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. 2002 May 1;21(9):2198-206.
doi: 10.1093/emboj/21.9.2198.

tcBid promotes Ca(2+) signal propagation to the mitochondria: control of Ca(2+) permeation through the outer mitochondrial membrane

György Csordás  1 , Muniswamy Madesh, Bruno Antonsson, György Hajnóczky
Affiliations

tcBid promotes Ca(2+) signal propagation to the mitochondria: control of Ca(2+) permeation through the outer mitochondrial membrane

György Csordás et al. EMBO J. .

Abstract

Calcium spikes established by IP(3) receptor-mediated Ca(2+) release from the endoplasmic reticulum (ER) are transmitted effectively to the mitochondria, utilizing local Ca(2+) interactions between closely associated subdomains of the ER and mitochondria. Since the outer mitochondrial membrane (OMM) has been thought to be freely permeable to Ca(2+), investigations have focused on IP(3)-driven Ca(2+) transport through the inner mitochondrial membrane (IMM). Here we demonstrate that selective permeabilization of the OMM by tcBid, a proapoptotic protein, results in an increase in the magnitude of the IP(3)-induced mitochondrial [Ca(2+)] signal. This effect of tcBid was due to promotion of activation of Ca(2+) uptake sites in the IMM and, in turn, to facilitation of mitochondrial Ca(2+) uptake. In contrast, tcBid failed to control the delivery of sustained and global Ca(2+) signals to the mitochondria. Thus, our data support a novel model that Ca(2+) permeability of the OMM at the ER- mitochondrial interface is an important determinant of local Ca(2+) signalling. Facilitation of Ca(2+) delivery to the mitochondria by tcBid may also support recruitment of mitochondria to the cell death machinery.

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Figures

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Fig. 1. tcBid induces selective permeabilization of the OMM and promotes propagation of the IP3-induced [Ca2+]c signal to the mitochondria in suspensions of permeabilized RBL-2H3 cells. (A) The permeabilized cells were treated with tcBid (200 nM for 5 min) or solvent, then centrifuged, and the supernatants (cytosol) and pellets (membrane) were resolved by 15% SDS–PAGE followed by immunoblotting for cytochrome c (left panel). To evaluate the respective roles of cell permeabilization and tcBid in cytochrome c release, cells were exposed to varying concentrations of digitonin for 5 min prior to treatment with solvent or tcBid (100 nM for 5 min) (right panel). (B) ΔΨm was monitored in suspensions of permeabilized cells incubated in the presence of tcBid (red) or solvent (black). Additions were: tcBid (200 nM), CaCl2 (2 µM, 2Ca), IP3 (10 µM) and uncoupler (Unc; FCCP/oligomycin 5 µg/ml of each). Inset: tcBid (20 nM)-induced mitochondrial depolarization in the presence of oligomycin. Incubations were carried out in the presence of cytochrome c (10 µM; purple trace), cyclosporin A (5 µM; blue trace) or solvent (red trace). (C) Cytosolic [Ca2+] was followed with rhod2/FA added to the medium (lower panel) and [Ca2+]m was measured using compartmentalized fura2FF (upper panel). tcBid- (200 nM), CaCl2- (2 µM, 2Ca) and IP3- (10 µM) induced [Ca2+]c and [Ca2+]m responses were recorded in the absence or presence of uncoupler (Unc; FCCP/oligomycin 5 µg/ml of each). The data are representative of 3–7 different experiments.
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Fig. 2. No effect of tcBid on delivery of Tg- and Ca2+-induced sustained [Ca2+]c signals to the mitochondria. Cytosolic [Ca2+] was followed with rhod2/FA added to the medium (lower panel) and [Ca2+]m was measured using compartmentalized fura2FF (upper panel). tcBid (200 nM, red) or solvent (black) was added to the permeabilized cells 5 min before treatment with either Tg (2 µM) or CaCl2 (10 µM, 10Ca) in the absence or presence of uncoupler (Unc; FCCP/oligomycin 5 µg/ml of each). These data are representative of three different experiments.
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Fig. 3. tcBid enhances the net mitochondrial Ca2+ uptake evoked by IP3-induced Ca2+ release. (A) Net mitochondrial Ca2+ uptake evoked by IP3 (10 µM), Tg (2 µM) and CaCl2 (10 µM, 10Ca) in the absence (black traces) and presence of tcBid (200 nM, red traces). To calculate net mitochondrial Ca2+ accumulation, the IP3-induced [Ca2+]c signal was subtracted from a parallel experiment carried out in the presence of uncoupler. The traces represent mean values calculated for experiments performed with 3–7 different cell cultures (IP3, seven; Tg, three; and 10Ca, three), and each experiment was carried out in duplicate. (B) Rate of mitochondrial Ca2+ uptake during IP3-induced Ca2+ mobilization in the absence (black) or presence of tcBid (red). Decay of the global [Ca2+]c signal (60 s) was normalized to the initial peak value as follows: decay rate (%/min) = {([Ca2+]c-peak – [Ca2+]c-60 s)/[Ca2+]c-peak} × 100. Calculations were carried out with the data obtained in the absence (left) and presence of uncoupler. The data represent means ± SE from seven different experiments.
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Fig. 4. tcBid facilitates the effect of IP3 on permeability of the mitochondrial Ca2+ uptake sites. (A) Fluorescence quenching was initiated by addition of 50 µM MnCl2 (Mn2+) to fura2FF-loaded permeabilized cells. Permeabilized cells were pre-treated with tcBid (200 nM for 5 min, traces b and d) or solvent (traces a and c). The traces show: no IP3 addition (a and b), IP3 added (c and d) 10 s after Mn2+ (left), 10 s (290 s, middle left), 30 s (270 s, middle right) and 60 s (240 s, right) before Mn2+, respectively. Fura2FF fluorescence (F) was normalized to the initial fluorescence (F0). The free Mn2+ (∼3 µM, buffered by ATP) exceeds the Kd for Mn2+ binding to fura2FF by 2–3 orders of magnitude, which ensures essentially stochiometric quench of compartmentalized fura2FF as Mn2+ enters the mitochondrial matrix. In the lower row, the IP3-dependent component of the Mn2+ quench responses was obtained by subtraction from a parallel quench response in the absence of IP3 (–tcBid, a–c; +tcBid, b–d), and non-linear regression (single exponential) fits were calculated for the first 30 s after Mn2+ addition (solid lines). (B) Pool size and rate constant calculated for the IP3-dependent Mn2+ quench obtained in the absence (grey) and presence of tcBid (red). Calculations were carried out using the traces shown in (A, lower row). Thus, the pool size of the IP3-dependent Mn2+ quench is expressed as a fraction of the initial fluorescence (ΔF/F0). The rate constant was calculated for a 30 s period (from 300 to 330 s, Mn2+ added at 300 s) and is expressed as s–1 Bi-exponential kinetics appeared to provide a better fit to a few recordings of Mn2+ quench in control cells pre-treated with IP3 for 30 and 60 s, but for calculation of the rate constants we used the single exponential kinetic that gave an excellent fit to most of the traces. The symbols show the results with two different cell cultures; each experiment was carried out in duplicate.
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Fig. 5. Local calcium signalling is less sensitive than cytochrome c release to tcBid, suggesting a role for _targeting of the OMM pores to the ER–mitochondrial interface. (A) Dose–response for cytochrome c release evoked by tcBid. Cytosolic samples were generated by rapid filtration of the cells. The data are representative of two different experiments. (B) Dose–response for tcBid-induced potentiation of the IP3-dependent [Ca2+]m signal (upper) and for enhancement of the mitochondrial Ca2+ uptake rate (lower). The data represent means ± SE from 3–5 experiments.
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