Intracellular Ca²⁺ is regulated within three major compartments: the cytosol, the endoplasmic reticulum and mitochondria. This Chapter reviews the mechanisms involved in handling of Ca²⁺ within these compartments with reference to potential strategies for neuroprotection. In the cytosol, Ca²⁺ buffering has a major influence on Ca²⁺ signals. Cytosolic Ca²⁺-binding proteins such as CB28 participate in Ca²⁺ buffering and may have a role in resistance to neurotoxicity. In the endoplasmic reticulum, a number of proteins are involved in Ca²⁺ uptake, lumenal buffering or release, and these may be of value as potential targets for therapeutic intervention. Mitochondria are receiving increasing attention for their role in Ca²⁺ storage and signaling, and as key players in the processes leading to cell death following Ca²⁺ overload. An improved understanding of how Ca²⁺ is controlled within these intracellular compartments, and how these compartments interact, will be important for neuroprotective strategies.

Introduction

The management of Ca²⁺ inside cells is sometimes referred to as Ca²⁺ homeostasis, but this understates the dynamic role of Ca²⁺ as a signaling molecule. Ca²⁺ transients encode information about neuronal activity via their amplitude, timing and subcellular location. This signaling system exploits a steep gradient of Ca²⁺concentration ([Ca²⁺]) between the cytosol (resting level ˜100 nM) and the extracellular fluid and endoplasmic reticulum (ER), in which [Ca²⁺] is three or four orders of magnitude higher. The gradients allow generation of rapid rises of cystolic [Ca²⁺], but have energetic costs and carry risks of Ca²⁺ overload. A complex set of mechanisms has evolved to allow active Ca²⁺ signaling while avoiding toxicity.

The impact of Ca²⁺ influx through the plasma membrane on the resulting rise in free cytosolic Ca²⁺ concentration ([Ca²⁺]_c) depends upon cytosolic buffering, the rates of removal from cytosol by sequestration into organelles (mainly ER and mitochondria), and extrusion from the cell via the plasma membrane. In addition, Ca²⁺ that enters may influence subsequent Ca²⁺ influx by inactivating voltage- or receptor-operated channels in the plasma membrane, or by activating K channels that reduce membrane excitability, or may affect Ca²⁺ release from intracellular stores.

Cytosolic Ca²⁺ buffering

A consistent finding across many cell types, including neurons, is that the transient rises of free cytosolic Ca²⁺ concentration ([Ca²⁺]_c) during Ca²⁺ influx represent only a tiny proportion of Ca²⁺ that enters the cell. The remainder does not contribute to the signal because it is bound to high-affinity Ca²⁺ binding sites (buffers) within the cytosol. Typical estimates suggest that for neurons only one in every 100–400 molecules of added Ca²⁺ remains free.^1,2 In some neurons such as Purkinje cells of the cerebellum that are adapted for high Ca²⁺ fluxes, the proportion that remains free may be as low as one in several thousand.³

Cytosolic Ca²⁺ buffering has a major impact on the spatial distribution of Ca²⁺ signals. Ca²⁺ diffuses much more slowly in cytosol than in unbuffered media, because of frequent encounters with Ca²⁺-binding proteins that are either fixed or of lower mobility. Its range of messenger action is therefore likely to be short relative to unbuffered molecules such as IP₃.⁴ Much of the signaling action of Ca²⁺ probably occurs via highly localized and steep rises in [Ca²⁺], for example immediately under the plasma membrane around Ca²⁺ channels, or at close associations between ER and mitochondria.

The cellular constituents that contribute the bulk of the cytosolic Ca²⁺ buffering have not been conclusively identified, but a large group of proteins belonging to the "EF-hand" family are known to be specialized for Ca²⁺-binding.^5–8 Some members of this family, such as calmodulin, are ubiquitous across cell types and operate primarily as Ca²⁺ sensors. These undergo conformational change on binding Ca²⁺ and thereby regulate the activity of target proteins. Other EF-hand proteins, such as calbindin_28kDa (CB28), parvalbumin (PV) and calretinin, are found in specific types of neurons and there is evidence that these function (at least in part) as Ca²⁺ buffers. In rat dorsal root ganglion neurons, introduction of CB28 or PV reduces the rate of rise and the peak of the [Ca²⁺]_c transient.⁹ Pituitary tumor (GH3) cells transfected with CB28 show smaller increases in [Ca²⁺]_c for a given Ca²⁺ influx.¹⁰ In Purkinje neurons, which normally contain both CB28 and PV, a fast component of the cytosolic Ca²⁺ transient is increased in CB28-null mutant mice.¹¹ CB28, PV and calretinin are generally regarded as mobile cytosolic buffers, but about one-third of the CB28 and calretinin in neurons is associated with insoluble cellular structures.^12,13

Ca²⁺ buffering and neuroprotection

The possibility that certain Ca²⁺-binding proteins might confer neuroprotection by buffering intracellular Ca²⁺ has generated considerable interest. CB28 and PV have received the most attention because they are expressed only in specific subtypes of neuron. Questions therefore arose about whether their presence or level of expression might be associated with resistance to excitotoxic or degenerative processes.

Several correlative studies indicated that neurons containing CB28 or PV were spared in some pathological conditions. In the hippocampal formation, the neurons most susceptible to damage from experimental seizures are the hilar interneurons and CA3 pyramidal cells, which lack CB28 or PV.¹⁴ In Parkinson's disease, the subgroup of dopamine neurons in the substantia nigra that express CB28 are relatively spared.¹⁵ The same neurons are resistant to MPTP toxicity in animal studies.¹⁶ However, other findings have suggested a more complex picture. In neurodegenerative conditions such as Parkinson's, Alzheimer's and Pick's diseases, the loss of neurons containing Ca²⁺ binding proteins varies across different brain areas (for a review see ref. ¹⁷). Furthermore, within a given region, vulnerability may depend more on the type of insult than the presence of Ca²⁺-buffering proteins. For example, CA3 pyramidal cells do not express CB28 or PV but are more resistant to ischemia than CB28-containing neurons elsewhere in the hippocampus.¹⁸

Findings from investigations in CB28-transgenic mice¹¹ have tended to downplay or even contradict a neuroprotective role for CB28. Surprisingly, in models of ischemia CB28 null mutants suffer less hippocampal damage than wild types.¹⁹ The CB28 null mutants are not more vulnerable than wild types to kainate-induced excitotoxicity in the hippocampus (although heterozygosity confers some resistance²⁰). Substantia nigra dopamine neurons in the CB28-deficient mice are not more susceptible to degeneration following MPTP administration; the same population of cells is lost regardless of genotype.²¹ Similarly, transgenic mice deficient in PV, or double knockouts for PV/CB28 or PV/calretinin, are no more vulnerable than wild types to kainate excitotoxicity in the hippocampus.²²

As a result of these studies, it has been suggested that a downregulation of CB28 may be neuroprotective. The loss of CB28 from dentate granule cells that is associated with seizures may reflect an adaptive response by the neuron to reduce its vulnerability to further threat.^23–25 Indeed, a recent electrophysiological study of human dentate granule cells removed from patients with hippocampal sclerosis confirmed that loss of CB28 is associated with a reduced Ca²⁺ influx during trains of action potentials,²⁵ which would be consistent with a neuroprotective response.

The data from transgenic animals remain controversial, in part because the life-long lack of the deficient protein may allow the opportunity for developmental compensation. Therefore a substantial body of work continues to be based on the premise that some advantage may be gained by increasing the expression of CB28 in vulnerable neurons. Sapolsky's group, in particular, is reporting success in using herpes simplex viral vectors to express (or overexpress) CB28. In cultured hippocampal neurons, this confers resistance to hypoglycemic and glutaminergic insults.^26–28 In the whole animal, injections into the hippocampus lead to increased survival of neurons following kainic acid or 3-acetylpyridine neurotoxicity,²⁹ while striatal injections promote neuronal survival after middle cerebral artery occlusion.³⁰ Other groups have shown that cell culture lines transfected with CB28 have improved survival when challenged with NMDA^31,32 or that cultured motor neurons rendered particularly sensitive to glutamate by expression of a mutant Cu/Zn-superoxide dismutase are protected by coexpression of CB28.³³ On the other hand, there have been conflicting reports about whether hippocampal neurons that naturally express CB28 in cultures are more robust,^34,35 and in one study the overexpression of PV in mouse neocortical neurons was found to enhance neurotoxicity at some concentrations of NMDA.³⁶

An alternative approach to the manipulation of intracellular Ca²⁺ buffering involves the use of exogenous Ca²⁺ chelators, e.g., BAPTA and its analogs. An early study of interneurons in the dentate hilus showed that these neurons, which usually deteriorated under prolonged stimulation, were protected by injection of BAPTA via the recording electrode.³⁷ This prompted further investigation into whether cell-permeant forms of BAPTA and its analogs might have efficacy as neuroprotective agents. BAPTA-AM and Quin-2 were shown to be taken up by spinal neurons in vitro and to reduce or delay neuronal death.^38,39 In vivo intravenous infusion of BAPTA-AM resulted in delivery of the chelator to the brain, affected the electrophysiology of hippocampal neurons as expected for a Ca²⁺ buffer, and reduced cortical infarct volume in a model of focal cerebral ischemia.³⁸ DP-b99 (DP-BAPTA), a variant of BAPTA designed to be preferentially active in lipid environments, is claimed to show efficacy in animal models and is under commercial development as a neuroprotective drug for cerebral ischemia (D-Pharm Ltd.; and see http://www.strokecenter.org/trials/ints/intPage95.htm). However, it should be noted that not all studies have reported positive findings with exogenous chelators. In cultured hippocampal neurons, BAPTA-AM reduced the peak magnitudes of [Ca²⁺]_c transients evoked by brief applications of excitatory amino acids, but also slowed the recovery to baseline. With longer exposure to agonists, the peak [Ca²⁺]_c responses were not reduced, and excitotoxicity was either increased or similar to controls.^40,41

The inconsistency of the above studies indicates that the actions of both endogenous and exogenous Ca²⁺ buffers are complex and still poorly understood. Their neuroprotective efficacy will depend upon many factors that vary with cell type and circumstance. For example, their accessibility to different subcellular compartments may be variable so that they buffer Ca²⁺ entry by some pathways more than others; they likely influence the mobility of intracellular Ca²⁺, thereby affecting the rate and distance of Ca²⁺ diffusion, and the reduction of transient peaks of [Ca²⁺]_c needs to be balanced against the consequences of longer durations of raised [Ca²⁺]_c. Ca²⁺ buffers may have important actions via binding of other ions, e.g., Zn²⁺, and the majority of Ca²⁺-buffering proteins may well have additional Ca²⁺­ sensor properties. As the field evolves it will become clearer how these factors impact on the various animal models and their applicability to human neuroprotection.

The endoplasmic reticulum Ca²⁺ store

The ER is an elaborate membrane-enclosed network that extends from the nuclear envelope to the periphery of the cell, which in neurons includes axons and synaptic terminals, dendrites and spines. Its functions include the folding and assembly of newly-formed proteins, lipid synthesis and an important role in Ca²⁺ storage and signaling. The ER forms a connected network with a continuous lumen (also continuous with the nuclear envelope), but it does show some regional specialization; areas most prominently associated with ribosomes, and therefore protein synthesis, are termed rough ER and the remainder is known as smooth ER. Although the entire ER is believed to function as a Ca²⁺ store, some regional variation has been shown in the distribution of Ca²⁺ uptake pumps and release channels, and also in concentrations of stored Ca²⁺ (reviewed in ref. ⁴² and ⁴³).

In neurons, most of the Ca²⁺ destined for uptake into the ER is probably admitted into the cell via voltage-gated Ca²⁺ channels, i.e., influx depends upon electrical activity of the plasma membrane. However, the importance of another route of Ca²⁺ entry, studied extensively in non-excitable cells, is not yet apparent for neurons. Termed capacitative or store-operated Ca²⁺ entry, this process is activated by a fall in [Ca²⁺] within the ER. It is uncertain whether the signal from the ER involves a diffusible messenger (putative calcium influx factor) or a mechanical linkage at regions where the ER is closely apposed to the plasma membrane.⁴⁴ The plasma membrane channels responsible for Ca²⁺ entry via this route have been characterized electrophysiologically in a number of non-excitable cells, and a strong candidate for the molecular identity of the channel has been reported recently.⁴⁵ This putative store-operated Ca²⁺ channel, CaT1, is a member of the TRP family⁴⁶ and is expressed in a number of tissues including brain.^47,48 It seems likely that a number of closely-related channels have similar roles.

Ca²⁺ is taken up from the cytosol into the ER by sarcoplasmic-endoplasmic reticulum Ca²⁺ ATPases (SERCAs). The SERCA family originates from three genes giving rise to multiple splice variants, with SERCA2b and SERCA3a being expressed in brain.⁴⁹ SCRCAa hydrolyze one ATP molecule for every two Ca²⁺ ions they pump into the ER lumen, with the different isoforms varying in Ca²⁺ affinity and rate of Ca²⁺ uptake.

Within the ER the majority of Ca²⁺ is buffered by Ca²⁺-binding proteins. The most prominent of these in neurons is calreticulin which binds Ca²⁺ with low affinity (_Kd ˜2 mM) but high capacity (>20 mol Ca²⁺ per mol protein.^50,51) Other proteins that contribute to Ca²⁺ buffering in the ER lumen include endoplasmin, BiP and protein disulfide isomerase. These molecules, including calreticulin, have a dual role in that they also assist with protein folding and assembly. Estimates suggest that when the ER stores are filled their total [Ca²⁺] is about 5–10 mM and their free [Ca²⁺] is 100–700 mM.⁵²

The two main pathways for Ca²⁺ release from the ER are via IP₃ receptors (IP₃R) and ryanodine receptors (RyR). Both are Ca²⁺ channels composed of four subunits. IP₃R subunits are encoded by at least three genes (with splice variants), and the complete IP₃R is activated by IP₃ and also modulated by cytosolic Ca²⁺. RyR originate from three genes and are gated by Ca²⁺ and in some cells by cADP-ribose. For both IP₃R and RyR the sensitivity to [Ca²⁺]_c is bell-shaped so that channel activity is promoted within a certain range; the actual values and degree of sensitivity vary with the multiple ribosome receptor isoforms. IP₃R and RyR show some differences of distribution within the brain. For example, IP₃R predominate over RyR in cerebellar Purkinje neurons and hippocampal CA1 pyramidal cells, whereas the reverse is true in the dentate gyrus and CA3 regions of the hippocampus.⁵³

The role of the ER in Ca²⁺ homeostasis and Ca²⁺ signaling in neurons clearly involves much more than Ca²⁺ storage or uptake so as to maintain low [Ca²⁺]_c. The properties of the IP₃R and RyR endow them with the ability to act as a source of Ca²⁺ transients (waves or spikes) within the cytosol. Ca²⁺ release from the ER can be triggered by the diffusion of IP₃ from distant sites of generation at the plasma membrane, in response to the activation of phospholipase C by extracellular stimuli. Alternatively, where the ER is near enough to a source of elevated [Ca²⁺]_c, e.g., where it is closely apposed to plasma membrane that contains Ca²⁺ channels, RyR or IP₃ channel opening is triggered or enhanced in a process known as Ca²⁺-induced Ca²⁺ release (CICR). The Ca²⁺ released from a localized area of ER may then promote further release from neighboring regions to form a propagating wave that spreads along the ER. Thus, the ER in neurons should be thought of as an active excitable organelle that promotes the integration and communication of Ca²⁺ signals with the cell.⁵⁴

Given that the neuron has ample machinery at the plasma membrane to generate Ca²⁺ transients, the ER must provide some advantages for Ca²⁺ signaling. As a source of Ca²⁺ signals, it has been suggested that the ER facilitates rises of free cytosolic Ca²⁺ deep within the cell, out of the range of the limited diffusion of Ca²⁺ entering from the plasma membrane. In addition, the extracellular space immediately surrounding many neurons may be of such limited volume as to be a poor source of Ca²⁺.⁵⁶ As a means of terminating Ca²⁺ signals efficiently, the ER has in its favor a larger surface area than the plasma membrane and lower energetic costs because Ca²⁺ uptake is not opposed by a membrane potential.⁵⁵

The endoplasmic reticulum and neuroprotection

A potential link between disturbances of ER homeostasis and pathological conditions is shown by experiments in which ER Ca²⁺ stores are artificially depleted. Agents such as thapsigargin and cyclopiazonic acid, which inhibit SERCA, promote store emptying. A number of studies have reported that this leads to suppression of protein synthesis, activation of stress genes, and ultimately apoptosis, a picture seen in various ischemic/neurotoxic conditions (reviewed in refs. ^57–59). It is likely that both the low [Ca²⁺] within the ER and the high [Ca²⁺] associated with excessive or prolonged store emptying have deleterious effects, which might contribute to a pathological scenario. In the ER, insufficient levels of intraluminal Ca²⁺ interfere with protein processing, at least in part because the chaperone activity of calreticulin and other ER proteins is Ca²⁺ dependent.^51,60 In the cytoplasm potential targets for released Ca²⁺ include proteases, endonucleases, phospholipases and other Ca²⁺-sensitive proteins that have been implicated in neurotoxicity.⁶¹ Adequate ER functioning presumably requires an appropriate environment of cytoplasmic Ca²⁺ within a certain range, which might explain why cellular vulnerability seems to increase at both high and low extremes of [Ca²⁺]_c.⁶²

Drugs that influence the Ca²⁺-handling machinery of the ER might provide opportunities for therapeutic intervention. For example, dantrolene, which blocks release of Ca²⁺ from the ER via RyR, has shown some neuroprotective efficacy in experimental models. It was reported to reduce glutamate-induced cytotoxicity in cultured cerebral cortical neurons,⁶³ to prevent seizure-induced cell death in rat hippocampal slices,⁶⁴ and to protect against death of hippocampal and cerebral cortical neurons following brain ischemia in gerbils and rats.^65,66

Another approach is to identify proteins that would be potential targets for drugs because of their role in ER Ca²⁺ homeostais. For example, calreticulin has been suggested to have anti-apoptotic properties because inhibition of its expression increases ionomycin-induced cell death.^67,68 Another protein with evidence for anti-apoptotic activities is Bcl-2. This oncogenic protein resides in the membranes of the ER and nuclear envelope, and in the outer mitochondrial membrane. Its normal function in the ER membrane is unknown at present, but alterations in ER Ca²⁺ homeostasis have been described in cultured cells overexpressing Bcl-2. Transfected cells, unlike controls, were reported to maintain ER Ca²⁺ uptake and protein processing in the presence of thapsigargin,⁷⁰ to upregulate SERCA⁷¹ or (in contrast) to show reduced loading of ER Ca²⁺ stores.⁷² Interpretation of these findings is currently difficult, and it is unclear how they relate to the actions of the Bcl-2 family of proteins in mitochondria (see below).

Mitochondria and Ca²⁺ homeostasis

Mitochondria receive attention in any consideration of cell death or neuroprotection because of their key role in energy metabolism and because of the potentially damaging consequences of the reactive oxygen species that are generated as a by-product. However in the last few years there has been a change in perspective so that mitochondria are now seen as increasingly important for their influence on intracellular Ca²⁺ homeostasis.^73–75

Ca²⁺is taken up into mitochondria via a uniporter in the inner membrane driven by a membrane potential of 150–200 mV (negative inside). The membrane potential is generated by H extrusion associated with mitochondrial respiration. A rise of inner mitochondrial [Ca²⁺] activates dehydrogenases in the citric acid cycle, thereby linking energy metabolism with cellular activity. The relatively low affinity of the uniporter raised the conundrum that the rise in bulk [Ca²⁺]_c in stimulated cells, peaking at several mM, seemed insufficient to allow significant mitochondrial uptake. Accumulating evidence now points to a close functional relationship between mitochondria and the ER. At regions where these are in close apposition, microdomains of high [Ca²⁺]_c are formed by release from the ER, and mitochondrial uptake becomes significant.⁷⁴ In addition, the pattern of Ca²⁺ signals may be important with uptake being more efficient when mitochondria are exposed to pulses of Ca²⁺.⁷⁶

An important question is whether mitochondrial Ca²⁺ handling goes beyond a signaling function for regulation of activity within the organelle itself, to additionally influence signals within the cytoplasm. There is now substantial evidence that this is the case, although with some variation between cells. Uptake of Ca²⁺ into mitochondria appears to be of sufficient rapidity and magnitude to act as a buffer so that recovery from Ca²⁺ transients is accelerated. Ca²⁺ is then released from mitochondria over a slower time course due to action of the mitochondrial Na-Ca²⁺ exchanger, resulting in a prolongation of the raised [Ca²⁺]_c after the initial recovery.⁷⁷ One example of the functional importance of this phenomenon is the contribution of persistently raised [Ca²⁺]_c in presynaptic terminals to post-tetanic potentiation.⁷⁸ Another consequence of mitochondrial Ca²⁺ release may be a feedback influence on nearby ER Ca²⁺ channels, thereby affecting the propagation of cytosolic Ca²⁺ waves.^74,75

Mitochondria, neurotoxicity and neuroprotection

An emerging body of evidence implicates Ca²⁺ overload of mitochondria, perhaps coupled with other factors such as nitric oxide or reactive oxygen species production as a crucial factor in the progression towards neuronal death (for detailed reviews see refs. ^75,79–81). It is hypothesized that overloading of the mitochondria with Ca²⁺ has two important consequences: (a) loss of the proton gradient across the inner mitochondrial membrane and therefore cessation of ATP production and (b) release into the cytoplasm of cytochrome c and other proactive substances, with subsequent activation of caspases. The degree to which these occur might tip the balance between necrosis and apoptosis. With intense insult the loss of ATP might be sufficient to cause rapid necrosis, whereas lesser injuries may allow survival of enough mitochondria to produce energy for the delayed apoptotic process.

A leading hypothesis states that a key step in the pathological loss of function in mitochondria involves formation of a permeability transition pore (PTP) across the inner and outer membranes.⁷⁹ An abrupt opening of this pore, promoted by Ca²⁺, oxidative stress, falling ATP concentration or other factors, is believed to cause mitochondrial depolarization, loss of proton gradient, and therefore uncoupling of oxidative phosphorylation. An associated increase in permeability of the outer membrane allows release of cytochrome c and other substances, e.g., apoptosis-inducing factor, from the inter-membrane space into the cytoplasm, with subsequent initiation of the apoptotic cascade. The putative makeup of the PTP is a complex of at least three proteins that forms at regions of close contact between the inner and outer mitochondrial membranes. These are the voltage-dependent ion channel (VDAC) in the outer membrane, the adenine nucleotide translocase (ANT) in the inner membrane (which exchanges ADP and ATP), and cyclophilin-D in the mitochondrial matrix. Whether formation of these components into the PTP occurs only under pathological conditions or whether transient openings might be important contributors to Ca²⁺ signaling⁷³ is controversial.

The hypothesis that the PTP is involved in cell death indicates possible targets for neuroprotective agents. For example, cyclosporin A is one drug known to bind to cyclophilin-D and to inhibit PTP opening. Mitochondrial depolarization and cell death in cultured neurons exposed to glutamatergic agonists are ameliorated or prevented by cyclosporin A.^82-84 Cyclosporin A injected into rat hippocampus has been reported to reduce the necrosis caused by forebrain ischemia.⁸⁵ However, cyclosporin A also binds to cyclophilins in the cytoplasm and ER so definitive attribution of its site of action awaits analogs that are specific for mitochondrial cyclophilin-D.⁷⁵ Another strategy might be to act further upstream by preventing loading of the mitochondria with Ca²⁺. In cultured neurons, Stout et al⁸⁶ showed that pre-emptive depolarization of mitochondrial membranes with FCCP inhibited mitochondrial Ca²⁺ uptake and reduced glutamate neurotoxicity. A further potential target is the Ca²⁺ uptake uniporter which still awaits molecular characterization and development of blockers.

The Bcl-2 family of proteins has emerged as important regulators of apoptosis, and a major part of their action is proposed to occur in mitochondria (for reviews see refs. ^87–89). Of this group, Bcl-2, Bcl-x_L and several others are anti-apoptotic whereas a number of structurally similar relatives promote cell death. Most members of the family have a hydrophobic C-terminal segment which localizes them to the cytoplasmic aspect of organellar membranes. Bcl-2 and Bcl-x_L are of particular interest as potential drug targets because they appear to oppose processes that disrupt mitochondrial function. When Bcl-2 or Bcl-x_L are applied as recombinant protein to isolated mitochondria, or over-expressed in cultured cells, they inhibit cytochrome c release in the face of apoptotic stimuli.^90-94 Consistent with these findings, over-expression of Bcl-2 in neuronal cell lines reduce cell death induced by a variety of factors,^95,96 and when over-expressed in transgenic mice, cortical infarcts induced by middle cerebral artery occlusion are smaller than in wild-type animals.⁹⁷ The mechanism of action of the Bcl-2 family of proteins is still uncertain, but it has been proposed that the anti-apoptotic members, e.g., Bcl-2 and Bcl-x_L, associate with the mitochondrial pore complex and stabilize its normal function in a way that inhibits pathological PTP activity.

Future challenges

This short review gives a simplified and rather linear overview of the cellular machinery involved in Ca²⁺ handling. By linear, I mean that the components (cytosolic buffers, ER and mitochondria) are considered in succession, whereas a major task for the future will be to understand how these function together in a working system. The close relationship between ER and mitochondria, for example, appears to be crucial for mitochondrial Ca²⁺ uptake, and therefore requires investigation of still smaller spatial and temporal domains. This makes heavy demands on technological development and also on our ability to construct useful models of interacting systems. Further complicating the picture, the majority of components are multifunctional. For example, Ca²⁺-binding proteins can have both buffer and sensor actions; ER is involved in Ca²⁺ signaling, protein and lipid processing; and Ca²⁺ mitochondrial uptake is important for enzyme modulation and for shaping cytosolic Ca²⁺ transients. Of course, none of these functions is independent. To add to the picture, there are other players in the Ca²⁺ game. Some, such as the Golgi apparatus⁹⁸ and nuclear envelope, may have a minor but distinct role; others, e.g., a large fraction of the buffering capacity of the cytosol, have a major role but are still not clearly defined. It remains a challenging and worthwhile enterprise for the basic scientist to reveal and recognize those key components of the Ca²⁺ handling system that may ultimately become useful targets for therapeutic intervention.

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