398
Mitochondrial
oversight of cellular Ca*+ signaling
Donner F Babcock* Mitochondria, sequester
the metabolic
and release
export
of Ca*+
needs.
Recent
play
a major
and Bertil Hillet
powerhouses
large amounts
helps to adjust
energy
advances
that mitochondrial
show
role in normal
Ca*+
Figure
of the cell, can
of Can+. This import production
1
and
to cellular
H+
Ca2+ fluxes
signaling.
Addresses Department School
of Physiology
of Medicine,
and Biophysics,
Box 357290,
Seattle,
University Washington
of Washington 98195-7290,
USA *e-mail:
[email protected] +e-mail:
[email protected] Current
Opinion
in Neurobiology
1998,
8:398-404
http://biomednet.com/elecref/0959438800800398 0 Current
Biology
Ltd ISSN
0959-4388
Abbreviations
AYlll
mitochondrial
Ca, Cam CCCP
free [Caz+] in cytosol free [Ca’J+] in mitochondria carbonyl cyanide m-chlorophenylhydrazone
membrane
potential
CGP-37157
7-chloro-3,5-dihydro-5-phenyl-1
CsA FCCP
2-on cyclosporin A carbonyl cyanide
H-4,1 -benzothiazepine-
p-trifluoromethoxyphenylhydrazone
Introduction Early studies on isolated mitochondria demonstrated their extraordinary ability to take up Caz+ added to the bathing solution [l-S]. The uptake is attributable to electrophoretic entry through a Ca2+-selective channel - the mitochondrial Caz+ uniporter - driven by the large inside-negative membrane potential (AY,) of energized mitochondria (Figure 1). Uptake can be blocked by ruthenium red and stops if membrane potential is discharged by application of protonophores such as carbonyl cyanide m-chlorophenylhydrazone (CCCP) or carbonyl cyanine p-trifluoromethoxyphenylhydrazone (FCCP). On the other hand, export of Caz+ from isolated mitochondria is typically Na+-dependent and mediated by a Na+/Caz+ exchanger. In addition, Caz+ can be liberated more catastrophically through opening of a high-conductance permeability transition pore [3,5]. Much work on isolated mitochondria has explored mechanisms controlling the uniporter, this exchanger, and the transition pore and their relation to mitochondrial energy production. A new phase of research began with the clear discovery by Thayer and Miller and others [6-81 that mitochondrial CaZ+ transport can have a significant impact on the time course of cytoplasmic Caz+ (Ca,) transients in living neurons. They found that ruthenium red or the uncouplers CCCP and FCCP inhibited a process that blunts an imposed rise of Ca, and prolongs the final return of
Current Op~nm in Neurobiology
Pathways of Ca2+ and H+ transport across the inner mitochondrial membrane. Indicated are some selective inhibitors. e.t.c., electron transport chain; F, Fo, ATP synthase; mCU, mitochondrial Ca2+ uniporter; mNCE, mitochondrial Na+/Caz+ exchanger; mPTP, mitochondrial permeability transition pore; RuR, ruthenium
red.
Ca, to resting levels (Figure 2). It became apparent that mitochondrial Caz+ uptake could be fast compared with other cellular Caz+-clearance mechanisms, so that mitochondria might be a significant buffer for cytoplasmic Ca2+. The subsequent gradual export of Caz+ from mitochondria would prolong the final return to rest. This recent work has begun to explore the cellular conditions under which mitochondria modulate Ca, and how this affects Ca2+-dependent phenomena such as secretion and synaptic transmission.
Molecules transport
involved in mitochondrial
Ca2+
The outer mitochondrial membrane seems readily permeable to small molecules, so the machinery for Caz+ transport (Figure 1) lies primarily at the inner membrane. Unlike Ca2+-transport devices of the plasma membrane, these still have not been definitively identified electrophysiologically, purified as proteins, or their genes cloned. Instead, they are known largely through flux studies and pharmacology. Uptake of Caz+ by isolated mitochondria has the properties of entry through an ion channel. Estimated flux through the uniporter exceeds 10,000 ions per second, somewhat greater than that of the fastest known pumps and exchangers. The Caz+ import depends directly upon AY’,, and the flux is downhill. A steep dependence of
Mitochondrial
Figure
2
% (nM) 4000
CCCP c
CCCP
A
15 min
A
oversight
of cellular
Ca2+ signaling
Babcock
and Hike
399
limit that is sensitive to pro-oxidants, inhibitors of the adenine nucleotide translocator, and other agents. The cyclophilin ligand cyclosporin A (CsA) or its more selective 4MeVal-analog [16] prevents or delays the permeability transition. When open, the pore allows passage of ions and small molecules (11.5 kDa), suggesting a pore size of -3 nm. Under some circumstances the pore may open transiently in a ‘low-conductance mode’ to allow more selective ionic fluxes [17*]. Possible molecular correlates of the transition pore are a multiconductance ‘megachannel’ detected in patches of mitochondrial inner membrane [S] and a high relative molecular weight (>400 kDa) complex found in extracts of brain mitochondria [l&19].
A Current Opimon I” Neurobwlog~
Early evidence for rapid clearance of cytoplasmic Ca’J+ into the mitochondria of a neuron. The time course of Ca, is measured with indo-l dye loaded in the cytoplasm of a rat dorsal root ganglion cell. The cell is depolarized three times for 30 s (arrowheads) to apply large Ca, loads. The first load is cleared rapidly to a plateau level near 500 nM that persists for 8 min. The second load is cleared similarly, but then 1 PM CCCP applied after 3 min releases an enormous bolus of CaQ+ from mitochondria into the cytoplasm, showing that most of the Ca2+ load had been taken into mitochondria. In the continuous presence of CCCP, the initial clearance of the third load is much slowed, but there is no plateau. Modified from [71.
Ca2+-uptake rates upon bathing [Caz+] suggests that the channel opens only if Ca, rises. Cytosolic components including ATP, Pi, and Mg2+ can prevent or modulate its opening [9]. On the other hand, export of Ca*+ is usually uphill, driven by entry of more than two Na+ ions per Caz+ [lo], and is blockable by the inhibitor CGP-37157 or the less selective but more readily available clonazepam [11,12*,13]. In a ‘cytosolic’ medium containing ATP, brain mitochondria remove external Ca2+ to a Na+-dependent ‘set-point’ of -300 nM [14] that apparently represents kinetic equilibrium between residual Caz+ uptake through the uniporter and Caz+ export by the Na+/Caz+ exchanger.
What are the consequences of Ca2+ import by isolated mitochondria? Electrogenic entry of Ca2+ via the uniporter directly decreases AY, and briefly interrupts ATP synthesis. Sufficient elevation of mitochondrial free [Caz+] (Cam) activates dehydrogenases of the tricarboxylic acid cycle thereby increasing NAD(P)H production, electron transport chain fluxes and ATP synthesis. One could say that mitochondrial energy production anticipates periods of energy demand by attending to the Cam signal. The transport model of Magnus and Keizer [15*] reproduces several of the expected features for a modest Ca2+ challenge using published kinetic parameters. Knowledge of the molecular nature of the permeability transition response that is evoked by larger Ca2+ challenges is limited. The transition pore seems to open when the mitochondrial Ca2+ load exceeds some
Mitochondria phenomena
in cellular Ca2+ signaling:
initial
Textbook descriptions of cellular Ca2+ metabolism focus on transporters and ion channels of the plasma membrane and the reticular CaZ+ stores. Mitochondria have recently came to the fore in studies on isolated neurons [6-8,20-261. Cytoplasmic Ca2+ was monitored optically as cells were depolarized by KCl, by electric currents or by neurotransmitters to induce Ca2+ entry. Typically, Ca, rose to much higher levels during Ca2+ entry if mitochondrial Ca2+ uptake was inhibited in a variety of ways, but the final stages of recovery of Ca, were often hastened (Figure 2). Application of CCCP or FCCP some seconds after Ca2+ loading released a bolus of Caz+ into the cytoplasm - presumably from the mitochondria. Lowering the cytosolic [Na+] or pharmacological inhibition of the mitochondrial Na+/Caz+ exchanger had little effect on early Ca, clearance but hastened late Ca, recovery. Similar results have been obtained with smooth muscle [27], chromaffin [28,29**], and anterior pituitary [30] cells. The clear implication of this work is that mitochondria can be a major sink for Caz+ clearance when Ca, rises into the low micromolar levels, and that Ca2+ is subsequently slowly extruded from mitochondria via the Na+/Caz+ exchanger. In this view, mitochondria speed the initial fall of Ca, from high values (> 500 nM) by Ca2+ uptake and then prolong the final phase of recovery by continuing to feed out accumulated Caz+ into the cytoplasm.
Testing the role of mitochondria Ca2+ clearance
in cellular
These ideas have now been tested in several ways and are being accepted. An important initial control was to ensure that Ca2+ clearance was not being slowed by CCCP and FCCP simply because the cell had run out of ATP Three kinds of evidence argue against that explanation. First, experiments and solution changes can be done rapidly, and the effects of the protonophores are evident within < 5 s of their application, Second, early clearance of Ca, is still delayed greatly by treatment with CCCP when oligomycin is present to minimize mitochondrial consumption of cytosolic ATP Finally, the experiments can be done using whole-cell pipettes containing millimolar levels of ATP to provide a continuous supply of energy.
400
Signalling mechanisms
Mitochondria will be significant for cellular Caz+ handling only if they can transport Ca*+ at rates comparable to or exceeding those of other Caz+-transporting devices in the same cell. Partial quantitative comparisons of transport rates have been done in sympathetic ganglion cells [S] and more complete comparisons in isolated chromaffin cells [28]. In chromaffin cells, mitochondrial Ca2+ uptake at Ca, = 1 pM is four times faster than the sum of transports by the Na+/Ca*+ exchanger of the plasma membrane and by the Ca2+-ATPases of the plasma membrane and reticular Caz+ stores. Thus, it is clearly dominant even during relatively modest physiological Ca, elevations so that 80% of the initial Ca*+ load is cleared first into the mitochondria. Similar tests will need to be done in other types of cells as each may be different.
If significant Caz+ is accumulated in mitochondria, effects on mitochondrial physiology and on indicators of mitochondrial calcium (Car,,) should be observed. An early study with single sensory neurons [31] showed changes in the NAD(P)H autofluorescence that reports metabolism and in rhodamine 123 fluorescence that reports AY,. Consistent with studies of isolated mitochondria, evidence that a Caz+ signal is conveyed from cytoplasm to mitochondria and exerts positive control on NAD(P)H production is now particularly strong for hepatocytes [32] and cardiac muscle [13,33*].
Ca, in living cells has now been monitored directly in several ways with optical indicators. In one of the first, Miyata et al. [34] showed that after selective quenching of cytosolic indo-l fluorescence with MnZ+, a residual compartmentalized signal reported a rise of Ca, of single cardiomyocytes in response to electrical stimulation of the cell. Others have relied on imaging to focus on the signals produced from mitochondrial regions that were identified by other means [35,36”]. Ideally one might use some physiological property of mitochondria to target CaZ+-selective reporters directly to that compartment. Rizzuto et a/. [37] used a mitochondrial import sequence to develop a targeted version of the photoprotein aequorin that monitors Ca, in fields of transfected cells. Their probe reported that Ca, rises transiently upon mobilization of reticular Caz+ stores and upon induced Caz+ entry. Another approach takes advantage of the tendency for membrane-permeant cations to accumulate in the very negative mitochondrial matrix compartment. I n particular, the cationic acetoxymethyl ester of the Caz+ probe rhod-2 accumulates in mitochondria of living cells (like its parent chromophore rhodamine 123) and there generates a trapped Ca, indicator [32]. The rhod-2 signal reports that Ca, rises in a variety of cell types when Ca, is elevated by Caz+ entry [29**,38,39*] or by mobilization of reticular stores [29**,32,36**,40-42]. Others have examined signals from the mitochondrially localized, but non-quantitative probe chlortetracycline Caz+ content at 117'1,or have assessed mitochondrial
single time points by protonophore-induced [7,25,28,39*] or Caz+-ionophore-mediated [43*] release into the cytosol. Results have been most complete with chromaffin cells [29**]. These cells can be loaded with rhod-2 in the mitochondria and Calcium Green in the cytoplasm and then subjected to voltage-clamp steps of membrane potential to inject controlled Ca2+ loads into the cytoplasm. In Figure 3, a 0.5 s depolarization makes an immediate elevation in Ca,, which decays in about 3 s as much of the Caz+ is cleared into mitochondria. Calcium also rises quickly in the mitochondria but takes about 60s to be extruded. A subsequent exposure to CCCP causes loss of residual Ca2+ from mitochondria and a gain in the cytoplasm. With CCCP still present, a second cytoplasmic CaZ+ load is not well taken up into mitochondria and takes much longer than 3s for its initial clearance from the cytoplasm via other routes. In other experiments, the export of accumulated mitochondrial Caz+ can be slowed considerably by inhibitors of the mitochondrial Na+/Caz+ exchanger: CGP-37157 and clonazepam [11,12*,13,22,26,29”]. The precise levels of Ca, at rest and during loading have been difficult to determine. Usually the best calibrations of Ca2+ indicators are done in sitlr rather than by extrapolation from bulk measurements in a fluorimeter. There are at least two problems here. First, it is hard to achieve known free [Ca*+] in tiny organelles. Second, individual mitochondria are typically so small that in viva measurements usually represent averages from many mitochondria each with a different Ca,, perhaps including levels that saturate the dye. Most authors find that the resting Caz+ level in mitochondria is about the same as the resting level in cytoplasm, about 1OOnM. Reported elevations of Ca, upon Ca, challenge range from 400-10,000 nM, reflecting methodological differences we believe. Much of the Ca*+ in the mitochondrial matrix must be bound. From our calibrations for rhod-2 (Figure 3) and the large quantity of Caz+ calculated to enter mitochondria, we estimate a Caz+-binding ratio (bound/free) in mitochondria of -5000 [29**], some 50 times that of cytosol but similar to Ca*+-binding ratios determined elsewhere for isolated mitochondria.
Conclusions
and future directions
Accepting that Caz+ does enter mitochondria in a major way, what is this uptake for? Clearly it will limit the spread of micromolar elevations of Ca, within the cell, maintaining the strong local character of Ca*+ signaling, and cutting off the spread of exocytosis and of Caz+dependent inactivation of various CaZ+ entry channels [23,25,39*]. Slow export from mitochondria will extend the period during which Ca, remains modestly elevated (to 150-500nM) after a bout of Caz+ signaling, prolonging the period of activation of Caz+-dependent enzymes and of processes dependent on residual Ca2+ such as
Mitochondrial
Figure
3 1
(a) Ca, (nM)
oversight
of cellular Gas+ signaling
Babcock and Hille
401
messengers [47,48”], such as the pyridine nucleotide metabolites cyclic ADP-ribose (cADPR) or nicotinic acid adenine dinucleotide phosphate (NAADP), that affect other Caz+ signaling systems.
700 r
CCCP
2 min
A hallmark of the permeability transition pore studied in isolated mitochondria is the complex modulation of its sensitivity to the Caz+ content of these organelles. The possibility is now being considered [17*] that the threshold Caz+ content required for opening of the pore is reached during normal cellular responses. Even more attention is being focused on possible opening of the pore or more selective alterations in mitochondrial ionic permeability as key events in ischemia/reperfusion injury, in neurodegenerative disease and in the commitment to apoptosis [21,49-52,53’,54’].
W % (nM) 1000
500
a
-.
Oligomycin
Apoptosis or programmed cell death is required for normal neural development and provides a line of defense against invasive or malignant challenge. It may also underlie some neurodegenerative diseases and other pathologies. Interest in apoptosis increased upon recognition that at least two of its mediators, the ‘caspases’ and Bcl-family proteins, are evolutionarily conserved from nematode to man. Mitochondrial involvement in the commitment to apoptosis was suggested by the preferential localization of Bcl-2 to these organelles, by the early changes in AY, that are elicited by many apoptotic stimuli [49,55,56], and by a release of cytochrome c from the mitochondrial intermembrane compartment, which may evoke activation of caspases.
Current Opmion in Neurobiolog)
Reversible transfer of Ca2+ from cytoplasm to mitochondria. A rat chromaffin cell is examined in a perforated-patch preparation after co-loading with two Can+- sensitive dyes: (a) rhod-2 in the mitochondria reports Car,, and (b) Calcium Green in the cytoplasm reports Ca,. The cell is depolarized twice for 0.5 s (arrowheads) to impose modest Ca, loads. After the first pulse, the cytoplasmic load is cleared in 3s into mitochondria and is exported from mitochondria over a 60 s period. Application of 2 pM CCCP releases residual mitochondrial Cal+ into the cytoplasm and prevents much Can+ from being taken up into mitochondria in the second load. Consequently, Ca, takes 30 s to be cleared by other routes. Oligomycin (3 mM) was present from the beginning to block consumption of cytosolic ATP by reverse-mode operation of the ATP synthase. Modified from [29**1.
gene expression and post-tetanic potentiation of synaptic transmission [44**]. In addition, elevation of Cam will raise the rate of energy’production in the mitochondria. It is quite possible, as some have speculated, that mitochondria occupy special positions near sources or sinks of Ca2+, perhaps allowing preferential exchanges of Ca2+ between organelles. Indeed, mitochondrial Ca2+ uptake is important for Caz+ oscillations and propagation of Caz+ waves [30,41,45,46*]. Like reticular Caz+ stores, mitochondria may generate signals that reflect their filling state. For example, they are possibly sources of
The sequence and mechanistic details of these events are not yet clear. In several systems apoptotic responses are altered by CsA or other agents that target the transition pore [21,52,53*]. These observations favor a model in which opening of the pore causes swelling of the mitochondrial matrix, rupture of the outer membrane and nonselective release of cytochrome c. Overexpression of Bcl-2 or Bcl-XL protects against various apoptotic stimuli [52,53*,54’,55,56] and mitochondria from such cells have greatly increased Ca2+ uptake capacity [57], suggesting that Bcl-2 inhibits opening of the permeability transition pore. It remains possible that cytochrome c is released selectively through a macromolecular assembly of Bcl-family proteins; Bcl-XL resembles the bacterial pore-forming proteins diphtheria toxin and colicins in structure. Recombinant anti-apoptotic Bcl-2 and pro-apoptotic Bax both form channels in lipid bilayers or vesicles [58-611, and apoptotic stimuli evoke translocation of Bax to mitochondria [62*]. However, other recent evidence indicates that Bcl-family proteins evoke mitochondrial swelling and rupture by controlling ionselective permeability [54-l. Regardless of the outcome of such debates, continued study of the involvement of mitochondria in apoptosis will almost certainly increase our knowledge of their roles in normal cellular Caz+ signaling as well.
402
Signalling mechanisms
Acknowledgements
16.
We thank Joe Howard and Ed Kaftan for critical reading of this review. The authors are supported by National Institutes of Health grants AR17803 and HD12629, and by the WM Keck Foundation.
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