhythmias, we generated three CPVT-causing mutants of hCSQ2 (R33Q, L167H, and D307H) and two non-pathological mutants (T66A and V76M) and investigated the effect of these mutations. In addition, we determined the crystal structure of the corresponding wild-type hCSQ2 to gain insight into the structural effects of those mutations. Our data show clearly that all three CPVT-related mutations lead to significant reduction in Ca²⁺-binding capacity in spite of the similarity of their secondary structures to that of the wild-type hCSQ2. Light-scattering experiments indicate that the Ca²⁺-dependent monomer-polymer transitions of the mutants are quite different, confirming that the linear polymerization behavior of CSQ is linked directly to its high-capacity Ca²⁺ binding. R33Q and D307H mutations result in a monomer that appears to be unable to form a properly oriented dimer. On the other hand, the L167H mutant has a disrupted hydrophobic core in domain II, resulting in high molecular aggregates, which cannot respond to Ca²⁺. Although one of the non-pathological mutants, T66A, shares characteristics with the wild-type, the other null mutant, V76M, shows significantly altered Ca²⁺-binding and polymerization behaviors, calling for careful reconsideration of its status.     

Characterization of Ca2+-Dependent Protein-Protein Interactions within the Ca2+ Release Units of Cardiac Sarcoplasmic Reticulum

In the heart, excitation-contraction (E-C) coupling is mediated by Ca²⁺ release from sarcoplasmic reticulum (SR) through the interactions of proteins forming the Ca²⁺ release unit (CRU). Among them, calsequestrin (CSQ) and histidine-rich Ca²⁺ binding protein (HRC) are known to bind the charged luminal region of triadin (TRN) and thus directly or indirectly regulate ryanodine receptor 2 (RyR2) activity. However, the mechanisms of CSQ and HRC mediated regulation of RyR2 activity through TRN have remained unclear. We first examined the minimal KEKE motif of TRN involved in the interactions with CSQ2, HRC and RyR2 using TRN deletion mutants and in vitro binding assays. The results showed that CSQ2, HRC and RyR2 share the same KEKE motif region on the distal part of TRN (aa 202–231). Second, in vitro binding assays were conducted to examine the Ca²⁺ dependence of protein-protein interactions (PPI). The results showed that TRN-HRC interaction had a bell-shaped Ca²⁺ dependence, which peaked at pCa4, whereas TRN-CSQ2 or TRN-RyR2 interaction did not show such Ca²⁺ dependence pattern. Third, competitive binding was conducted to examine whether CSQ2, HRC, or RyR2 affects the TRN-HRC or TRN-CSQ2 binding at pCa4. Among them, only CSQ2 or RyR2 competitively inhibited TRN-HRC binding, suggesting that HRC can confer functional refractoriness to CRU, which could be beneficial for reloading of Ca²⁺ into SR at intermediate Ca²⁺ concentrations.     

Perspective: The List of Potential Volume-sensitive Chloride Currents Continues to Swell (and Shrink)

The channel of the glutamate N-methyl-d-aspartate receptor (NMDAR) transports Ca²⁺ approximately four times more efficiently than that of Ca²⁺-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPAR). To investigate the basis of this difference in these glutamate receptors (GluRs), we measured the ratio of Cs⁺ efflux and Ca²⁺ influx in recombinant NMDAR and Ca²⁺-permeable AMPAR channels expressed in human embryonic kidney 293 (HEK 293) cells over a wide voltage range. At any one potential, this biionic flux ratio was measured by quantifying the total charge and the charge carried by Ca²⁺ using whole-cell currents and fluorometric techniques (dye overload) with Cs⁺ internally and Ca²⁺ externally (1.8 or 10 mM) as the only permeant ions. In AMPAR channels, composed of either GluR-A(Q) or GluR-B(Q) subunits, the biionic flux ratio had a biionic flux-ratio exponent of 1, consistent with the prediction of the Goldman-Hodgkin-Katz current equation. In contrast, for NMDAR channels composed of NR1 and NR2A subunits, the biionic flux-ratio exponent was ∼2, indicating a deviation from Goldman-Hodgkin-Katz. Consistent with these results, in NMDAR channels under biionic conditions with high external Ca²⁺ and Cs⁺ as the reference ions, Ca²⁺ permeability (P_Ca/P_Cs) was concentration dependent, being highest around physiological concentrations (1–1.8 mM; P_Ca/P_Cs ≈ 6.1) and reduced at both higher (110 mM; P_Ca/P_Cs ≈ 2.6) and lower (0.18 mM; P_Ca/P_Cs ≈ 2.2) concentrations. P_Ca/P_Cs in AMPAR channels was not concentration dependent, being around 1.65 in 0.3–110 mM Ca²⁺. In AMPAR and NMDAR channels, the Q/R/N site is a critical determinant of Ca²⁺ permeability. However, mutant AMPAR channels, which had an asparagine substituted at the Q/R site, also showed a biionic flux-ratio exponent of 1 and concentration-independent permeability ratios, indicating that the difference in Ca²⁺ transport is not due to the amino acid residue located at the Q/R/N site. We suggest that the difference in Ca²⁺ transport properties between the glutamate receptor subtypes reflects that the pore of NMDAR channels has multiple sites for Ca²⁺, whereas that of AMPAR channels only a single site.     

Ca2+/H+ exchange,lumenal Ca2+ release and Ca2+/ATP coupling ratios in the sarcoplasmic reticulum ATPase

The Ca²⁺ transport ATPase (SERCA) of sarcoplasmic reticulum (SR) plays an important role in muscle cytosolic signaling, as it stores Ca²⁺ in intracellular membrane bound compartments, thereby lowering cytosolic Ca²⁺ to induce relaxation. The stored Ca²⁺ is in turn released upon membrane excitation to trigger muscle contraction. SERCA is activated by high affinity binding of cytosolic Ca²⁺, whereupon ATP is utilized by formation of a phosphoenzyme intermediate, which undergoes protein conformational transitions yielding reduced affinity and vectorial translocation of bound Ca²⁺. We review here biochemical and biophysical evidence demonstrating that release of bound Ca²⁺ into the lumen of SR requires Ca²⁺/H⁺ exchange at the low affinity Ca²⁺ sites. Rise of lumenal Ca²⁺ above its dissociation constant from low affinity sites, or reduction of the H⁺ concentration by high pH, prevent Ca²⁺/H⁺ exchange. Under these conditions Ca²⁺ release into the lumen of SR is bypassed, and hydrolytic cleavage of phosphoenzyme may yield uncoupled ATPase cycles. We clarify how such Ca²⁺pump slippage does not occur within the time length of muscle twitches, but under special conditions and in special cells may contribute to thermogenesis.     

Differential alterations in ATP-supported calcium transport activities of sarcoplasmic reticulum and sarcolemma of aging myocardium

Two membrane fractions, one enriched in sarcoplasmic reticulum and the other enriched in sarcolemma, were isolated from the myocardium of young (3–4-months-old) and aged (24–25-months old) rats. ATP-supported Ca²⁺ binding and accumulating activities as well as (Mg²⁺ + Ca²⁺)-ATPase activities of these membrane fractions were studied in an effort to determine the influence of age on the Ca²⁺ pump function of the two myocardial membrane systems. Sarcoplasmic reticulum from aged hearts showed significantly reduced (approx. 50%) rates of ATP-supported (oxalate-facilitated) Ca²⁺ accumulation compared to sarcoplasmic reticulum from young hearts; the amount of Ca²⁺ accumulated by this membrane of aged heart at steady state was also lower. On the other hand, sarcolemma from aged hearts displayed 2-fold higher rates of ATP-supported Ca²⁺ accumulation compared to sarcolemma from young hearts; at steady state, sarcolemma from aged hearts accumulated significantly higher amounts of Ca²⁺ than did sarcolemma from young hearts. Similar age-related differences were also observed in the ATP-dependent Ca²⁺ binding activities of the two membranes, determined in the absence of oxalate. The divergent age-associated changes in Ca²⁺ binding and accumulating activities of sarcoplasmic reticulum and sarcolemma were seen at varying Ca²⁺ concentrations (0.24–39.1 μM).With either membrane, kinetic analysis showed 2-fold age-related differences in the V values for ATP-supported Ca²⁺ accumulation (V (nmol Ca²⁺/mg protein per min): sarcoplasmic reticulum — young, 119 ± 8; aged, 59 ± 5; sarcolemma — young, 11 ± 2; aged, 21 ± 3); the concentrations of Ca²⁺ required for half-maximal velocities did not differ significantly with age (K_0.5 for Ca²⁺ (μM): sarcoplasmic reticulum — young, 2.5 ± 0.20; aged, 2.9 ± 0.25; sarcolemma — yount, 2.7 ± 0.25; aged, 3.2 ± 0.30). Kinetic parameters of ATP-dependent Ca²⁺ binding also indicated that the velocity of Ca²⁺ binding but not the concentration of Ca²⁺ required for half-maximal binding was altered due to aging. At identical Ca²⁺ concentrations, the combined Ca²⁺ accumulating activity of sarcoplasmic reticulum and sarcolemma from aged hearts was significantly lower (38–47%) than the combined Ca²⁺ accumulating activity of the two membranes from young hearts. No significant age-related differences were observed in the ATP-independent (passive) Ca²⁺ binding (or accumulation) by sarcoplasmic reticulum and sarcolemma, the (Mg²⁺ + Ca²⁺)-ATPase activities of these membranes, their polypeptide composition or relative purity. These results indicate that differential alterations occur in the ATP-supported Ca²⁺ pump activities of sarcoplasmic reticulum and sarcolemma in aging myocardium and such alterations may be due to age-associated changes in the efficacy of coupling ATP hydrolysis to Ca²⁺ transport. Further, the age-related increment in the Ca²⁺ pump activity of sarcolemma is inadequate to fully compensate for the diminished Ca²⁺ pump activity of sarcoplasmic reticulum. It is, therefore, suggested that deterioration of the Ca²⁺ pump function of sarcoplasmic reticulum may contribute to the increased relaxation time observed in aging heart.     

Diversity of mitochondrial Ca signaling in rat neonatal cardiomyocytes: Evidence from a genetically directed Ca probe,mitycam-E31Q

I_Ca-gated Ca²⁺ release (CICR) from the cardiac SR is the main mechanism mediating the rise of cytosolic Ca²⁺, but the extent to which mitochondria contribute to the overall Ca²⁺ signaling remains controversial. To examine the possible role of mitochondria in Ca²⁺ signaling, we developed a low affinity mitochondrial Ca²⁺ probe, mitycam-E31Q (300–500 MOI, 48–72 h) and used it in conjunction with Fura-2AM to obtain simultaneous TIRF images of mitochondrial and cytosolic Ca²⁺ in cultured neonatal rat cardiomyocytes. Mitycam-E31Q staining of adult feline cardiomyocytes showed the typical mitochondrial longitudinal fluorescent bandings similar to that of TMRE staining, while neonatal rat cardiomyocytes had a disorganized tubular or punctuate appearance. Caffeine puffs produced rapid increases in cytosolic Ca²⁺ while simultaneously measured global mitycam-E31Q signals decreased more slowly (increased mitochondrial Ca²⁺) before decaying to baseline levels. Similar, but oscillating mitycam-E31Q signals were seen in spontaneously pacing cells. Withdrawal of Na⁺ increased global cytosolic and mitochondrial Ca²⁺ signals in one population of mitochondria, but unexpectedly decreased it (release of Ca²⁺) in another mitochondrial population. Such mitochondrial Ca²⁺ release signals were seen not only during long lasting Na⁺ withdrawal, but also when Ca²⁺ loaded cells were exposed to caffeine-puffs, and during spontaneous rhythmic beating. Thus, mitochondrial Ca²⁺ transients appear to activate with a delay following the cytosolic rise of Ca²⁺ and show diversity in subpopulations of mitochondria that could contribute to the plasticity of mitochondrial Ca²⁺ signaling.     

Ca2+-induced Ca2+ Release in Chinese Hamster Ovary (CHO) Cells Co-expressing Dihydropyridine and Ryanodine Receptors

Combined patch-clamp and Fura-2 measurements were performed on chinese hamster ovary (CHO) cells co-expressing two channel proteins involved in skeletal muscle excitation-contraction (E-C) coupling, the ryanodine receptor (RyR)-Ca²⁺ release channel (in the membrane of internal Ca²⁺ stores) and the dihydropyridine receptor (DHPR)-Ca²⁺ channel (in the plasma membrane). To ensure expression of functional L-type Ca²⁺ channels, we expressed α₂, β, and γ DHPR subunits and a chimeric DHPR α₁ subunit in which the putative cytoplasmic loop between repeats II and III is of skeletal origin and the remainder is cardiac. There was no clear indication of skeletal-type coupling between the DHPR and the RyR; depolarization failed to induce a Ca²⁺ transient (CaT) in the absence of extracellular Ca²⁺ ([Ca²⁺]_o). However, in the presence of [Ca²⁺]_o, depolarization evoked CaTs with a bell-shaped voltage dependence. About 30% of the cells tested exhibited two kinetic components: a fast transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) (the first component; reaching 95% of its peak <0.6 s after depolarization) followed by a second increase in [Ca²⁺]_i which lasted for 5–10 s (the second component). Our results suggest that the first component primarily reflected Ca²⁺ influx through Ca²⁺ channels, whereas the second component resulted from Ca²⁺ release through the RyR expressed in the membrane of internal Ca²⁺ stores. However, the onset and the rate of Ca²⁺ release appeared to be much slower than in native cardiac myocytes, despite a similar activation rate of Ca²⁺ current. These results suggest that the skeletal muscle RyR isoform supports Ca²⁺-induced Ca²⁺ release but that the distance between the DHPRs and the RyRs is, on average, much larger in the cotransfected CHO cells than in cardiac myocytes. We conclude that morphological properties of T-tubules and/or proteins other than the DHPR and the RyR are required for functional “close coupling” like that observed in skeletal or cardiac muscle. Nevertheless, some of our results imply that these two channels are potentially able to directly interact with each other.     

Bax Inhibitor-1-mediated Ca leak is decreased by cytosolic acidosis

Bax Inhibitor-1 (BI-1) is an evolutionarily conserved six-transmembrane domain endoplasmic reticulum (ER)-localized protein that protects against ER stress-induced apoptotic cell death. This function is closely connected to its ability to lower steady-state ER Ca²⁺ levels. Recently, we elucidated BI-1's Ca²⁺-channel pore in the C-terminal part of the protein and identified the critical amino acids of its pore. Based on these insights, a Ca²⁺-channel pore-dead mutant BI-1 (BI-1^D213R) was developed. We determined whether BI-1 behaves as a bona fide H⁺/Ca²⁺ antiporter or as an ER Ca²⁺-leak channel by investigating the effect of pH on unidirectional Ca²⁺-efflux rates. At pH 6.8, wild-type BI-1 expression in BI-1^−/− cells increased the ER Ca²⁺-leak rate, correlating with its localization in the ER compartment. In contrast, BI-1^D231R expression in BI-1^−/−, despite its ER localization, did not increase the ER Ca²⁺-leak rate. However, at pH < 6.8, the BI-1-mediated ER Ca²⁺ leak was blocked. Finally, a peptide representing the Ca²⁺-channel pore of BI-1 promoting Ca²⁺ flux from the ER was used. Lowering the pH from 6.8 to 6.0 completely abolished the ability of the BI-1 peptide to mediate Ca²⁺ flux from the ER. We propose that this pH dependence is due to two aspartic acid residues critical for the function of the Ca²⁺-channel pore and located in the ER membrane-dipping domain, which facilitates the protonation of these residues.     

Effects of adenosine diphosphate on Ca fluxes and Ca accumulation of sarcoplasmic reticulum

Ca²⁺ transport by sarcoplasmic reticulum vesicles was examined by incubating sarcoplasmic reticulum vesicles (0.15 mg/ml) at 37°C in, either normal medium that contained 0.15 M sucrose, 0.1 M KCl, 60 μM CaCl₂, 2.5 mM ATP and 30 mM Tes at pH 6.8, or a modified medium for elimination of ADP formed from ATP hydrolysis by including, in addition, 3.6 mM phosphocreatine and 33 U/ml of creatine phosphokinase. In normal medium, Ca²⁺ uptake of sarcoplasmic reticulum vesicles reached a plateau of about 100 nmol/mg. In modified medium, after this phase of Ca²⁺ uptake, a second phase of Ca²⁺ accumulation was initiated and reached a plateau of about 300 nmol/mg. The second phase of Ca²⁺ accumulation was accompanied by phosphate uptake and could be inhibited by ADP. Since, under these experimental conditions, there was no significant difference of the rates of ATP hydrolysis in normal medium and modified medium, extra Ca²⁺ uptake in modified medium but not in normal medium could not be explained by different phosphate accumulation in the two media. Unidirectional Ca²⁺ influx of sarcoplasmic reticulum near steady state of Ca²⁺ uptake was measured by pulse labeling with ⁴⁵Ca²⁺. The Ca²⁺ efflux rate was then determined by subtracting the net uptake from the influx rate. At the first plateau of Ca²⁺ uptake in normal medium, Ca²⁺ influx was balanced by Ca²⁺ efflux with an exchange rate of 240 nmol/mg per min. This exchange rate was maintained relatively constant at the plateau phase. In modified medium, the Ca²⁺ exchange rate at the first plateau of Ca²⁺ uptake was about half of that in normal medium. When the second phase of Ca²⁺ uptake was initiated, both the influx and efflux rates started to increase and reached a similar exchange rate as observed in normal medium. Also, during the second phase of Ca²⁺ uptake, the difference between the influx and efflux rates continued to increase until the second plateau phase was approached. In conditions where the formation of ADP and inorganic phosphate was minimized by using a low concentration of sarcoplasmic (7.5 μg/ml) and/or using acetyl phosphate instead of ATP, the second phase of Ca²⁺ uptake was also observed. These data suggest that the Ca²⁺ load attained by sarcoplasmic reticulum vesicles during active transport is modulated by ADP accumulated from ATP hydrolysis. ADP probably exerts its effect by facilitating Ca²⁺ efflux, which subsequently stimulates Ca²⁺ exchange.     

Immunolocalization of the sarcolemmal Ca2+/Mg2+ ecto-ATPase (myoglein) in rat myocardium

Cardiac plasma membrane Ca²⁺/Mg²⁺ ecto-ATPase (myoglein) requires millimolar concentrations of either Ca²⁺ or Mg²⁺ for maximal activity. In this paper, we report its localization by employing an antiserum raised against the purified rat cardiac Ca²⁺/Mg²⁺ ATPase. As assessed by Western blot analysis, the antiserum and the purified immunoglobulin were specific for Ca²⁺/Mg²⁺ ecto-ATPase; no cross reaction was observed towards other membrane bound enzymes such as cardiac sarcoplasmic reticulum Ca²⁺-pump ATPase or sarcolemmal Ca²⁺-pump ATPase. On the other hand, the cardiac Ca²⁺/Mg²⁺ ecto-ATPase was not recognized by antibodies specific for either cardiac sarcoplasmic reticulum Ca²⁺-pump ATPase or plasma membrane Ca²⁺-pump ATPase. Furthermore, the immune serum inhibited the Ca²⁺/Mg²⁺ ecto-ATPase activity of the purified enzyme preparation. Immunofluorescence of cardiac tissue sections and neonatal cultured cardiomyocytes with the Ca²⁺/Mg²⁺ ecto-ATPase antibodies indicated the localization of Ca²⁺/Mg²⁺ ecto-ATPase in association with the plasma membrane of myocytes, in areas of cell-matrix or cell-cell contact. Staining for the Ca²⁺/Mg²⁺ ecto-ATPase was not cardiac specific since the antibodies detected the presence of membrane proteins in sections from skeletal muscle, brain, liver and kidney. The results indicate that Ca²⁺/Mg²⁺ ecto-ATPase is localized to the plasma membranes of cardiomyocytes as well as other tissues such as brain, liver, kidney and skeletal muscle.     

Excitation-contraction coupling in a pre-vertebrate twitch muscle: The myotomes of Branchiostoma lanceolatum

Summary The segmented trunk muscle (myotome muscle) of the lancelet (Branchiostoma lanceolatum), a pre-vertebrate chordate, was studied in order to gain information regarding the evolution of excitation-contraction (EC) coupling.Myotome membrane vesicles could be separated on isopycnic sucrose gradients into two main fractions, probably comprising solitary microsomes and diads of plasma membrane and sarcoplasmic reticulum, respectively. Both fractions bound the dihydropyridine PN 200/110 and the phenylalkylamine (–)D888 (devapamil) while specific ryanodine binding was observed in the diad preparation only. Pharmacological effects on Ca²⁺ currents measured under voltage-clamp conditions in single myotome fibers included a weak block by the dihydropyridine nifedipine and a shift of the voltage dependences of inactivation and restoration to more negative potentials by (–)D888. After blocking the Ca²⁺ current by cadmium in voltage-clamped single fibers, the contractile response persisted and a rapid intramembrane charge movement could be demonstrated. Both responses exhibited a voltage sensitivity very similar to the one of the voltage-activated Ca²⁺ channels.Our biochemical and electrophysiological results indicate that the EC coupling mechanism of the protochordate myotome cell is similar to that of the vertebrate skeletal muscle fiber: Intracellular Ca²⁺ release, presumably taking place via the ryanodine receptor complex, is under control of the cell membrane potential. The sarcolemmal Ca²⁺ channels might serve as voltage sensors for this process.We thank Drs. H.Ch. Lüttgau and L.M.G. Heilmeyer, Jr. for stimulating discussions during the work, Dr. N.R. Brandt for helpful suggestions, and Drs. A.H. Caswell and M. Michalak for their generous gifts of antibodies. We also thank Ms. P. Goldmann, Mr. R. Schwalm, and Mr. U. Siemen for technical support and Ms. E. Linnepe for editorial help. This work was supported by grant G1 72/1-5 of the Deutsche Forschungsgemeinschaft. R. Benterbusch was recipient of a scholarship by the Studienstiftung des Deutschen Volkes.     

Oxygen free radicals and calcium homeostasis in the heart

Many experiments have been done to clarify the effects of oxygen free radicals on Ca²⁺ homeostasis in the hearts. A burst of oxygen free radicals occurs immediately after reperfusion, but we have to be reminded that the exact levels of oxygen free radicals in the hearts are yet unknown in both physiological and pathophysiological conditions. Therefore, we should give careful consideration to this point when we perform the experiments and analyze the results. It is, however, evident that Ca²⁺ overload occurs when the hearts are exposed to an excess amount of oxygen free radicals. Though ATP-independent Ca²⁺ binding is increased, Ca²⁺ influx through Ca²⁺ channel does not increase in the presence of oxygen free radicals. Another possible pathway through which Ca²⁺ can enter the myocytes is Na⁺?Ca²⁺ exchanger. Although, the activities of Na⁺?K⁺ ATPase and Na⁺?H⁺ exchange are inhibited by oxygen free radicals, it is not known whether intracellular Na⁺ level increases under oxidative stress or not. The question has to be solved for the understanding of the importance of Na⁺?Ca²⁺ exchange in Ca²⁺ influx process from extracellular space. Another question is ‘which way does Na⁺?Ca²⁺ exchange work under oxidative stress? Net influx or efflux of Ca^2+?’ Membrane permeability for Ca²⁺ may be maintained in a relatively early phase of free radical injury. Since sarcolemmal Ca²⁺-pump ATPase activity is depressed by oxygen free radicals, Ca²⁺ extrusion from cytosol to extracellular space is considered to be reduced. It has also been shown that oxygen free radicals promote Ca²⁺ release from sarcoplasmic reticulum and inhibit Ca²⁺ sequestration to sarcoplasmic reticulum. Thus, these changes in Ca²⁺ handling systems could cause the Ca²⁺ overload due to oxygen free radicals.     

ITPRs/inositol 1,4,5-trisphosphate receptors in autophagy: From enemy to ally

ITPRs (inositol 1,4,5-trisphosphate receptors), the main endoplasmic reticulum (ER) Ca²⁺-release channels, were originally proposed as suppressors of autophagy. Yet, new evidence has accumulated over recent years supporting a crucial, stimulatory role for ITPRs in driving the autophagic flux. Here, we provide an integrated view on how ITPR-mediated Ca²⁺ signaling can have a dual impact on autophagy, depending on the characteristics of the spatio-temporal Ca²⁺ signals, including the existence of ER-mitochondrial and ER-lysosomal Ca²⁺ signaling microdomains.     

Functional abnormalities in iPSC‐derived cardiomyocytes generated from CPVT1 and CPVT2 patients carrying ryanodine or calsequestrin mutations

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia characterized by syncope and sudden death occurring during exercise or acute emotion. CPVT is caused by abnormal intracellular Ca²⁺ handling resulting from mutations in the RyR2 or CASQ2 genes. Because CASQ2 and RyR2 are involved in different aspects of the excitation‐contraction coupling process, we hypothesized that these mutations are associated with different functional and intracellular Ca²⁺ abnormalities. To test the hypothesis we generated induced Pluripotent Stem Cell‐derived cardiomyocytes (iPSC‐CM) from CPVT1 and CPVT2 patients carrying the RyR2^R420Q and CASQ2^D307H mutations, respectively, and investigated in CPVT1 and CPVT2 iPSC‐CM (compared to control): (i) The ultrastructural features; (ii) the effects of isoproterenol, caffeine and ryanodine on the [Ca²⁺]_i transient characteristics. Our major findings were: (i) Ultrastructurally, CASQ2 and RyR2 mutated cardiomyocytes were less developed than control cardiomyocytes. (ii) While in control iPSC‐CM isoproterenol caused positive inotropic and lusitropic effects, in the mutated cardiomyocytes isoproterenol was either ineffective, caused arrhythmias, or markedly increased diastolic [Ca²⁺]_i. Importantly, positive inotropic and lusitropic effects were not induced in mutated cardiomyocytes. (iii) The effects of caffeine and ryanodine in mutated cardiomyocytes differed from control cardiomyocytes. Our results show that iPSC‐CM are useful for investigating the similarities/differences in the pathophysiological consequences of RyR2 versus CASQ2 mutations underlying CPVT1 and CPVT2 syndromes.     

Overexpression of SERCA2 ATPaase in vascular smooth muscle cells treated with oxidized low density lipoprotein

Oxidized low density lipoprotein (oxLDL) has been identified as a potentially important atherogenic factor. Atherosclerosis is characterized by the accumulation of lipid and calcium in the vascular wall. OxLDL plays a significant role in altering calcium homeostasis within different cell types. In our previous study, chronic treatment of vascular smooth muscle cells (VSMC) with oxLDL depressed Ca²⁺ _i homeostasis and altered two Ca²⁺ release mechanisms in these cells (IP₃ and ryanodine sensitive channels). The purpose of the present study was to further define the effects of chronic treatment with oxLDL on the smooth muscle sarcoplasmic reticulum (SR) Ca²⁺ pump. One of the primary Ca²⁺ uptake mechanisms in VSMC is through the SERCA2 ATPase calcium pump in the sarcoplasmic reticulum. VSMC were chronically treated with 0.005-0.1 mg/ml oxLDL for up to 6 days in culture. Cells treated with oxLDL showed a significant increase in the total SERCA2 ATPase content. These changes were observed on both Western blot and immunocytochemical analysis. This increase in SERCA2 ATPase is in striking contrast to a significant decrease in the density of IP₃ and ryanodine receptors in VSMC as the result of chronic treatment with oxLDL. This response may suggest a specific adaptive mechanism that the pump undergoes to attempt to maintain Ca²⁺ homeostasis in VSMC chronically exposed to atherogenic oxLDL.     

The actin cytoskeleton differentially regulates NG115-401L cell ryanodine receptor and inositol 1,4,5-trisphosphate receptor induced calcium signaling pathways

Regulation of bi-directional communication between intracellular Ca²⁺ pools and surface Ca²⁺ channels remains incompletely characterized. We report Ca²⁺ release mediated by inositol 1,4,5-trisphosphate receptor (IP₃R) and ryanodine receptor (RyR) pathways is diminished under actin cytoskeleton disruption in NG115-401L (401L) neuronal cells, yet despite truncated Ca²⁺ release, Ca²⁺ influx was not significantly altered in these experiments. However, disruption of cortical actin networks completely abolished IP₃R induced Ca²⁺ release, whereas RyR-mediated Ca²⁺ release was preserved, albeit attenuated. Moreover, cortical actin disruption completely abolished IP₃R and RyR linked Ca²⁺ influx even though Ca²⁺ pool sensitivities were different. These findings suggest discrete Ca²⁺ store/Ca²⁺ channel coupling mechanisms in the IP₃R and RyR pathways as revealed by the differential sensitivity to actin perturbation.     

Mechanisms Underlying Leakage of Calcium from the Endoplasmic Reticulum of Acinar Cells of the Submandibular Salivary Gland

In the resting state, the Ca²⁺ concentration in agonist-sensitive intracellular stores reflects the balance between active uptake of Ca²⁺, which is mediated by Ca²⁺-ATPase (SERCA), and passive leakage of Ca²⁺. The mechanisms underlying such a leakage in cells of the submaxillary salivary gland were not studied. In our experiments, we examined possible pathways of passive leakage of Ca²⁺ from the endoplasmic reticulum (ER) of acinar cells obtained from the rat submaxillary salivary gland; direct measurements of the concentration of Ca²⁺ in the ER ([Ca²⁺]_ER) using a low-affinity calcium-sensitive dye, mag-fura 2/AM, were performed. The cellular membrane was permeabilized with the help of β-escin (40 μg/ml); the Ca²⁺ concentration in the cytoplasm ([Ca²⁺] _i ) was clamped at its level typical of the resting state (∼100 nM) using an EGTA/Ca²⁺ buffer. Incubation of permeabilized acinar cells in a calcium-free intracellular milieu, as well as application of thapsigargin, resulted in complete inhibition of the uptake of Ca²⁺ with the involvement of SERCA. This effect was observed 1 min after the beginning of superfusion of the cells with the corresponding solutions and was accompanied by the leakage of Ca²⁺ from the ER; this is confirmed by a gradual drop in the [Ca₂₊]_ER. Such a leakage of Ca²⁺ remained unchanged in the presence of thapsigargin, heparin, and ruthenium red; therefore, it is not mediated by SERCA, inositol 1,4,5-trisphosphate-sensitive receptors (InsP₃R), or ryanodine receptors (RyRs). At the same time, an antibiotic, puromycin (0.1 to 1.0 mM), which disconnects polypeptides from the ER-ribosome translocon complex, caused intensification of passive leakage of Ca²⁺ from the ER. This effect did not depend on the functioning of SERCA, InsP₃R, or RyR. Therefore, passive leakage of Ca²⁺ from the ER in acinar cells of the submaxillary salivary gland is realized through pores of the translocon complex of the ER membrane. Neirofiziologiya/Neurophysiology, Vol. 37, No. 4, pp. 339–346, July–August, 2005.     

Functional Coupling of Ryanodine Receptors to KCa Channels in Smooth Muscle Cells from Rat Cerebral Arteries

The relationship between Ca²⁺ release (“Ca²⁺ sparks”) through ryanodine-sensitive Ca²⁺ release channels in the sarcoplasmic reticulum and K_Ca channels was examined in smooth muscle cells from rat cerebral arteries. Whole cell potassium currents at physiological membrane potentials (−40 mV) and intracellular Ca²⁺ were measured simultaneously, using the perforated patch clamp technique and a laser two-dimensional (x–y) scanning confocal microscope and the fluorescent Ca²⁺ indicator, fluo-3. Virtually all (96%) detectable Ca²⁺ sparks were associated with the activation of a spontaneous transient outward current (STOC) through K_Ca channels. A small number of sparks (5 of 128) were associated with currents smaller than 6 pA (mean amplitude, 4.7 pA, at −40 mV). Approximately 41% of STOCs occurred without a detectable Ca²⁺ spark. The amplitudes of the Ca²⁺ sparks correlated with the amplitudes of the STOCs (regression coefficient 0.8; P < 0.05). The half time of decay of Ca²⁺ sparks (56 ms) was longer than the associated STOCs (9 ms). The mean amplitude of the STOCs, which were associated with Ca²⁺ sparks, was 33 pA at −40 mV. The mean amplitude of the “sparkless” STOCs was smaller, 16 pA. The very significant increase in K_Ca channel open probability (>10⁴-fold) during a Ca²⁺ spark is consistent with local Ca²⁺ during a spark being in the order of 1–100 μM. Therefore, the increase in fractional fluorescence (F/F_o) measured during a Ca²⁺ spark (mean 2.04 F/F_o or ∼310 nM Ca²⁺) appears to significantly underestimate the local Ca²⁺ that activates K_Ca channels. These results indicate that the majority of ryanodine receptors that cause Ca²⁺ sparks are functionally coupled to K_Ca channels in the surface membrane, providing direct support for the idea that Ca²⁺ sparks cause STOCs.