Subplasmalemmal Mitochondria Modulate the Activity of Plasma Membrane Ca2+-ATPases
2005; Elsevier BV; Volume: 280; Issue: 52 Linguagem: Inglês
10.1074/jbc.m510279200
ISSN1083-351X
AutoresMaud Frieden, Serge Arnaudeau, Cyril Castelbou, Nicolas Demaurex,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoMitochondria are dynamic organelles that modulate cellular Ca2+ signals by interacting with Ca2+ transporters on the plasma membrane or the endoplasmic reticulum (ER). To study how mitochondria dynamics affects cell Ca2+ homeostasis, we overexpressed two mitochondrial fission proteins, hFis1 and Drp1, and measured Ca2+ changes within the cytosol and the ER in HeLa cells. Both proteins fragmented mitochondria, decreased their total volume by 25-40%, and reduced the fraction of subplasmalemmal mitochondria by 4-fold. The cytosolic Ca2+ signals elicited by histamine were unaltered in cells lacking subplasmalemmal mitochondria as long as Ca2+ was present in the medium, but the signals were significantly blunted when Ca2+ was removed. Upon Ca2+ withdrawal, the free ER Ca2+ concentration decreased rapidly, and hFis1 cells were unable to respond to repetitive histamine stimulations. The loss of stored Ca2+ was due to an increased activity of plasma membrane Ca2+-ATPase (PMCA) pumps and was associated with an increased influx of Ca2+ and Mn2+ across store-operated Ca2+ channels. The increased Ca2+ influx compensated for the loss of stored Ca2+, and brief Ca2+ additions between successive agonist stimulations fully corrected subsequent histamine responses. We propose that the lack of subplasmalemmal mitochondria disrupts the transfer of Ca2+ from plasma membrane channels to the ER and that the resulting increase in subplasmalemmal [Ca2+] up-regulates the activity of PMCA. The increased Ca2+ extrusion promotes ER depletion and the subsequent activation of store-operated Ca2+ channels. Cells thus adapt to the lack of subplasmalemmal mitochondria by relying on external rather than on internal Ca2+ for signaling. Mitochondria are dynamic organelles that modulate cellular Ca2+ signals by interacting with Ca2+ transporters on the plasma membrane or the endoplasmic reticulum (ER). To study how mitochondria dynamics affects cell Ca2+ homeostasis, we overexpressed two mitochondrial fission proteins, hFis1 and Drp1, and measured Ca2+ changes within the cytosol and the ER in HeLa cells. Both proteins fragmented mitochondria, decreased their total volume by 25-40%, and reduced the fraction of subplasmalemmal mitochondria by 4-fold. The cytosolic Ca2+ signals elicited by histamine were unaltered in cells lacking subplasmalemmal mitochondria as long as Ca2+ was present in the medium, but the signals were significantly blunted when Ca2+ was removed. Upon Ca2+ withdrawal, the free ER Ca2+ concentration decreased rapidly, and hFis1 cells were unable to respond to repetitive histamine stimulations. The loss of stored Ca2+ was due to an increased activity of plasma membrane Ca2+-ATPase (PMCA) pumps and was associated with an increased influx of Ca2+ and Mn2+ across store-operated Ca2+ channels. The increased Ca2+ influx compensated for the loss of stored Ca2+, and brief Ca2+ additions between successive agonist stimulations fully corrected subsequent histamine responses. We propose that the lack of subplasmalemmal mitochondria disrupts the transfer of Ca2+ from plasma membrane channels to the ER and that the resulting increase in subplasmalemmal [Ca2+] up-regulates the activity of PMCA. The increased Ca2+ extrusion promotes ER depletion and the subsequent activation of store-operated Ca2+ channels. Cells thus adapt to the lack of subplasmalemmal mitochondria by relying on external rather than on internal Ca2+ for signaling. Mitochondria are intracellular organelles that play a key role in Ca2+ signaling. Mitochondria take up and release Ca2+, and physiological increases in the cytosolic free Ca2+ concentration, [Ca2+]cyt, 2The abbreviations used are: [Ca2+]cytcytosolic [Ca2+][Ca2+]ERendoplasmic reticulum [Ca2+][Ca2+]mitmitochondrial [Ca2+]CGP 37157(7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one)ERendoplasmic reticulumGFPgreen fluorescent proteinPMCAplasma membrane Ca2+-ATPaseRFPmitmitochondrial red fluorescent proteinSERCAsarco/endoplasmic reticulum Ca2+ ATPaseYC4.1ERyellow cameleon targeted to the ERYC3.6pmyellow cameleon targeted to the plasma membrane.2The abbreviations used are: [Ca2+]cytcytosolic [Ca2+][Ca2+]ERendoplasmic reticulum [Ca2+][Ca2+]mitmitochondrial [Ca2+]CGP 37157(7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one)ERendoplasmic reticulumGFPgreen fluorescent proteinPMCAplasma membrane Ca2+-ATPaseRFPmitmitochondrial red fluorescent proteinSERCAsarco/endoplasmic reticulum Ca2+ ATPaseYC4.1ERyellow cameleon targeted to the ERYC3.6pmyellow cameleon targeted to the plasma membrane. are transmitted to the mitochondrial matrix as changes in [Ca2+]mit. [Ca2+]mit signals during cell stimulation activate mitochondrial dehydrogenases and boost ATP production, but excessive [Ca2+]mit increases can damage mitochondria and trigger apoptosis (1Demaurex N. Distelhorst C. Science. 2003; 300: 65-67Crossref PubMed Scopus (297) Google Scholar, 2Scorrano L. Oakes S.A. Opferman J.T. Cheng E.H. Sorcinelli M.D. Pozzan T. Korsmeyer S.J. Science. 2003; 300: 135-139Crossref PubMed Scopus (1220) Google Scholar). On the other hand, Ca2+ handling by mitochondria has numerous effects on cell Ca2+ homeostasis. By taking up Ca2+, mitochondria generate microdomains of low Ca2+ that enable the full activation of Ca2+ channels that are normally inhibited at high Ca2+ concentrations (3Hoth M. Fanger C.M. Lewis R.S. J. Cell Biol. 1997; 137: 633-648Crossref PubMed Scopus (462) Google Scholar, 4Malli R. Frieden M. Osibow K. Graier W.F. J. Biol. Chem. 2003; 278: 10807-10815Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). In addition, mitochondria have been shown to recycle the Ca2+ released from the ER back to this intracellular Ca2+ store, preventing the Ca2+ depletion of the ER (5Arnaudeau S. Kelley W.L. Walsh Jr., J.V. Demaurex N. J. Biol. Chem. 2001; 276: 29430-29439Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar), and to relay the Ca2+ entering across plasma membrane Ca2+ channels directly to the ER, short circuiting the cytosol (6Malli R. Frieden M. Osibow K. Zoratti C. Mayer M. Demaurex N. Graier W.F. J. Biol. Chem. 2003; 278: 44769-44779Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Mitochondria thus not only integrate intracellular Ca2+ signals to produce the appropriate metabolic response but also control the fluxes of Ca2+ inside cells to shape Ca2+ signals.Ca2+ enters mitochondria down its electrochemical gradient via a Ca2+ uniporter, using the negative mitochondrial membrane potential generated by the electron transport chain, and is released from mitochondria by a Na+/Ca2+ exchanger or by the opening of the mitochondrial permeability transition pore. Because of the low Ca2+ affinity of the mitochondrial Ca2+ uniporter, micromolar Ca2+ concentrations are required for efficient Ca2+ uptake into mitochondria. Such high Ca2+ concentrations occur only transiently in cells at the mouth of Ca2+ channels, implying that mitochondria are in close proximity to plasma membrane Ca2+ entry channels or to intracellular Ca2+ release channels (7Rizzuto R. Pinton P. Carrington W. Fay F.S. Fogarty K.E. Lifshitz L.M. Tuft R.A. Pozzan T. Science. 1998; 280: 1763-1766Crossref PubMed Scopus (1769) Google Scholar). Accordingly, morphological and functional evidence indicate that mitochondria are very close and are possibly physically connected to Ca2+ release channels on the endoplasmic reticulum (8Filippin L. Magalhaes P.J. Di Benedetto G. Colella M. Pozzan T. J. Biol. Chem. 2003; 278: 39224-39234Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 9Szabadkai G. Rizzuto R. FEBS Lett. 2004; 567: 111-115Crossref PubMed Scopus (123) Google Scholar).Mitochondria are structurally complex organelles that move, fuse, and divide within cells (10Yaffe M.P. Science. 1999; 283: 1493-1497Crossref PubMed Scopus (416) Google Scholar, 11Griparic L. van der Bliek A.M. Traffic. 2001; 2: 235-244Crossref PubMed Scopus (113) Google Scholar, 12Legros F. Lombes A. Frachon P. Rojo M. Mol. Biol. Cell. 2002; 13: 4343-4354Crossref PubMed Scopus (495) Google Scholar). This constant remodeling regulates the morphology, number, and function of mitochondria and ensures the proper transmission of mitochondria to daughter cells during cell division. In most cells, mitochondria fusion and fission are usually balanced, leading to the formation of a tubular network of interconnected mitochondria. This cable-like architecture facilitates the transfer of the mitochondrial membrane potential (ΔΨm) from oxygen-rich to oxygen-poor cellular regions (13Skulachev V.P. Trends Biochem. Sci. 2001; 26: 23-29Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar) and allows the rapid transmission of Ca2+ signals along the interconnected mitochondria (14Ichas F. Jouaville L.S. Mazat J.P. Cell. 1997; 89: 1145-1153Abstract Full Text Full Text PDF PubMed Scopus (646) Google Scholar). Disruption of the mitochondrial network by excessive mitochondrial fission or impaired fusion is a common feature of apoptosis, and the breakdown of the mitochondrial network is associated with a decrease in mitochondrial respiration, an increase in the production of reactive oxygen species, and the loss of the mitochondrial DNA (10Yaffe M.P. Science. 1999; 283: 1493-1497Crossref PubMed Scopus (416) Google Scholar).Mitochondrial fusion and fission reactions are regulated by a highly evolutionary conserved set of dynamin-related proteins. Mitochondrial fission is operated by Drp1, a GTPase located in the cytosol that is recruited to mitochondria by hFis1, a small adapter protein located on the outer mitochondrial membrane (15Smirnova E. Shurland D.L. Ryazantsev S.N. van der Bliek A.M. J. Cell Biol. 1998; 143: 351-358Crossref PubMed Scopus (571) Google Scholar, 16James D.I. Parone P.A. Mattenberger Y. Martinou J.C. J. Biol. Chem. 2003; 278: 36373-36379Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar). Mitochondrial fusion is regulated by the transmembrane GTPases mitofusin and OPA-1, located on the outer and inner mitochondrial membrane, respectively (reviewed in Ref. 17Bossy-Wetzel E. Barsoum M.J. Godzik A. Schwarzenbacher R. Lipton S.A. Curr. Opin. Cell Biol. 2003; 15: 706-716Crossref PubMed Scopus (363) Google Scholar). By overexpressing or ablating the genes coding for these mitochondrial fission and fusion proteins, it is now possible to selectively manipulate the mitochondrial network toward fusion or fission. Using this strategy, we previously showed that the overexpression of hFis1 induced a complete fragmentation of the mitochondrial network in HeLa cells (18Frieden M. James D. Castelbou C. Danckaert A. Martinou J.C. Demaurex N. J. Biol. Chem. 2004; 279: 22704-22714Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). The fragmented mitochondria clustered around the nucleus but retained a normal membrane potential and pH and normally took up the Ca2+ released from the ER upon stimulation with agonists.In the present study, we investigated the impact of a hFis1 or Drp1-induced mitochondrial fragmentation on the cytosolic and ER Ca2+ homeostasis. Our data show that the lack of subplasmalemmal mitochondria in cells overexpressing hFis1 or Drp1 is associated with an increased activity of plasma membrane Ca2+ pumps, which, in turn, induces a faster Ca2+ depletion of the ER and a subsequent activation of store-operated Ca2+ influx. These defects are balanced as long as Ca2+ is present in the extracellular medium and become apparent only when Ca2+ is removed. The lack of mitochondria below the plasma membrane thus renders cells more dependent on extracellular Ca2+ for signaling.EXPERIMENTAL PROCEDURESCell Culture and Transfection—HeLa cells were grown on minimum essential medium supplemented with 2 mm l-glutamine, 50 μg/ml streptomycin, and 50 units/ml penicillin along with 10% heat-inactivated fetal calf serum and were maintained at 37 °C under 5% CO2. For experiments, cells were plated on 25-mm diameter glass coverslips 2-3 days before use. After reaching 40-60% of confluence, cells were transiently transfected with the different plasmids using the TransFast reagent according to the protocol supplied by the manufacturer (Promega). For measurements of [Ca2+]cyt, hFis1 or Drp1 were co-transfected with a GFP targeted to the nucleus in order to identify cells expressing the mitochondrial fission proteins. All experiments were performed between 16 and 20 h after transfection with hFis1, and after 48 h with Drp1. To measure endoplasmic reticulum Ca2+, [Ca2+]ER, cells were transfected with the cameleon YC4.1 targeted to the endoplasmic reticulum (YC4.1ER; a gift from Dr. R. Y. Tsien, University of California at San Diego) and 24 h later with hFis1.Materials—Minimum essential medium, fetal calf serum, penicillin, and streptomycin were obtained from Invitrogen. Histamine and thapsigargin were obtained from Sigma. The acetoxymethyl ester form of Fura-2 (Fura-2/AM) and MitoTracker Red were obtained from Molecular Probes Europe. TransFast transfection reagent was purchased from Promega (Catalysis AG).Cysotolic Ca2+ Measurements—Experiments were performed in Hepes-buffered solution containing 140 mm NaCl, 5 mm KCl, 1 mm MgCl2, 2 mm CaCl2, 20 mm Hepes, and 10 mm glucose, pH 7.4, with NaOH. The Ca2+-free solution contained 1 mm EGTA instead of CaCl2 except in the Mn2+ quench experiments, where no EGTA was added. The low Na+ solution contained 121 mm N-methyl-d-glucamine, 19 mm NaCl, 5 mm KCl, 1 mm MgCl2, 2 mm CaCl2, 20 mm Hepes, and 10 mm glucose, pH 7.4, with HCl. Glass coverslips were mounted in a thermostatic chamber (Harvard Apparatus, Holliston, MA) equipped with gravity feed inlets and a vacuum outlet for solution changes. Cells were imaged on an Axiovert S100 TV using a 100×, 1.3 numerical aperture, oil immersion objective (Carl Zeiss AG, Feldbach, Switzerland). Cells were loaded for 30 min with 2 μm Fura-2/AM at room temperature in the dark, washed twice, and equilibrated for 15-20 min to allow de-esterification. To monitor [Ca2+]cyt, cells were alternatively excited at 340 and 380 nm with a monochromator (DeltaRam, Photon Technology International Inc., Monmouth Junction, NJ) through a 430 DCLP dichroic mirror, and emission was monitored through a 510WB40 filter (Omega Optical, Brattleboro, VT). Transfected cells were recognized by the fluorescence of the nuclear-targeted GFP (480-nm excitation, 535-nm emission), and the characteristic morphology of mitochondria (globular versus tubular) was verified by imaging the MitoTracker labeling (577-nm excitation, 590-nm emission). For this purpose, cells were loaded prior the experiments with 500 nm MitoTracker Red for 90 s and washed 2-3 times with experimental buffer. To minimize cross-talk of the nuclear GFP fluorescence into the 380 nm Fura-2 signal, we only selected cells with a moderate nuclear fluorescence, and the region of the cytosol used to estimate [Ca2+]cyt did not include the nucleus. Fluorescence emission was imaged using a cooled, 16-bit, charge-coupled device, back-illuminated, frame transfer Micro-Max camera (Princeton Instruments, Roper Scientific, Trenton, NJ). Image acquisition and analysis were performed with the MetaFluor 6.2 software (Universal Imaging, West Chester, PA).ER Ca2+ Measurements—HeLa cells transfected with the YC4.1ER were excited at 430 nm through a 455 DRLP dichroic mirror (Omega Optical), and emissions were collected alternatively at 475 and 535 nm (475DF15 and 535DF25, Omega Optical) through emission filters mounted on a filter wheel (Ludl Electronic Products, Hawthorn, NY). To recognize the fragmented aspect of the mitochondria due to hFis1 overexpression, HeLa cells were loaded with 500 nm MitoTracker Red prior to the experiments. A low intensity threshold was applied to define the fluorescent signal associated with the ER. The total fluorescent area was taken for measurement.Measurements of Mitochondria Morphology.—Cells were imaged on an Axiovert 200M microscope equipped with an array laser confocal spinning disk (QLC100; VisiTech, Sunderland, UK) using a 63×, 1.4 numerical aperture, oil-immersion objective (Carl Zeiss AG). Cells were alternately excited at 488 and 568 nm to image the green emission (525/50 nm) from the nuclear targeted enhanced GFP (EGFP) or the membrane-targeted yellow cameleon YC3.6pm and the red (615/60 nm) emission from MitoTracker Red or RFPmit. These fluorescent images were acquired using a cooled, 12-bit, charge-coupled device camera (CoolSnap HQ; Roper Scientific, Trenton, NJ) operated by the Metamorph 6.2 software (Universal Imaging). Optical slices of 300-nm step size z sections were acquired, and the green and red channels were further deconvoluted with the Huygens algorithm (Scientific Volume Imaging BV, Hilversum, The Netherlands). Three-dimensional rendering of the deconvoluted z stacks (shadow projections or cross-sections) was done with the Imaris 4.2 software (Bitplane AG, Zurich, Switzerland). For measurements of mitochondrial volume, complete series of the deconvoluted z stacks were processed with the "measure volume" routine from the Metamorph software after application of a low intensity threshold on the MitoTracker Red or RFPmit fluorescence. The percentage of membrane volume colocalized with mitochondria was calculated in the whole cell volume using the colocalization option from the Imaris software after restriction of each fluorescence signal by a low intensity threshold to define the respective structures.Measurements of Ca2+ Entry—Mn2+ (100 μm) was substituted for Ca2+ to estimate the ion flux through Ca2+ entry channels according to the Mn2+-quench technique (19Demaurex N. Monod A. Lew D.P. Krause K.H. Biochem. J. 1994; 297: 595-601Crossref PubMed Scopus (81) Google Scholar). Cells were excited at 356-358 nm, which corresponded to the isosbestic point of Fura-2. The rate of fluorescence decrease reflects the rate at which Mn2+ enters the cells, and the slope during the first 1-2 min was used as an indicator for Ca2+ entry.Statistics—Unless indicated, statistical analysis was performed using the unpaired Student's t test.RESULTSEffects on hFis1 and Drp1 on Mitochondria Distribution—To study the impact of hFis1 or Drp1 overexpression on mitochondria morphology, we analyzed the structure of mitochondria by confocal imaging. HeLa cells were co-transfected with a nuclear targeted GFP together with the fission protein, labeled with MitoTracker Red, and imaged on a spinning wheel confocal microscope. As shown in Fig. 1, overexpression of either hFis1 or Drp1 induced the fragmentation of mitochondria, but the organelles redistributed very differently inside the cells. hFis1-fragmented mitochondria clustered around the nucleus, whereas Drp1-fragmented mitochondria remained scattered throughout the cell. Interestingly, the expression of the fission proteins was associated with a reduction in the mitochondrial volume (Fig. 1, bottom right panel). The total volume labeled with MitoTracker in the confocal stacks was reduced by 39% upon the expression of hFis1 and by 25% upon the expression of Drp1. A 34% reduction in mitochondrial volume was also observed upon the expression of hFis1 using RFPmit as a mitochondrial marker. The reduction in mitochondrial volume was not associated with a change in cellular volume (data not shown). Thus, the two fission proteins not only caused the fragmentation of mitochondria but also reduced the total mitochondrial content. In addition, the two proteins redistributed mitochondria differently within cells, with hFis1 inducing a collapse of mitochondria around the nucleus, whereas Drp1-fragmented mitochondria remained distributed throughout the cell.We showed previously that hFis1 expression removed mitochondria from the cell periphery (18Frieden M. James D. Castelbou C. Danckaert A. Martinou J.C. Demaurex N. J. Biol. Chem. 2004; 279: 22704-22714Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar), but this analysis has not been performed with Drp1. Because of the different fragmentation pattern obtained with Drp1, mitochondria could still be close to the plasma membrane in the z axis in our fairly thin HeLa cells. To establish the location of mitochondria relative to the plasma membrane, cells were transfected with a fluorescent protein bearing the membrane anchor sequence CAAX of ki-Ras (YC3.6pm) (20Nagai T. Yamada S. Tominaga T. Ichikawa M. Miyawaki A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 10554-10559Crossref PubMed Scopus (849) Google Scholar). As shown in Fig. 2 (green channel), the membrane-targeted probe was expressed at the cell periphery and in filopodial and lamellipodial structures. In control cells, mitochondria decorated the inner leaflet of the plasma membrane, as seen in the xz and yz cross-section images. In contrast, in cells expressing hFis1 or Drp1, mitochondria were not distributed evenly below the plasma membrane. As a result, large contiguous regions of the plasma membrane were devoid of neighboring mitochondria. Using the complete series of the z stacks, we performed a quantitative analysis of the interactions between mitochondria and the plasma membrane (Fig. 2, bottom right panel). The fraction of the plasma membrane bearing mitochondria decreased from 10.1 ± 2.5 to 2.5 ± 0.3 and 2.9 ± 0.4% upon hFis1 and Drp1 expression, respectively (p < 0.01). Thus, both fission proteins reduced the fraction of the plasma membrane bearing mitochondria by 4-fold.FIGURE 2Effect of hFis1 and Drp1 overexpression on mitochondria location. HeLa cells were transfected with hFis1 or Drp1 cDNA together with a membrane-targeted yellow cameleon YC3.6pm, labeled with MitoTracker Red, and imaged as in Fig. 1. Images are xy, xz, and yz cross-sections of the two fluorescence channels, shown as green/red overlay. The cross-hairs indicate the location of the sectioning within the stack. Scale bar, 10 μm. Bottom right, the extent of co-localization between the MitoTracker and membrane labeling is expressed as a percentage of the total membrane volume. Statistics were performed on 20-40 sections from six control, six hFis1, and six Drp1-expressing cells. Bars are mean ± S.E.; *, p < 0.01, Mann-Whitney test.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effects of hFis1 on Histamine-induced Cytosolic Ca2+ Elevations—To evaluate the impact of the mitochondrial redistribution on the cytosolic Ca2+ signals, HeLa cells were challenged with the Ca2+-mobilizing agonist histamine or with the SERCA inhibitor thapsigargin, and changes in the cytosolic Ca2+ concentration were measured with Fura-2. The effects of hFis1, which induced the most dramatic mitochondrial redistribution, were analyzed in detail. The resting Ca2+ concentrations were not different between control and hFis1-overexpressing cells, but the responses elicited by histamine differed markedly depending on the presence of external Ca2+ (Fig. 3). When Ca2+ was present in the extracellular medium the application of a low dose of histamine (1 μm) elicited Ca2+ elevations of similar amplitude and duration in control and hFis1-overexpressing cells (Δratio was 0.77 ± 0.08 and 0.75 ± 0.10 for control and overexpressing cells, n = 18 and n = 14, respectively; Fig. 3A). In contrast, in the absence of external Ca2+ the histamine-induced Ca2+ elevation was reduced by 40% in hFis1-expressing cells (Δratio was 0.53 ± 0.04 versus 0.95 ± 0.04, n = 32 and n = 77, p < 0.0001; Fig. 3B). An even more severe reduction was observed when cells were kept for a longer duration in Ca2+-free medium before the application of histamine (15 min; Fig. 3C). The integrated Ca2+ responses measured in control and hFis1-expressing cells, expressed as the area under the curve, are illustrated in Fig. 3E for the different protocols. The Ca2+ responses were similar in the presence of Ca2+ but were reduced by 60% when Ca2+ was omitted for 2 min and by 80% when Ca2+ was omitted for 15 min. Interestingly, a normal response was observed in Ca2+-free medium when hFis1-expressing cells were stimulated with supramaximal doses of histamine (50 μm; data not shown). However, in all conditions tested (1 and 50 μm histamine in Ca2+ or Ca2+-free medium), the percentage of cells displaying an oscillatory response was lower in hFis1 overexpressers, suggesting that these cells had a defect in intracellular Ca2+ handling.FIGURE 3Effect of hFis1 overexpression on cytosolic Ca2 + responses. HeLa cells were loaded with 2μm Fura-2 and stimulated with histamine or thapsigargin. A, responses elicited by the application of 1 μm histamine to cells maintained in a medium containing 2 mm Ca2+. Traces are averages of 18 and 14 recordings for control and hFis1, respectively. B and C, responses elicited by the application of 1 μm histamine to cells maintained in Ca2+-free medium for 2 min (B, n = 77 and 32 for control and hFis1, respectively) or for 15 min (C, n = 21 and 15 for control and hFis1, respectively). D, cells maintained in Ca2+-free medium for 2 min were stimulated with 1 μm thapsigargin (TG). Traces are the average of 17 and 12 recordings for control and hFis1, respectively. E, statistical evaluation of the Ca2+ increase induced by histamine, expressed as the area under the curve (A.U.C.). Bars are mean ± S.E.; *, p < 0.05. F, statistical evaluation of the Ca2+ increase induced by 1 μm thapsigargin done under normal (145 mm) and low (19 mm) extracellular Na+ conditions, expressed as the area under the curve (A.U.C.). Bars are mean ± S.E; *, p < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To verify that the reduced Ca2+ response was due to a decreased release of Ca2+ from intracellular stores, cells were stimulated with 1 μm thapsigargin in the absence of extracellular Ca2+ to indirectly assess the ER Ca2+ content. As shown in Fig. 3D, the thapsigargin-induced response of hFis1-expressing cells was reduced by 58%, suggesting that the Ca2+ content of the ER was reduced by an equivalent amount. ER Ca2+ content was also assessed at low extracellular Na+ concentration (19 mm NaCl) to prevent the activity of the Na+/Ca2+ exchanger. In this condition, the response to thapsigargin was reduced by 47% in hFis1-overexpressing cells (Fig. 3F).Effects of hFis1 on ER Ca2+ Content—To study the effect of hFis1 on ER Ca2+ handling, we directly measured the free ER Ca2+ concentration, [Ca2+]ER, using the genetically encoded yellow cameleon probe YC4.1ER. Cells were sequentially exposed to a Ca2+-free medium and then to a maximal dose of 50 μm histamine followed by 1 μm thapsigargin to release Ca2+ from the ER (Fig. 4A). Neither the resting [Ca2+]ER levels nor the [Ca2+]ER levels attained after extensive store depletion with histamine and thapsigargin differed significantly between control and hFis1-overexpressing cells (resting: 0.984 ± 0.018 with n = 33 and 1.035 ± 0.034 with n = 18 for untransfected and hFis1, respectively; depleted: 0.904 ± 0.014 with n = 11 and 0.876 ± 0.026 with n = 10 for untransfected and hFis1, respectively). However, detailed analysis of the kinetics of Ca2+ release revealed that the [Ca2+]ER decreased more rapidly in hFis1-overexpressing cells upon switching to a Ca2+-free medium and during the application of histamine (Fig. 4A, first two arrows from the left). Consequently, the drop in [Ca2+]ER elicited by Ca2+ removal or by histamine application was significantly larger in hFis1-overexpressing cells (Ca2+ removal: 0.008 ± 0.002 with n = 33 and 0.026 ± 0.007 with n = 18 for untransfected and hFis1, respectively, with p < 0.05; histamine application: 0.038 ± 0.006 with n = 11 and 0.069 ± 0.010 with n = 10 for untransfected and hFis1, respectively, with p < 0.05; Fig. 4B). These data indicate that the [Ca2+]ER is decreasing more rapidly in hFis1-overexpressing cells upon the removal of extracellular Ca2+ and during cell stimulation with agonists.FIGURE 4Effect of hFis1 overexpression on ER Ca2+ homeostasis. HeLa cells were transiently transfected with the ER-targeted cameleon probe YC4.1ER and 1 day later with hFis1. A, original recordings of ER Ca2+ following transition from Ca2+ to Ca2+-free medium and subsequent stimulation with 50 μm histamine and 1 μm thapsigargin. B, statistical evaluation of the ER Ca2+ decrease elicited by Ca2+ removal and by application of 50 μm histamine (n = 11 and 10 for control and hFis1, respectively). Bars are mean ± S.E.; *, p < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effects of hFis1 on Plasmalemmal Ca2+ Fluxes—Because both the cytosolic and the ER [Ca2+] were decreasing faster in hFis1 cells kept in Ca2+-free medium even without agonist stimulation, we assessed whether the fluxes of Ca2+ at the plasma membrane were altered. To test the basal activity of the Ca2+ extrusion and Ca2+ influx machinery, we used a simple Ca2+ switch protocol, alternating the bath solution from Ca2+-containing to Ca2+-free media. As shown in Fig. 5, A and B, Ca2+ removal caused a more pronounced [Ca2+]cyt decrease in hFis1 overexpressers, whereas the readdition of Ca2+ produced a faster and larger [Ca2+]cyt increase in these cells. The increased Ca2+ entry was confirmed by using the Mn2+ quench technique. The addition of 100 μm Mn2+ to cells kept for 3 min in nominal Ca2+-free medium caused a significantly faster decrease in Fura-2 fluoresc
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