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Release of mitochondrial Ca²⁺ via the permeability transition activates endoplasmic reticulum Ca²⁺ uptake ¹

Department of Physiology, The University of Melbourne, Victoria 3010, Australia

²Correspondence. E-mail: davidaw{at}unimelb.edu.au

SPECIFIC AIMS

Regulatory interactions between the endoplasmic reticulum (ER) and the mitochondria in the control of intracellular free Ca²⁺ [Ca²⁺]_i may be important in the control of many cell functions, especially those involved in initiating cell death. We used targeted Ca²⁺ sensors (cameleons) to investigate the movement of Ca²⁺ between the ER and mitochondria of intact cells and focused on the role of the mitochondrial permeability transition (MPT) in this interaction. We hypothesized that release of Ca²⁺ from mitochondria in response to a known MPT agonist (atractyloside) would cause release of ER Ca²⁺, perpetuating cellular Ca²⁺ overload and cell death.

PRINCIPAL FINDINGS

1. Targeting and Ca²⁺ sensitivity of cameleons
The cDNA for yellow cameleon 2.1 (YC2.1) and ER targeted cameleon-3ER (Cam3ER) were provided by Roger Tsien (Howard Hughes Medical Institute, University of California, San Diego). We constructed a mitochondrial targeted YC2.1 (YC2.1mito) by amplifying YC2.1 from pCDNA3 by PCR and subcloning it into Invitrogen’s pCMV/myc/mito mitochondrial targeting vector. HEK cells were plated onto poly-L-lysine coated glass coverslips and transfected with one of the above sensors using Qiagen’s Effective Reagent. Cells were imaged with a Bio-Rad MRC-1024 laser (100 mW argon ion) scanning confocal microscope. Cyclopiazonic acid (CPA) caused indirect ER Ca²⁺ release by blocking sarcoendoplasmic reticulum Ca²⁺ ATPases primarily responsible for ER Ca²⁺ uptake. Addition of CPA to the bathing solution induced loss of ER Ca²⁺, verifying the ability of YC3ER to sense such changes. To examine the ability of YC2.1mito to detect changes in mitochondrial Ca²⁺, HEK293 cells expressing YC2.1mito were bathed in atractyloside to activate the MPT pore. As expected, pore opening allowed equilibration of Ca²⁺ (and other solutes <1.5 kDa) across the mitochondrial membranes, which was seen as a release of mitochondrial Ca²⁺. The bath solution was changed to one containing a Ca²⁺ ionophore (25 µM 4Br-A23187) and 2.5 mM CaCl₂. Additional atractyloside (0.25 mM) opened the MPT pore and allowed large amounts of Ca²⁺ to enter the mitochondrial matrix, a change detected by YC2.1mito. Addition of 25 mM EGTA to the bathing solution significantly reduced external Ca²⁺ and subsequently mitochondrial Ca²⁺ levels. This experimental sequence demonstrated the ability of the cameleons to provide fluorescence signals, albeit of relatively narrow dynamic range, that could by used to sense changes in organelle Ca²⁺ in these conditions.

2. Atractyloside activates the mitochondrial permeability transition pore resulting in loss of mitochondrial Ca²⁺
We used the calcein-cobalt quenching method for monitoring MPT activation. Cells were incubated with calcein-AM (1 µM) for 10 min, the medium replaced with one containing CoCl₂ (1 mM) for 60 min to quench accessible (cytoplasmic) fluorescence. Cells were rinsed with fresh medium before imaging. Upon opening of the MPT pore, calcein was released from the mitochondrial matrix, resulting in redistribution of the fluorescence. Using this technique, we confirmed MPT pore activation by atractyloside. Upon opening of the pore, calcein release was evident over the ensuing few minutes (Fig. 1 B), with a time course similar to the release of mitochondrial Ca²⁺ (Fig. 1C ). Cyclosporin A inhibited MPT activation by atractyloside. This provided further evidence that Ca²⁺ was lost through an MPT-based mechanism.

View larger version (28K): [in this window] [in a new window]   Figure 1. Activation of the MPT by atractyloside in HEK293 cells. MPT activation using the calcein-cobalt quenching technique was determined by dividing the fluorescence of a mitochondrial area (M) by that of a nuclear area (N) within an individual cell (A). Data were normalized to the initial fluorescence levels at the beginning of an experiment. A) Comparative images of a group of cells at the beginning and conclusion of an experiment. Note the increases in nuclear fluorescence and loss of mitochondrial fluorescence after atractyloside treatment. Scale bars: 10 µm. B) MPT activation after 0.25 mM atractyloside (indicated by bar) in control cells and those pretreated with cyclosporin A (20 µM, 30 min). Data traces for atractyloside alone represent the average response of 7 cells in the imaging field (t=343.4±15.1 s) and are representative of a total of 5 individual experiments investigating 28 cells. The atractyloside response in the presence of cyclosporin A is the average response of 3 cells in the imaging field (data representative of 3 individual experiments with a total of 11 cells). C) Loss of mitochondrial Ca2+ in cells expressing YC2.1mito after 0.25 mM atractyloside (indicated by bar) in control cells and those pretreated with cyclosporin A (20 µM, 30 min). Data trace for atractyloside alone is the average of 14 cells in the imaging field (t=82.1±4.0 s) (data representative of 7 individual experiments investigating 31 cells). The data trace for atractyloside in the presence of cyclosporin A is the average of 11 cells in the imaging field (representative of 2 individual experiments investigating 24 cells).

3. Released mitochondrial Ca²⁺ is initially sequestered by endoplasmic reticulum before eventually being released to the cytosol
We hypothesized that Ca²⁺ released via the MPT would activate further ER Ca²⁺ release through a Ca²⁺-induced Ca²⁺ release-type mechanism. Instead, the ER initially took up Ca²⁺ over a 40 s period and then released it (Fig. 2 A). If ER Ca²⁺ uptake was first inhibited with CPA, opening of the MPT failed to induce changes in ER Ca²⁺ concentration (Fig. 2B ).

View larger version (13K): [in this window] [in a new window]   Figure 2. Effect of MPT activation and subsequent mitochondrial Ca2+ release on ER Ca2+ dynamics. A) Ca2+ from the mitochondria on MPT activation with 0.25 mM atractyloside is initially absorbed by the ER, but subsequently lost. Data presented are the average of 13 cells (t=313.4±8.9 s) (data representative of 3 experiments investigating 20 cells). B) CPA (25 µM) releases ER Ca2+ (t=35.9±3.5 s) and inhibits ER Ca2+ uptake. Under these conditions, atractyloside is unable to activate ER Ca2+ uptake as seen in panel A (average of 9 cells in the imaging field: data representative of 3 experiments investigating 27 cells).

4. Release of stored ER Ca²⁺ activates the mitochondrial permeability transition pore, leading to loss of mitochondrial Ca²⁺
The absence of response in the presence of CPA could be due to prior MPT activation by raised cytosolic Ca²⁺. Excess cytosolic Ca²⁺ is normally absorbed by mitochondria via an electrogenic Ca²⁺ uniporter. The subsequent increase in matrix Ca²⁺ would have the potential to activate the MPT in turn. Addition of 25 µM CPA activated MPT pores, as evidenced by loss of mitochondrial calcein (Fig. 3 A), with a time course similar to that evident for MPT activation by atractyloside (described earlier). In the majority of cells, CPA induced the immediate loss of mitochondrial Ca²⁺ (Fig. 3B ).

View larger version (27K): [in this window] [in a new window]   Figure 3. Schematic diagram of ER–mitochondrial Ca2+ interactions after MPT activation. MPT activation (e.g., by atractyloside) induces release of Ca2+ (1) and potentially other substances from mitochondria. Elevation of intracellular Ca2+ stimulates Ca2+-ATPases located in ER and cell membranes (2), thus removing Ca2+ from the cytosolic space. Excessive release of Ca2+ or an unknown factor from mitochondria activates IP3 receptors (IP3R) causing further release of ER Ca2+ (4) and cell death.

CONCLUSION

Atractyloside was used in the present study to induce the MPT in intact cells. Activation of the MPT in intact cells was confirmed by monitoring release of mitochondrially trapped calcein and inhibiting the response with cyclosporin A. Calcein was able to pass through the pore like most commercially available chemically based Ca²⁺ sensors due to low molecular mass (<1.5 kDa, the molecular cutoff for MPT pores). Therefore, these Ca²⁺ sensors have been problematic in investigations of MPT Ca²⁺ loss. The cameleons are much larger molecules that remained trapped in the mitochondrial matrix. After MPT activation with atractyloside, mitochondrial Ca²⁺ equilibrates across the mitochondrial membranes within 300 s. This suggests that in these conditions HEK293 cells maintain a functionally significant Ca²⁺ gradient between mitochondria and cytosol that ‘fuels’ the movements of Ca²⁺. Though clearly an important observation, there are few published reports to provide a reference base for this result. In separate experiments with HEK293 and other cell types (cultured osteosarcoma cells), we observed (with fluo-3) distinct cytosolic Ca²⁺ transients in response to atractyloside addition (unpublished data), an observation in support of this contention.

The released mitochondrial Ca²⁺ is subsequently absorbed into the ER by Ca²⁺-ATPases (SERCA). However, Ca²⁺ uptake proceeded only transiently before net ER Ca²⁺ release was evident. The mechanism by which Ca²⁺ was eventually lost from the ER is not known. Here we propose two possible explanations. First, it is possible that the local extramitochondrial Ca²⁺ concentration after the MPT is high enough to activate adjacent ER ryanodine receptors. Second, MPT activation is reported to release proapoptotic factors, including cytochrome c and apoptosis-inducing factor, which trigger caspase activation and apoptosis. These factors or others may act directly or via cell death pathways to activate ER Ca²⁺ release. The ER Ca²⁺ release mechanism could involve a member of the bcl-2 family (bcl-2, bax, etc.) that has been demonstrated to form Ca²⁺ permeable ion channels in lipid bilayers and localize to ER membranes. Our future investigations will address these possibilities.

We have used organelle-targeted cameleon Ca²⁺ sensors to provide new information on the movement of Ca²⁺ via the mitochondrial permeability transition pore. We first constructed a mitochondrial targeted sensor based on second-generation yellow cameleon 2 (termed YC2.1). The advantage of YC2.1 is the greatly reduced pH sensitivity of YFP due to the introduction of V68L and Q69K mutations. This is of particular importance for a mitochondrial Ca²⁺ sensor due to the pH gradient across the mitochondrial membranes. YC2.1 has biphasic Ca²⁺ sensing properties, with dissociation constants (K_d) 100 nM and 4.3 µM, which are appropriate for mitochondrial Ca²⁺ sensing. On the other hand, YC3 (ER targeted) has monophasic Ca²⁺ sensing properties with a K_d of 1.5 µM. Although the YFP of YC3 is pH sensitive, there is no reported pH gradient between the ER and cytoplasm. In preliminary experiments, the cameleons were used as ratiometric indicators of Ca²⁺ by monitoring CFP and YFP fluorescence emissions. However, in the present study, we instead excited CFP (548 nm) and collected the YFP fluorescence due to RET since movement artifacts and redistribution or loss of sensor were minimal. The absolute fluorescence emission changes of the cameleons are still small and their sensitivity is fairly low compared to traditional chemically based Ca²⁺ sensors such as fluo-3 and Fura-2. Newer Ca²⁺ sensors based on circularly permutated GFP such as ‘camgaroos’ and ‘pericams’ offer promise, but await independent evaluation.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0828fje; to cite this article, use FASEB J. (May 8, 2002) 10.1096/fj.01-0828fje.

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