Ru360 or ruthenium red injection showed that injected Ca2+ was partly taken up by mitochondria, but Ca2+ entering through channels was not. External pH was changed using a mixture of three buffers to minimise changes in buffering power. With depolarisation-induced Ca2+ or Ba2+ loads the ΔpHs were not changed significantly over the pH range 6.5–8.5. With Ca2+ injections into cells with mitochondrial uptake blocked the ΔpHs were significantly smaller at pH 8.5 than at 7.5, but this could be explained in part by the slower rate of activity of the PMCA. Low intracellular pH also changed the ΔpHs responses to Ca2+ injection, but not significantly. Again this may have been due to reduced pump activity at low pHi. I conclude that in snail neurones the PMCA coupling ratio is either insensitive or much less sensitive to pH than in red blood cells or barnacle muscle.To explore the effects of both external and internal pH (pH In nerve cells two ATPase pumps are involved in removing Ca2+ from the cytoplasm. These are the sarco-endoplasmic reticulum calcium ATPase (SERCA) which drives uptake into the endoplasmic reticulum and the plasma-membrane calcium ATPase (PMCA) which extrudes Ca2+ across the plasma membrane. As first shown by Niggli et al. many years ago the latter couples the efflux of Ca2+ to the uptake of H+ ions 2+ pump driven by Na+ influx rather than by ATP, the Na+/Ca2+ exchanger, but in many nerve cells the PMCA is the principle Ca2+ extrusion mechanism at low Ca2+ loads Cell calcium must be kept very low to allow fast and efficient intracellular Ca2+ extrusion and H+ uptake at normal pH levels by the PMCA remains controversial. While the early workers 2+:H+ ratio was 1:2, some subsequent work has suggested fewer H+ ions are transported 2+:2H+. In contrast, in snail neurones I recently found that the ratio under normal conditions is 1Ca2+:2H+In snail neurones the PMCA is the only mechanism for calcium extrusion from the cell body o) on the coupling ratio have been studied in red blood cells and barnacle muscle, but not in nerve cells. In red blood cell PMCA preparations the Ca2+:H+ ratio is changed by pHo from about 1:2 at pH 6.5 to 1:0 at pH 8.5 i) have not apparently been investigated in any cell. The quantity of H+ ions pumped into neurones by the PMCA is important because many channels and carriers are sensitive to small pH changes The effects of extracellular pH  were measured. The two other processes that might change surface pH, namely intracellular pH regulation and H+ channels, were inhibited or kept inactivated. Normal pHi regulation was inhibited by removal of bicarbonate +/H+ exchanger + channels were kept closed by avoiding large depolarisations 2+ was not taken up by mitochondria, cells were injected with ruthenium red o I measured the ΔpHs for equal depolarisations or Ca2+ injections at different pHo values. For the experiments on pHi effects I measured the ΔpHs induced by equal Ca2+ injections while changing pHi by HCl injection or application and removal of CO2/bicarbonate. With pHo between 7.5 and 8.5 I have found significant changes in the PMCA-induced ΔpHs only with injected Ca2+ loads. Allowing for variation in the pump rate with pH, these findings suggest that the snail neurone PMCA coupling ratio is little changed by external or internal pH.To investigate the effects of pH on the coupling ratio in nerve cells I have extended my recent experiments 22.1H. aspersa2+ or Ba2+ snail Ringer's solution, or by iontophoretic injection of Ca2+ ions. The changes in surface pH (ΔpHs) resulting from the subsequent extrusion of Ba2+ or Ca2+ were measured using pH-sensitive microelectrodes which were pressed against the surface of the chosen cell. The effects of changing pHo were assessed by changing the superfusate pH using a mixture of three different buffers (with different dissociation constants) to minimise changes in buffering power. To reduce pHi, H+ ions were injected by iontophoresis, while to increase pHi the preparation was first equilibrated with bicarbonate-buffered saline and then returned to normal snail Ringer's solution. The consequent loss of accumulated intracellular bicarbonate as CO2 caused a pHi increase of 0.4–0.5 units. In some experiments changes in intracellular Ca2+ were followed with a Ca2+-sensitive microelectrode (CaSM).Experiments were done on large (150–250 μm diameter) neurones in isolated sub-oesophageal ganglia of the common snail, 2.2An aestivating snail was killed by rapid removal of the circumoesophageal ring of ganglia, and the large cells on the dorsal side of the suboesophageal ganglia exposed as previously described 2.32, 5 MgCl2, and 20 Hepes, titrated with NaOH to pH 7.5. Solutions of pH 6.5, 7.5 and 8.5 with a low buffering power had 2 or 5 mM of Pipes, Hepes and Taps, with additional NaCl to maintain tonicity. pH 9.5 Ringer was buffered with 20 mM CHES. Ba Ringer solutions had the same ionic composition but with BaCl2 replacing CaCl2. The CO2 Ringer solution was the same as normal except that it had 20 mM NaHCO3 instead of Hepes, was bubbled with 2.5% CO2 in air and contained 0.1 mM NaH2PO4.The normal snail Ringer solution contained (mM): 80 NaCl, 4 KCl, 7 CaCl−1Ruthenium red was dissolved in 0.1 M KCl at 10 mg ml2.42 with tips broken by touching a pin in the bath to give resistances of 5–10 MΩ. For H+ injection microelectrodes were pulled from quartz tubing filled with 1 M HCl and left with tips intact.Conventional micropipettes were pulled from 1.2 mm filamented borosilicate glass tubing and backfilled with 1 M CsCl for passing clamp current or recording membrane potential. Microelectrodes for iontophoretic injection were filled with 0.1 M CaClIntracellular CaSMs were made from quartz glass and pHSMs from borosilicate glass 2.5Potentials from the conventional microelectrodes, CaSMs and pHSMs were recorded as voltages referred to membrane potential, with the voltages from the pHSMs converted to pH before display. Potentials from the surface pHSMs were referred to the bath potential. Results were discarded if on withdrawal of an electrode its potential in Ringer solution had changed by more than 7 mV.Potentials from the voltage-recording microelectrodes were led via preamplifiers in the Faraday cage to an 8-pole Bessel filter and recorded at 20 Hz on a PC via a CED micro 1401 interface and Spike 2 data collection program . The clamp current was recorded at 100 Hz as above.VCa or pH records generated by electronic pickup were partially erased, and the clamp current records were in some cases restricted in range. Data are presented as means ± S.E.M. of n observations. The statistical significance of observed differences was determined by a paired two-sample two-tailed t-test for means. Differences between means were considered significant when P < 0.05.Figures were prepared from the CED data after loading into Microsoft Excel. Spikes in the 33.12+]i in the face of a continuous influx even in a cell voltage-clamped at −50 mV. To investigate the coupling between Ca2+ extrusion and H+ uptake I have chosen to stimulate the PMCA with a brief, repeated, additional load of Ca2+ or Ba2+ ions. I assume that the coupling does not change with rate of activity. A Ca2+ load can be delivered in several ways. The simplest way is by a brief depolarisation to open Ca2+ channels, while a second more controllable method is by iontophoretic injection of Ca2+ ions The PMCA is continually active, maintaining the normal low [Ca+ while an injected Ca2+ load was extruded. With a number of assumptions, including that all the injected Ca2+ was extruded, I was able to assess possible changes in coupling ratio. This last assumption, however, proved untenable, as was indeed suggested by earlier work with pressure-injected Ca2+2+, but not Ca2+ entering through channels, was taken up by mitochondria, as shown in i and VCa  and stimulated the PMCA by both depolarisations and Ca2+ injections. The first three depolarisations each caused an increase in VCa of 13 mV (i.e. an increase in intracellular Ca2+) erased and a small fall in pHi. Similarly the two iontophoretic injections of Ca2+ each caused an increase in VCa of 5 mV. Although the pHi decrease was larger for the two injections than for the depolarisations, the increase in VCa for the injections was smaller than for the depolarisations. I then injected Ru360, which blocks Ca2+ uptake by mitochondria VCa increase seen with an injection, to 19 mV, but did not alter the depolarisation-induced Ca2+ increases. The pHi responses were not obviously changed.  Similar results were obtained with ruthenium red. This suggests that a large fraction of the injected Ca2+ was taken up by the mitochondria.In initial experiments I measured the intracellular pH change resulting from the uptake of H2+ uptake blockers also increased the surface pH changes induced by injection. Such ΔpHs result from the H+ being pumped into the cell as the Ca2+ is extruded s depends on the location of the pH electrode, the local buffering power and the activity of the PMCA. If most of the injected Ca2+ is taken up by the mitochondria there will be little surface pH increase, as shown in 2+, and both pHs and VCa measured. About 7 min after the start of the recording shown, ruthenium red was injected sufficient to make the cell clearly pink.  I then resumed alternating the methods of delivering a Ca2+ load. The Ru360 had little effect on either the ΔpHs or Ca2+ increases following depolarisations, but increased both the Ca2+ transients and, spectacularly, the ΔpHs following Ca2+ injections.Mitochondrial CaVCa transient following injection by an average factor of 2.56 ± 0.57. In three experiments blocking uptake increased the surface pH changes by an average of 2.3 ± 1.1 times. On the other hand the blockers had no effect on the responses of VCa and surface pH to depolarisations. Presumably injections deliver a high concentration of Ca2+ deep in the cell, close to the mitochondria, while entry through channels delivers a lower concentration of Ca2+ all round the cell periphery, where the PMCA is located. I have therefore subsequently done experiments with Ca2+ injections only on cells injected with ruthenium red or Ru360.In a total of five similar experiments I found that blocking mitochondrial uptake increased the size of the 2+ uptake by the endoplasmic reticulum. It was without effect, after stores were empty, on any of the responses to depolarisation or injection .I have also tested cyclopiazonic acid (CPA), which blocks Casee also . It ther3.22+ solutions was 7.5, 8.5 and 6.5. Each depolarisation initiated a ΔpHs of about 0.025 units. Both the increase and decrease in superfusate pH from normal pH 7.5 appeared to reduce the size of the surface pH changes by 5–15%.2+ Ringer after an initial period in normal Ca2+ Ringer. Ba2+ is extruded by the PMCA more slowly than Ca2+2+ does not activate K+ channels, I was also able to estimate the Ba2+ influx from the charge needed to depolarise the membrane. In this experiment the depolarisations were of 20, 30, 40 and 50 mV for 5 s in each series, with the first and last series in normal Ca2+ Ringer. The increasing size of the depolarisations was designed to reveal at what potential the H+ channels opened. Again the ΔpHs were smaller in pH 6.5 and 8.5 than in 7.5, but the transient caused by the largest depolarisation in pH 8.5 were contaminated with H+ efflux through channels, as shown by the brief surface acidification.2+ and five in low buffered Ba2+ Ringer are compared in s responses to the same size depolarisation , averaging several measurements at each pH where possible. I have then calculated the ΔpHs for pH 6.5 and 8.5 as ratios of that seen at pH 7.5, and averaged these ratios for all experiments. The results show that both a pH increase and a decrease from normal apparently reduce the size of the surface pH changes, although not significantly.The results from six experiments in low buffered Ca2+ or Ba2+ entry at pH 6.5, since Ca2+ channels are well-known to be inhibited by low pH 2+ influx from the clamp current, since Ca2+ entry activates K+ channels. With Ba2+ entry, however, few K+ channels are opened, so the charge carried in by the Ba2+ is likely to be close to the total clamp current during the depolarisations 2+ influx is likely to be about 20% less than at pH 7.5.One factor that may contribute to this is a reduced Ca3.32+ injected by a given charge is unlikely to be influenced by external pH, so any variation in ΔpHs with pH will probably reflect a change in PMCA rate of activity or coupling between Ca2+ extrusion and H+ uptake. s and VCa responses to a series of Ca2+ injections made at different external pH values. The cell had been earlier injected with ruthenium red. The peak ΔpHs occurred some seconds after the end of the injection, since the injection site was deep in the cell and the diffusion of ions from the injection site is likely to take some time s responses at pH 8.5 were clearly smaller than at pH 7.5, and were followed by an acid overshoot. The quantity of Ca+VCa after depolarisation-induced Ca2+ loads. VCa record at a value of VCa which passed through all the recoveries My results show that depolarisations provide a consistent way of stimulating the PMCA, which generates surface pH increases as H2+ load and the extent of its extrusion by the PMCA, the accuracy of its measurement with a CaSM at a point in the cell, and the extent to which the surface pH change is proportional to the H+ uptake. I also assume that the PMCA coupling ratio does not change with rate of activity, since this has not been reported for any ATPase. I have made no attempt to measure the coupling ratio itself, being concerned only with its possible alteration.The validity of my measurements, and their relevance to the PMCA coupling ratio, depends on a number of assumptions. These concern the constancy of the Ca4.12+ it is likely that low pHs reduces Ca2+ influx; the sensitivity of Ca2+ channels to pH has indeed been known for many years 2+ by the same charge are not changed by either pH or time. Following my experiments with ruthenium red and Ru360, I have assumed that the pHs increase generated by a depolarisation-induced Ca2+ or Ba2+ load was generated only by the PMCA. Similarly, where a cell has been injected with Ru360 or ruthenium red I assume that most of the injected Ca2+ is rapidly extruded, and not sequestered. In previous work with very large pressure injections of CaCl2 it was concluded i decreases observed were generated largely by mitochondrial Ca2+/H+ exchange with some possible contribution from Ca2+ extrusion. Others have reported that mitochondria are involved in buffering Ca2+ loads in rat neurones I have assumed that repeated depolarisation-induced loads are relatively unaffected by time or pH. From the measurements of the charge carried by Ba2+ and Ca2+ recoveries after injection or loading by depolarisation i. Such an assumption cannot be relied on, however, at low pHi values: my previous recordings of VCa show that at low pHi values the PMCA does not always return the VCa to its pre-injection level.Previous recordings of intracellular Ba2+ uptake by the endoplasmic reticulum is little changed by intracellular acidification, release is inhibited 2+ that is rapidly pumped out by the PMCA, but I found no effect of CPA, which blocks Ca2+ uptake by the endoplasmic reticulum. All my measurements on the effects of pHi were made as pHi fell, so that release from the endoplasmic reticulum would be unlikely while I was measuring the injection-induced ΔpHs. I also found that CPA had no effect on the pH or VCa changes following Ca2+ loads.I have shown that while Ca4.2VCa after an injection or a depolarisation. The VCa was recorded at a single point inside each neurone, close to the cell membrane on the far side of the cell. Thus its recovery can be only an indirect measure of pump rate. Previous experiments with the CaSM method do suggest, however, that the decline in VCa can be fitted closely by models including intracellular diffusion and pump rate To assess the PMCA activity I measured the recovery of 4.3i regulation, was active only at a low level since bicarbonate was absent during PMCA activation. (I added bicarbonate only to accelerate pHi recovery from large acidifications, as in s overshoots seen at pHo 8.5 in i regulation activated by the transiently large H+ gradient before the H+ diffused away from the interior of the cell membrane. They suggest that residual pHi regulating mechanisms may be driven by a local pH gradient caused by the PMCA. If this was activated rapidly it would reduce the size of the observed ΔpHs. I avoided using inhibitors of pH regulation since they are only slowly reversible and tend to have multiple effects. The second non-PMCA pathway is the H+ channel, opened by large depolarisations o 8.5 I did see evidence of H+ channel opening, e.g. in + efflux produced a brief spike in pHs which may well have reduced the total PMCA effect. Measurements showing such signs of H+ efflux were excluded from the analysis. From experiments on surface pH reported earlier s changes produced by very large depolarisations are proportional to H+ flux across the cell membrane via H+ channels and to external buffering power. Previously such measurements were made at essentially the same pH. Those reported here, however, were done in three solutions of different pH using buffers with pK values of 6.8, 7.5 and 8.4. These gave a relatively constant buffering power between pH 6.5 and 8.5. Thus the same ΔpHs in this range should represent that same flux of H+ ions. This makes no allowance for a possible contribution of fixed buffers on the cell surface, about which little is known. Perhaps they make a significant contribution at low pH values.As well as the PMCA, two other membrane pathways may influence surface pH. The first, pHs at pH 8.5, then the increased pump rate at pH 6.5 should theoretically make the ΔpHs much larger. Failure to demonstrate this possibly suggests that the peak rate of the PMCA, which determines the size of the ΔpHs, was limited by availability of ATP or another factor.If the reduction in PMCA activity at high pH explains in part the reduction in the ΔpH4.4+ ions pumped in for each Ca2+ extruded was about 1.9 at pHo 6.3, but that this number fell to zero as pHo was increased to 8.0 + that were available outside the cell, the more were pumped in. With barnacle muscle similar findings have been reported o of 6.5 the authors estimated that 3H+ were taken up for each Ca2+ extruded, while at pH 8.2 only 1H+ ion was pumped in. No previous study has been published on the effects of pHi on the coupling ratio.These findings differ from those reported for the effects of extracellular pH on the coupling ratio of the PMCA of red blood cells and barnacle muscle fibres. With red blood cells it was reported that the number of H2+ efflux with 45Ca. For the red blood cell studies the H+ uptake of a large number of cells was measured by clamping extracellular pH and measuring how much acid must be added as the 45Ca was pumped out i of a single muscle fibre was followed with a microelectrode, and measured buffering power used to estimate H+ uptake 2+ or H+ movement across the cell membrane. External Na+ and Ca2+ were both removed. My experiments were done with normal Na+ and Ca2+ gradients, on single voltage-clamped cells to minimise passive ion fluxes. Cells were unavoidably loaded with Cl− via the microelectrodes, but it is hard to see how this would influence the PMCA.The sensitivity of the PMCA to pH may well be different in neurones. It is also worth noting that my methods were quite different from those used previously, and perhaps involved less disruption to the normal physiology of the cell. Both previous reports measured Ca+, as well as for its molecular mechanism.If my assumptions are correct, I conclude that in my preparation the PMCA coupling ratio is relatively insensitive to changes in either intracellular or extracellular pH. This has important implications for the power of the PMCA as a source of intracellular HThere are no conflicts of interest.


Background: Mushroom tyrosinase, a copper containing enzyme, modifies growth and survival of tumor cells. Mushroom tyrosinase may foster apoptosis, an effect in part due to interference with mitochondrial function. Erythrocytes lack mitochondria but are able to undergo apoptosis-like suicidal cell death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine-exposure at the erythrocyte surface. Signaling involved in the triggering of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i) and activation of sphingomyelinase with subsequent formation of ceramide. The present study explored, whether tyrosinase stimulates eryptosis. Methods: Cell volume has been estimated from forward scatter, phosphatidylserine-exposure from annexin V binding, [Ca2+]i from Fluo3-fluorescence, and ceramide abundance from binding of fluorescent antibodies in flow cytometry. Results: A 24 h exposure to mushroom tyrosinase (7 U/mL) was followed by a significant increase of [Ca2+]i, a significant increase of ceramide abundance, and a significant increase of annexin-V-binding. The annexin-V-binding following tyrosinase treatment was significantly blunted but not abrogated in the nominal absence of extracellular Ca2+. Tyrosinase did not significantly modify forward scatter. Conclusions: Tyrosinase triggers cell membrane scrambling, an effect, at least partially, due to entry of extracellular Ca2+ and ceramide formation. The observations disclose that exposure to tyrosinase stimulates erythrocyte cell membrane scrambling, an effect paralleled by and at least in part secondary to increase of [Ca2+]i.The present study explored, whether tyrosinase influences i). Thus, [Ca2+]i was determined utilizing Fluo3 fluorescence. To this end, erythrocytes were loaded with Fluo3-AM and Fluo3 fluorescence determined in FACS analysis following prior incubation in Ringer solution without or with tyrosinase. As shown in Cell membrane scrambling is stimulated by increase of cytosolic Ca2+ entry was required for the effect of tyrosinase on cell membrane scrambling. Erythrocytes were exposed to 7 U/mL tyrosinase for 24 h, either in the presence of 1 mM Ca2+, or in the absence of Ca2+, and the presence of Ca2+ chelator EGTA (1 mM). As illustrated in 2+. However, even in the absence of extracellular Ca2+ tyrosinase still increased the percentage annexin V binding erythrocytes.An additional series of experiments was performed testing whether extracellular Ca2+]i, ceramide abundance at the erythrocyte surface was determined utilizing an anti-ceramide antibody. As illustrated in In order to test whether tyrosinase increased the formation of ceramide, which is known to trigger eryptosis even without increase of [Ca2+ activity ([Ca2+]i) and ceramide formation.The present study uncovers that tyrosinase stimulates cell membrane scrambling leading to phosphatidylserine translocation to the erythrocyte surface. Treatment of human erythrocytes with 5 U/L tyrosinase is further followed by increase of cytosolic Ca2+]i, tyrosinase did not decrease but tended to increase the erythrocyte forward scatter. The increase of [Ca2+]i were expected to activate Ca2+ sensitive K+ channels [+ exit, cell membrane hyperpolarization, Cl− exit and, thus, cellular loss of KCl with osmotically obliged water [2+ sensitive K+ channels, blocks the Cl− channels or stimulates some other mechanism increasing cell volume. Notably, the ionomycin induced erythrocyte shrinkage was not abrogated in the presence of tyrosinase. Excessive erythrocyte swelling may eventually result in rupture of the cell membrane leading to release of cellular hemoglobin, which is filtered in renal glomerula and subsequently occludes renal tubules [Despite its effect on [Cachannels  with subed water . Possibl tubules .Eryptosis is followed by removal of the defective erythrocytes, Phosphatidylserine at the surface of eryptotic cells binds to the respective receptors of phagocytosing cells leading to subsequent engulfment of the affected erythrocytes . AccordiPhosphatidylserine exposing erythrocytes may further adhere to the vascular wall by binding of phosphatidylserine at the erythrocyte surface to endothelial CXCL16/SR-PSO . The adhin vitro at a hematocrit of 0.4% in Ringer solution containing (in mM) 125 NaCl, 5 KCl, 1 MgSO4, 32 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 5 glucose, 1 CaCl2; pH 7.4 at 37 °C for 48 h. Where indicated, erythrocytes were exposed to mushroom tyrosinase  at the indicated concentrations. In Ca2+-free Ringer solution, 1 mM CaCl2 was substituted by 1 mM glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA).Leukocyte-depleted erythrocytes were kindly provided by the blood bank of the University of Tübingen. The study is approved by the ethics committee of the University of Tübingen (184/2003V). Erythrocytes were incubated 2 and then stained with Annexin-V-FITC  in this solution at 37 °C for 20 min under protection from light. In the following, the forward scatter (FSC) of the cells was determined, and annexin-V fluorescence intensity was measured with an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur . Following treatment with Ca2+-free Ringer solution, care was taken to measure annexin V binding rapidly enough to avoid triggering of cell membrane by the addition of 5 mM Ca2+.After incubation under the respective experimental condition, 50 µL cell suspension was washed in Ringer solution containing 5 mM CaCl2 and 5 µM Fluo-3/AM. The cells were incubated at 37 °C for 30 min and washed twice in Ringer solution containing 5 mM CaCl2. The Fluo-3/AM-loaded erythrocytes were resuspended in 200 µL Ringer. Then, Ca2+-dependent fluorescence intensity was measured with an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur.After incubation erythrocytes were washed in Ringer solution and then loaded with Fluo-3/AM  in Ringer solution containing 5 mM CaClFor the determination of ceramide, a monoclonal antibody-based assay was used. After incubation, cells were stained for 1 h at 37 °C with 1 µg/mL anti ceramide antibody  in PBS containing 0.1% bovine serum albumin (BSA) at a dilution of 1:5. The samples were washed twice with PBS-BSA. Subsequently, the cells were stained for 30 min with polyclonal fluorescein isothiocyanate (FITC) conjugated goat anti-mouse IgG and IgM specific antibody  diluted 1:50 in PBS-BSA. Unbound secondary antibody was removed by repeated washing with PBS-BSA. The samples were then analyzed by flow cytometric analysis with an excitation wavelength of 488 nm and an emission wavelength of 530 nm.t-test as appropriate. n denotes the number of different erythrocyte specimens studied. As different erythrocyte specimens used in distinct experiments are differently susceptible to triggers of eryptosis, the same erythrocyte specimens have been used for control and experimental conditions.Data are expressed as arithmetic means ± SEM. As indicated in the figure legends, statistical analysis was made using ANOVA with Tukey’s test as post-test and 2+ entry, which in turn triggers cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface.Tyrosinase stimulates Ca


Activation of these non-selective channels also results in a Na+ influx. This localised increase in Na+ levels can potentially switch the Na+/Ca2+ exchanger into reverse mode and so result in a further influx of Ca2+. The aim of this study was to characterise the expression and physiological function of the Na+/Ca2+ exchanger in cultured human bronchial smooth muscle cells and determine its contribution to agonist induced Ca2+ influx into these cells.Agonist stimulation of airway smooth muscle (ASM) results in IP+/Ca2+ exchanger) homologues in cultured human bronchial smooth muscle cells was determined by reverse transcriptase PCR. The functional activity of reverse mode NCX was investigated using a combination of whole cell patch clamp, intracellular Ca2+ measurements and porcine airway contractile analyses. KB-R7943 (an antagonist for reverse mode NCX) and target specific siRNA were utilised as tools to inhibit NCX function.The expression profile of NCX (which encodes the Na+ loading and addition of extracellular Ca2+ induced an outwardly rectifying current which was augmented following stimulation with histamine. This outwardly rectifying current was inhibited by 10 μM KB-R7943 (an antagonist of reverse mode NCX1) and was reduced in cells incubated with siRNA against NCX1. Interestingly, this outwardly rectifying current was also inhibited following knockdown of STIM1, suggesting for the first time a link between store operated cation entry and NCX1 activation. In addition, 10 μM KB-R7943 inhibited agonist induced changes in cytosolic Ca2+ and induced relaxation of porcine peripheral airways.NCX1 protein was detected in cultured human bronchial smooth muscle cells (HBSMC) cells and NCX1.3 was the only mRNA transcript variant detected. A combination of intracellular Na2+ homeostasis and link store depletion via STIM1 directly with NCX activation.Taken together, these data demonstrate a potentially important role for NCX1 in control of Ca Cells were plated in 48 well plates and loaded with Fluo-4AM (Molecular probes) for 1 hour at room temperature in DMEM supplemented with 10% FCS, 2 mM glutamine and 2.5 mM probenecid . After loading, cells were washed with hanks balanced saline solution (HBSS) containing 10 mM HEPES, 2.5 mM probenecid, 2 mM CaCl2 and 1 mM MgCl2. After a 10 second baseline, cells were stimulated with either bradykinin  or histamine  for a total time period of 2 minutes. 10 μM KB-R7943 or equivalent DMSO vehicle control was added to the cells shortly before assay. Ca2+ imaging was performed on the spinning disk confocal microscope system. Confocal images (1 image/second) were collected at 525 nm emission after excitation at 491 nm. Volocity analysis software (Improvision) was used to analyze the images. Each experiment incorporated at least 5 repeat wells per condition and each experiment was repeated at least 4 times. Data are presented as changes in mean fluorescence intensity (FI) compared with the baseline and peak fluorescence values was used as an estimation of changes in [Ca2+]i.Intracellular Ca2+ to the one containing 2 mM Ca2+ with or without reduced 5 mM Na+ as previously described [2+-free solution as previously described [2, 0 mM or 2 mM CaCl2, 10 μM nifedipine, 20 μM ouabain, 2 μM ryanodine, 100 μM niflumic acid and 5 mM HEPES (pH = 7.4). The electrode solution contained: 120 mM Cs-methanesulfonate, 20 or 40 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, 10 mM HEPES (pH = 7.2 adjusted with cesium hydroxide and methanesulfonic acid). The intracellular free Ca2+ concentration  was 14 nM. Pipettes were drawn from borosilicate glass and had a resistance of 5-8MΩ when filled with internal solution. Average junction potentials of ~-10 mV between bath solution and electrode solution were generated and corrected before data acquisition by use of Patchmaster. Experimental drugs were delivered through a pressurised puffer pipette positioned 50 μm around the cells followed by a separate pressurised pipette used to wash out drugs . The bath solution was gravity perfused constantly from a 50 ml syringe at a flow rate of ~1 ml/minute. Cells were held at a membrane potential of -40 mV and current-voltage relationships were analyzed every 1 s from voltage ramps from -100 to +100 mV (1 Hz) at a rate of 0.5 V·s-1. Currents were filtered at 1 kHz and sampled at 4 KHz. Capacitance and series resistance readings were continuously monitored and the recording was discarded if a significant change occurred in either parameter. Current density at -80 mV and +80 mV were calculated and plotted against time following re-addition of Ca2+. By convention, positive (outward) currents taken at +80 mV reflect reverse-mode NCX-1 currents as the stoichiometry of this exchanger is 3Na+:1Ca2+ [2+-free solution were subtracted in all groups for the purpose of comparison between groups (as stated in figure legends). Measurements were taken at 41 seconds unless otherwise stated .Whole porcine lungs were obtained from a local abattoir (Nottingham). An isolated, distal lobe was inflated to ~90% capacity with 2% agarose  solution (in HBSS) by inserting a tube via the trachea; the airways were cleared of agarose by injecting a pulse of air. Slices were cut ~130 μM thick using a vibrotome tissue slicer . Lung slices were transferred to DMEM, supplemented with 2 mM L-glutamine and penicillin-streptomycin and incubated for 2~3 days at 37°C/5% COUnless otherwise stated, statistical significance between groups was assessed by unpaired T tests where *p < 0.05, **p < 0.01 were considered significant.RT-PCR revealed that only NCX1 was present in HBSMCs Figure . Human bOutwardly rectifying or reverse mode NCX1 currents in HBSMCs were recorded using the modified whole cell voltage clamp technique as previously described ,22,23.2+-free extracellular solution containing 140 mM Na+. Extracellular Ca2+ was applied to the Na+ loaded cell along with physiological (140 mM) or reduced (5 mM) Na+ to induce an outwardly rectifying current mediated by reverse mode NCX1 i.e. 3Na+ efflux and 1Ca2+ influx according to the stoichiometry of this exchanger (3Na+:1Ca2+). The intracellular solution was buffered to low Ca2+ (~14 nM), and high Na+ (20 mM) which favors reverse mode NCX1. Under these conditions, perfusion with Ca2+ free extracellular solution with 140 mM Na+ induced a current, with a linear current-voltage relationship and a reversing potential of ~ 0 mV . A typical example of the current activated by extracellular application of 2 mM Ca2+ is illustrated in Figure + was reduced from 140 mM to 5 mM , the outwardly rectifying current was significantly augmented. The peak current density following extracellular 2 mM Ca2+ re-addition with 5 mM Na+ was increased by 243% compared to that with 140 mM Na+. This increase in outwardly rectifying current was also abolished by 10 μM KB-R7943 . Outwardly rectifying currents following re-addition of extracellular Ca2+ were markedly enhanced in the presence of HA  activates a non-selective current in HBSMCs which is mediated by STIM1 [2+/ATPase and mediates passive depletion of intracellular store Ca2+. However, the protocol we describe in this present study includes a prolonged perfusion with Ca2+ free extracellular buffer in the presence of ryanodine and so the intracellular stores are likely to be already depleted. This may go some way to explain why the inclusion of CPA did not significantly enhance the NCX1 outwardly rectifying current proposed to be mediated by Na+ influx through SOC channels.These results demonstrate that an outwardly rectifying current mediated by NCX1 is activated by HA in cultured HBSMCs. We next tested the hypothesis that Naby STIM1 . Indeed,2+ free extracellular buffer and the addition of HA induced a Na+ mediated current which was completely abolished following siRNA mediated knockdown of STIM1 , HBSMCs respond to both BK and HA with a reduced intracellular Ca2+ signal. Peak BK induced fluorescence signals were reduced by 41% (n = 6) and peak HA signals were reduced by 25% (n = 4).We next investigated the effects of 10 μM KB-R7943 on CaThe effects of KB-R7943 on porcine airway contraction were next investigated. Lung slices were prepared as described in the methods section. Small peripheral airways (approximately 150 μm in diameter) with visibly beating cilia were selected for experimentation. After establishing a baseline where the airway was fully relaxed, 1 μM ACh or 10 μM HA was perfused over the lung slice to induce a sustained contraction. Following this, increasing additive concentrations of KB-R7943 (0.1 μM -100 μM), in the continual presence of agonist was perfused over the slice. KB-R7943 dose dependently relaxed the pre-contracted airways, with a noticeable relaxation occurring from 10 μM KB-R7943. Figure 2+ influx in HBSMCs. We also provide additional evidence in support of an existing hypothesis whereby Na+ influx through SOC channels activates reverse mode NCX; to our knowledge we are the first to show a link between STIM1 mediated Na+ influx and the activation of NCX1.This present study provides, for the first time, molecular and electrophysiological data as well as cellular and tissue pharmacological evidence supporting the presence of a functional NCX1 protein that can operate in reverse mode and thus presents an additional mechanism of CaWe initially confirmed expression of NCX1 mRNA and protein in HBSMCs. The splice variant we identified was NCX1.3 and is consistent with previous findings . We coul2+ signals in these cells. NCX1.1 is the splice variant expressed in cardiomyocytes and has been extensively studied and its electrophysiological properties are well characterized [Following confirmation of the expression of NCX1.3 in cultured HBSMCs, we then studied the functional role of this protein and its contribution to agonist evoked Cacterized . Few stu+ through perfusion with a Ca2+ free extracellular buffer and the addition of ouabain. Reverse mode NCX1 was subsequently activated via re-addition of 2 mM extracellular Ca2+. The outwardly rectifying current displayed typical characteristics to that of Na+ efflux through NCX1 and were inhibited by KB-R7943 (a relatively selective inhibitor of reverse mode NCX1). These currents were greatly enhanced by stimulation with the contractile agonist HA and these HA enhanced currents were inhibited by KB-R7943. However, because at high concentrations KB-R7943 can have effects on other channels, we also used knockdown of NCX1 by siRNA, with similar results. Perfusion with Ca2+ free extracellular buffer activates a SOC like current prior to the activation of the NCX1 mediated outwardly rectifying current. Interestingly, inhibition of this SOC current by prior knockdown of STIM1 also inhibited HA induced NCX1 currents. As far as we are aware, these are the first data that provide evidence of a functional link between STIM1 mediated SOC activation and reverse mode NCX1 activation.Experimental conditions were adjusted to favour the activation of reverse mode NCX1 currents. Cells were first loaded with Na2+ signals and contraction of ASM. The addition of 10 μM KB-R7943 almost completely abolished NCX1 sensitive currents. However, in comparison the equivalent concentration of inhibitor only marginally inhibited HA induced Ca2+ signals. The experimental conditions used to yield the electrophysiology results were designed to optimise and isolate NCX1 currents. Experimental conditions implemented in Figure 2+ influx pathways (insensitive to NCX1 inhibition) would have been activated. Inhibition of reverse mode NCX1 by KB-R7943 resulted in dose dependent relaxation of pre-contracted small airways, illustrating the functional importance of NCX1 in maintaining contraction. Addition of KB-R7943 also inhibited both BK and HA induced intracellular Ca2+ signals in HBSMCs.The electrophysiology data provided evidence of a functionally active NCX1 that is activated following HA induced SOC activation. We then further tested its physiological relevance through measurement of agonist induced Ca2+ handling within ASM is likely to contribute to inappropriate contraction, a major symptom of asthma. The understanding of how intracellular Ca2+ is regulated under physiological and pathophysiological situations forms an important aspect in the search for new therapeutic targets for the treatment of asthma. In recent years, there have been major advances in the understanding of Ca2+ homeostasis driven in part by the identification of STIM1 and ORAI1 as critical regulators of Ca2+ influx in many tissue types. In HBSMCs, we have shown that the interaction between STIM1 and ORAI1 plays an important role in the activation of SOC channels [3 mediated Ca2+ store release, resulting in store depletion of Ca2+. This in turn activates SOC channels via a STIM1 dependent mechanism. Activation of SOC channels permits influx of Na+ . This localized increase of intracellular Na+ results in membrane depolarization that activates reverse mode NCX1 to further promote Ca2+ influx.Abnormal Cachannels . The pre2+ homeostasis in ASM. This role is dependent on STIM1 activation of SOC following intracellular SOC depletion. Whilst other Ca2+ entry mechanisms may also play a part in Ca2+ influx, these data suggest targeting NCX may provide a novel therapeutic strategy in diseases such as asthma.In summary, we provide evidence demonstrating that reverse mode NCX1 plays a role in the control of CaASM: airway smooth muscle; BK: bradykinin; FCS: foetal calf serum; HA: histamine; HBSMC: human bronchial smooth muscle cells; ROC: receptor operated cation; SOC: store operated cation; SR: sarcoplasmic reticulum.The authors declare that they have no competing interests.BL and SP designed research, performed experiments and wrote the paper, JF performed lung slice experiments and IH supervised, contributed to experimental design and data interpretation. All authors read and approved of the final manuscript.


Our model includes a realistic 3D geometry of a single t-tubule and its surrounding half-sarcomeres for rabbit ventricular myocytes. The effects of spatially distributed membrane ion-transporters , and stationary and mobile Ca2+ buffers  are also considered. We used a coupled reaction-diffusion system to describe the spatio-temporal concentration profiles of free and buffered intracellular Ca2+. We obtained parameters from voltage-clamp protocols of L-type Ca2+ current and line-scan recordings of Ca2+ concentration profiles in rabbit cells, in which the sarcoplasmic reticulum is disabled. Our model results agree with experimental measurements of global Ca2+ transient in myocytes loaded with 50 μM Fluo-3. We found that local Ca2+ concentrations within the cytosol and sub-sarcolemma, as well as the local trigger fluxes of Ca2+ crossing the cell membrane, are sensitive to details of t-tubule micro-structure and membrane Ca2+ flux distribution. The model additionally predicts that local Ca2+ trigger fluxes are at least threefold to eightfold higher than the whole-cell Ca2+ trigger flux. We found also that the activation of allosteric Ca2+-binding sites on the Na+/Ca2+ exchanger could provide a mechanism for regulating global and local Ca2+ trigger fluxes in vivo. Our studies indicate that improved structural and functional models could improve our understanding of the contributions of L-type and Na+/Ca2+ exchanger fluxes to intracellular Ca2+ dynamics.The transverse tubular system of rabbit ventricular myocytes consists of cell membrane invaginations (t-tubules) that are essential for efficient cardiac excitation-contraction coupling. In this study, we investigate how t-tubule micro-anatomy, L-type Ca The SR provides Ca2+ for the troponin C (TnC) myofilament protein, thereby activating and regulating myocyte contraction  in the cell interior  in isolated rabbit ventricular myocytes. Pharmaceutical disruption of SR activity enabled them to examine the contributions of LCC and NCX to Ca2+ trigger flux, which otherwise would have been masked by SR-bound fluxes from RyRs and the SR Ca2+ ATPase (SERCA). The study suggests that at positive voltages (Vm = +30 mV), the trigger flux of Ca2+ is greater than estimates from a simple summation of the fluxes through LCCs and Ca2+ entry via reverse NCX mode. The authors hypothesized that openings of LCCs increase local ([Ca2+]i) near the NCX protein complex, which activates Ca2+-binding sites on NCX and results in an increase of Ca2+ influx via the exchanger.To investigate relationships between ion fluxes via LCCs and NCXs at voltages corresponding to the early AP plateau, Sobie et al.  recently2+ signaling and excitation-contraction coupling (ECC) in cellular and sub-cellular environments. To this end, whole-cell models have suggested an intimate relationship between Ca2+, Na+, K+ ionic fluxes, and Ca2+ transient in rabbit ventricular myocytes  in rabbit ventricular myocytes exhibits a simple topology that more closely resembles the t-system of canine and human ventricular myocytes . We included equations describing the voltage-dependent background Ca2+ leak and the sarcolemmal Ca2+ ATPase pump  were considered  diffuse and react throughout the cytoplasm. The outer sarcolemma and sarcomere box faces were subject to reflective boundary conditions.The effects of four exogenous and endogenous Cad Figure C. The end Figure . The freILCC, INCX, IPump, and ILeak. Formulation of INCX was presented as the product of an electrochemical (ΔE) and an allosteric factor  used here was from Shannon et al. (KmCaAct) was fitted to 0.29 vs. 0.256 μM in Shannon et al.  of 0.1 μM, and buffers in equilibrium. The extracellular Ca2+ and Na+ concentrations were 2 and 140 mM, respectively, and remained constant. The voltage-clamp protocols  were derived from data in rabbits  was converted to Ca2+ flux (see Eq. Cm/Vcell ∼ 4.54 pF/pL) in adult rabbit ventricular myocytes  in the presence of 50 μM Fluo-3 and [Na+]i of 10 mM  is in reverse mode at both applied voltages with 10 mM [Na+]i . In the presence of 10 mM [Na+]i, our results demonstrate that: (1) the overall scale of INCXsarc was ∼5.6-fold greater than INCXglobal; (2) the increase in INCXsarc during ILCC upstroke relative to INCXglobal was much faster; (3) while INCXglobal monotonically increased over the entire simulation, local peaks in INCXsarc are predicted at 15 ms. Moreover, in absence of [Na+]i, the model predicts a sharp reversal in INCXsarc at 0 mV, whereas INCXsarc had a more gradual and monotonically decreasing flux.Figure dF/dtmax) quantifies the maximum inward Ca2+ flux. By normalizing dF/dtmax and ILCC to 1.0, it was suggested that the relative contribution of global NCX flux can be estimated  and at +50 mV, a ∼30% reduction in d[Ca2+]global/dtmax is predicted relative to a measured 50% decline.The whole-cell trigger flux reported by Sobie et al.  were very different: 3 vs. 9 μM/s at 0 mV; 1.3 vs. 2.5 μM/s at +50 mV  to probe contributions of the realistic surface and t-tubule shape to local Ca2+ profiles. The calculated line-scan images and local Ca2+ time-courses are shown in Figures +]i non-uniform Ca2+ distributions for both voltages are predicted . The contribution of a LCC cluster located ∼2 μm away from upper surface is evident as a spike in the line-scan images. At +50 mV a slight Ca2+ gradient is predicted in transverse cell direction  follow the same trends as the global Ca2+ transients.In Sobie’s et al.  experime2+ transients in the proximity of a t-tubule  differ considerably with respect to local cytosolic Ca2+ transients. Adjacent to the LCC cluster located at the t-tubule distal end . Toward the cell surface, the trends depend on the applied voltage . At 0 mV, the [Ca2+]i peak at the cell surface is nearly half of the transient at the LCC cluster and occurs ∼10 ms later. At +50 mV, the sub-membrane [Ca2+]i initially increases faster than the cytosolic analog (for t < 20 ms) and thereafter [Ca2+]i increases at the same rate as local cytosolic. We should mention here, that such local Ca2+ signals are difficult to resolve experimentally due to optical blurring and noise. Thus, our modeling study is one more example that computational models may serve as powerful tools for prediction and analysis on how local Ca2+ dynamics is regulated.The model also predicts that local Cad Figure A, the lo+]i-influenced [Ca2+]i transient is shown in Movie 2+ gradient in the narrow sub-sarcolemmal region. In these movies, the LCC clusters are clearly evident as localized regions of enhanced local [Ca2+]i. Comparison of the 0 vs. +50 mV cases (top and bottom rows) demonstrates that at both voltages [Ca2+]i increases heterogeneously in transverse cell direction as suggested by experiment  Ca2+ gradient traveling from the external membrane to the cell interior is predicted at both voltages when LCCs were continuously distributed along the t-tubule (Movie right column); and (2) lesser inwardly propagating Ca2+ gradient was observed at +50 mV when using the non-allosteric model of NCX .The most compelling evidence of the voltage and i profiles  ∼ 3 μM/s, INCXglobal(+50mV) ∼ 8 μM/s, INCXsarc(0mV) ∼ 9 μM/s, INCXsarc(+50mV) ∼ 22 μM/s]. At 0 mV, when the LCC flux was dominant, a modest decrease in d[Ca2+]mouth/dtmax (2.5 vs. 3 μM/s) was predicted. At +50 mV, when NCX contributes a significantly larger fraction of the total Ca2+ transient, the effects of NCX allostery were more evident. Assumption of a non-allosteric NCX model resulted in a approximately twofold drop in d[Ca2+]mouth/dtmax and lesser inwardly propagating Ca2+ gradient at +50 mV (Movie lower left panel vs. Movie lower left panel). These data reveal that the allosteric catalysis of NCX can augment the local trigger fluxes as well . A possible reason for the negligible predicted effects of NCX allostery on d[Ca2+]end/dtmax and d[Ca2+]global/dtmax, which is at odds with experimental estimates of the global trigger fluxes , intracellular Na+ diffusion and buffering, and distributions of Na+ channels and Na+/K+ pumps along the sarcolemma we may further examine the role of NCX allostery due to LCC opening, especially early in the AP. We further anticipate that the allosteric interaction between NCX and LCCs will be more nuanced during an AP, given that the fast Na+ promotes activation of the exchanger before many LCCs have opened  the linear t-tubule topology and the punctuate spatial distribution of LCC along the sarcolemma function to inhibit inwardly propagating Ca2+ gradients; (2) local trigger fluxes of Ca2+ are at least threefold to eightfold higher than whole-cell Ca2+ trigger flux; and (3) the activation of allosteric Ca2+-binding sites on Na+/Ca2+ exchanger could provide a mechanism that regulates local and global Ca2+ trigger fluxes in vivo. We concluded that improved models representing the localized positions and the functional diversity of LCC clusters could help to improve our understanding of ILCC and INCX contributions to global and local Ca2+ trigger fluxes.We developed and validated a 3D reaction-diffusion model of CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.http://www.frontiersin.org/Computational_Physiology_and_Medicine/10.3389/fphys.2012.00351/abstractThe Supplementary Material for this article can be found online at Supplementary Movie S1+ on spatial Ca2+ dynamics in the presence of allosteric catalysis and 50 μM Fluo-3Effects of voltage and intracellular Na.Click here for additional data file.Supplementary Movie S22+ dynamics in the presence of allosteric catalysis, 50 μM Fluo-3 and 10 mM [Na+]iEffects of continuous LCC current distribution along the t-tubule on spatial Ca.Click here for additional data file.Supplementary Movie S3+ on spatial Ca2+ dynamics in the absence of allosteric catalysis and presence of 50 μM Fluo-3Effects of voltage and intracellular Na.Click here for additional data file.Supplementary Movie S42+ dynamics at 0 mV in the presence of allosteric catalysis, 50 μM Fluo-3 and 10 mM [Na+]iEffects of LCC cluster placement along t-tubule on spatial Ca.Click here for additional data file.Supplementary Movie S52+ dynamics at +50 mV in the presence of allosteric catalysis, 50 μM Fluo-3 and 10 mM [Na+]iEffects of changes in NCX allosteric constant and maximum NCX rate on spatial Ca.Click here for additional data file.


In this review we focus on the role of intracellular inorganic phosphate (Pi) whose concentration can increase rapidly from around 5–30 mM during intense fatigue. Studies from skinned muscle fibers show that these changes substantially impair myofibrillar performance although the effects are strongly temperature dependent. Increased Pi can also cause reduced Ca2+ release from the SR and may therefore contribute to the reduced activation. In a recent study, we have measured both Pi and Ca2+ release in a blood-perfused mammalian preparation and the results from this preparation allows us to test the extent to which the combined effects of Pi and Ca2+ changes may contribute to fatigue.Muscle fatigue is the decline in performance of muscles observed during periods of intense activity. ATP consumption exceeds production during intense activity and there are multiple changes in intracellular metabolites which may contribute to the changes in crossbridge activity. It is also well-established that a reduction in activation, either through action potential changes or reduction in Ca Muscle fatigue can be defined as the decline of muscle performance associated with intense muscle activity. Many mechanisms contribute to this decline, from psychological factors within the cortex down to the state of individual proteins inside the muscle. To study mechanisms in detail it is usually necessary to use a simplified preparation and a well-defined pattern of stimulation. One approach has been to use isolated small preparations of muscle and stimulate with intermittent, maximal, isometric tetani; obviously central  mechanisms do not occur in this preparation and the effects of changes in blood flow are also absent. Another approach is to use human subjects and induce fatigue by maximal or near-maximal contractions repeated regularly. In suitable muscles both force and metabolite changes can be measured simultaneously and repeatedly. Two limitations to this approach are that both the force and the metabolites are usually averaged across a muscle which consists of various fiber types with different properties. In addition the degree of central vs. peripheral fatigue is often uncertain in human subjects. Given the interpretative difficulties of these two approaches, a third approach is of great importance; application of known metabolite changes to skinned muscle preparations. In this preparation, the metabolites are defined and can be changed at will and the fiber type can be determined. Extensive data is available documenting how the performance of the contractile proteins is affected by individual metabolite changes.i) contributes to the changes in crossbridge behavior, (2) consider the evidence that phosphate contributes to the reduced Ca2+ release, (3) examine the correlation between force and Pi in fatiguing muscles, and (4) describe a new model of fatigue in which both Pi and intracellular tetanic Ca2+ concentration ([Ca2+]i) can be measured and analyse how these two factors in combination contribute to the force decline. This type of analysis involves many assumptions and can only give a general indication of the magnitude of the contribution that might arise from these sources. It is accepted that many other factors, with both positive and negative effects, contributed to the overall effect.In this short review we will (1) discuss the evidence that inorganic phosphate  to represent the levels that may occur in near-maximally activated tetani. Even at high Ca2+, the relation between force and Pi declines because of the effect of Pi on maximum Ca2+-activated force and this effect is largest at low Pi and decreases as Pi increases. At lower Ca2+ levels the relationship falls more steeply as the effect of changes in pCa50  add to the reduced maximum Ca2+-activated force.Ruegg et al. , workingi Figure . In mammi on skinned fibers are quantitatively and qualitatively different when repeated at near physiological temperatures  Pi has relatively little effect on force  does the force fall substantially and the effect is least at low Pi and greater as Pi increases.The early work on skinned muscle cited above was performed at 10–20°C and a major recent development was the discovery that the effects of both pH and Pi Figure . We haves Figure . Note ths Figure , there ie Figure . Only whi and H+ depress muscle force and when applied simultaneously the effect is larger and generally assumed to be multiplicative. Nosek et al.  occurs between them. The molecule is illuminated with a wavelength that excites CFP which then emits at its characteristic wavelength. In the presence of Ca2+, FRET occurs between CFP and YFP so that some fraction of the emitted light is now at the YFP emission wavelength. Thus, the ratio of YFP/CFP emitted fluorescence is Ca2+ sensitive.The data in Figure 2+]i typically shows no significant change during this fatigue protocol. Tetanic [Ca2+]i falls during fatigue and recovers afterwards. If the difference between tetanic and resting [Ca2+]i is defined as 100%, then the average tetanic ratio during fatigue declined to 67%. The ratio then recovers with a timecourse similar to that of force. We have not calibrated our YFP/CFP ratios but data from Miyawaki et al. (2+ should be linear for our indicator (YC2) over the lower part of its range.Figure i et al.  suggest 2 and extraction with perchloric acid. Pi was measured in the supernatant using 31P-NMR spectroscopy. The relation between force and Pi in control (upper leftmost point) and various types of fatigue is depicted in Figure i and force over the whole range of force outputs. In order to decide to what extent changes in Pi can explain the reduction in force one needs to compare data in Figure 2+ in order to make the comparison. (2) Both the force and the metabolic measurements are averaged from the whole muscle which will consist of a mixture of fiber types. Percival et al.  and the fatigued Pi (15 mM). Our Ca2+ measurements are not calibrated but, since we stimulate at a frequency that gives maximal force, we can assume that Ca2+ is at least supramaximal on the appropriate Pi curve  reading from Figure 2+]i should be ~pCa = 5.9 giving a tetanic [Ca2+]i = 1.26 μM. This point is marked on Figure 2+ measurements we found that tetanic [Ca2+]i reduced to 67% in the fatigued muscle giving a tetanic [Ca2+]i of 0.83 μM (pCa 6.08). Simultaneously during fatigue Pi rises to 15 mM so the expected force for these conditions is given by the empty circle (22% control force). Thus, under these assumptions, the combination of Pi and [Ca2+]i predict a force of 22% in our fatigue protocol whereas the measured fatigue force was 49%. This analysis assumes that both the fast and slow fibers had similar changes in Ca2+ and Pi and that the properties of the slow and fast fibers are similar with respect to Pi. On the first point, Karatzaferi et al.  compared to the Type IIA fibers (71 mmol/kg dry weight). This suggests that Pi would be higher than the average in Type IIB fibers and correspondingly lower in the Type IIA fibers. On the second point Debold et al.  and 20% less in the Type IIA fibers (12 mM). The half shaded circles  becomes 64%. Thus, a 25% change in our estimated Ca2+ leads to large (64 vs. 21%) estimate in the fatigued force. Furthermore, we have no information on the tetanic Ca2+ in Type IIA fibers or how it might change during fatigue. Our conclusion as present is that the combination of declining Ca2+ and rising Pi during fatigue are probably capable of explaining much of the observed force reduction. To make this estimate more precise we would need calibrated Ca2+ measurements in both fiber types and more details on the metabolite changes in different fiber types.To understand how Pi et al.  measuredd et al.  state thi and Ca2+ are important in fatigue. A second possibility is that the difference in Pi is greater between the two fiber types. A further possibility is that the Ca2+ changes occurring during fatigue are smaller in the Type IIA fibers and larger in the Type IIB fibers which would be expected if the Ca2+ changes are a function of the Pi changes as discussed earlier.An unresolved difficulty with these calculations is to explain the observation of Larsson et al.  showing i in muscle during fatigue suggest that the combination of the increase in Pi and the decrease in tetanic Ca2+ are capable of explaining much of the observed decline in force. This analysis is highly dependent on skinned fiber data obtained at 30°C and more data at 37°C would be desirable. Another area of uncertainty is the level of tetanic Ca2+ in the various fibers types and the extent to which this changes during fatigue protocols. While the mechanism of change in tetanic Ca2+ remains uncertain, a strong possibility is that the change in Pi is instrumental in the reduction of tetanic Ca2+ suggesting that evolution has utilized one metabolite to reduce both the myofibrillar performance and the Ca2+ release. The many other factors that modulate fatigue would appear to do so by fine-tuning the substantial reduction in force caused by the interactions between Pi and myofibrillar proteins and the Ca2+ release mechanism.Current data on the changes in PThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Consequently, cytoplasmic free Ca2+ is tightly regulated through the co-ordinated action of primary and secondary Ca2+ transporters. Identifying selective inhibitors of Ca2+ transporters is key towards understanding their physiological role as well as having therapeutic potential, therefore screening systems to facilitate the search for potential inhibitors are a priority. Here, the methodology for the expression of a Calcium membrane transporter that can be scaled to high throughputs in yeast is presented.Calcium  was expressed in the yeast Saccharomyces cerevisiae and its activity monitored by the bioluminescence from apoaequorin triggered by divalent cations, such as calcium, magnesium and manganese.The 2+, Mg2+ and Mn2+ in yeast mutants lacking the homologue yeast antiporter Vcx1p. In the scalable format of 96-well culture plates pharmacological assays with a cation antiporter inhibitor allowed the measurement of inhibition of the Ca2+ transport activity of PfCHA conveniently translated to the familiar concept of fractional inhibitory concentrations. Furthermore, the cytolocalization of this antiporter in the yeast cells showed that whilst PfCHA seems to locate to the mitochondrion of P. falciparum, in yeast PfCHA is sorted to the vacuole. This facilitates the real-time Ca2+-loading assays for further functional and pharmacological studies.Bioluminescence assays demonstrated that PfCHA effectively suppressed induced cytoplasmic peaks of CaS. cerevisiae and luminescence-based detection of cytoplasmic cations as presented here offer a tractable system that facilitates functional and pharmacological studies in a high-throughput format. PfCHA is shown to behave as a divalent cation/H+ antiporter susceptible to the effects of cation/H+ inhibitors such as KB-R7943. This type of gene expression systems should advance the efforts for the screening of potential inhibitors of this type of divalent cation transporters as part of the malaria drug discovery initiatives and for functional studies in general.The functional expression of PfCHA in 2+ as well as Mg2+ and Mn2+ lend itself to high-throughput and quantitative settings for pharmacological screening and functional studies.The expression and activity of the PfCHA detected in yeast by a bioluminescence assay that follows the levels of cytoplasmic Ca They include erythrocyte invasion [in vivo synchronicity in the erythrocytic cycle [2+ is tightly maintained at 50-150 nM [P. falciparum include two Ca2+ ATPases, a low-affinity transporter PfATP4 [2+ ATPase PfATP6 [2+ in Plasmodium has been found in acidic compartments  [2+ sequestration has also been observed in the malaria parasite’s mitochondrion [2+ pumps, low-affinity secondary transporters that facilitate the membrane transport of Ca2+ and other divalent cations  into organelles or through plasma membrane using a proton (in lower eukaryotes and plants) gradient in the opposite direction (Ca2+/H+ exchangers or antiporters) are known to mediate the dissipation of cytoplasmic peaks of Ca2+[P. falciparum Ca2+/H+ antiporter (PfCHA) homologue to the family of CAtion eXchangers  [X. laevis as a divalent cation /H+ exchanger [Cainvasion -3, in viic cycle , togetheic cycle -7. As in0-150 nM ,9. In eur PfATP4  and a hie PfATP6 ,12. Intr.4-2 μM) ,14. Ca2+hondrion . Besidess of Ca2+,18. In txchanger .Saccharomyces cerevisiae is a highly developed and widely used model organism. Furthermore, S. cerevisiae has become a model for eukaryotic Ca2+ homeostasis [vcx1Δ (VaCuolar Ca2+/H+ eXchanger) gene knock-out mutant. A bioluminescence apoaequorin reporter system has been used to allow the detection of cytoplasmic Ca2+ in vcx1Δ where PfCHA is shown to be able to re-establish Ca2+ mobilisation from cytoplasm. In the apoaequorin system aequorin catalyses the oxidation of an imidazolopyrazinone (coelenterazine) upon Ca2+ binding and light is emitted from the oxidized and excited state of this chromophore that exists tightly bound to aequorin. In vitro, Ca2+ binding to apoaequorin triggers the emission of blue light (469 nm), although other activating cations such as trivalent lanthanides [2+ and Mg2+ are also known to bind aequorin via three EF-hand Ca2+-binding sites [eostasis ,22. In tthanides  and divang sites .S. cerevisiae the exchanger is sorted to the vacuole. This finding offers further practical advantages for the studies of a membrane transporter such as PfCHA since yeast vacuoles are their main Ca2+ storage compartments. Yeast is an attractive organism for recombinant protein production as it combines highly developed genetic systems and ease of use with reductions in time and costs. Moreover, due to the challenging nature of expressing functional membrane proteins a yeast expression system for PfCHA is a valuable tool for further functional studies and pharmacological screens. To this extent, the 96-well format was used to further demonstrate divalent cation  transport by PfCHA in vcx1Δ yeast cells and present an inhibition assay with a cation antiporter inhibitor as a proof of concept of the opportunities offered by this expression system for the search of PfCHA inhibitors.Interestingly, whilst in the parasite PfCHA is reported to be sorted to the mitochondrion , in S. cP. falciparum 3D7 was extracted with Trizol (Invitrogen) following manufacturer’s protocol using parasites harvested from standard cultures [NotI and BamHI restriction sites obtained from Euroscarf. Primers for PFF0170w were 5’-ATGGTTATGGGTAGAGTTC and 5’-TTATGATGTATCAAACCAG. The S. cerevisiae VCX1 gene was directly cloned into pGREG505 using the following primers: 5’-gaattcgatatcaagcttatcgataccgtcgacaGGCTGCTGATAGCAAATAAA and 5’- gcgtgacataactaattacatgactcgaggtcgacGAATTTCTGCGCTACTGTTC. pGREG505 vector has the LEU2 selectable marker and the GAL1 promoter. Escherichia coli TOP10 cells (Invitrogen) were routinely used as recombinant plasmids host. For bioluminescence measurements of cytosolic Ca2+ yeast cells were transformed with a plasmid carrying the aequorin gene [ADH3 promoter was kindly provided by David M. Bedwell (University of Alabama at Birmingham). Reverse transcription PCR and real-time PCR with iQ SYBR Green Supermix (Bio-Rad) in yeast were performed using total RNA extracted with RiboPure-Yeast  following manufacturer's instructions. cDNA was synthesized using ThermoScript (Invitrogen), and PCR amplifications using Platinum Taq High Fidelity (Invitrogen).Total RNA from cultures . Gene secultures  and GenBcultures . The polcultures  between rin gene . This geSaccharomyces cerevisiae haploid strain Y03825  was obtained from the European Saccharomyces cerevisiae Archives for Functional Analysis (Euroscarf) [2+ antiporter gene VCX1  resistant phenotype. BY4741n is the isogenic and haploid parental strain (Euroscarf Y00000). Yeast cells were transformed by the DMSO-based method [Yeast roscarf) . In Y038YDL128w, ) is repld method . Yeast wd method .et al[URA3 selection marker) expresses the soluble calcium-binding protein aequorin [600/ml. The cultures were then washed twice in SD medium and grow a further four hours in SD with 2% (w/v) galactose to induced gene expression. Ten OD600 units of cells were resuspended in 180 μl of test medium –TM- (0.17% (w/v) YNB, 2 mM EGTA, 40 mM MES-Tris, pH 6.5) after two washes with the same medium. To equilibrate cytosolic aequorin with its allosteric group, 20 μl of 0.6 mM coelenterazine (Fluka Biochemica 07372) dissolved in methanol were added and the cell suspension incubated for 30 min at room temperature. Cells were then washed twice in TM. The final pellet was resuspended in 1 ml of TM and allowed to equilibrate for 30 min at room temperature. After detecting the baseline light emission 1 ml of 1 M of CaCl2 was added. Final concentrations of CaCl2 were 50 mM or 500 mM in 2 ml total volume. For the multi-well plate experiments 0.1 OD600 were plated in 0.1 ml volumes and 1 M of the divalent cation salts  were added in 0.1 ml to make 0.5 M final concentrations in 0.2 ml total volume. Light emission was collected from the 2 ml assays with a CliniLumat Berthold luminometer at intervals of 2 seconds or from the 0.2 ml multi-well assays with a LumiStarOmega luminometer (BMG Labtech) plate reader at 0.5 seconds intervals. Where indicated the samples were incubated with dimethylsulphoxide (DMSO) alone or with Bafilomycin A1 dissolved in DMSO  or KB-R7943 dissolved in DMSO  for 30 minutes at room temperature prior to the addition of CaCl2.Apoaequorin assays were adapted from Miseta et al. pSEY8 vaequorin . Cells cet al[et al[Yeast membranes were extracted and purified as in Fisher et al. Indirec al[et al. Polyclovcx1Δ has a significant defect in the rapid Ca2+ sequestration response to high cytosolic levels of this cation [VCX1 genes as well as the empty vector control were transformed into vcx1Δ. For the detection of cytosolic Ca2+ by bioluminescence, these strains were subsequently transformed with a vector carrying the apoaequorin gene (pSEY8-AEQ). Positive colonies were selected for growth in the absence of leucine and uracil and in the presence of 400 μg/ml of geneticin in SD medium. The presence of the recombinant vectors was verified and their RNA expression monitored by reverse transcription and PCR amplification , vcx1Δ carrying the recombinant pGREG505-VCX1 (vcx1Δ + VCX1), and vcx1Δ carrying the recombinant vector pGREG505 with PfCHA (vcx1Δ + PfCHA). All four strains responded with a sharp elevation in cytosolic Ca2+ levels when 50 mM CaCl2 was added . Four d2+ shock the luminescence of the samples were registered for 8 seconds to establish the basal line. As expected vcx1Δ mutant showed the highest peak of cytosolic Ca2+ level and the reference BY4741n strain presented the lowest signal with a 10-fold difference between them. Mutant vcx1Δ cells expressing the homologue VCX1 closely followed the Ca2+ levels of BY4741n. Significantly, vcx1Δ cells expressing the heterologous PfCHA also followed the extrusion of cytosolic Ca2+ after the initial peak. Although as anticipated less efficiently than the homologue VCX1 gene . Replacing D-galactose with D-glucose, and thereby inhibiting the transcription of genes under the GAL1 promoter of the pGREG505 vector, impaired the expression of both VCX1 and PfCHA  when even higher concentrations (500 mM) of CaCl2 were applied as external Ca2+  Ca2+ shock experiments were carried out as described above with 500 mM CaCl2. For all the strains, bafilomycin A1 treatment resulted in a reduced ability of the yeast cells to recover from the Ca2+ challenge , treatment with bafilomycin A1 also affected the ability of PfCHA-carrying yeast cells to recover from the Ca2+ challenge   or the recombinant vectors pGREG505–VCX1 (vcx1Δ + VCX1) and pGREG505-PfCHA (vcx1Δ + PfCHA) were processed for apoaequorin assays as described in Methods. The parental strain BY4741n was not used for assays in the multi-well plates as the mutant carrying the homologue VCX1 gene behaved equivalently in the Ca2+ loading assays presented above. In 96-well plates yeast cells were distributed in 0.1 ml per well containing a number of cells equivalent to 0.1 OD unit (OD600). After establishing the base line for 5 seconds Ca2+, Mg2+ or Mn2+ were injected as 1 M chloride salts on to the selected wells (0.1 ml) to render 0.5 M final concentrations. The luminescence signals were recorded for 60 seconds at intervals of 0.5 seconds. The luminescence peaks were sharper and the area under the curves was smaller than those originated by the larger cell volumes and densities used previously with equivalent final concentrations of Ca2+  by comparison to the vector only control demonstrating the functionality of the assays in the multi-well format . Noteworthy, the initial functional characterization of PfCHA as a Ca2+ and Mn2+ transporter did not present direct transport activity of Mg2+ by PfCHA when expressed in X. laevis oocytes [2+ was inferred from competition studies against 45Ca2+ transport. Here, the yeast expression system seems to substantiate the evidence of Mg2+ being a substrate for PfCHA-mediated transport. On the other hand, the yeast Vcx1p antiporter is known to lack Mn2+ transport [2+ cytosolic peak in the cells expressing the gene product of VCX1  had a subsequently bolus of Ca2+   Figure D. The raaequorin ,41. Thus+ Figure D. The ma+ Figure D. The Ca2+ at slower rates when cells had been pre-loaded with Mg2+. Yeast Vcx1p dissipated the Ca2+ signal 13 times slower on average (means 0.32 RLUs/second vs 0.025 RLUs/second) in the presence of Mg2+, with PfCHA rates of Ca2+ clearance falling to 63 times on average (means 0.44 RLUs/second vs 0.007/second) significantly slower when in the presence of Mg2+. Clearance rates for Ca2+ were calculated from the highest point to the first point of the plateau for each curve (Ca2+ only versus Ca2+ after Mg2+ load). The delayed response to cytoplasmic Ca2+ is interpreted as the time required for Ca2+ to displace Mg2+ from the aequorin EF-hand Ca2+-binding sites, and the slower clearance rates as the inhibitory effects of Mg2+ on the transport of Ca2+ by Vcx1p and PfCHA. Interestingly, by comparison PfCHA seems to be a more proficient transporter of Mg2+ given that a higher proportion was cleared from cytoplasm by PfCHA (Ca2+ peak lower in cells loaded with Mg2+). Also, PfCHA seems to have a higher affinity for Mg2+ than the yeast Vcx1p since the rate of Ca2+ sequestration of cytosolic Ca2+ in cells loaded with Mg2+ was most affected in the cells expression PfCHA. Pre-loading with Mn2+ was less informative due to the overlapping patterns with the signal generated by Ca2+ (data not shown). This is observed as the result of Mn2+ occupying a EF-hand Ca2+-binding site different from the two sites that Ca2+ binds in aequorin [PfCHA as well as Vcx1p, however, sequestered Caaequorin ,24.2+/H+ antiporter inhibitors KB-R7943 was used to illustrate an inhibitory assay for PfCHA using the yeast expression system. KB-R7943 is an inhibitor of Na+/Ca2+ antiporters [X. laevis oocytes [2+ at 20 μM. Here yeast cultures were incubated for 30 minutes at room temperature in several concentrations of KB-R7943 after the cells had been treated with coelenterazine as in Methods. The effect of this inhibitor on the sequestration of cytosolic Ca2+ was apparent in all three experimental groups of the vcx1Δ knock-out strain carrying the empty vector pGREG505 (vcx1Δ + p505) or expressing VCX1 (vcx1Δ + VCX1) and PfCHA (vcx1Δ + PfCHA)  at which their transport was inhibited by 50%  was calculated to be 7.5 μM, 12.5 μM and 17.5 μM of KB-R7943 for vcx1Δ + pGREG505, vcx1Δ + VCX1 and vcx1Δ + PfCHA, respectively  as a divalent cation reporter delivers a suitable screening setting for high throughput search for molecules with inhibitory activity against P. falciparum’s Ca2+/H+ antiporter PfCHA.In the absence of specific Caiporters -44 althoiporters . More re oocytes  where itvcx1Δ mutants carrying pGREG505-PfCHA showed a fluorescent signal derived from the vacuole (tonoplast)  raised against a 14 aa synthetic peptide representing residues 73-86 of PfCHA, indirect immunolocalization assays were performed in the transformed yeast. Yeast X. laevis, in S. c2+ generated when influx temporarily exceeds efflux usually functions as an intracellular signal. Calcium-proton exchangers are low-affinity cytosolic export systems that coupled to the thermodynamically downhill exchange flux of H+ dissipate cytosolic increases of Ca2+[2+ energized by the electrochemical gradient established by proton pumps such as H+-ATPases and H+-pyrophosphatases [P. falciparum however, the CAX homologue PfCHA seems to localize and perform Ca2+ (as well as Mn2+) exchange in the mitochondrion of this parasite [2+ from the matrix when the mitochondrion is going through transient Ca2+ overloads [A transient rise in cytosolic free Cas of Ca2+. PfCHA iphatases . Accordiphatases  and yeasphatases  where thparasite . In thisverloads .S. cerevisiae, the apoaequorin luminescent response to divalent cations was used as a reporter system to follow cytosolic levels of Ca2+, Mg2+ and Mn2+[2+ homeostasis [2+ transporters such as the A. thaliana Ca2+/H+ antiporters (AtCAX) [2+, Mg2+ and Mn2+ in the yeast vcx1Δ mutant and that this activity was sensitive to inhibition of the H+ transmembrane gradient by a vacuolar-ATPase inhibitor as well as the Na+/H+ antiporter and TRP channels inhibitor KB-R7943. The levels of cytosolic Ca2+, Mg2+ and Mn2+ reported by the apoaequorin luminescence show apparent and significant cytosolic dissipation of all three of these divalent cations by PfCHA in vcx1Δ cells. Taken together, the functional data and immunolocalization point at PfCHA to behave as a divalent cation/H+ exchanger that in yeast functions as a vacuolar membrane transporter. As a consequence of the role of the proton gradient in the function of this type of calcium exchanger potential inhibitors of PfCHA will have to be tested for their own capacity to inhibit proton pumps.In order to detect functional PfCHA in  and Mn2+. Apoaequeostasis ,46, incl (AtCAX) . Here, P2+ is sequestered within the yeast vacuole in stable complexes (e.g. with polyphosphates) as well as free Ca2+[2+ in yeast is maintained through the complementary action of Vcx1p [2+ ATPase Pmc1p [2+ transporter that responds to sudden increments of the cytosolic Ca2+ by rapid sequestration of this cation into the vacuole in exchange for two protons down a proton gradient (vacuolar pH 5.5-6.2 vs cytoplasmic pH 7.0) [In yeast it is estimated that at least 90% of the total 1.5-4 mM of cellular Cafree Ca2+ allowingof Vcx1p ,49 and tse Pmc1p . As with pH 7.0) ,51.S. cerevisiae, cells have to be exposed to seemingly excessive concentrations of these cations  in order to reach measurable cytosolic transient elevations of these ions. When exposed to 50 mM S. cerevisiae cytosolic levels of Ca2+ are measured to be in the region of 250-290 nM  that increases further to 320-350 nM when the external Ca2+ is raised to 400 mM [vcx1Δ mutant undergoes transient elevations slightly higher with cytosolic calcium concentrations of approximately 400 nM and 510 nM when in the presence of 50 mM and 400 mM of Ca2+, respectively [2+[2+ reported for other CAX proteins (up to 25μM) [vcx1Δ background  . NonetheS. cerevisiae. In S. cerevisiae proteins are sorted to the vacuole by at least four different pathways [vs cell surface proteins in the early stages of the secretory pathway, (ii) endocytic engulfing of material from the plasma membrane, (iii) cytoplasm-to-vacuole targeting pathways that do not transit the early stages of the secretory pathway, and (iv) the de facto inheritance of vacuolar material by daughter cells during cell division. However, in S. cerevisiae membrane proteins lacking motifs or otherwise protein structure information for cellular localization are predominantly delivered to the vacuole membrane [a priori the destination of a membrane transporter in a heterologous expression system. In the yeast expression system as reported here it is speculated that PfCHA is sorted by a default mechanism to the yeast vacuole [The localization of PfCHA to the yeast vacuole is not altogether surprising as this organelle seems to be the default localization for foreign proteins in pathways : (i) sormembrane ,54. Moremembrane , it is n vacuole .S. cerevisiae is by far the most advantageous gene expression system since it is relatively inexpensive to culture and it is the most genetically tractable eukaryotic system with excellent recombinant DNA tools. Additionally, recent advances seeking to overcome the difficulties intrinsic to the expression of foreign proteins in yeast, particularly membrane proteins, have rendered transgenic strains with altered ribosomal content that seems to tolerate and much improve heterologous gene overexpression [S. cerevisiae and its localization to the yeast vacuole permits a very tractable system for further functional and pharmacological studies as exemplified here in multi-well whole-cell cation transport and inhibitory assays.In eukaryotic cell hosts, the yeast pression ,57. The P. falciparum. Particularly membrane transporters such as PfCHA that are still very seldom successfully expressed in heterologous hosts. Moreover, the bioluminescence assay for the detection of cytoplasmic Ca2+ as well as Mg2+ and Mn2+ lends itself to high-throughput and quantitative settings.Yeast offers a cost-effective and user-friendly gene expression system for further functional studies and pharmacological screens of proteins from Plasmodium falciparum Ca2+/H+ antiporter; VCX1: Saccharomyces cerevisiae; Ca2+/H+ antiporter; RLU: Relative Light Units.PfCHA: The authors declare that they have no competing interests.JES-S designed the study and performed experiments. JES-S and GAB wrote the initial draft of the manuscript. SAW and GAB edited and revised the manuscript, and provided materials and equipment. All authors read and approved the final manuscript.PfCHA and VCX1 RNA yeast expression. A. The expression of Plasmodium PfCHA as well as yeast VCX1 was observed by reverse transcription PCR using total RNA from the reference strain BY4741n, the vcx1Δ mutant carrying the expression plasmid only (p505) and the same mutant carrying the recombinant plasmid with either VCX1 or PfCHA. Forward and reverse primer position and sequences: PfCHA (64 - 488nt) , VCX1 (269 - 666nt) , and yeast S. cerevisiae actin YFL039c (130 - 533 nt) . B. Real-time PCR using total RNA from the vcx1Δ mutant carrying recombinant plasmids with VCX1 or PfCHA following the amplification of either a fragment or the full gene for VCX1–666 bp and VCX1-1236 bp respectively. Likewise for the PfCHA (488 bp fragment or entire gene of 1321 bp). Primers for total length genes as in Methods. Evidently, RNA levels of PfCHA as full size gene are present around three-fold lower than its homologous counterpart VCX1 while the difference in the levels of shorter (truncated) transcribed fragments is only half that difference.Click here for filePhenotypic rescue of yeast Saccharomyces cerevisiae vcx1Δ inhibited in presence of glucose. Conditions and strains as in Figure GAL1 promoter present in pGREG505.Click here for file


We therefore systematically tested this hypothesis on healthy male FBFN1 rats fed for four weeks with isocaloric, 10% fat-containing diets supplemented with 1, 3, or 10% fish oil (FO). High resolution mass spectrometric analysis confirmed expected diet-driven increases in docosahexaenoic acid  and eicosapentaenoic acid  in sera, liver and nonsynaptosomal brain mitochondria. We further evaluated the resistance of brain and liver mitochondria to Ca2+ overload and prooxidants. Under these conditions, neither mitochondrial resistance to Ca2+ overload and prooxidants nor mitochondrial physiology is altered by diet, despite the expected incorporation of DHA and EPA in mitochondrial membranes and plasma. Collectively, the data eliminate one of the previously proposed mechanism(s) that n-3 PUFA induced augmentation of mitochondrial resistance to the oxidant/calcium-driven dysfunction. These data furthermore allow us to define a specific series of follow-up experiments to test related hypotheses about the effect of n-3 PUFAs on brain mitochondria.Omega-3 polyunsaturated fatty acids (n-3 PUFAs) block apoptotic neuronal cell death and are strongly neuroprotective in acute and chronic neurodegeneration. Theoretical considerations,  In mammals, the central nervous system (CNS) has the second highest concentration of lipids after adipose tissue. Lipids play a critical role in neuronal membrane function as well as in enzyme, receptor, and ion channel activities , 2. One +, K+-ATPase), production of neurotransmitters and brain peptides, gene expression, intensity of inflammation, and synaptic plasticity , which were followed by monitoring the fluorescence intensity of Ca-Green-5N (125 nM) (Invitrogen) at excitation and emission wavelengths of 482 and 535 nm, respectively. Mitochondria were challenged to single or multiple Ca2+ additions. For liver mitochondria, each addition was 20–40 nmol Ca2+/mg mitochondrial protein; for brain mitochondria, each addition was 200 nmol/mg protein. Mitochondrial calcium retention capacity (CRC) was determined as amount of Ca2+ sequestered by mitochondria without incurring structural damage and expressed in nmol/mg protein. The redox state of pyridine nucleotides in the mitochondrial suspension was followed by monitoring NADH autofluorescence at excitation and emission wavelengths of 350 and 450 nm, respectively.Mitochondrial CaMitochondrial swelling was measured as a function of light scattering at excitation and emission wavelengths of 587 nm or by a standard spectroscopic assay on a plate reader at 540 nm.Original respiration and fluorimeter-based data were analyzed using Origin v.8.0 (OriginLab) software. Data were normalized to protein concentration and expressed in corresponding units.2+-induced MPT A via Ca2+ uniporter. The formation of an intermediate state [MH]I, on the other hand, is a complex function of the number of Ca2+ ions absorbed by the mitochondrion and expressed as k2 = k2  ′ × ([Ca2+]M/[MH]A)n. Here, [Ca2+]M is the concentration of Ca2+ that has been absorbed by mitochondria; thus, the ratio [Ca2+]M/[MH]A is essentially an average number of Ca2+ ions absorbed per active mitochondrion. Parameter n is an apparent order of the k2 step with respect to Ca2+, and k2  ′ is the reaction constant of this step. Kinetic parameter k2 is related to the ability of the Ca2+ sequestered by mitochondria to facilitate the formation/assembly of the MPT pore intermediate. Functionally, the intermediate represents MPT pore assembled but still closed. In our model, parameter n interpreted as an order of reaction reflects quantitative characteristics of MPT intermediate formation in respect to Ca2+. Therefore, any change in n would be expected to correlate with an ability of mitochondria to resist Ca2+-induced MPT.The rate t-test (Origin 8.0). The significance of data was considered at P value 0.05. Experiments were performed 5-6 times.Data were presented as the mean +/− the standard error of the median (SEM). Group comparisons of the effects of diet modulation on mitochondrial parameters were determined by a two-sample Rats consumed 14.1 ± 3.1 g of food a day and gained 4.85 ± 1.55 g of body weight a day for 28 days. Food intake and body weight gain of all the groups were not significantly different.By using LC-MS profiling, we were able to monitor the n-3 and n-6 free fatty acids—EPA, DHA, AA and the AA, synthesis intermediate DGLA  in both the serum and mitochondrial samples. This method did not provide absolute quantitative amounts of the fatty acids in each sample; however, it provided a means to quantitatively monitor these species across the biological samples. The study gives information regarding relative amounts of species present and allows for direct comparisons across samples to be performed , 49. TheV2), state 3 (V3), state 4 (V4), FCCP-stimulated, or uncoupled respiration (Vunc)). The substrate glutamate/malate was used to stimulate respiration initiated at complex I and the substrate succinate + rotenone was used to stimulate respiration initiated at complex II.Respiratory parameters of brain and liver mitochondria isolated from tested animal groups were measured in different metabolic states: , with succinate as a substrate , which are prooxidative ketoaldehydes [3/4, RCR3/2, and Vunc/ADP also were lower in the presence of IsoKs in all tested animal groups, but this observation was similar for all diets  of nonsynaptosomal brain mitochondria isolated from control group of animals. Further assessment of brain mitochondria function suggested no statistically significant difference between tested groups. However, mitochondria isolated from animals fed FO 1% or 3% diets showed a weak tendency to be more resistant to a Ca2+ challenge (2+ retention capacity (CRC), that is, the ability to sequester Ca2+ without incurring structural damage. Cyclosporine (Cs A), the prototypical MPT pore inhibitor, increased the CRC in all tested groups, with potentially enhanced protection in the groups fed FO 1% and 3% diets (μM IsoKs) resulted in a significant reduction of the CRC in all tested groups (μM) also significantly reduced the CRC in all groups; the effect was again more pronounced in mitochondria isolated from 10% FO group. Mitochondria showed no diet-dependent difference in susceptibility to the prooxidative agent tBH.hallenge . Resista3% diets . Prometh3% diets . Incubatd groups , as we h2+ induced MPT . The analysis of the rate of the formation of an MPT intermediate (k2-step) revealed that FO diets had no statistically significant changes in the resistance of mitochondria to Ca2+   , demonst2+ and prooxidative agents (tBH and IsoKs). Only slight trend towards protective effects, insufficient to underlie the reported neuroprotective effects of n-3 PUFA supplementation, were ever seen with lower levels of supplementation . The lowest dose used in the present study, that is, the 1% FO diet that corresponds to an acceptable intake in humans showed no effect on the mitochondrial parameters tested. Recent study [The data presented contradict our proposed hypothesis, suggesting another explanation must link dietary omega-3 fatty acids with the inhibition of cell death cascades that involve mitochondria. One possibility is that other mitochondrial pathways  are involved. Although this explanation is feasible, the injury used in experiments discussed earlier , 44, 45 nt study  suggestsnt study , 52.2+ than we usually observed in our routine experiments with identical rats fed diets that have a lower percentage of total fat (4.5% versus 10% in the present study). Specifically, CRC for brain mitochondria isolated from animals fed with 4.5% is equal to 250–300 nmol of Ca2+/mg protein, whereas CRC for tested in the present study animal groups vary from 360 to 460 nmol of Ca2+/mg protein. Kinetic analysis done on the Ca2+ induced MPT in liver mitochondria isolated from animals fed with 4.5% of fat revealed Ca2+ requirement (kinetic parameter n) to be about 2.27 ± 0.05 [n (up to 3.1). Such an increase was found upon application of promethazine [2+ or promethazine increased n, thereby increasing the resistance of mitochondria to Ca2+-induced MPT. On the other hand, addition of very low (0.5 nM) concentration of FCCP decreased n (not shown) facilitating MPT induction via a decrease of the ΔΨ, which is in line with data published earlier [With respect to total dietary fat content, we noted that brain and liver mitochondria isolated from control animals in this study exhibited less sensitivity to Ca7 ± 0.05 , while tethazine  or in thethazine  that welin vitro model of ischemic injury (Ca2+overloading and prooxidant exposure), despite the observed changes in plasma and mitochondrial fatty acid content, suggests that increased resistance to Ca2+- and oxidant-mediated mitochondrial damage is not central to the well-documented neuroprotection induced byn-3 PUFAs. Investigation of other potential mechanisms, such as extramitochondrial targets and a potential role of total fat, will require further study.In conclusion, we tested and rejected one of the possible mechanisms linking n-3 PUFA dietary supplementation and brain mitochondria functional parameters and injury mechanisms. The absence of a protective effect of diet in an The tables S1 and S2 belong to the supplementation in references 48,49. The supplementation of the present paper has Table S1 and ARRIVE as mentioned in “Materials and Methods” section 2.2. Pointed online address did not work.Click here for additional data file.