Leucine Zipper EF Hand-containing Transmembrane Protein 1 (Letm1) and Uncoupling Proteins 2 and 3 (UCP2/3) Contribute to Two Distinct Mitochondrial Ca²⁺ Uptake Pathways

Abstract

Cytosolic Ca²⁺ signals are transferred into mitochondria over a huge concentration range. In our recent work we described uncoupling proteins 2 and 3 (UCP2/3) to be fundamental for mitochondrial uptake of high Ca²⁺ domains in mitochondria-ER junctions. On the other hand, the leucine zipper EF hand-containing transmembrane protein 1 (Letm1) was identified as a mitochondrial Ca²⁺/H⁺ antiporter that achieved mitochondrial Ca²⁺ sequestration at small Ca²⁺ increases. Thus, the contributions of Letm1 and UCP2/3 to mitochondrial Ca²⁺ uptake were compared in endothelial cells. Knock-down of Letm1 did not affect the UCP2/3-dependent mitochondrial uptake of intracellularly released Ca²⁺ but strongly diminished the transfer of entering Ca²⁺ into mitochondria, subsequently, resulting in a reduction of store-operated Ca²⁺ entry (SOCE). Knock-down of Letm1 and UCP2/3 did neither impact on cellular ATP levels nor the membrane potential. The enhanced mitochondrial Ca²⁺ signals in cells overexpressing UCP2/3 rescued SOCE upon Letm1 knock-down. In digitonin-permeabilized cells, Letm1 exclusively contributed to mitochondrial Ca²⁺ uptake at low Ca²⁺ conditions. Neither the Letm1- nor the UCP2/3-dependent mitochondrial Ca²⁺ uptake was affected by a knock-down of mRNA levels of mitochondrial calcium uptake 1 (MICU1), a protein that triggers mitochondrial Ca²⁺ uptake in HeLa cells. Our data indicate that Letm1 and UCP2/3 independently contribute to two distinct, mitochondrial Ca²⁺ uptake pathways in intact endothelial cells.

Introduction

With the introduction of sophisticated techniques that allowed direct measurements of mitochondrial Ca²⁺ signals in intact cells (1–6), the strong functional and even physical interaction of mitochondria with their cellular environment became evident (7–9). This interaction appeared to be crucial for the organelle's capability to decode and integrate cellular Ca²⁺ signals, which is an essential feature of cell signaling. Notably, convergences between mitochondria and other membrane structures allow the generation of high Ca²⁺ domains at sites of mitochondrial Ca²⁺ uptake (10, 11). It is believed that during physiological cell stimulation such high Ca²⁺ domains enable mitochondria to locally sequester Ca²⁺ via a low Ca²⁺-sensitive mitochondrial Ca²⁺ uniporter (MCU) that was characterized as a highly selective Ca²⁺ ion channel (12). Notably, besides this low Ca²⁺-sensitive MCU, modes of high sensitive mitochondrial Ca²⁺ uptake that operate at submicromolar Ca²⁺ ranges have been convincingly reported (13, 14). However it is not clear whether or not mitochondrial Ca²⁺ uptake is accomplished by a unique ubiquitous pathway that works at modes of different Ca²⁺ sensitivities. Alternatively, mitochondria might be equipped with different Ca²⁺ uptake machineries that achieve Ca²⁺ sequestration at different Ca²⁺ concentrations. Although the exact identity of the proteins that actually achieve Ca²⁺ transport into the mitochondrial matrix is still unclear, several recent findings confirm the latter assumption: 1) two different mitochondrial Ca²⁺ influx currents (15) and pathways (16) could be recently identified in one given cell, 2) uncoupling proteins 2 and 3 (UCP2/3)² were described to be involved in mitochondrial Ca²⁺ uptake in intact cells (17), 3) with the mitochondrial calcium uptake 1 (MICU1) protein a novel modulator of mitochondrial Ca²⁺ uptake was recently described in HeLa cells (18), and 4) the leucine zipper EF hand-containing transmembrane protein 1 (Letm1) was identified as a mitochondrial Ca²⁺/H⁺ exchanger that achieves a slow but highly sensitive mitochondrial Ca²⁺ loading (19). Moreover, evidence was provided that mitochondrial Ca²⁺ uptake depends on the mode and source of Ca²⁺ mobilization (14, 20, 21).

Based on recent data that indicate that UCP2/3-dependent mitochondrial Ca²⁺ uptake is involved in the rather low Ca²⁺-sensitive mitochondrial uptake of intracellularly released Ca²⁺ but not that of entering Ca²⁺ (16, 22), and the findings that Letm1 operates as a highly sensitive Ca²⁺ uptake mechanism (19), this study was designed to investigate the particular contribution of Letm1 and UCP2/3 to mitochondrial Ca²⁺ uptake from the two major Ca²⁺ sources (i.e. intracellular Ca²⁺ release as well as store-operated Ca²⁺ entry, SOCE) in endothelial cells. Finally, we tested the function of MICU1 to complement an assessment of the individual role of the three most promising putative contributors to mitochondrial Ca²⁺ uptake in endothelial cells.

EXPERIMENTAL PROCEDURES

Materials

Dulbecco's modified Eagle's medium (DMEM), 2,5-di-tert-butylhydrochinone (BHQ), histamine, 2-deoxy-d-glucose, oligomycin, choline chloride, and digitonin were purchased at Sigma-Aldrich (Vienna, Austria). Fetal calf serum and media supplements were obtained from PAA Laboratories (Pasching, Austria). Fura-2/AM was ordered from Molecular Probes Europe (Leiden, Netherlands) and Transfast® reagent from Promega (Mannheim, Germany). All other chemicals were from Roth (Karlsruhe, Germany).

Cell Culture, Constructs, and Transfection

The human umbilical vein endothelial cell line, EA.hy926 passage at ≥45 stably expressing ratiometric pericam-mito (RP-mt) was used for this study. Cells were cultured in DMEM containing 10% FCS, 1% HAT (5 mm hypoxanthin, 20 μm aminopterin, 0.8 mm thymidine), 50 units/ml penicillin, 50 μg/ml streptomycin, and kept at 37 °C in 5% CO₂ atmosphere. 2–4 days before experiments cells were plated on 30 mm glass cover slips. After reaching ∼80% of confluence, cells were co-transfected with different plasmids and siRNAs using Transfast® according to the protocol supplied by the manufacturer.

Buffers and Solutions

Cells were loaded with Fura-2/AM and rested prior to experiments in a Hepes-buffered solution containing (in mm): 135 NaCl, 5 KCl, 2 CaCl₂, 1 MgCl₂, 10 Hepes acid, 2.6 NaHCO₃, 0.44 KH₂PO₄, 0.34 Na₂HPO₄, 10 d-glucose, 0.1% vitamins, 0.2% essential amino acids, and 1% penicillin/streptomycin; pH was adjusted to 7.4 with NaOH. For experiments in intact cells the Ca²⁺-containing experimental buffer (EB) was composed of (in mm): 138 NaCl, 5 KCl, 2 Ca₂Cl, 1 MgCl₂, 10 d-glucose, and 10 Hepes acid; pH was adjusted to 7.4 with NaOH. For experiments in Ca²⁺-free solution, EB containing 1 mm EGTA instead of Ca²⁺ was used. For experiments in partially permeabilized cells, cells were perfused with 3 μm digitonin for 3 min in a high KCl buffer containing (in mm): 110 KCl, 0.5 KH₂PO₄, 1 MgCl₂, 20 Hepes acid, 0.03 EGTA, 5 succinate, 10 d-glucose; pH was adjusted to 7.4 with KOH. Mitochondrial Ca²⁺ uptake was triggered by the actual intracellular Ca²⁺ concentration ([Ca²⁺]_a set to 174 ± 18 nm (n = 17) (referred as “low Ca²⁺”) or to 921 ± 119 nm (n = 17) (referred as “high Ca²⁺”): [Ca²⁺]_a was calculated from Fura-2 signals using the following equation as recently described (22): [Ca²⁺]_a = 350 nm* (F^Ca − F^min)/(F^max − F^Ca). To verify the role of the plasma membrane Ca²⁺ ATPase (PMCA), cells were stimulated with 100 μm histamine and 15 μm BHQ in a low sodium buffer (LSB) composed of (in mm): 19 NaCl, 119 choline chloride, 5 KCl, 2 CaCl₂ or 1 EGTA, 1 MgCl₂, 10 d-glucose, and 10 Hepes acid; pH was adjusted to 7.4 with KOH. For experiments using the perforated patch clamp technique the standard external solution contained (in mm): 145 NaCl, 5 KCl, 1.2 MgCl₂, 10 HEPES, 10 d-glucose, 2.4 CaCl₂. In Ca²⁺-free solutions, MgCl₂ was increased to 2.2 mm and 1 mm EGTA was added. Patch pipettes were filled with a solution containing (mm): 100 KAsp, 40 KCl, 10 HEPES, 2 MgCl_2, 0.2 EGTA.

Isolation of Total RNA and cDNA Synthesis

Total RNA from EA.hy926 cells was isolated with the peqGold Total RNA kit (Peqlab, Erlangen). 2–3 μg of RNA were subsequently reverse transcribed to cDNA using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Lincoln, CA).

Gene Verification

Detection of human Letm1 (Letm1, GenBank^TM accession no. NM_12318.2) and human MICU1 (MICU1, GenBank^TM accession no. NM_006077.2) in EA.hy926 was performed by RT-PCR. Letm1 was identified using a forward primer at position 1082 (5′-AGTTCCTCCAGGACACCATC-3′) and a reverse primer at position 1612 (5′-TCTGCAGTGTGGACTTGAGC-3′). For the verification of MICU1 (GenBank^TM accession no. NM_006077.2) the forward primer at position 418 (5′-CCTGGTGAAGCAGAAGTGTT-3′) and the reverse primer at position 1151 (5′-CTCAATGCAGTGTCCACATC-3′) were used.

RNAi Design

According to already published siRNA sense sequences for Letm1 (19) and for MICU1 (18), two different siRNAs were tested in EA.hy926 cells for both genes, Letm1 and MICU1 versus a non-functional Control siRNA (Control): AGGUAGUGUAAUCGCCUUGtt; sense sequence for Letm1 siRNA1 (si1-Letm1): UCCACAUUUGAGACUCAGUtt and siRNA2 (si2-Letm1): AUGUUCCAUUUGGCUGCUGtt; sense sequence for MICU1 siRNA1 (si1-MICU1): GCAAUGGCGAACUGAGCAAUAtt and siRNA2 (si2-MICU1): GCAGCUCAAGAAGCACUUCAAtt. Silencing of UCP2/3 was performed using siRNAs as described and validated previously (16, 17).

Validation of siRNAs

Knock-down efficiency of functional siRNAs against human Letm1 (Ambion, Cambridgeshire, UK) or human MICU1 (Microsynth, Balgach, Switzerland) were validated individually and in combination by real-time quantitative-PCR (RTq-PCR) versus the Control siRNA (Microsynth, Balgach, Switzerland). 48 h after cell transfection with the respective siRNAs, mRNA was isolated and reverse transcribed. RTq-PCR was performed using specific primer pairs for human Letm1 (5′-TGTTCTTCAAGGCCATCTCC-3′, 5′-TGTTGCTGTGAAGCTCTTCC-3′), for human MICU1 (5′-CAGGTTCAGAGCATCATTCG-3′, 5′-GAACACAAGCCAGACTTGAG-3′), and QuantiTect® Primer Assays (Qiagen, Hilden, Germany), for human UCP2 (Cat. No.: QT00014140) for human UCP3 (Cat. No.: QT00017220) and for human GAPDH (Cat. No.: QT01192646) as housekeeping gene. RTq-PCR was performed with a LightCycler® 480 System (Roche, Basel, Switzerland) using the QuantiFast SYBR Green PCR kit (Qiagen).

Plasmid Constructs

Vectors for Letm1 overexpression were purchased form GeneCopoeia^TM (Rockville, MD). For mitochondrial Ca²⁺ measurements the plasmid encoding the untagged Letm1 (Letm1; Cat. No.: EX-W0230-M02) was used for transfection in a ratio 3:1 with a nuclear-targeted GFP (nls-GFP). Visualization of Letm1 was done with a vector expressing mCherry C-terminally fused to Letm1 (Letm1-mCherry; Cat. No.: EX-W0230-M56). UCP3 overexpression was achieved as previously shown (16). For ATP measurements the novel FRET-based ATP indicator AT1.03 for the cytosolic ATP and its mitochondrial-targeted version mt AT1.03 (23) for mitochondrial ATP were used.

Cytosolic Ca²⁺ and Ba²⁺ Measurements

Changes in [Ca²⁺]_cyto and [Ba²⁺]_cyto were monitored using Fura-2/AM as previously described (24, 25). Addition of Ba²⁺ to SOCE-activated cells was performed with EB using 10 mm BaCl₂ instead of 2 mm CaCl₂.

Mitochondrial Ca²⁺ and pH Measurements with Ratiometric Pericam-mito

Cells stably expressing ratiometric-pericam-mito (RP-mt) (3) were used to monitor [Ca²⁺]_mito and [H⁺]_mito simultaneously. RP-mt was excited at either 430 nm or 485 nm with a high-speed polychromator system VisiChrome (Visitron Systems, Puchheim, Germany). Emitted light was recorded at 535 nm using the 535AF26 emission filter from Omega Optical (Brattleboro, VT). [Ca²⁺]_mito was expressed as 1-F₄₃₀/F₀ as previously shown (17, 26). Changes in pH were expressed as 1 − F₄₈₅/F₀, where F₄₈₅ is the fluorescence (485 nm excitation) at a given time and F₀ is the mean fluorescence of 30–60 individual measurements collected at the beginning of recordings (27). Experiments were performed at room temperature. Rates of acquisition were between 1.04 and 2.66 s and exposure times were 600–800 ms.

FRET-based Cytosolic and Mitochondrial ATP Measurements

For ATP measurements cells were transiently transfected with the FRET-based ATP indicators AT1.03 or mt AT1.03 to measure changes in cytosolic or mitochondrial ATP levels, respectively (23). The sensor was excited at 430 nm using a high-speed polychromator system VisiChrome (Vistitron Systems, Puchheim, Germany) and emission was collected at 535 and 480 nm (Versatile Filter Wheel Systems, Vistitron Systems, Puchheim, Germany).

Patch Clamp Recordings

Membrane potential was recorded using the perforated patch-clamp technique in a current clamp mode (28–32). For membrane perforation, nystatin (300 μm) was included into the pipette solution. Membrane potential was recorded using a List EPC7 amplifier (HEKA, Lambrecht/Pfalz, Germany). Borosilicate glass pipettes were pulled with a Narishige puller (Narishige Co. Ltd, Tokyo, Japan), fire-polished and had a resistance of 4–6 MΩ. The signals obtained were low pass filtered at 1 kHz, and digitized with a sample rate of 10 kHz using a Digidata 1200A A/D converter (Axon Instruments, Foster City, CA). Data collection and analysis were performed using Clampex and Clampfit software of pClamp 9 (Axon Instruments, Molecular Devices, Sunnyvale, CA).

Confocal Microscopy

High resolution imaging of cells expressing Letm1-mCherry and ratiometric pericam-mito (RP-mt) was performed using a Nipkow-disk-based array confocal laser scanning microscope (ACLSM) as described previously (17, 33). The ACLSM consisted of a Zeiss Axiovert 200 m (Zeiss 100×/1.45 oil objective, Zeiss Microsystems, Jena, Germany), equipped with VoxCell Scan® (VisiTech, Sunderland, UK), and an air-cooled argon ion laser system (series 543, CVI Melles Griot, CA). The laser line 488 nm was used to excite RP-mt, whereas alternatively wavelength 561 nm was used to excite Letm1-mCherry. Emitted light was collected at 535 nm (535AF26; Omega Optical, Brattleboro, VT) for RP-mt or 620 nm (Omega Optical) for Letm1-mCherry using a high resolution CCD camera (Photometrics CoolSNAPfx-HQ, Roper Scientific, Tucson, AZ). Acquisition and analysis were performed with Metamorph 6.2r6 (Universal Imaging, Visitron Systems, Puchheim, Germany).

Statistics

Statistical data are presented as mean ± S.E. Analysis of variance (ANOVA) and Scheffe's post hoc F test were used for evaluation of the statistical significance. p < 0.05 was defined significant.

RESULTS

Letm1 Is Expressed in Endothelial Cells and mRNA Levels of Letm1 Can Be Efficiently Reduced by a Combination of Two siRNAs

Using respective primers (see “Experimental Procedures”), the expression of Letm1 was verified in the human umbilical vein endothelial cell line EA.hy926 (Fig. 1A). Two siRNA sequences were tested alone and in combination for the knock-down efficiency in human endothelial cells. The siRNAs reduced the mRNA level of Letm1 by 33.6 ± 4.7 (n = 3) and 69.4 ± 7.5 (n = 3) %, respectively. The combination of both siRNAs achieved the highest knock-down efficiency (85.7 ± 1.1%; n = 3), while they had no effect on the expression levels of UCP2 and UCP3 (Fig. 1B), thus, this combination was subsequently used for all experiments.

FIGURE 1.

Detection and siRNA validation of human Letm1 on the mRNA level of Ea.hy926 cells. Panel A, RT-PCR using specific primers for Letm1 mRNA (see “Experimental Procedures”) yielded a clear 530 bp product amplification. Panel B, points of applications of 2 different siRNAs (see “Experimental Procedures”) against Letm1 are illustrated within the open reading frame of the mRNA of Letm1. Efficiency of siRNA-mediated Letm1 knock-down was verified by real time quantitative-PCR after transfection of siRNA1 (si1-Letm1, n = 3) or siRNA2 (si2-Letm2, n = 3) against Letm1 individually or both in combination (si1/si2-Letm1, n = 3) versus Control siRNA (Control, n = 3). Data are expressed in % of the maximal response in Control. *, p = 0.013; **, p = 0.0019; ***, p < 0.0001 versus Control. mRNA expression levels of either UCP2 or UCP3 were not influenced by the knock-down of Letm1 using the sample that was treated with both siRNAs against Letm1.

Knock-down of Letm1 and UCP2/3 Exhibit Different Inhibitory Patterns on Mitochondrial Ca²⁺ Uptake

In our previous work using the same type of cells, considerable differences in the contribution of UCP2/3 to mitochondrial Ca²⁺ sequestration were described that basically depend on the source of the Ca²⁺ supply (i.e. intracellular Ca²⁺ release from the ER or entering Ca²⁺) (16, 22). Therefore, the impact of a knock-down of Letm1 on mitochondrial uptake of intracellularly released Ca²⁺ and Ca²⁺ that is entering the cell via the store-operated Ca²⁺ entry (SOCE) was tested. For comparison, the same type of protocol was performed with cells treated with siRNAs against UCP2/3. Intriguingly, mitochondrial Ca²⁺ signals of cells reduced of either Letm1 or UCP2/3 were very different. In cells that were transiently transfected with siRNA against Letm1, no inhibitory effect on mitochondrial Ca²⁺ sequestration in response to intracellular Ca²⁺ release was found (Fig. 2A). The decay of the mitochondrial Ca²⁺ signal in cells treated with siRNA against Letm1 appeared to be slightly but not significantly slower, indicating that knock-down of Letm1 to some extent affects the mitochondrial Ca²⁺ extrusion process. This observation possibly points to the proposed function of mitochondrial Ca²⁺/H⁺ antiport of Letm1, which might secondly contributes to the organelle's Na⁺ homeostasis. In contrast knock-down of Letm1 strongly reduced mitochondrial uptake of entering Ca²⁺ by ∼80% (Fig. 2B). Notably, this inhibitory pattern of knock-down of Letm1 was opposite to that of UCP2/3, which reduced mitochondrial Ca²⁺ uptake of intracellularly released but not entering Ca²⁺ (Fig. 2, A and B).

FIGURE 2.

Knock-down of UCP2/3 exclusively reduced the mitochondrial Ca²⁺ uptake from intracellularly released Ca²⁺ whereas Letm1 knock-down strongly diminished mitochondrial Ca²⁺ accumulation only upon SOCE. Endothelial cells that stably express RP-mt cells were transiently co-transfected with nuclear GFP and either Control siRNA (Control: n = 6, 14 cells), or siRNA against Letm1 (siLetm1: n = 6, 12 cells), or siRNA against UCP2 and UCP3 (siUCP2/3: n = 6, 17 cells). Mitochondrial Ca²⁺ was measured with RP-mt. Panel A, knock-down of UCP2/3 but not that of Letm1 diminished mitochondrial Ca²⁺ sequestration in response to intracellular Ca²⁺ release. *, p < 0.05 versus Control. Panel B, knock-down of Letm1 but not that of UCP2/3 blunted mitochondrial uptake of entering Ca²⁺. *, p < 0.05 versus Control.

Both, Letm1 and UCP2/3-dependent Mitochondrial Ca²⁺ Signals Are Accompanied by an Acidification of the Mitochondrial Matrix

Our data so far indicate that Ca²⁺ released from the ER rapidly enters mitochondria mainly via a UCP2/3-dependent but Letm1-independent Ca²⁺ uniport. In contrast slow mitochondrial sequestration of entering Ca²⁺ appears to be primarily accomplished by Letm1, which was supposed to function as a Ca²⁺/H⁺ antiporter (19). Consequently differences of the mitochondrial proton concentration ([H⁺]_mito) in response to intracellular Ca²⁺ release and Ca²⁺ entry were evaluated by using mitochondrial-targeted pericam that offers the possibility to measure changes of [Ca²⁺]_mito and [H⁺]_mito simultaneously (19, 26, 27). Mitochondrial Ca²⁺ elevation induced by either ER Ca²⁺ release or Ca²⁺ entry was always accompanied by increase of [H⁺]_mito (supplemental Fig. S2). These findings are in line with a recent report demonstrating decreases in mitochondrial pH that were triggered by cytosolic Ca²⁺ elevations (34). Notably, during Ca²⁺ entry mitochondrial acidification strictly correlated temporally with the raise of [Ca²⁺]_mito, while the increase of [H⁺]_mito upon ER Ca²⁺ release occurred delayed from the mitochondrial Ca²⁺ signal (supplemental Fig. S2). Knock-down of Letm1 did not affect changes in [Ca²⁺]_mito and [H⁺]_mito that were induced by ER Ca²⁺ mobilization. However, in line with the data described above, cells treated with siRNA against Letm1 showed reduced changes in [Ca²⁺]_mito and [H⁺]_mito during SOCE (supplemental Fig. S2).

The Knock-down of the Mitochondrial Ca²⁺ Transporter Does Not Affect Cytosolic and Mitochondrial Basal ATP Levels

To asses cellular ATP levels, cytosolic, and mitochondrial ATP levels were recorded using FRET-based ATP sensors that are referred to as AT1.03 and mtAT1.03, respectively (23) (supplemental Fig. S1). Basal mitochondrial ATP levels were found to be significantly lower than the cytosolic ATP content in EA.hy926 cells (Fig. 3A), which is in line with a recent report introducing AT1.03 and mtAt1.03 investigating ATP levels of HeLa cells (23). Neither the knock-down of Letm1 nor that of UCP2/3 affected basal cytosolic or mitochondrial ATP levels (Fig. 3A). Similarly, the energetic activity of the cells that was indicated by the drop of cytosolic and mitochondrial ATP levels in response to 2-deoxy-d-glucose and oligomycin was not affected by knock-down of Letm1 or UCP2/3 (Fig. 3B).

FIGURE 3.

Knock-down of Letm1 did not affect basal cytosolic and mitochondrial ATP levels or the cell's energetic activity. Endothelial cells were transiently co-transfected with either the cytosolic ATP sensor AT1.03 or with the mitochondrial targeted mt AT1.03 together with the respective siRNAs, control siRNA (Control), siRNA against Letm1 (siLetm1) or siRNA against UCP2/3 (siUCP2/3). Panel A, basal cytosolic and mitochondrial ATP levels were neither affected by the knock-down of Letm1 nor by the knock-down of UCP2/3. Left columns represent the average ratio (F₅₃₅/F₄₈₀) of cytosolic ATP levels ([ATP]_cyto) at resting conditions for control cells (Control; n = 20, 28 cells) and cells transfected with either siRNA against Lemt1 (siLetm1; n = 14, 15 cells) or UCP2/3 (siUCP2/3; n = 9, 14 cells). Right columns show mitochondrial ATP levels ([ATP]_mito) for control cells (n = 29, 32 cells), for siLetm1 (n = 15, 15 cells) or siUCP2/3 (n = 11, 17 cells). *,#,Δ, p < 0.05 between respective cytosolic and mitochondrial [ATP]. Panel B, knock-down of Letm1 or UCP2/3 did not influence the cell's energetic activity that was initialized as shown in supplemental Fig. S1. Changes in ATP levels were calculated and expressed as Δ_max of Ratio (F₅₃₅/F₄₈₀)/R₀ values representing Δ[ATP]_cyto (left columns) for control (n = 13, 13 cells), siLetm1 (n = 6, 11 cells) or siUCP2/3 (n = 4, 7 cells) and of Δ[ATP]_mito (right columns) for control (n = 11, 11 cells), siLetm1 (n = 5, 6 cells), or siUCP2/3 (n = 5, 6 cells) upon cell treatment with 10 mm 2-deoxy-d-glucose and 2 μm ologomycin.

Letm1 and UCP2/3 Independently Contribute to Different Mitochondrial Ca²⁺ Uptake Pathways in Endothelial

To test whether or not Letm1 contributes to the same mitochondrial Ca²⁺ uptake machinery than UCP2/3, the expression of these proteins was simultaneously reduced by transient transfection of a mixture of all respective siRNAs. The knock-down of Letm1 did not further reduce mitochondrial Ca²⁺ sequestration of intracellularly released Ca²⁺ in cells with a knock-down of UCP2/3 (Fig. 4A). In line with these findings, knock-down of UCP2/3 failed to further attenuate mitochondrial Ca²⁺ uptake of entering Ca²⁺ in cells lacking Letm1 (Fig. 4B).

FIGURE 4.

The combined knock-down of Letm1 and UCP2/3 did not further reduce mitochondrial Ca²⁺ uptake. Stable expressing RP-mt cells were transiently co-transfected with nuclear GFP and either Control siRNA (Control: n = 6, 14 cells) or siRNA against Letm1 (siLetm1: n = 6, 12 cells) or siRNA against UCP2 and UCP3 (siUCP2/3: n = 6, 17 cells) or both, siRNA against Letm1 and siRNA against UCP2 and UCP3 (siUCP2/3 siLetm1: n = 6, 17 cells). Panel A, double knock-down of Letm1 and UCP2/3 did not further reduce mitochondrial Ca²⁺ sequestration in response to intracellular Ca²⁺ release compared with that achieved by siRNA against UCP2/3 alone. *,#, p < 0.05 versus Control. Panel B, double knock-down of Letm1 and UCP2/3 exhibit not more inhibitory effect than the siRNA against Letm1 alone. Curves for Control and siUCP2/3 are re-plotted from Fig. 2. *,#, p < 0.05 versus Control.

To test whether or not UCP3 is able to compensate the diminution of Letm1, UCP3 was overexpressed in cells treated with siRNAs against Letm1. Notably, the expected augmentation of mitochondrial uptake of intracellularly released Ca²⁺ in UCP3 overexpressing cells was very robust and was not affected by Letm1 knock-down (Fig. 5A). As previously shown an overexpression of UCP3 almost doubled mitochondrial Ca²⁺ uptake of Ca²⁺ that enters the cells via SOCE (Fig. 5B). Notably, this increase of [Ca²⁺]_mito in cells overexpressing UCP3 remained unaffected by a knock-down of Letm1 (Fig. 5B).

FIGURE 5.

The effect of an overexpression of UCP3 on mitochondrial Ca²⁺ sequestration was not affected by the knock-down of Letm1. Stable expressing RP-mt cells were transiently co-transfected with nuclear GFP and either Control siRNA (Control: n = 6, 16 cells), or siRNA against Letm1 (siLetm1: n = 6, 17 cells). Overexpression of UCP3 was accomplished by co-transfecting the nuclear GFP with a plasmid coding for UCP3 in a ratio 1:3 and either Control siRNA (UCP3: n = 6, 15 cells), or siRNA against Letm1 (UCP3 siLetm1: n = 6, 17 cells). Mitochondrial Ca²⁺ was measured with RP-mt. Panel A, knock-down of Letm1 did not influence increased mitochondrial Ca²⁺ uptake from intracellular released Ca²⁺ upon UCP3 overexpression. Panel B, overexpression of UCP3 induced a strong mitochondrial Ca²⁺ entry via SOCE that was not affected by the knock-down of Letm1. Data are expressed as 1 − F₄₃₀/F₀ values. *, #, ≪, p < 0.05 versus Control.

UCP3 Overexpression Rescues a Diminished SOCE in Cells Treated with siRNA against Letm1

Mitochondrial Ca²⁺ uptake was shown to facilitate SOCE (35–39). Thus we examined the impact of Letm1 knock-down on SOCE-induced cytosolic Ca²⁺ signals. Indeed, diminution of Letm1 expression reduced the cytosolic Ca²⁺ elevation in response to SOCE, while the transient increase of [Ca²⁺]_cyto elicited by ER Ca²⁺ mobilization remained unaffected (Fig. 6A). Notably, knock-down of Letm1 reduced the SOCE induced cytosolic Ca²⁺ signal by ∼20%, while the respective mitochondrial Ca²⁺ signal was reduced by almost 80%. This disparity is in line with a recent report showing that the impact of mitochondrial Ca²⁺ handling on SOCE fades with the strength of Ca²⁺ entry in this particular cell line (40). However, an overexpression of UCP3 in cells treated with siRNA against Letm1, in which an augmented mitochondrial Ca²⁺ load in response to SOCE was observed (Fig. 5B), completely restored SOCE-induced elevation of [Ca²⁺]_mito (Fig. 6A).

FIGURE 6.

Knock-down of Letm1 reduced the SOCE-induced cytosolic Ca²⁺ elevation that was rescued by the overexpression of UCP3, while Letm1 knock-down did not affect basal membrane potential. Panel A, cells were transiently co-transfected with nuclear GFP and either Control siRNA (Control: n = 14, 64 cells) or siRNA against Letm1 (siLetm1: n = 14, 66 cells), or together with a plasmid coding for UCP3 in a ratio 1:3 and either Control siRNA (UCP3: n = 14, 66 cells) or siRNA against Letm1 (UCP3 siLetm1: n = 6, 17 cells). Cytosolic Ca²⁺ was subsequently measured after loading cells with Fura-2/AM. *, p < 0.05 versus Control. Panels B–E, columns and curves representing the average V_m of perforated patch-clamp recordings. Panel B, membrane potential at rest was recorded in controls (n = 22) and siLetm1 (n = 16). Panel C, peak hyperpolarization was measured after stimulation with 100 μm histamine and 15 μm BHQ in controls (n = 6) or siLetm1 (n = 7). Panel D, membrane potential was monitored after readdition of Ca²⁺ in controls (n = 6) or siLetm1 (n = 6). Panel E, slope was calculated from Ca²⁺-induced peak hyperpolarization to 1 min thereafter in controls (n = 6) or siLetm1 (n = 6). *, p < 0.05 versus control. Panel F, cytosolic Ba²⁺ entry was measured in Fura-2/AM-loaded cells after the induction of maximal intracellular Ca²⁺ mobilization with 100 μm histamine and 15 μm BHQ in nominal Ca²⁺-free solution. Columns represent the average Δ_max ratio (F₃₄₀/F₃₈₀) in controls (n = 14) or siLetm1 (n = 14) that was induced by addition of 2 mm Ca²⁺ to histamine/BHQ prestimulated cells. Data were analyzed from curves presented in panel A, right columns represent Δ_max ratio (F₃₄₀/F₃₈₀) induced by the addition of 10 mm Ba²⁺ to ER Ca²⁺-depleted cells under control conditions (Control; n = 11) or in cells ablated from Letm1 (siLetm1; n = 11).

To test whether or not the reduction of SOCE due to Letm1 knock-down was caused by a possible effect on the plasma membrane potential and/or Ca²⁺-triggered membrane hyperpolarization, electrophysiological recordings were performed. Letm1 knock-down had no effect on either the resting membrane potential (Fig. 6B) or peak hyperpolarization in response to Ca²⁺ readdition to histamine/BHQ prestimulated cells (Fig. 6, C and D). However, in Letm1 knock-down cells plasma membrane hyperpolarization upon Ca²⁺ addition to prestimulated cells was more transient (Fig. 6D) and repolarization occurred faster (Fig. 6E), thus, indicating that the knock-down of Letm1 yields attenuation of the maintenance of SOCE probably by the lack of the mitochondrial Ca²⁺ buffering capacity.

In agreement with this assumption, diminution of Letm1 had no effect on Ba²⁺, which serves as Ca²⁺ surrogate for the SOCE but does not exhibit its inhibitory action on the SOC channels (Fig. 6F). These data further indicate that Letm1 knock-down had no effect on SOCE activation mechanisms but rather reduced its maintenance by the lack of mitochondrial Ca²⁺ buffering.

Letm1 Knock-down Does Not Affect Plasma Membrane Ca²⁺ ATPase (PMCA) Activity

Though knock-down of Letm1 did not affect cellular ATP levels, changes of cellular Ca²⁺ signals may also occur due to alterations in PMCA activity. Thus, PMCA activity was tested according to a protocol of R. S. Lewis' group that measures the decay of cytosolic Ca²⁺ upon removal of extracellular Ca²⁺ (41). We performed similar experiments in the presence of a the SERCA inhibitor BHQ, the IP3-stimulating agonist histamine, and in low Na⁺ concentration to avoid ER Ca²⁺ refilling and Ca²⁺ extrusion via the plasma membrane Na⁺/Ca²⁺ exchanger, respectively. These experiments revealed no effect of Letm1 knock-down on PMCA activity (supplemental Fig. S3).

In Contrast to UCP2/3, Letm1 Overexpression Fails to Improve Mitochondrial Ca²⁺ Uptake

As already shown in Fig. 4, overexpression of UCP3 yielded strong elevation in mitochondrial uptake of Ca²⁺ independently from the source it was delivered (i.e. intracellular Ca²⁺ release or entering Ca²⁺ via SOCE). In order to test whether an overexpression of Letm1 exhibits similar effects than that of UCP3, two Letm1 overexpression vectors according to that previously published by Jiang et al. (19) were designed. To verify targeting of Letm1, a mCherry-fusion construct was used that revealed targeting of overexpressed Letm1 to the mitochondria (Fig. 7A). Notably, neither the expression of mCherry fused Letm1 nor that of the wild-type protein had any obvious effect on mitochondrial Ca²⁺ accumulation in response to intracellular Ca²⁺ release and Ca²⁺ influx (Fig. 7B).

FIGURE 7.

Overexpression of Letm1 co-localized with mitochondria but did not affect mitochondrial Ca²⁺ uptake from any resources. Plasmids encoding for Letm1-mCherry or Letm1 were transiently transfected in endothelial cells that stably express RP-mt. Panel A, mitochondria were visualized using RP-mt (left panel), Letm1 that was N-terminally tagged to mCherry was imaged to test protein targeting (middle panel). The overlay shows Letm1-mCherry exclusively targeted to the mitochondria (right panel). Panel B, overexpression of Letm1 did not affect mitochondrial sequestration of either intracellularly released or entering Ca²⁺. Cells were co-transfected with Letm1 together with a nuclear-targeted GFP in a ratio 3:1 (Letm1: n = 9, 26 cells) or nuclear-targeted GFP alone (Control: n = 9, 25 cells).

Letm1 and UCP3 Differ in Terms of Their Ca²⁺ Sensitivity

In view of the data described above that point to a distinct contribution of UCP2/3 and Letm1 to two separate mitochondrial Ca²⁺ uptake routes, the Ca²⁺ sensitivity of Letm1- and UCP2/3-dependent mitochondrial Ca²⁺ uptake was tested in digitonin-permeabilized cells. Under conditions of low Ca²⁺ application (i.e. 174 ± 18 nm cytosolic free Ca²⁺; n = 17) (22) knock-down of Letm1 completely abolished mitochondrial Ca²⁺ sequestration, while the knock-down of UCP2/3 had no effect (Fig. 8A). In line with the experiments shown in Fig. 4B, overexpression of UCP3 boosted mitochondrial Ca²⁺ uptake even under conditions of Letm1 knock-down and established a large Ca²⁺ sequestration that did not differ from the signal in cells expressing Letm1 (Fig. 8A).

FIGURE 8.

siRNA-mediated knock-down of Letm1 and UCP2/3 had oppositional effects on mitochondrial Ca²⁺ uptake in digitonin-permeabilized cells by applying low and high Ca²⁺ concentrations, while UCP2/3 overexpression rescued mitochondrial Ca²⁺ uptake in cells with Letm1 knock-down. Stable expressing RP-mt cells were transiently co-transfected with nuclear GFP and either Control siRNA (Control), siRNA against Letm1 (siLetm1), siRNA against UCP2/3 (siUCP2/3), or siRNA against UCP2/3 and Letm1 (siUCP2/3 + Letm1). Overexpression of UCP3 was achieved by co-transfecting the nuclear GFP with a plasmid coding for UCP3 in a ratio 1:3 and either Control siRNA (UCP3), or siRNA against Letm1 (UCP3 siLetm1). Mitochondrial Ca²⁺ was measured with RP-mt. Data are expressed as % of Control under each individual condition. Panel A, average curves of mitochondrial Ca²⁺ uptake upon the addition of either low Ca²⁺ concentration or high Ca²⁺ concentration in mild digitonin-permeabilized cells (Control, black curves; siLetm1, blue curves; UCP3, light gray curves; UCP3 + siLetm1, red curves; siUCP2/3 light gray curves; siUCP2/3 + siLetm1, green curves). Panel B, maximum mitochondrial Ca²⁺ accumulation according to the average curves of panel A was calculated 3.4 min after the application of low Ca²⁺ concentration and 1.8 min after applying high Ca²⁺ concentration. Columns represent the average of mitochondrial Ca²⁺ signals upon the addition of the low Ca²⁺ concentration (Control, left white column, n = 4, 12 cells; siLetm1, left blue column, n = 4, 12 cells; UCP3, left dark gray column, n = 4, 12 cells; UCP3 + siLetm1, left red column, n = 4, 12 cells; siUCP2/3, left light gray column, n = 4, 13 cells; siUCP2/3 + siLetm1, left green column, n = 4, 13 cells), and the high Ca²⁺ concentration (Control, right white column, n = 4, 10 cells; siLetm1, right blue column, n = 4, 17 cells; UCP3, right dark gray column, n = 4, 16 cells; UCP3 + siLetm1, right red column, n = 4, 13 cells; siUCP2/3, right light gray column, n = 8, 22 cells; siUCP2/3 + siLetm1, right green column, n = 8, 30 cells). *, p < 0.05 versus the respective Control; #, p < 0.05 between siLetm1 and UCP3 + siLetm1; §, p < 0.05 between siUCP2/3 and siUCP2/3 + siLetm1; and Δ, p < 0.05 between siLetm1 and siUCP2/3 + Letm1.

Challenging the permeabilized cells with a high Ca²⁺ concentration (i.e. 921 ± 119 nm cytosolic free Ca²⁺; n = 17) (22) revealed mitochondrial Ca²⁺ uptake that was not affected by Letm1 knock-down but was markedly impaired in cells treated with siRNA against UCP2/3. Overexpression of UCP3 boosted mitochondrial uptake of high Ca²⁺ independently from the expression level of Letm1 (Fig. 8B). These data are in line with the findings in intact cells (Figs. 2&4) and confirm the idea of two separate mitochondrial Ca²⁺ uptake pathways: the Letm1-dependent pathway achieves mitochondrial Ca²⁺ sequestration of small capacity at relative low Ca²⁺ concentrations, while the UCP2/3-dependent mitochondrial Ca²⁺ uptake requires higher cytosolic Ca²⁺ concentrations to establish a high capacity Ca²⁺ uptake route into the organelle.

Despite Its Expression in Endothelial Cells, MICU1 Appears Not to Be Involved in Mitochondrial Ca²⁺ Sequestration in This Particular Cell Type

To verify the contribution of MICU1 to mitochondrial Ca²⁺ sequestration in the endothelial cell line used, the expression of MICU1 was tested using RT-PCR (Fig. 9A). Hence the efficiencies of the two recently published siRNAs against MICU1 (18) were measured (Fig. 9B). Because these experiments revealed best knock-down efficiency by a combination of both siRNAs, such approach was used in all upcoming experiments regarding MICU1 knock-down. In contrast to the knock-down of either Letm1 or UCP2/3, siRNA-mediated diminution of MICU1 mRNA levels had no effect on mitochondrial sequestration of intracellularly released as well as entering Ca²⁺ (Fig. 9C). Moreover, the combination of MICU1 knock-down with that of either Letm1 or UCP3 did not have any effect on mitochondrial Ca²⁺ uptake compared with that observed in MICU1-containing cells with the respective knock-down of either Letm1 or UCP2/3 (Fig. 9D).

FIGURE 9.

siRNA-mediated knock-down of MICU1 efficiently reduced mRNA levels but had no effect on mitochondrial Ca²⁺ sequestration in endothelial cells. Panel A, RT-PCR using specific primers (see “Experimental Procedures”) resulted in a 733 bp product amplification, approving the expression of MICU1 in the human endothelial cell line used in this study. Panel B, MICU1 silencing using 2 different siRNA (single and combined). Efficiency of siRNA-mediated MICU1 knock-down was verified by real time quantitative-PCR after transfection of siRNA1 (si1-MICU1, n = 3) or siRNA2 (si2-MICU1, n = 3) against MICU1 individually or both in combination (si1/si2-MICU1, n = 3) versus Control siRNA (Control, n = 3). Data are expressed in % of Control. ***, p < 0.0001 versus Control. Panel C, mitochondrial Ca²⁺ uptake was not affected by MICU1 knock-down. Stably expressing RP-mt endothelial cells were transiently co-transfected with a plasmid for expression of nuclear-targeted GFP and either a Control siRNA (Control n = 4, 14 cells) or siRNA against MICU1 (MICU1 n = 4, 14 cells). Panel D, knock-down of MICU1 did not affect mitochondrial Ca²⁺ uptake upon intracellular Ca²⁺ release or SOCE. Mitochondrial Ca²⁺ uptake was visualized by RP-mt in endothelial cells transiently expressing nuclear-targeted GFP and the respective siRNAs: Control siRNA (Control, plain white column, n = 4, 14 cells), siRNAs against Letm1 (siLetm1, plain blue columns, n = 4, 14 cells); siRNAs against MICU1 (siMICU1, plain dark red columns, n = 4, 14 cells); siRNAs against UCP2/3 (siUCP2/3, plain light gray columns, n = 4, 12 cells), siRNAs against UCP2/3 and MICU1 (siUCP2/3 + siMICU1, dark red-striped light gray columns, n = 4, 15 cells) or siRNA against Letm1 and MICU1 (siLetm1 + siMICU1, dark red-striped blue columns, n = 4, 16 cells). Left panel, maximal intracellular Ca²⁺ mobilization in response to 100 μm histamine and 15 μm BHQ in a nominal Ca²⁺-free solution. Columns represent the average Δ_max 1−F−F₄₃₀/F₀ of mitochondrial Ca²⁺ uptake upon ER Ca²⁺ release. *, p < 0.05 versus Control and #, p < 0.05 between siMICU1 and siUCP2/3 + siMICU1. Right panel, columns represent the average Δ_max 1−F₄₃₀/F₀ of mitochondrial Ca²⁺ uptake after the addition of 2 mm Ca²⁺ to cells prestimulated with histamine and BHQ. *, p < 0.05 versus Control and #, p < 0.05 between siMICU1 and siLetm1 + siMICU1.

DISCUSSION

Despite intensive investigations over more than a decade, the molecular identity of the mitochondrial Ca²⁺ uniporter could not be resolved entirely so far. During recent years, siRNA-based screening approaches have highlighted basically three proteins that have been found to be essential for or to contribute to mitochondrial Ca²⁺ uptake in intact cells: UCP2/3 (17), Letm1 (19), and MICU1 (18). However, in these studies mitochondrial Ca²⁺ uptake was tested under certain conditions and in distinct cell types like endothelial cells, HeLa cells (17, 18), and Drosophila Schneider 2 cells (19). As in other subsequent studies using different cell types and/or approaches these results were challenged, the current consensus suggests that these proteins might modulate mitochondrial Ca²⁺ uptake rather than to contribute directly to this phenomenon (42–45). Importantly, in cardiac myocytes two electrophysiological distinct Ca²⁺ uptake currents could be verified (15) that differ in terms of the Ca²⁺ range they are active, their capacity and sensitivity to ruthenium red. In agreement with this landmark publication, in our previous work, evidence was provided for the co-existence of at least two molecularly distinct mitochondrial Ca²⁺ uptake routes in the endothelial cell line EA.hy926 (16, 22, 46).

As previously published, the siRNA-mediated knock-down of UCP2/3 yielded strong reduction of mitochondrial Ca²⁺ sequestration upon intracellular Ca²⁺ release while no effect was found on mitochondrial uptake of Ca²⁺ that entered the endothelial cells via SOCE (16, 22), thus, pointing to an exclusive contribution of this particular transporter to mitochondrial Ca²⁺ sequestration at ER-mitochondria junctions in wild type endothelial cells. In this study, knock-down of Letm1, had no effect on mitochondrial Ca²⁺ uptake at ER-mitochondria junctions, but strongly diminished mitochondrial sequestration of entering Ca²⁺, thus, indicating that the UCP2/3- and Letm1-dependent Ca²⁺ signals account for mitochondrial Ca²⁺ uptake from distinct sources (i.e. ER-derived intracellular Ca²⁺ release and SOCE). Our findings that the knock-down of Letm1 had no effect on Ba²⁺ entry, PMCA activity, membrane potential or basal ATP levels but diminished cytosolic Ca²⁺ elevation in response to SOCE is in line with previous reports on the considerable contribution of mitochondrial Ca²⁺ uptake/buffering for the activity/maintenance of store-operated Ca²⁺ channels (7, 26, 35–40, 47).

Because the combination of Letm1 and UCP2/3 knock-down just reflected the additive combination of the effects of the individual siRNAs, these particular mitochondrial Ca²⁺ uptake routes appear to be independent from each other. This assumption was further supported by our findings that under conditions of Letm1 knock-down, the effect of UCP3 overexpression remained unaffected. Notably, under such conditions, the overexpression of UCP3 compensated the lack of Letm1 in terms of mitochondrial sequestration of entering Ca²⁺. These data are in agreement with our previous findings showing that overexpression of UCP2/3 establishes a respective mitochondrial Ca²⁺ uptake route also for entering Ca²⁺, thus, pointing to the expression level of UCP3 as being the bottleneck for the establishment of a respective mitochondrial Ca²⁺ uptake.

The particular contribution of Letm1- and UCP2/3-dependent mitochondrial Ca²⁺ uptake routes to either entering Ca²⁺ or ER-released Ca²⁺ may indicate that these carriers achieve mitochondrial Ca²⁺ uptake at different Ca²⁺ concentrations. In this respect, the generation of high Ca²⁺ domains in the junction between the ER and the mitochondria to provide sufficient high Ca²⁺ levels to allow mitochondrial Ca²⁺ sequestration via the rather Ca²⁺ insensitive mitochondrial Ca²⁺ uniporter were frequently emphasized (9, 48, 49) and very recently convincingly approved (11, 50). Moreover, considerable differences in the kinetics and capacity of mitochondrial uptake of Ca²⁺ from the two major sources (i.e. intracellular Ca²⁺ release and Ca²⁺ entry via SOCE) in endothelial cells further confirmed these data and emphasized lower Ca²⁺ concentrations at the mitochondria surface under conditions of entering Ca²⁺ than within the ER-mitochondria junction (22). The present experiments using digitonin-permeabilized cells are in agreement with these previous assumptions and indicate that in endothelial mitochondria two Ca²⁺ uptake routes exist that work either at low or high Ca²⁺ concentrations. Our findings that the siRNA-mediated knock-down of Letm1 abolished mitochondrial Ca²⁺ uptake at low but not high Ca²⁺ exposure indicates the Letm1-dependent Ca²⁺ carrier to exclusively account for mitochondrial Ca²⁺ uptake under low Ca²⁺ conditions. Considering the previous reports that entering Ca²⁺ does not generate high Ca²⁺ domains at the mitochondria surface (11, 22) these findings suggest Letm1-dependent mitochondrial Ca²⁺ uptake to account for the organelle's sequestration of Ca²⁺ that enters the cell via the SOCE. In contrast to Letm1, UCP2/3 obviously accounts for mitochondrial Ca²⁺ uptake at high Ca²⁺ concentrations. However, upon overexpression UCP3 is able to compensate the lack of Letm1 even in regard of low Ca²⁺ exposure. It seems likely that even a low active UCP2/3-dependent carrier under low Ca²⁺ load achieves mitochondrial Ca²⁺ load simply because of the largely increased amount of Ca²⁺-carrying proteins. Notably, while the overexpression of UCP3 (and UCP2) established an increased mitochondrial Ca²⁺ uptake, an overexpression of Letm1 was without effect. Though its localization into the mitochondria was clearly approved, these findings may result from a non-functional Letm1 upon overexpression. However, as both the wild type Letm1 (transfection was controlled by co-expression of nuclear targeted GFP) as well as the FP-fusion construct failed to exhibit any effect on mitochondrial Ca²⁺ signaling, this possibility appears rather unlikely. On the other hand, Letm1 might essentially depend on (a) distinct protein(s) that are the rate-limiting factors for mitochondrial Ca²⁺ uptatke. Such multi-protein complex for mitochondrial Ca²⁺ uptake was also postulated for the UCP2/3-dependent Ca²⁺ uptake route (42, 52).

Considering that both the UCP2/3- and Letm1-dependent mitochondrial Ca²⁺ carriers might be established by a multi-protein complex rather than by the individual proteins alone, a protein that was very recently described to be involved in the regulation of mitochondrial Ca²⁺ uptake in intact cells, MICU1 (18, 43, 53), attracts great attention. However, though mRNA levels from MICU1 could be found in the endothelial cells type used in this study, approved siRNA-mediated MICU1 knock-down did not affect mitochondrial Ca²⁺ sequestration by either Letm1- or UCP2/3-dependent pathways. Accordingly, these data suggest MICU1 to be not involved in Letm1- and UCP2/3-dependent mitochondrial Ca²⁺ transport in this particular cell type. Moreover, in intact and in permeabilized cells, a knock-down of UCP2/3 could not entirely prevent mitochondrial Ca²⁺ sequestration to intracellular Ca²⁺ release and high Ca²⁺ load, respectively. Notably neither knock-down of Letm1 nor that of MICU1 further reduced mitochondrial Ca²⁺ sequestration under these conditions. Though the remaining uptake might be due to an insufficient knock-down of UCP2/3 or a modulator role rather than a carrier function of UCP2/3 in this process (7, 42), the existence of a UCP2/3-, Letm1- and MICU1-independent Ca²⁺ carrier cannot be excluded. In this respect, proteins that may not serve as Ca²⁺ carrier under physiological conditions may allow/facilitate Ca²⁺ influx into the organelle under such artificial Ca²⁺ stress conditions (e.g. NCX_mito or ANT) (42, 51).

The present findings demonstrate that at least two molecularly distinct mitochondrial Ca²⁺ uptake pathways co-exist in endothelial cells. The distinct mitochondrial Ca²⁺ uptake routes appear to be independent from the recently described modulator protein MICU1 but essentially depend on either Letm1 or UCP2/3. While further studies are necessary to investigate the specific role of each individual Ca²⁺ uptake route in physiology and pathology, this work explains mitochondrial Ca²⁺ uptake to be not a unitary process but to be established by distinct molecules, thus providing the opportunity to verify the particular contribution of each individual mitochondrial Ca²⁺ transporter to distinct physiological and pathological conditions in various cell types.