SLP-2 negatively modulates mitochondrial sodium-calcium exchange

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Article

Reference

SLP-2 negatively modulates mitochondrial sodium-calcium exchange

DA CRUZ, Sandrine, et al.

Abstract

Mitochondria play a major role in cellular calcium homeostasis. Despite decades of studies, the molecules that mediate and regulate the transport of calcium ions in and out of the mitochondrial matrix remain unknown. Here, we investigate whether SLP-2, an inner membrane mitochondrial protein of unknown function, modulates the activity of mitochondrial Ca(2+) transporters. In HeLa cells depleted of SLP-2, the amplitude and duration of mitochondrial Ca(2+) elevations evoked by agonists were decreased compared to control cells. SLP-2 depletion increased the rates of calcium extrusion from mitochondria. This effect disappeared upon Na(+) removal or addition of CGP-37157, an inhibitor of the mitochondrial Na(+)/Ca(2+) exchanger, and persisted in permeabilized cells exposed to a fixed cytosolic Na(+) and Ca(2+) concentration. The rates of mitochondrial Ca(2+) extrusion were prolonged in SLP-2 over-expressing cells, independently of the amplitude of mitochondrial Ca(2+) elevations. The amplitude of cytosolic Ca(2+) elevations was increased by SLP-2 depletion and decreased by SLP-2 over-expression. These data show that SLP-2 [...]

DA CRUZ, Sandrine, et al . SLP-2 negatively modulates mitochondrial sodium-calcium exchange. Cell Calcium , 2010, vol. 47, no. 1, p. 11-18

DOI : 10.1016/j.ceca.2009.10.005 PMID : 19944461

Available at:

http://archive-ouverte.unige.ch/unige:18371

Disclaimer: layout of this document may differ from the published version.

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Contents lists available atScienceDirect

Cell Calcium

j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / c e c a

SLP-2 negatively modulates mitochondrial sodium–calcium exchange

Sandrine Da Cruz

^a,1,2

, Umberto De Marchi

^b,1

, Maud Frieden

^b

, Philippe A. Parone

^a,2

, Jean-Claude Martinou

^a

, Nicolas Demaurex

^b,∗

aDepartment of Cell Biology, University of Geneva, Switzerland

bDepartment of Cell Physiology and Metabolism, University of Geneva, 1 Michel-Servet, CH-1211 Geneva 4, Switzerland

a r t i c l e i n f o

Article history:

Received 24 June 2009

Received in revised form 27 October 2009 Accepted 30 October 2009

Available online 26 November 2009

Keywords:

Mitochondria Calcium signaling Sodium–calcium exchange Stomatin

a b s t r a c t

Mitochondria play a major role in cellular calcium homeostasis. Despite decades of studies, the molecules that mediate and regulate the transport of calcium ions in and out of the mitochondrial matrix remain unknown. Here, we investigate whether SLP-2, an inner membrane mitochondrial protein of unknown function, modulates the activity of mitochondrial Ca²⁺transporters. In HeLa cells depleted of SLP-2, the amplitude and duration of mitochondrial Ca²⁺elevations evoked by agonists were decreased compared to control cells. SLP-2 depletion increased the rates of calcium extrusion from mitochondria. This effect disappeared upon Na⁺removal or addition of CGP-37157, an inhibitor of the mitochondrial Na⁺/Ca²⁺

exchanger, and persisted in permeabilized cells exposed to a ﬁxed cytosolic Na⁺and Ca²⁺concentration.

The rates of mitochondrial Ca²⁺extrusion were prolonged in SLP-2 over-expressing cells, independently of the amplitude of mitochondrial Ca²⁺elevations. The amplitude of cytosolic Ca²⁺elevations was increased by SLP-2 depletion and decreased by SLP-2 over-expression. These data show that SLP-2 modulates mitochondrial calcium extrusion, thereby altering the ability of mitochondria to buffer Ca²⁺and to shape cytosolic Ca²⁺signals.

1. Introduction

Mitochondria are eukaryotic organelles that play a major role in cellular calcium homeostasis. Mitochondrial calcium fluxes are involved in the regulation of several physiological processes includ- ing energy metabolism, insulin secretion, synaptic transmission, cardiac contraction and cell death[1–3]. The transport of calcium ions in and out of the mitochondria involves crossing two membranes. The mitochondrial outer membrane (MOM) is relatively permeable to ions in general and calcium transport is mediated by the non-selective voltage-dependent anion channel VDAC[4]. Con- versely, calcium fluxes across the mitochondrial inner membrane (MIM) are tightly regulated, since the MIM is highly impermeable to ions, and requires a variety of specific transport systems[5], see [1]for a recent review. Calcium uptake, for example, is primarily mediated by the mitochondrial uniporter[6], but has also been shown to occur through the “rapid-mode calcium uptake” (RaM) channel and the ryanodine receptor isoform 1 (mRyR) in excitable cells[7,8]. Mitochondrial calcium efflux mainly happens through

∗ Corresponding author. Tel.: +41 22 379 5399; fax: +41 22 379 5338.

E-mail address:Nicolas.Demaurex@unige.ch(N. Demaurex).

1These authors contributed equally to this work.

2Current address: Ludwig Institute for Cancer Research, University of California, San Diego, 9500 Gilman drive, La Jolla, CA 92093-0670, United States.

the sodium/calcium exchanger (mNCE) but has been shown to be also mediated by a calcium/proton antiporter (mHCE) and the per- meability transition pore (PTP)[9]. Although mitochondrial calcium channels have been studied extensively using a range of biochemical approaches and by electrophysiology[6,10], their molecular identity remains unknown. The uncoupling proteins UCP2 and UCP3 have been shown to be essential for mitochondrial Ca²⁺

uptake, suggesting that these molecules might be part of the Ca²⁺

uniporter of mitochondria[11]. However, this claim is disputed and awaits conﬁrmation[12,13]. So far, no candidate has been proposed for the mNCE.

Stomatin is a plasma membrane protein that is found in a large number of organisms ranging from mammals to bacteria. Studies on patients suffering from over-hydrated hereditary stomatocytosis, a rare autosomal dominant hemolytic anemia, have shown that stomatin was absent from red blood cells of these patients. Inci- dentally, these red blood cells have an increased cationic leak and lyse prematurely[14]. These results have suggested that stomatin is involved in the regulation of cation channel activities. These conclu- sions were supported by studies inC. elegansshowing that stomatin homologues interact with degenerin/sodium channels and modulate their activity[15,16]. Furthermore, stomatin was shown to interact with acid-sensing ion channels and to alter their gating in mammalian cells[17]. Stomatins are the founding members of a family of proteins called stomatin-like proteins which include SLP-

doi:10.1016/j.ceca.2009.10.005

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12 S. Da Cruz et al. / Cell Calcium47 (2010) 11–18

1, 2 and 3[18,19]. A function has not yet been ascribed to these three proteins but it is known that SLP-2 is expressed in a range of mammalian tissues, notably the plasma membrane of erythro- cytes[20]. Phylogenic analysis revealed that SLP-2 was acquired through the mitochondrial endosymbiosis and belongs to a different lineage than other stomatin-like proteins[21]. SLP-2 is highly expressed in several types of human tumors[22], and high SLP-2 levels are associated with decreased survival of patients suffering from primary invasive breast cancer[23], suggesting a role for SLP-2 in tumorigenesis.

In a proteomic study from our laboratory, we identified SLP-2 as a component of the MIM[24]. The same protein was also detected in similar studies of human and plant mitochondria[25,26]. We then confirmed that SLP-2 is attached to the MIM[27]. SLP-2 plays an important role in mitochondria. SLP-2 interacts with MFN2, a component of the mitochondrial fusion machinery[28], and is required for the stress-induced hyperfusion of mitochondria[29]. SLP-2 reg- ulates the stability of the mitochondrial chaperones prohibitins 1 and 2, of the respiratory chain complexes I and IV[27], and of the long isoform of Opa1[29]. Since SLP-2 appears to play an important scaffolding role in the MIM and is related to stomatin, a molecule known to regulate ion channels, we investigated whether SLP-2 can modulate the activity of mitochondrial ion channels. In this study, we show that SLP-2 is involved in the regulation of mitochondrial calcium homeostasis. Mitochondrial Ca²⁺release was delayed in SLP-2 over-expressing cells and accelerated in cells depleted of the protein, both in intact cells stimulated with histamine or in permeabilized cells exposed to known amounts of Ca²⁺. Altogether we show that SLP-2 may modulate mitochondrial calcium efflux by negatively regulating the mNCE.

2. Materials and methods 2.1. Cell culture and reagents

HeLa cells were cultured in DMEM + 10% fetal bovine serum, 100 U/ml penicillin, 0.1 mg/ml streptomycin and 2 mM glutamine and maintained in 5% CO₂ at 37^◦C. Tissue culture plates were obtained from Nunc (Roskilde) and all other cell culture reagents were obtained from Sigma (Buchs). Histamine was from Sigma and CGP-37157 from Tocris.

2.2. RNA interference

shRNA against SLP-2 was directly synthesized in cells using the recently developed pRETRO vector (generously provided by Dr. Agami and previously described in [30]. Nucleotides 697–715 of SLP-2 were chosen as targets for RNA interference.

To generate pRETRO shSLP-2 RNA, the pRETRO vector was digested with BglII and HindIII and the annealed oligos (5GATCC- CCGGCTGAACAGATAAATCAGTTCAAGAGACTGATTTATCTGTTCAG- CCTTTTTGGAAA3) and (5AGCTTTTCCAAAAAGGCTGAACAGATA- AATCAGTCTCTTGAACTGATTTATCTGTTCAGCCGGG3) were ligated into the vectors. Similarly, as a control we generated a pRETRO Luc shRNA, which targets the Luciferase transcripts. HeLa cells were plated on 10 cm dishes and transfected with the appropriate pRETRO constructs using Ca/phosphate method. 24 h after transfection, puromycin (Calbiochem) was added at 3␮g/ml and the cells were incubated for a further 24 h. After puromycin incubation the cells were washed and left to recover for a further 72 h before being collected and processed for Western blotting.

2.3. Cell lysis

Whole cells were lysed for 20 min on ice in lysis buffer (10 mM Hepes, 300 mM KCl, 5 mM MgCl₂, 1 mM EGTA, 1% Nonidet P-40,

0.1% SDS, pH 7.4) supplemented with protease inhibitors (Roche).

The lysate was centrifuged at 10,000×gfor 10 min and the protein content of the supernatant determined using a modiﬁed Brad- ford assay (Bio-Rad). 15␮g of total protein was loaded per lane of SDS/PAGE.

2.4. Electrophoresis and Western blotting

Standard SDS-PAGE was performed according to Laemmli. Fol- lowing electrophoresis, proteins were blotted to nitrocellulose membranes. Immunoreactive material was visualized by chelumi- nescence (ECL, Pierce) according to manufacturer’s instructions.

2.5. Antibodies

Anti-SLP-2 antibody was produced by immunizing rabbits with synthetic peptide with a sequence CRKRATVLESEGTRES. The mon- oclonal the anti-GAPDH was from Abcam and the anti-Tubulin from Chemikon International.

2.6. Mitochondrial Ca²⁺measurements

HeLa cells were plated on 25 mm diameter glass cover-slips and co-transfected with a construct expressing a ratiometric pericam probe targeted to mitochondria (RP3.1_mit, gift from Dr. Atsushi Miyawaki) and pRETRO constructs at a 2:1 ratio using Ca/phosphate method. 24 h after transfection, puromycin (Calbiochem) was added at 3␮g/ml and the cells were incubated for a further 24 h.

After puromycin incubation the cells were washed and left to recover for a further 72 h before Ca²⁺assays. For over-expressed proteins, HeLa cells were co-transfected with the appropriate construct (pCI or pCI-SLP2-HA) and a construct expressing the mitochondrial-targeted Ca²⁺probe 4mtD3cpV at a 2:1 ratio using TransFast transfection reagent (Promega). Ca²⁺assays were performed 48 h after transfection. Cover-slips were mounted in a thermostatic chamber (Harvard Apparatus) and experiment performed in Hepes-buffered solution containing 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 20 mM Hepes, 10 mM Glucose, pH 7.4 with NaOH.

Cells were imaged on an Axiovert s100 TV using a 100×, 1.3 NA oil-immersion objective (Zeiss). Fluorescence emission was imaged using a cooled, 16-bit CCD back-illuminated frame trans- fer MicroMax camera (Princeton Instruments). Image acquisition and analysis were performed with the Metafluor 6.2 software (Uni- versal Imaging). For RP3.1_mit tranfections, cells were excited at 410 and 480 nm, and emission was collected at 535 nm (535DF45, Omega Optical) through a 505DCXR (Omega Optical) dichroic mirror. Changes in mitochondrial Ca²⁺are shown as (1−F/F0) because RP3.1_mitfluorescence at410 nm= 410 nm decreases with increasing Ca²⁺concentrations. For 4mtD3cpV transfections, cells were excited at 430 nm with a monochromator (DeltaRam, PTI, Mon- mouth Junction, NJ) through a 455-nm dichroic mirror and imaged sequentially at 475 and 535 nm using a filter wheel (455DRLP, 475DF15, and 535DF25, Omega Optical, Brattleboro, VT).

The maximal calcium efflux rates were calculated by perform- ing a first order derivative on the data obtained during the first minute of the decay phase of the calcium response. Calculation was performed with Microsoft Excel after smoothing of the original recordings, and the maximal value taken for each individual recording.

2.7. Cytosolic Ca²⁺measurements

HeLa cells were transfected with the pRETRO constructs using Ca/Phosphate method. 24 h after transfection, puromycin (Cal- biochem) was added at 3␮g/ml and the cells were incubated for

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a further 24 h. After puromycin incubation the cells were washed and left to recover for a further 72 h before being analyzed for Ca²⁺

assays. For over-expressed SLP-2, HeLa cells were transfected as described above with the appropriate construct (pCI, pCI-SLP-2) and 48 h after transfection, the cells were analyzed for Ca²⁺assays.

Glass cover-slips were mounted in a thermostatic chamber (Harvard Apparatus) and the cells were imaged as described above. HeLa cells were loaded for 30 min with 2␮M Fura-2/AM at room temperature in the dark, washed twice and equilibrated for 15 min to allow de-esteriﬁcation. The experiment was performed in Hepes-buffered solution containing 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 20 mM Hepes, 10 mM glucose, pH 7.4 with NaOH. To monitor the [Ca²⁺]_c, cells were alternatively excited at 340 and 380 nm with a monochromator (DelatRam; Photon Tech- nology International) through a 430 DCLP dichroic mirror. Emission was monitored through a 510WB40 ﬁlter (Omega Optical).

2.8. Permeabilized cells

Cells were washed with high K⁺intracellular buffer (IB), containing 130 mM KCl, 10 mM NaCl, 1 mM MgCl2, 5 mM K2HPO4, 1 mM MgATP, 5 mM glucose, 5 mM succinate, 20 mM Hepes (pH 7.2 at 37^◦C), supplemented with 0.2 mM EGTA and 0.075 mM CaCl₂. For permeabilisation, 5␮M digitonin was added for 5 min and the cells then kept for 3 min in IB buffer before the addition of CaCl₂. Free [Ca²⁺] was calculated with the Maxchelator program (http://maxchelator.stanford.edu/).

2.9. Mitochondrial membrane potential (m) measurements

Cells were loaded with TMRM (Molecular Probes, 20 nM, 45 min, 37^◦C, dissolved in Hank’s Balanced Salt Solution) in the presence of verapamil (20␮M) to avoid TMRM extrusion by the

MDR. Cells were excited at 545 nm and fluorescence collected through an LP 590 long pass filter. Changes inmwere expressed as the ratio of the fluorescence in mitochondria divided by the cytosolic fluorescence (F_mito/Fcyto), measured in the same cells. At the end of the recording the protonophore carbonylcyanide- p-trifluoromethoxy phenylhydrazone (FCCP) was used to dissipatem.

2.10. Statistics

The signiﬁcance of differences between means was established using the Student’st-test for unpaired samples (^*p< 0.05;^**p< 0.01;

***p< 0.001).

3. Results

To investigate whether changes in SLP-2 expression levels could alter mitochondrial Ca²⁺fluxes, we used RNA interference to down- regulate the expression of SLP-2 (see Section 2). As shown in Fig. 1A, the shRNA targeting SLP-2 efficiently reduced SLP-2 protein levels at 120 h post-transfection, whereas levels of tubulin and GAPDH remained constant. The effects of the down-regulation of SLP-2 expression on mitochondrial Ca²⁺dynamics were then assessed using a genetically encoded Ca²⁺-sensitive probe targeted to mitochondria, the “pericam” probe RP3.1_mit. The fluorescence of RP3.1_mitdecreases with increasing concentrations of Ca²⁺when excited at 410 nm, enabling to obtain a semi-quantitative measure of the free Ca²⁺concentration within the mitochondrial matrix, [Ca²⁺]_mit. HeLa cells were co-transfected with RP3.1_mitand either the SLP-2 or control shRNA construct and cultured for 120 h before [Ca²⁺]_mitrecordings. As shown inFig. 1B, the amplitude and duration of the [Ca²⁺]_mitelevations evoked by histamine in HeLa cells

Fig. 1.Mitochondrial Ca²⁺responses in cells depleted of SLP-2. (A) HeLa cells were transfected with the Ctrl shRNA (pRETRO Luc shRNA) or the SLP-2 shRNA (pRETRO SLP-2 shRNA) constructs. 120 h post-transfection, 15␮g of proteins from the cell extract were analyzed by Western blotting. The levels of endogenous SLP-2 were assessed using the anti-SLP-2 antibody. Tubulin and GAPDH were used as loading controls. (B and C) HeLa cells were transiently co-transfected with the mitochondrial calcium probe RP3.1mitand the indicated shRNA constructs for 120 h. (B) Original recordings of HeLa cells stimulated with 100␮M histamine in Ca²⁺-containing medium. (C) Quantiﬁcation of the effects of the SLP-2 shRNA on the amplitude of the [Ca²⁺]mitsignal evoked by histamine (top), the integrated [Ca²⁺]mitresponse (middle, area under the curve) and the maximal mitochondrial Ca²⁺efﬂux rate (bottom, derived from the decaying phase of the [Ca²⁺]mitsignal). Bars are mean±S.E.M. of 38 and 55 cells for Ctrl and SLP-2 shRNA, respectively.

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Fig. 2.Effect of CGP-37157 on the [Ca²⁺]mitresponses of SLP-2 knockdown cells. HeLa cells were transiently co-transfected with RP3.1mitand either control or SLP-2 shRNA for 120 h. Cells were pre-incubated for 2 min with 10␮M CGP-37157 (+CGP) or Hepes-buffered solution before stimulation with histamine. (A and B) Original recordings of HeLa cells stimulated with 100␮M histamine. Traces obtained in the absence of CGP-37157 are shown for comparison. (C) Statistical evaluation of SLP-2 shRNA effects on the [Ca²⁺]mitsignal amplitude (top) and maximal mitochondrial Ca²⁺efﬂux rates (bottom) measured with or without CGP-37157 (data fromFig. 1). Bars are mean±S.E.M. of 38 vs. 55 and 21 vs. 15 Ctrl and SLP-2 shRNA cells untreated and treated with CGP-37157, respectively.

depleted of SLP-2 were markedly reduced compared to cells transfected with the control shRNA (Peak amplitude: 0.16±0.01 in SLP-2 shRNA cells vs. 0.22±0.01 in control cells). As a result, the total amount of Ca²⁺that transited through the mitochondria, assessed by integrating the Ca²⁺responses during the 90 s after histamine stimulation, was decreased by 41% in HeLa cells transfected with the SLP-2 shRNA (Fig. 1C; AUC: 7.51±0.57 in SLP-2 shRNA cells vs. 12.81±1.01 in control cells). Detailed analysis of the kinetics of the [Ca²⁺]_mitsignal revealed that the maximal rate of Ca²⁺extrusion from mitochondria, measured during the decaying phase of the [Ca²⁺]_mitsignal, was increased in SLP-2 depleted cells compared to control transfectants (Fig. 1C;F/dt= 0.111±0.005 in SLP-2 shRNA cells vs. 0.089±0.007 in control cells). Thus, mitochondria from SLP-2 depleted cells released Ca²⁺faster than mitochondria from control cells. This behaviour was not due to a reduction of the cytosolic Ca²⁺elevations (see below). These results indicate that down-regulating the expression of SLP-2 reduced the amplitude and duration of [Ca²⁺]_mitresponses, possibly by increasing the rate of Ca²⁺extrusion from mitochondria.

The negative membrane potential of respiring mitochondria drives the entry of Ca²⁺ions into mitochondria. To test whether the reduced [Ca²⁺]_mitsignal could be due to a reduced MIM potential (m) in cells depleted of SLP-2, we measured the m

in cells transfected with control and SLP-2 shRNA. As shown in supplemental Fig. S1, no difference could be observed in them

of control and SLP-2 shRNA cells, either at rest or during the addition of histamine to mobilize Ca²⁺, or after addition of the protonophore FCCP to dissipatem. These results were con- ﬁrmed by ﬂow cytometric analysis of the whole cell population stained with TMRE (data not shown). These data show that depleting cells of SLP-2 did not affect them, thus suggesting that the alterations in mitochondrial Ca²⁺transport in those cells could not be attributed to differences inm.

To study whether depletion of SLP-2 altered mitochondrial Ca²⁺

entry or extrusion, we used the speciﬁc inhibitor of the mNCE, CGP- 37157[31,32]. As shown inFig. 2, treatment of cells with 10␮M CGP-37157 transformed the transient [Ca²⁺]_mit elevation evoked by histamine into a sustained [Ca²⁺]_mitelevation, indicating that Ca²⁺was effectively trapped into the mitochondrial matrix. In these conditions the rate of Ca²⁺extrusion from mitochondria, measured as inFig. 1, was identical between SLP-2 depleted cells and control cells (Fig. 2C, bottom panel). This suggests that the increased Ca²⁺

efﬂux rate of SLP-2 depleted cells was due to an increased activity of the mNCE. However, despite near complete abrogation of mitochondrial Ca²⁺extrusion, CGP-37157 did not restore the amplitude of the [Ca²⁺]_mitelevation evoked by histamine in SLP-2 depleted cells to levels observed in control cells (0.17±0.02 vs. 0.24±0.01;

Fig. 2C, top panel). This indicates that increased Ca²⁺extrusion from mitochondria was not responsible for the blunted amplitude of the [Ca²⁺]_mitelevation induced by histamine.

Two transporters catalyze the extrusion of Ca²⁺ from mitochondria, a Na⁺/Ca²⁺ exchanger inhibited by CGP-37157 and a H⁺/Ca²⁺antiporter reportedly insensitive to CGP-37157. To conﬁrm that the increased mitochondrial Ca²⁺extrusion of SLP-2 depleted cells was due to increased activity of the Na⁺/Ca²⁺exchanger, we measured [Ca²⁺]_mitelevations in the absence of Na⁺. To avoid con- founding effects due to reversal of the plasma membrane Na⁺/Ca²⁺

exchanger, which in the absence of external Na⁺can catalyze the entry of Ca²⁺ions into cells, all the experiments were performed in Ca²⁺-free medium. As shown inFig. 3, Na⁺ removal mimicked the effects of CGP-37157. The duration of the [Ca²⁺]_mitsignal was greatly prolonged, and the Ca²⁺efﬂux rates greatly reduced, but the amplitude of the [Ca²⁺]_mitelevation remained blunted in SLP-2 depleted cells. Importantly, the differences in mitochondrial Ca²⁺

efﬂux rates, which persisted in Na⁺-rich, Ca²⁺-free solutions, disappeared entirely in Na⁺-free, Ca²⁺-free solutions. These data conﬁrm

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Fig. 3.Effect of Na⁺removal on the [Ca²⁺]mitresponses of SLP-2 knockdown cells.

HeLa cells were transiently co-transfected with RP3.1mitand either control or SLP-2 shRNA for 120 h. (A) Original recordings of HeLa cells stimulated with 100␮M histamine in Na⁺-free medium. (B and C) Statistical evaluation of SLP-2 shRNA effects on the [Ca²⁺]mitsignal amplitude and maximal mitochondrial Ca²⁺efﬂux rates, measured in Na⁺-free and Na⁺-rich conditions. Bars are mean±S.E.M. of 23 and 21 cells in Na⁺-free and of 14 and 17 cells in Na⁺-rich conditions for Ctrl and SLP-2 shRNA, respectively.

that the mNCE is the main mechanism responsible for the increased mitochondrial Ca²⁺ extrusion observed in SLP-2 depleted cells.

Moreover, they indicate that the defect in mitochondrial Ca²⁺handling associated with SLP-2 expression is independent of the source of Ca²⁺ions that fuel mitochondria. In Ca²⁺-free solutions, only the Ca²⁺that is released from intracellular Ca²⁺stores contributes to the measured [Ca²⁺]_mitsignal.

Although SLP-2 was identiﬁed in a proteome screen from mitochondria, the protein might be expressed in other cellular membranes and thus alter mitochondrial Ca²⁺extrusion indirectly.

For instance, SLP-2 depletion might increase the cytosolic Na⁺concentration, thereby favoring mitochondrial Ca²⁺extrusion through the Na⁺/Ca²⁺ exchanger by increasing the driving force for Na⁺ entry. To test this possibility, we measured [Ca²⁺]_mit elevations in cells permeabilized with digitonin and maintained at a ﬁxed cytosolic Na⁺concentration of 10 mM. As shown inFig. 4, robust [Ca²⁺]_mitelevations were evoked by the addition of 5␮M Ca²⁺to permeabilized cells. Remarkably, all the alterations observed in intact cells depleted of SLP-2 persisted in permeabilized cells. The amplitude of the [Ca²⁺]_mitresponse was decreased and the kinetics of [Ca²⁺]_mitrecovery increased in permeabilized cells depleted of SLP-2. These data establish that SLP-2 alters mitochondrial Na⁺/Ca²⁺exchange at the level of mitochondria and not via alterations in cytosolic sodium or calcium handling.

To further examine the role of SLP-2 in the regulation of mitochondrial Ca²⁺ homeostasis, we investigated the effects of SLP-2 over-expression on [Ca²⁺]_mit responses. HeLa cells were co-transfected either with SLP-2 or the empty expression vector together with the ratiometric “cameleon” probe 4mtD3cpv, and [Ca²⁺]_mitmeasured 48 h after transfection. As shown inFig. 5, the maximal amplitude of the [Ca²⁺]_mitsignal was slightly but not sig- niﬁcantly higher in SLP-2 expressing cells compared to control cells.

However, the duration of the [Ca²⁺]_mit signal in those cells was

Fig. 4. Mitochondrial Ca²⁺responses in permeabilized cells depleted of SLP-2.

HeLa cells were transiently co-transfected with the mitochondrial calcium probe 4mtD3cpv and the indicated shRNA constructs for 120 h. After permeabilization with digitonin, [Ca²⁺]mitwas measured in intracellular buffer (IB) containing 10 mM Na⁺. (A) Original [Ca²⁺]mitrecordings of permeabilized HeLa cells during the addition of 5␮M free Ca²⁺. (B) Statistical evaluation of SLP-2 shRNA effects on the [Ca²⁺]mitresponse amplitude (left), the integrated [Ca²⁺]mitresponse (middle, area under the curve) and the maximal mitochondrial Ca²⁺efﬂux rates (right). Bars are mean±S.E.M of 50 and 34 cells for Ctrl and SLP-2 shRNA, respectively.

Fig. 5. Mitochondrial Ca²⁺responses in cells over-expressing SLP-2. HeLa cells were transiently co-transfected with a mitochondrial calcium probe and either pCI (=Ctrl) or SLP-2 constructs. (A) Original recordings of HeLa cells stimulated with 30␮M histamine, recorded with the ratiometric mitochondrial calcium probe 4mtD3cpv.

(B) Statistical evaluation of SLP-2 cDNA effects on the [Ca²⁺]mitsignal amplitude (left) integrated [Ca²⁺]mitresponse (middle) and maximal mitochondrial Ca²⁺efﬂux rates (right). Bars are mean±S.E.M. of 56 and 43 cells for control and SLP-2 cDNA, respectively.

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prolonged compared to control cells, and the integrated mitochondrial Ca²⁺responses measured during the histamine stimulation was increased by 51% (Fig. 5B, middle panel). This altered kinetics reﬂected a reduction in the maximal rate of Ca²⁺extrusion from mitochondria, from 0.028±0.012 in SLP-2 over-expressing cells to 0.062±0.031 in control cells (Fig. 5B, right panel). Thus, mitochondria from cells over-expressing SLP-2 released Ca²⁺more slowly than mitochondria from control cells, an effect opposite to the one measured in SLP-2 depleted cells.

Interestingly, SLP-2 over-expression did not increase the amplitude of the [Ca²⁺]_mit response, whereas SLP-2 depletion altered both the amplitude and the kinetics of [Ca²⁺]_mit responses. The decreased amplitude of the [Ca²⁺]_mitsignal in cells depleted of SLP- 2 renders a direct comparison of Ca²⁺efflux rates difficult, because efflux rates depend on the mitochondrial Ca²⁺gradient. To enable a quantitative comparison of the data obtained in cells enriched or depleted of SLP-2, we analyzed the recovery rates as a function

Fig. 6.Mitochondrial Ca²⁺efﬂux rates as a function of the [Ca²⁺]mitsignal amplitude.

Maximal efﬂux rates and [Ca²⁺]mitsignal amplitude were determined at the same time point. The data were then aggregated for different ranges of [Ca²⁺]mitvalues to express the efﬂux rates as a function of [Ca²⁺]mit. (A) Rate vs. [Ca²⁺]mitamplitude rela- tionship measured in intact cells over-expressing SLP-2 (circles) or depleted of SLP-2 (triangles). Data are derived from the experiments shown inFigs. 1 and 5, dotted lines are linear regression through the data. (B) Rate vs. [Ca²⁺]mitamplitude rela- tionship measured in permeabilized cells. Data are derived from the experiments shown inFig. 4.

of [Ca²⁺]_mit. As shown inFig. 6, the SLP-2 effects did not depend on the amplitude of the [Ca²⁺]_mitsignal. In intact cells, SLP-2 over- expression reduced Ca²⁺efflux rates at all but the smallest [Ca²⁺]_mit concentrations, and SLP-2 depletion increased Ca²⁺ efflux rates within this range of [Ca²⁺]_mitvalues (Fig. 6A). Similar results were obtained in permeabilized cells depleted of SLP-2, using a different genetically endoded indicator to measure [Ca²⁺]_mit(Fig. 6B). These data indicate that SLP-2 levels alter mitochondrial Ca²⁺efflux rates independently of the mitochondrial Ca²⁺ load. The SLP-2 effects thus primarily reflect altered mitochondrial Ca²⁺extrusion.

To assess whether the alterations in mitochondrial Ca²⁺handling caused by SLP-2 impacted on cell Ca²⁺ signaling, we also measured cytosolic Ca²⁺responses ([Ca²⁺]cyt). As shown inFig. S2, a larger [Ca²⁺]cyt response was observed in SLP-2 depleted cells compared to control cells, whereas a smaller [Ca²⁺]cyt response was observed in SLP-2 over-expressing cells. As observed with [Ca²⁺]_mitelevations, both the peak amplitude as well as the duration of the cytosolic Ca²⁺elevations were altered by the changes in SLP-2 expression levels. Interestingly, the cytosolic and mitochondrial Ca²⁺elevations were modulated by SLP-2 in an opposite manner. The larger [Ca²⁺]cytelevations of SLP-2 depleted cells were associated with smaller [Ca²⁺]_mitelevations, whereas the smaller [Ca²⁺]cytelevations of SLP-2 expressing cells were associated with larger [Ca²⁺]_mitelevations. The opposite alterations observed in the cytosol and in mitochondria are consistent with an altered Ca²⁺

sequestration by mitochondria, but not with altered cytosolic Ca²⁺

handling, which should cause parallel changes in the cytosol and mitochondria. This further confirms that SLP-2 specifically alters mitochondrial Ca²⁺fluxes.

4. Discussion

SLP-2, a newly discovered MIM protein, is highly homologous to stomatin, a protein known to regulate plasma membrane ion channels. This prompted us to investigate whether SLP-2 could fulﬁll a similar role for mitochondrial Ca²⁺channels. For this pur- pose, expression levels of SLP-2 were modulated in HeLa cells, by over-expression or RNA interference, and mitochondrial Ca²⁺

ﬂuxes were studied using ﬂuorescent mitochondrial-targeted Ca²⁺

probes. Here, we report that SLP-2 modulates mitochondrial Ca²⁺

homeostasis, possibly by inhibiting the mNCE activity. In SLP- 2 depleted cells, we observed a reduction in the amplitude and duration of the [Ca²⁺]_mitelevations. Therefore the capacity of mitochondria to store Ca²⁺upon histamine stimulation was decreased in those cells. An opposite effect was observed in SLP-2 expressing cells. Here, we observed an increase in the duration of the [Ca²⁺]_mit elevations and thus in the capacity of mitochondria to store Ca²⁺. In both cases, the converse alterations were observed in the cytosol.

The [Ca²⁺]_cytelevations were increased in SLP-2 depleted cells and decreased in SLP-2 expressing cells. Because a primary cytosolic Ca²⁺ defect is mirrored in mitochondria, this suggests that the mitochondrial defect is a cause, and not a consequence, of the cytosolic Ca²⁺defect. Accordingly, decreased mitochondrial Ca²⁺

storage capacity was observed in permeabilized cells exposed to known Ca²⁺and Na⁺concentrations. This indicates that SLP-2 acts at the level of mitochondria to alter the capacity of these organelles to store Ca²⁺.

One possible explanation for the altered capacity of mitochondria to store Ca²⁺could be that changes in SLP-2 levels alter the Ca²⁺buffering capacity of mitochondria. In the matrix, Ca²⁺is most likely to be bound to enzymes, membrane phospholipids and/or phosphate, thus forming insoluble hydroxyapatite[33,34]. There- fore it is possible that, for an unknown reason, depleting cells of SLP-2 alters one of these parameters. This would lead to a decrease in the level of Ca²⁺that can be retained within the organelle, thereby explaining the increase in Ca²⁺ efﬂux kinetics observed in cells

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transfected with the SLP-2 shRNA. Although we cannot exclude this possibility, our experimental evidences favor another explanation which implies an alteration of the activity of the mNCE. Following histamine stimulation the mitochondria recover basal levels of calcium in about 3 min and this is due to the efﬂux of calcium through the mNCE[9]. The molecular identity of this exchanger is so far unknown and there is little information about the proteins modulating its activity. A study by Yang et al.[35]suggested that protein kinase C (PKC) might act as a regulator of mNCE since its activation enhances mitochondrial Ca²⁺release. In addition, a chemical compound of the diltiazem family (CGP-37157) has been shown to inhibit the release of Ca²⁺from mitochondria, probably by acting directly on the mNCE. In our study, CGP-37157 was found to inhibit Ca²⁺release from mitochondria from SLP-2 depleted cells. A similar inhibition was observed in solutions devoid of sodium, conﬁrming that the increased Ca²⁺extrusion of SLP-2 depleted cells was mediated by the mNCE. Our data suggest that in SLP-2 depleted cells the activity of the mNCE is higher than in control cells.

We recently reported that SLP-2 is required for the stability of the pro-fusion protein Opa1 during stress-induced mitochondrial hyperfusion[29]. Depletion of SLP-2 accelerated the proteolytic cleavage of Opa1 and prevented the fusion of mitochondria induced by inhibitors of protein synthesis. SLP-2 depletion did not fragment mitochondria in non-stressed cells, but caused mitochondria to fragment during UV irradiation or exposure to actinomycin.

Here, we observed that mitochondria were partially fragmented in∼20% of our HeLa cells depleted of SLP-2 (data now shown).

In contrast, SLP-2 over-expression never altered the morphol- ogy of mitochondria. This suggests that SLP-2 depleted cells are more prone to mitochondrial fragmentation. Mitochondrial fragmentation impairs Ca²⁺propagation within mitochondria[36]and decreases mitochondrial Ca²⁺uptake by relocating mitochondria away from ER Ca²⁺release sites[37]. The tendency of mitochondria to fragment at low SLP-2 levels might thus contribute to the reduced mitochondrial Ca²⁺ uptake of SLP-2 depleted cells. The effects of SLP-2 on mitochondrial Ca²⁺ extrusion, on the other hand, appear independent from the fragmentation state of mitochondria. Mitochondrial fragmentation is not known to alter the activity of the mNCE, and most cells depleted of SLP-2 had normal mitochondria yet increased mitochondrial Ca²⁺ extrusion. Con- versely, all cells over-expressing SLP-2 had normal mitochondria but decreased mitochondrial Ca²⁺extrusion. Thus, the SLP-2 effects on mNCE are likely independent of mitochondrial ﬁssion.

One major question raised by our results concerns the mechanism by which SLP-2 may modulate the activity of the mNCE.

SLP-2 is a MIM protein which interacts with prohibitin and may play a role in regulating the stability of mitochondrial proteins, possibly like a chaperone. SLP-2 also prevents the proteolytic cleavage of Opa1. Therefore we can hypothesize that SLP-2 could regulate mitochondrial ion channels by controlling their stability. Various chaperones have been shown to interact and regulate the activity of ion channels of the plasma membrane[38,39]. Furthermore, chaperones have been shown to inhibit the activity of ion channels through a direct interaction or by interacting with intermediate partners[40,41]. In addition, stomatin, a homologue of SLP-2, is known to interact and negatively regulate mechanoreceptors, the ASIC3 channels in mammalian cells[17]and MEC4 inC. elegans [15,16]. In the nematode, this interaction is mediated by a well- characterized domain which is conserved in SLP-2[16]. SLP-2 has also been shown to contribute to the assembly and maintenance of multimolecular signaling complexes within lipid rafts[42,43].

Destabilization of signalsomes together with loss of Opa1 might alter the activity of the mNCE. Biochemical and genetic evidence indicate that SLP-2 stabilizes the long form of the pro-fusion protein Opa1[29]. Opa1 is not only required for fusion, but also for the formation of the cristae junction[44]. Loss of cristae junctions

might redistribute signaling molecules within the inner membrane of mitochondria and alter the activity of the mNCE. Altogether, this suggests that SLP-2 might act as a chaperone for mNCE. Stomatins have been immunoprecipitated with mechanoreceptors, suggesting a rather strong interaction between these two types of proteins.

Based on these data, we think that SLP-2 may represent an inter- esting tool to identify the mNCE by immunoprecipitation assays.

Acknowledgments

We are grateful to Cyril Castelbou for his help for the measurements of cytosolic calcium ﬂuxes, and for Sergei Startchik for help in the image analysis. This work was funded by grants from the Swiss National Science Foundation No. 3100A0-118393 (to ND) and 3100A0-109419/1 (to JCM).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, atdoi:10.1016/j.ceca.2009.10.005.

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