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1.
The large inner membrane electrochemical driving force and restricted volume of the matrix confer unique constraints on mitochondrial ion transport. Cation uptake along with anion and water movement induces swelling if not compensated by other processes. For mitochondrial Ca2+ uptake, these include activation of countertransporters (Na+/Ca2+ exchanger and Na+/H+ exchanger) coupled to the proton gradient, ultimately maintained by the proton pumps of the respiratory chain, and Ca2+ binding to matrix buffers. Inorganic phosphate (Pi) is known to affect both the Ca2+ uptake rate and the buffering reaction, but the role of anion transport in determining mitochondrial Ca2+ dynamics is poorly understood. Here we simultaneously monitor extra- and intra-mitochondrial Ca2+ and mitochondrial membrane potential (ΔΨm) to examine the effects of anion transport on mitochondrial Ca2+ flux and buffering in Pi-depleted guinea pig cardiac mitochondria. Mitochondrial Ca2+ uptake proceeded slowly in the absence of Pi but matrix free Ca2+ ([Ca2+]mito) still rose to ∼50 μm. Pi (0.001–1 mm) accelerated Ca2+ uptake but decreased [Ca2+]mito by almost 50% while restoring ΔΨm. Pi-dependent effects on Ca2+ were blocked by inhibiting the phosphate carrier. Mitochondrial Ca2+ uptake rate was also increased by vanadate (Vi), acetate, ATP, or a non-hydrolyzable ATP analog (AMP-PNP), with differential effects on matrix Ca2+ buffering and ΔΨm recovery. Interestingly, ATP or AMP-PNP prevented the effects of Pi on Ca2+ uptake. The results show that anion transport imposes an upper limit on mitochondrial Ca2+ uptake and modifies the [Ca2+]mito response in a complex manner. 相似文献
2.
Frank E. Nelson Stephen Hollingworth Lawrence C. Rome Stephen M. Baylor 《The Journal of general physiology》2014,143(5):605-620
The mating call of the Atlantic toadfish is generated by bursts of high-frequency twitches of the superfast twitch fibers that surround the swimbladder. At 16°C, a calling period can last several hours, with individual 80–100-Hz calls lasting ∼500 ms interleaved with silent periods (intercall intervals) lasting ∼10 s. To understand the intracellular movements of Ca2+ during the intercall intervals, superfast fibers were microinjected with fluo-4, a high-affinity fluorescent Ca2+ indicator, and stimulated by trains of 40 action potentials at 83 Hz, which mimics fiber activity during calling. The fluo-4 fluorescence signal was measured during and after the stimulus trains; the signal was also simulated with a kinetic model of the underlying myoplasmic Ca2+ movements, including the binding and transport of Ca2+ by the sarcoplasmic reticulum (SR) Ca2+ pumps. The estimated total amount of Ca2+ released from the SR during a first stimulus train is ∼6.5 mM (concentration referred to the myoplasmic water volume). At 40 ms after cessation of stimulation, the myoplasmic free Ca2+ concentration ([Ca2+]) is below the threshold for force generation (∼3 µM), yet the estimated concentration of released Ca2+ remaining in the myoplasm (Δ[CaM]) is large, ∼5 mM, with ∼80% bound to parvalbumin. At 10 s after stimulation, [Ca2+] is ∼90 nM (three times the assumed resting level) and Δ[CaM] is ∼1.3 mM, with 97% bound to parvalbumin. Ca2+ movements during the intercall interval thus appear to be strongly influenced by (a) the accumulation of Ca2+ on parvalbumin and (b) the slow rate of Ca2+ pumping that ensues when parvalbumin lowers [Ca2+] near the resting level. With repetitive stimulus trains initiated at 10-s intervals, Ca2+ release and pumping come quickly into balance as a result of the stability (negative feedback) supplied by the increased rate of Ca2+ pumping at higher [Ca2+]. 相似文献
3.
Xiaoli Zhao Daiju Yamazaki Ki Ho Park Shinji Komazaki Andoria Tjondrokoesoemo Miyuki Nishi Peihui Lin Yutaka Hirata Marco Brotto Hiroshi Takeshima Jianjie Ma 《The Journal of biological chemistry》2010,285(48):37370-37376
The sarcoplasmic reticulum (SR) of skeletal muscle contains K+, Cl−, and H+ channels may facilitate charge neutralization during Ca2+ release. Our recent studies have identified trimeric intracellular cation (TRIC) channels on SR as an essential counter-ion permeability pathway associated with rapid Ca2+ release from intracellular stores. Skeletal muscle contains TRIC-A and TRIC-B isoforms as predominant and minor components, respectively. Here we test the physiological function of TRIC-A in skeletal muscle. Biochemical assay revealed abundant expression of TRIC-A relative to the skeletal muscle ryanodine receptor with a molar ratio of TRIC-A/ryanodine receptor ∼5:1. Electron microscopy with the tric-a−/− skeletal muscle showed Ca2+ overload inside the SR with frequent formation of Ca2+ deposits compared with the wild type muscle. This elevated SR Ca2+ pool in the tric-a−/− muscle could be released by caffeine, whereas the elemental Ca2+ release events, e.g. osmotic stress-induced Ca2+ spark activities, were significantly reduced likely reflecting compromised counter-ion movement across the SR. Ex vivo physiological test identified the appearance of “alternan” behavior with isolated tric-a−/− skeletal muscle, i.e. transient and drastic increase in contractile force appeared within the decreasing force profile during repetitive fatigue stimulation. Inhibition of SR/endoplasmic reticulum Ca2+ ATPase function could lead to aggravation of the stress-induced alternans in the tric-a−/− muscle. Our data suggests that absence of TRIC-A may lead to Ca2+ overload in SR, which in combination with the reduced counter-ion movement may lead to instability of Ca2+ movement across the SR membrane. The observed alternan behavior with the tric-a−/− muscle may reflect a skeletal muscle version of store overload-induced Ca2+ release that has been reported in the cardiac muscle under stress conditions. 相似文献
4.
Zhenghang Zhao Richard Gordan Hairuo Wen Nadezhda Fefelova Wei-Jin Zang Lai-Hua Xie 《PloS one》2013,8(11)
Recent studies have suggested that mitochondria may play important roles in the Ca2+ homeostasis of cardiac myocytes. However, it is still unclear if mitochondrial Ca2+ flux can regulate the generation of Ca2+ waves (CaWs) and triggered activities in cardiac myocytes. In the present study, intracellular/cytosolic Ca2+ (Cai
2+) was imaged in Fluo-4-AM loaded mouse ventricular myocytes. Spontaneous sarcoplasmic reticulum (SR) Ca2+ release and CaWs were induced in the presence of high (4 mM) external Ca2+ (Cao
2+). The protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) reversibly raised basal Cai
2+ levels even after depletion of SR Ca2+ in the absence of Cao
2+ , suggesting Ca2+ release from mitochondria. FCCP at 0.01 - 0.1 µM partially depolarized the mitochondrial membrane potential (Δψ
m) and increased the frequency and amplitude of CaWs in a dose-dependent manner. Simultaneous recording of cell membrane potentials showed the augmentation of delayed afterdepolarization amplitudes and frequencies, and induction of triggered action potentials. The effect of FCCP on CaWs was mimicked by antimycin A (an electron transport chain inhibitor disrupting Δψ
m) or Ru360 (a mitochondrial Ca2+ uniporter inhibitor), but not by oligomycin (an ATP synthase inhibitor) or iodoacetic acid (a glycolytic inhibitor), excluding the contribution of intracellular ATP levels. The effects of FCCP on CaWs were counteracted by the mitochondrial permeability transition pore blocker cyclosporine A, or the mitochondrial Ca2+ uniporter activator kaempferol. Our results suggest that mitochondrial Ca2+ release and uptake exquisitely control the local Ca2+ level in the micro-domain near SR ryanodine receptors and play an important role in regulation of intracellular CaWs and arrhythmogenesis. 相似文献
5.
O. DeIbono 《The Journal of membrane biology》1995,146(1):91-99
Ca2+ transients and the rate of Ca2+ release (dCaREL/dt) from the sarcoplasmic reticulum (SR) in voltage-clamped, fast-twitch skeletal muscle fibers from the rat were studied with the double Vaseline gap technique and using mag-fura-2 and fura-2 as Ca2+ indicators. Single pulse experiments with different returning potentials showed that Ca2+ removal from the myoplasm is voltage independent. Thus, the myoplasmic Ca2+ removal (dCaREM/dt) was studied by fitting the decaying phase of the Ca2+ transient (Melzer, Ríos & Schneider, 1986) and dCaREL/dt was calculated as the difference between dCa/dt and dCaREM/dt. The fast Ca2+ release decayed as a consequence of Ca2+ inactivation of Ca2+ release. Double pulse experiments showed inactivation of the fast Ca2+ release depending on the prepulse duration. At constant interpulse interval, long prepulses (200 msec) induced greater inactivation of the fast Ca2+ release than shorter depolarizations (20 msec). The correlation (r) between the myoplasmic [Ca2+]i and the inhibited amount of Ca2+ release was 0.98. The [Ca2+]i for 50% inactivation of dCaREL/dt was 0.25 m, and the minimum number of sites occupied by Ca2+ to inactivate the Ca2+ release channel was 3.0. These data support Ca2+ binding and inactivation of SR Ca2+ release.This work was supported by Grant-in-Aid from the American Heart Association (National) and Muscular Dystrophy Association (USA). Part of this work was developed in Dr. Stefani's laboratory at Baylor College of Medicine. 相似文献
6.
Sarcoplasmic Reticulum K+ (TRIC) Channel Does Not Carry Essential Countercurrent during Ca2+ Release
Tao Guo Alma Nani Stephen Shonts Matthew Perryman Haiyan Chen Thomas Shannon Dirk Gillespie Michael Fill 《Biophysical journal》2013,105(5):1151-1160
The charge translocation associated with sarcoplasmic reticulum (SR) Ca2+ efflux is compensated for by a simultaneous SR K+ influx. This influx is essential because, with no countercurrent, the SR membrane potential (Vm) would quickly (<1 ms) reach the Ca2+ equilibrium potential and SR Ca2+ release would cease. The SR K+ trimeric intracellular cation (TRIC) channel has been proposed to carry the essential countercurrent. However, the ryanodine receptor (RyR) itself also carries a substantial K+ countercurrent during release. To better define the physiological role of the SR K+ channel, we compared SR Ca2+ transport in saponin-permeabilized cardiomyocytes before and after limiting SR K+ channel function. Specifically, we reduced SR K+ channel conduction 35 and 88% by replacing cytosolic K+ for Na+ or Cs+ (respectively), changes that have little effect on RyR function. Calcium sparks, SR Ca2+ reloading, and caffeine-evoked Ca2+ release amplitude (and rate) were unaffected by these ionic changes. Our results show that countercurrent carried by SR K+ (TRIC) channels is not required to support SR Ca2+ release (or uptake). Because K+ enters the SR through RyRs during release, the SR K+ (TRIC) channel most likely is needed to restore trans-SR K+ balance after RyRs close, assuring SR Vm stays near 0 mV. 相似文献
7.
Single channel properties of cardiac and fast-twitch skeletal muscle sarcoplasmic reticulum (SR) release channels were compared in a planar bilayer by fusing SR membranes in a Cs+-conducting medium. We found that the pharmacology, Cs+ conductance and selectivity to monovalent and divalent cations of the two channels were similar. The cardiac SR channel exhibited multiple kinetic states. The open and closed lifetimes were not altered from a range of 10–7 to 10–3 M Ca2+, but the proportion of closed and open states shifted to shorter closings and openings, respectively.However, while the single channel activity of the skeletal SR channel was activated and inactivated by micromolar and millimolar Ca2+, respectively, the cardiac SR channel remained activated in the presence of high [Ca2+]. In correlation to these studies, [3H]ryanodine binding by the receptors of the two channel receptors was inhibited by high [Ca2+] in skeletal but not in cardiac membranes in the presence of adenine nucleotides. There is, however, a minor inhibition of [3H]ryanodine binding of cardiac SR at millimolar Ca2+ in the absence of adenine nucleotides.When Ca2+-induced Ca2+ release was examined from preloaded native SR vesicles, the release rates followed a normal biphasic curve, with Ca2+-induced inactivation at high [Ca2+] for both cardiac and skeletal SR. Our data suggest that the molecular basis of regulation of the SR Ca2+ release channel in cardiac and skeletal muscle is different, and that the cardiac SR channel isoform lacks a Ca2+-inactivated site.This work was supported by research grants from the National Institutes of Health HL13870 and AR38970, and the Texas Affiliate of the American Heart Association, 91A-188. M. Fill was the recipient of an NIH fellowship AR01834. 相似文献
8.
Do Han Kim Young Sup Lee Arthur B. Landry III 《Molecular and cellular biochemistry》1992,114(1-2):105-108
Ca2+ release from skeletal sarcoplasmic reticulum (SR) could be regulated by at least three mechanisms: 1) Ca2+, 2) calmodulin, and 3) Ca2+/calmodulin-dependent phosphorylation. Bell-shaped Ca2+-dependence, of Ca2+ release from both actively- and passively-loaded SR vesicles suggest that opening and closing of the Ca2+ release channel could be regulated by [Ca2+
o] . The time- and concentration-dependent inhibition of Ca 2+ release from skeletal SR by calmodulin was also studied using passively-Ca2+ loaded SR vesicles. Up to 50% of Ca 2+ release was inhibited by calmodulin (0.01–0.5 µM); this inhibition required 5–15 min preincubation time. The hypothesis that Ca2+/calmodulin-dependent phosphorylation of a 60 kDa protein regulates Ca2+ release from skeletal SR was tested by stopped-flow fluorometry using passively-Ca2+-loaded SR vesicles. Approximately 80% of the initial rates of Ca2+-induced Ca2+ release was inhibited by the phosphorylation within 2 min of incubation of the SR with Mg·ATP and calmodulin. We identified two types of 60 kDa phosphoproteins in the rabbit skeletal SR, which was distinguished by solubility of the protein in CHAPS. The CHAPS-soluble 60 kDa phosphoprotein was purified by column chromatography on DEAE-Sephacel, heparin-agarose, and hydroxylapatite. Analyses of the purified protein indicate that the CHAPS-soluble 60 kDa protein is an isoform of phosphoglucomutase (PGM). cDNAs encoding isoforms of PGM were cloned and sequenced using synthetic oligonucleotides. Two types of PGM isoforms (Type I and Type 11) were identified. The translated amino acid sequences show that Type II isoform is SR-form. Our results are significant in terms of understanding evidence of an association of glycolytic and glycogenolytic enzymes with SR and a role in the regulation of SR functions. (Mol Cell Biochem 114: 105-108, 1992) 相似文献
9.
Kazuo Yamasaki Takashi Daiho Stefania Danko Hiroshi Suzuki 《The Journal of biological chemistry》2015,290(46):27868-27879
The mechanism whereby events in and around the catalytic site/head of Ca2+-ATPase effect Ca2+ release to the lumen from the transmembrane helices remains elusive. We developed a method to determine deoccluded bound Ca2+ by taking advantage of its rapid occlusion upon formation of E1PCa2 and of stabilization afforded by a high concentration of Ca2+. The assay is applicable to minute amounts of Ca2+-ATPase expressed in COS-1 cells. It was validated by measuring the Ca2+ binding properties of unphosphorylated Ca2+-ATPase. The method was then applied to the isomerization of the phosphorylated intermediate associated with the Ca2+ release process E1PCa2 → E2PCa2 → E2P + 2Ca2+. In the wild type, Ca2+ release occurs concomitantly with EP isomerization fitting with rate-limiting isomerization (E1PCa2 → E2PCa2) followed by very rapid Ca2+ release. In contrast, with alanine mutants of Leu119 and Tyr122 on the cytoplasmic part of the second transmembrane helix (M2) and Ile179 on the A domain, Ca2+ release in 10 μm Ca2+ lags EP isomerization, indicating the presence of a transient E2P state with bound Ca2+. The results suggest that these residues function in Ca2+ affinity reduction in E2P, likely via a structural rearrangement at the cytoplasmic part of M2 and a resulting association with the A and P domains, therefore leading to Ca2+ release. 相似文献
10.
Arturo Hernández-Cruz Ariel L. Escobar Nicolás Jiménez 《The Journal of general physiology》1997,109(2):147-167
The role of ryanodine-sensitive intracellular Ca2+ stores present in nonmuscular cells is not yet completely understood. Here we examine the physiological parameters determining the dynamics of caffeine-induced
Ca2+ release in individual fura-2–loaded sympathetic neurons. Two ryanodine-sensitive release components were
distinguished: an early, transient release (TR) and a delayed, persistent release (PR). The TR component shows
refractoriness, depends on the filling status of the store, and requires caffeine concentrations ≥10 mM. Furthermore, it is selectively suppressed by tetracaine and intracellular BAPTA, which interfere with Ca2+-mediated feedback loops, suggesting that it constitutes a Ca2+-induced Ca2+-release phenomenon. The dynamics of release is
markedly affected when Sr2+ substitutes for Ca2+, indicating that Sr2+ release may operate with lower feedback
gain than Ca2+ release. Our data indicate that when the initial release occurs at an adequately fast rate, Ca2+ triggers further release, producing a regenerative response, which is interrupted by depletion of releasable Ca2+ and
Ca2+-dependent inactivation. A compartmentalized linear diffusion model can reproduce caffeine responses:
When the Ca2+ reservoir is full, the rapid initial Ca2+ rise determines a faster occupation of the ryanodine receptor Ca2+ activation site giving rise to a regenerative release. With the store only partially loaded, the slower initial
Ca2+ rise allows the inactivating site of the release channel to become occupied nearly as quickly as the activating
site, thereby suppressing the initial fast release. The PR component is less dependent on the store''s Ca2+ content.
This study suggests that transmembrane Ca2+ influx in rat sympathetic neurons does not evoke widespread amplification by CICR because of its inability to raise [Ca2+] near the Ca2+ release channels sufficiently fast to overcome
their Ca2+-dependent inactivation. Conversely, caffeine-induced Ca2+ release can undergo considerable amplification especially when Ca2+ stores are full. We propose that the primary function of ryanodine-sensitive stores in
neurons and perhaps in other nonmuscular cells, is to emphasize subcellular Ca2+ gradients resulting from agonist-induced intracellular release. The amplification gain is dependent both on the agonist concentration and on
the filling status of intracellular Ca2+ stores. 相似文献
11.
Alain Lacampagne Michael G. Klein Martin F. Schneider 《The Journal of general physiology》1998,111(2):207-224
The modulation by internal free [Mg2+] of spontaneous calcium release events (Ca2+ “sparks”) from the sarcoplasmic reticulum (SR) was studied in depolarized notched frog skeletal muscle fibers using a laser scanning confocal microscope in line-scan mode (x vs. t). Over the range of [Mg2+] from 0.13 to 1.86 mM, decreasing the [Mg2+] induced an increase in the frequency of calcium release events in proportion to [Mg2+]−1.6. The change of event frequency was not due to changes in [Mg-ATP] or [ATP]. Analysis of individual SR calcium release event properties showed that the variation in event frequency induced by the change of [Mg2+] was not accompanied by any changes in the spatiotemporal spread (i.e., spatial half width or temporal half duration) of Ca2+ sparks. The increase in event frequency also had no effect on the distribution of event amplitudes. Finally, the rise time of calcium sparks was independent of the [Mg2+], indicating that the open time of the SR channel or channels underlying spontaneous calcium release events was not altered by [Mg2+] over the range tested. These results suggest that in resting skeletal fibers, [Mg2+] modulates the SR calcium release channel opening frequency by modifying the average closed time of the channel without altering the open time. A kinetic reaction scheme consistent with our results and those of bilayer and SR vesicle experiments indicates that physiological levels of resting Mg2+ may inhibit channel opening by occupying the site for calcium activation of the SR calcium release channel. 相似文献
12.
Sophia von Stockum Valentina Giorgio Elena Trevisan Giovanna Lippe Gary D. Glick Michael A. Forte Caterina Da-Rè Vanessa Checchetto Gabriella Mazzotta Rodolfo Costa Ildikò Szabò Paolo Bernardi 《The Journal of biological chemistry》2015,290(8):4537-4544
Mitochondria of Drosophila melanogaster undergo Ca2+-induced Ca2+ release through a putative channel (mCrC) that has several regulatory features of the permeability transition pore (PTP). The PTP is an inner membrane channel that forms from F-ATPase, possessing a conductance of 500 picosiemens (pS) in mammals and of 300 pS in yeast. In contrast to the PTP, the mCrC of Drosophila is not permeable to sucrose and appears to be selective for Ca2+ and H+. We show (i) that like the PTP, the mCrC is affected by the sense of rotation of F-ATPase, by Bz-423, and by Mg2+/ADP; (ii) that expression of human cyclophilin D in mitochondria of Drosophila S2R+ cells sensitizes the mCrC to Ca2+ but does not increase its apparent size; and (iii) that purified dimers of D. melanogaster F-ATPase reconstituted into lipid bilayers form 53-pS channels activated by Ca2+ and thiol oxidants and inhibited by Mg2+/γ-imino ATP. These findings indicate that the mCrC is the PTP of D. melanogaster and that the signature conductance of F-ATPase channels depends on unique structural features that may underscore specific roles in different species. 相似文献
13.
Giuseppe Inesi Francesco Tadini-Buoninsegni 《Journal of cell communication and signaling》2014,8(1):5-11
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. 相似文献
14.
Ben Short 《The Journal of general physiology》2021,153(2)
JGP study reveals that insufficient reuptake of calcium into the sarcoplasmic reticulum underlies arrhythmogenic variations in cardiac calcium transients.Ca2+ alternans (Ca-Alts) are beat-to-beat changes in the amplitude of the Ca2+ transients evoked in cardiomyocytes, which can lead to arrhythmias and sudden cardiac death. Ca-Alts can be induced by an elevated heart rate (tachycardia) or metabolic impairments such as ischemia or hypothermia, but the molecular mechanisms underlying the phenomenon are unclear. In this issue of JGP, Millet et al. reveal that Ca-Alts arise when SERCA pumps are unable to fully replenish Ca2+ levels in the SR (1).Jose Millet, Yuriana Aguilar-Sanchez, Ariel L. Escobar (left to right), and colleagues investigate the mechanisms underlying arrhythmogenic Ca-Alts. The FLOM technique shows how these beat-to-beat changes in Ca2+ transients can be induced in intact hearts by increased heart rate and local reductions in temperature produced by a cold finger. The researchers find that Ca-Alts result from insufficient replenishment of SR Ca2+ levels by SERCA pumps.“Ca-Alts are very arrhythmogenic,” says Ariel L. Escobar, a professor at the University of California, Merced. “If you develop these alternans, you have a very high chance of suffering ventricular fibrillation.”Yet the mechanisms underlying Ca-Alts remain unclear. Though they appear to involve changes in the amount of Ca2+ released from the SR (2,3,4), Ca-Alts could be triggered by variations in the duration of action potentials (APD-Alts) that stimulate calcium-induced calcium release, an incomplete recovery of the ryanodine receptor that releases Ca2+ from the SR, or incomplete refilling of the SR by SERCA ATPases.To investigate the phenomenon in more detail, Escobar and colleagues, including co-first authors Jose Millet and Yuriana Aguilar-Sanchez, developed a new technique called fluorescence local field optical mapping (FLOM), which uses optical conduits containing >70,000 optical fibers to map the fluorescence of calcium-sensitive or potentiometric dyes in the epicardium of intact mouse hearts. “This approach allows us to study the spatiotemporal dynamics of calcium and membrane potential changes in a functional heart,” Escobar explains.FLOM imaging confirmed that Ca-Alts can be induced by increased heart rate and/or global reductions in temperature, two conditions that also induce APD-Alts. More crucially, however, Escobar and colleagues used a small, crescent-shaped cold finger to show that local reductions in tissue temperature also induce Ca-Alts but do not cause APD-Alts, demonstrating that the two phenomena can be uncoupled and that Ca-Alts are not driven by changes in action potential duration.Because the crescent-shaped cold finger creates a temperature gradient within the epicardium, Escobar and colleagues were able to carefully analyze the temperature dependence of Ca2+ dynamics. The relaxation of Ca2+ transients becomes gradually slower at lower temperatures, and a thermodynamic analysis of this process suggested that it involves not only active mechanisms—such as the ATPases that pump Ca2+ into the SR—but also passive mechanisms such as diffusion and binding to cytosolic buffers.In contrast, the relatively steep temperature dependence of Ca-Alts indicated that they exclusively depend on an active process like SERCA-mediated Ca2+ reuptake into the SR. Indeed, Escobar and colleagues found that the Q10 temperature coefficient of Ca-Alts is remarkably similar to the Q10 of SERCA-mediated Ca2+ transport in vitro.To confirm the importance of Ca2+ reuptake in Ca-Alts, Escobar and colleagues treated hearts with the SERCA inhibitor Thapsigargin. Partial blockade of SERCA-mediated reuptake enhanced the level of Ca-Alts, the researchers found, indicating that Ca-Alts are induced when SERCA pumps fail to fully replenish SR Ca2+ stores between heart beats. This could occur when the heart is beating particularly fast or when the metabolic activity of cardiomyocytes is impaired by, for example, low temperatures.Escobar’s team is now developing a needle-shaped optical conduit that can be used to probe any layer within the ventricular wall. “We hope to measure Ca-Alts in each layer, including the endocardium where SERCA levels are lower and Ca-Alts tend to be initiated,” Escobar says. 相似文献
15.
Mitochondrial Ca2+ uptake exerts dual effects on mitochondria. Ca2+ accumulation in the mitochondrial matrix dissipates membrane potential (ΔΨm), but Ca2+ binding of the intramitochondrial enzymes accelerates oxidative phosphorylation, leading to mitochondrial hyperpolarization. The levels of matrix free Ca2+ ([Ca2+]m) that trigger these metabolic responses in mitochondria in nerve terminals have not been determined. Here, we estimated [Ca2+]m in motor neuron terminals of Drosophila larvae using two methods: the relative responses of two chemical Ca2+ indicators with a 20-fold difference in Ca2+ affinity (rhod-FF and rhod-5N), and the response of a low-affinity, genetically encoded ratiometric Ca2+ indicator (D4cpv) calibrated against known Ca2+ levels. Matrix pH (pHm) and ΔΨm were monitored using ratiometric pericam and tetramethylrhodamine ethyl ester probe, respectively, to determine when mitochondrial energy metabolism was elevated. At rest, [Ca2+]m was 0.22 ± 0.04 μM, but it rose to ∼26 μM (24.3 ± 3.4 μM with rhod-FF/rhod-5N and 27.0 ± 2.6 μM with D4cpv) when the axon fired close to its endogenous frequency for only 2 s. This elevation in [Ca2+]m coincided with a rapid elevation in pHm and was followed by an after-stimulus ΔΨm hyperpolarization. However, pHm decreased and no ΔΨm hyperpolarization was observed in response to lower levels of [Ca2+]m, up to 13.1 μM. These data indicate that surprisingly high levels of [Ca2+]m are required to stimulate presynaptic mitochondrial energy metabolism. 相似文献
16.
Adrian M. Duke Philip M. Hopkins Sarah C. Calaghan Jane P. Halsall Derek S. Steele 《The Journal of biological chemistry》2010,285(33):25645-25653
In malignant hyperthermia (MH), mutations in RyR1 underlie direct activation of the channel by volatile anesthetics, leading to muscle contracture and a life-threatening increase in core body temperature. The aim of the present study was to establish whether the associated depletion of sarcoplasmic reticulum (SR) Ca2+ triggers sarcolemmal Ca2+ influx via store-operated Ca2+ entry (SOCE). Samples of vastus medialis muscle were obtained from patients undergoing assessment for MH susceptibility using the in vitro contracture test. Single fibers were mechanically skinned, and confocal microscopy was used to detect changes in [Ca2+] either within the resealed t-system ([Ca2+]t-sys) or within the cytosol. In normal fibers, halothane (0.5 mm) failed to initiate SR Ca2+ release or Ca2+t-sys depletion. However, in MH-susceptible (MHS) fibers, halothane induced both SR Ca2+ release and Ca2+t-sys depletion, consistent with SOCE. In some MHS fibers, halothane-induced SR Ca2+ release took the form of a propagated wave, which was temporally coupled to a wave of Ca2+t-sys depletion. SOCE was potently inhibited by “extracellular” application of a STIM1 antibody trapped within the t-system but not when the antibody was denatured by heating. In conclusion, (i) in human MHS muscle, SR Ca2+ depletion induced by a level of volatile anesthetic within the clinical range is sufficient to induce SOCE, which is tightly coupled to SR Ca2+ release; (ii) sarcolemmal STIM1 has an important role in regulating SOCE; and (iii) sustained SOCE from an effectively infinite extracellular Ca2+ pool may contribute to the maintained rise in cytosolic [Ca2+] that underlies MH. 相似文献
17.
Lukas Jaroslaw Motloch Robert Larbig Tina Gebing Sara Reda Astrid Schwaiger Johannes Leitner Martin Wolny Lars Eckardt Uta C. Hoppe 《PloS one》2016,11(2)
Introduction
The possible role of UCP2 in modulating mitochondrial Ca2+-uptake (mCa2+-uptake) via the mitochondrial calcium uniporter (MCU) is highly controversial.Methods
Thus, we analyzed mCa2+-uptake in isolated cardiac mitochondria, MCU single-channel activity in cardiac mitoplasts, dual Ca2+-transients from mitochondrial ((Ca2+)m) and intracellular compartment ((Ca2+)c) in the whole-cell configuration in cardiomyocytes of wild-type (WT) and UCP2-/- mice.Results
Isolated mitochondria showed a Ru360 sensitive mCa2+-uptake, which was significantly decreased in UCP2-/- (229.4±30.8 FU vs. 146.3±23.4 FU, P<0.05). Single-channel registrations confirmed a Ru360 sensitive voltage-gated Ca2+-channel in mitoplasts, i.e. mCa1, showing a reduced single-channel activity in UCP2-/- (Po,total: 0.34±0.05% vs. 0.07±0.01%, P<0.05). In UCP2-/- cardiomyocytes (Ca2+)m was decreased (0.050±0.009 FU vs. 0.021±0.005 FU, P<0.05) while (Ca2+)c was unchanged (0.032±0.002 FU vs. 0.028±0.004 FU, P>0.05) and transsarcolemmal Ca2+-influx was inhibited suggesting a possible compensatory mechanism. Additionally, we observed an inhibitory effect of ATP on mCa2+-uptake in WT mitoplasts and (Ca2+)m of cardiomyocytes leading to an increase of (Ca2+)c while no ATP dependent effect was observed in UCP2-/-.Conclusion
Our results indicate regulatory effects of UCP2 on mCa2+-uptake. Furthermore, we propose, that previously described inhibitory effects on MCU by ATP may be mediated via UCP2 resulting in changes of excitation contraction coupling. 相似文献18.
Yasser Aboelkassem Jordan?A. Bonilla Kimberly?J. McCabe Stuart?G. Campbell 《Biophysical journal》2015,109(10):2101-2112
Although Ca2+ is the principal regulator of contraction in striated muscle, in vitro evidence suggests that some actin-myosin interaction is still possible even in its absence. Whether this Ca2+-independent activation (CIA) occurs under physiological conditions remains unclear, as does its potential impact on the function of intact cardiac muscle. The purpose of this study was to investigate CIA using computational analysis. We added a structurally motivated representation of this phenomenon to an existing myofilament model, which allowed predictions of CIA-dependent muscle behavior. We found that a certain amount of CIA was essential for the model to reproduce reported effects of nonfunctional troponin C on myofilament force generation. Consequently, those data enabled estimation of ΔGCIA, the energy barrier for activating a thin filament regulatory unit in the absence of Ca2+. Using this estimate of ΔGCIA as a point of reference (∼7 kJ mol−1), we examined its impact on various aspects of muscle function through additional simulations. CIA decreased the Hill coefficient of steady-state force while increasing myofilament Ca2+ sensitivity. At the same time, CIA had minimal effect on the rate of force redevelopment after slack/restretch. Simulations of twitch tension show that the presence of CIA increases peak tension while profoundly delaying relaxation. We tested the model’s ability to represent perturbations to the Ca2+ regulatory mechanism by analyzing twitch records measured in transgenic mice expressing a cardiac troponin I mutation (R145G). The effects of the mutation on twitch dynamics were fully reproduced by a single parameter change, namely lowering ΔGCIA by 2.3 kJ mol−1 relative to its wild-type value. Our analyses suggest that CIA is present in cardiac muscle under normal conditions and that its modulation by gene mutations or other factors can alter both systolic and diastolic function. 相似文献
19.
Recent studies have provided evidence that depolarization in the absence of extracellular Ca2+ can trigger Ca2+ release from internal stores in a variety of neuron subtypes. Here we examine whether postganglionic sympathetic neurons are able to mobilize Ca2+ from intracellular stores in response to depolarization, independent of Ca2+ influx. We measured changes in cytosolic ΔF/F0 in individual fluo-4 –loaded sympathetic ganglion neurons in response to maintained K+ depolarization in the presence (2 mM) and absence of extracellular Ca2+ ([Ca2+]e). Progressive elevations in extracellular [K+]e caused increasing membrane depolarizations that were of similar magnitude in 0 and 2 mM [Ca2+]e. Peak amplitude of ΔF/F0 transients in 2 mM [Ca2+]e increased in a linear fashion as the membrane become more depolarized. Peak elevations of ΔF/F0 in 0 mM [Ca2+]e were ~5–10% of those evoked at the same membrane potential in 2 mM [Ca2+]e and exhibited an inverse U-shaped dependence on voltage. Both the rise and decay of ΔF/F0 transients in 0 mM [Ca2+]e were slower than those of ΔF/F0 transients evoked in 2 mM [Ca2+]e. Rises in ΔF/F0 evoked by high [K+]e in the absence of extracellular Ca2+ were blocked by thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ ATPase, or the inositol 1,4,5-triphosphate (IP3) receptor antagonists 2-aminoethoxydiphenyl borate and xestospongin C, but not by extracellular Cd2+, the dihydropyridine antagonist nifedipine, or by ryanodine at concentrations that caused depletion of ryanodine-sensitive Ca2+ stores. These results support the notion that postganglionic sympathetic neurons possess the ability to release Ca2+ from IP3-sensitive internal stores in response to membrane depolarization, independent of Ca2+ influx. 相似文献
20.
V. K. Sharma V. Ramesh C. Franzini-Armstrong S-S Sheu 《Journal of bioenergetics and biomembranes》2000,32(1):97-104
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. 相似文献