An update on nuclear calcium signalling

Calcium (Ca²⁺) is a key player in signal transduction. It modulates diverse cellular activities ranging from fertilisation to cell death (Berridge et al., 2000). It is well known that different cell types use various elements from a broad Ca²⁺ signalling `toolkit' (Box 1); consequently, the characteristics of global Ca²⁺ signals, and their physiological effects, can vary considerably (Berridge et al., 2000). This might also be the case for nuclear Ca²⁺ signalling, which is essentially a complex form of local Ca²⁺ signalling. As described below, the mechanisms of nuclear Ca²⁺ signalling are extensive, and it is likely that there is considerable flexibility in how Ca²⁺ signals are triggered in the nucleus and in the downstream targets that are affected. In this Commentary, we discuss what is known about the origin and physiological significance of nuclear Ca²⁺ transients. In particular, we focus on the idea that the nucleus has an autonomous Ca²⁺ signalling system that can generate its own Ca²⁺ transients, which modulate processes such as gene transcription. We also discuss the role of nuclear pores and the nuclear envelope in controlling ion flux into the nucleoplasm. It should be noted that nuclear Ca²⁺ signalling has been the subject of previous reviews, some of which have presented different points of view (Bootman et al., 2000; Gerasimenko and Gerasimenko, 2004; Gomes et al., 2006; Lee et al., 1998; Santella and Carafoli, 1997).

It has been well established that Ca²⁺ signals occurring inside cells affect activities within the nucleus. For example, the amplitude and frequency of global cellular Ca²⁺ signals can regulate the transcription of various genes (Dolmetsch et al., 1998). But what is the significance of the nuclear component of these Ca²⁺ transients? Do patterns of nuclear Ca²⁺ signalling simply track cytosolic Ca²⁺ signalling, or are there independent nuclear Ca²⁺ signalling mechanisms that are necessary for proper cell function?

A precise method for analysing the function of nuclear Ca²⁺ is to specifically localise a buffering molecule within the nucleoplasm, which prevents changes in nuclear Ca²⁺ levels but does not affect cytosolic Ca²⁺ signals. Nathanson and colleagues employed this approach to examine the involvement of nuclear Ca²⁺ in regulating the proliferation of hepatocyte cell lines (Rodrigues et al., 2007). They observed that targeting the Ca²⁺-binding protein parvalbumin to the nucleus significantly reduced cell proliferation and altered the proportions of cells that were in the S and G₂-M phases of the cell cycle. By contrast, the expression of parvalbumin in the cytosol did not alter cell proliferation or cell cycle distribution. Most interestingly, the expression of the parvalbumin in the nucleus, but not in the cytosol, reduced the development of tumours when HepG2 cells were subcutaneously implanted into nude mice. Using a similar approach, the nuclear expression of parvalbumin was found to inhibit activation of the Elk1 transcription factor following stimulation of hepatoma cells with epidermial growth factor (EGF), whereas buffering cytosolic Ca²⁺ did not have the same effect (Pusl et al., 2002). Collectively, these data suggest that nuclear Ca²⁺signals are important for regulating cell proliferation in some contexts.

Ca²⁺ is a central player in many cellular signal-transduction cascades that modulate gene transcription. Signalling cascades mediate changes in gene expression by stimulating the translocation of transcription factors from the cytosol to the nucleus, or by causing the translocation and/or activation of enzymes that regulate the activity of nuclear transcription factors or the structure of chromatin. Although an increase in the concentration of nuclear Ca²⁺ might not be the initiating signal for such events, it can be necessary for gene transcription to occur or to be sustained. For example, nuclear Ca²⁺ can activate gene transcription via the nuclear factor of activated T cells (NFAT) family of proteins. In quiescent cells, NFAT is mainly found in the cytosol, but in response to an increase in cytosolic Ca²⁺, it rapidly (∼10 minutes) translocates to the nucleus (Tomida et al., 2003). The translocation of NFAT occurs following its dephosphorylation by the Ca²⁺-calmodulin-sensitive phosphatase calcineurin (Mellstrom et al., 2008), which exposes an intrinsic nuclear localisation sequence (NLS) in NFAT. NFAT and calcineurin associate in a Ca²⁺-dependent manner, and it has been reported that they translocate into the nucleus as a complex (Shibasaki et al., 1996). When Ca²⁺ is elevated in the nucleus, calcineurin remains active and associated with NFAT, and thereby sustains its transcriptional activity. Calcineurin mediates this effect by outcompeting kinases that promote NFAT re-phosphorylation (which would cause its nuclear export) and through a physical interaction that occludes a nuclear-export sequence (NES) in the NFAT protein.

An example of a nuclear-localised transcription factor that is regulated by both nuclear and cytosolic Ca²⁺ signals is cyclic AMP response element-binding protein (CREB). The signalling mechanisms that underlie CREB activation have been worked out particularly well in neurons, in which it has been established that depolarisation leads to the rapid phosphorylation of CREB on Ser133 by Ca²⁺-calmodulin-dependent kinase IV, which provides a necessary, but not sufficient, signal for CREB-mediated gene transcription (Dolmetsch et al., 2001). The triggering event is an increase in cytosolic Ca²⁺ levels in the immediate vicinity of L-type Ca²⁺ channels or N-methyl-D-aspartate receptors that are found in the synapse, at a site that is distal to the nucleus (Shaywitz and Greenberg, 1999). As CREB is only found in the nucleus, the phosphorylation of Ser133 is mediated by one of several Ca²⁺-sensitive signal transduction cascades that convey information from the remote synapses into the nucleus. In the nucleus, phosphorylated CREB binds additional proteins to form a transcriptionally active complex (Shaywitz and Greenberg, 1999). Although the synaptic Ca²⁺ signal does not need to propagate to the nucleus to induce phosphorylation of Ser133 on CREB, an increase in the level of nuclear Ca²⁺ is required for CREB-mediated gene transcription to occur. This is because additional Ca²⁺-dependent phosphorylation of CREB and its co-activators is required (Chawla et al., 1998; Kornhauser et al., 2002). Indeed, nuclear injection of an artificial Ca²⁺ buffer prevents CREB-mediated gene transcription, but not transcription induced by the serum-response element, which is sensitive to cytosolic Ca²⁺ buffering (Hardingham et al., 1997).

Another well-known target of nuclear Ca²⁺ signalling is the transcriptional regulator downstream regulatory element antagonist modulator (DREAM). It is well established that DREAM represses the expression of prodynorphin, an opiate-receptor precursor, and that mice that lack DREAM have a constitutive analgesic condition (Cheng et al., 2002). DREAM contains four Ca²⁺-binding EF hands (a widely expressed structural motif in proteins that bind Ca²⁺), and is directly regulated by an increase in nuclear Ca²⁺ levels (Mellstrom et al., 2008). When the concentration of Ca²⁺ in the nucleus is low, DREAM represses gene transcription by binding to DNA and displacing other transcriptional activators such as CREB (Ledo et al., 2002). An increase in the concentration of nuclear Ca²⁺ causes DREAM to dissociate from its DNA binding sites and thereby allows the transcription of its target genes.

Space limitations prevent a comprehensive discussion of the downstream targets of Ca²⁺ signalling pathways in the nucleus. However, it is evident from the in vitro and in vivo experiments described above that nucleoplasmic and cytosolic Ca²⁺ signals can synergistically control important cellular events. Furthermore, the nuclear component of global Ca²⁺ signals – or an independent increase in nuclear Ca²⁺ alone – can regulate specific nuclear processes.

Changes in nuclear Ca²⁺ concentration clearly impact on many cellular functions, but what are the mechanisms involved in regulating nuclear Ca²⁺ levels, and how are they related to those that operate in the cytosol? In the following section, we describe the mechanisms by which Ca²⁺ moves into and out of the nucleus and how intranuclear Ca²⁺ stores are triggered. Together, these mechanisms contribute to the regulation of Ca²⁺-dependent nuclear signalling pathways (Fig. 1).

To date, the most widely implicated mechanism for nuclear Ca²⁺ release is via the activation of inositol(1,4,5)-trisphosphate receptors (InsP₃Rs) (see Box 2). Evidence in support of a role for nuclear InsP₃Rs in regulating nuclear Ca²⁺ release has been obtained by using InsP₃-binding assays (Humbert et al., 1996), InsP₃R-specific antibodies (Leite et al., 2003; Malviya, 1994; Stehno-Bittel et al., 1995), electrophysiological recordings from nuclear envelope (NE) patches or from whole nuclei (Bustamante et al., 2000; Stehno-Bittel et al., 1995), nuclear microinjection of InsP₃ (Hennager et al., 1995; Huh et al., 2007; Santella et al., 2003), direct visualisation of InsP₃-evoked nuclear Ca²⁺ signals (Higazi et al., 2009), reconstitution of nuclear InsP₃Rs into lipid bilayers (Leite et al., 2003) and prevention of nuclear Ca²⁺ signals by using InsP₃R antagonists (Kumar et al., 2008). Furthermore, the addition of InsP₃ to nuclei that were isolated from Xenopus oocytes, Aplysia neurons, mammalian epithelial cells or pancreatic acinar cells (Bezin et al., 2008a; Gerasimenko et al., 1995; Quesada and Verdugo, 2005; Stehno-Bittel et al., 1995) has been shown to induce nucleoplasmic Ca²⁺ transients. Not all of these studies differentiated between expression of InsP₃Rs on the inner or outer NE. However, it is clear that InsP₃Rs are present on both the inner and outer NE, and that those channels expressed on the inner NE can trigger the release of Ca²⁺ from the NE lumen directly into the nucleoplasm. The actual proportion of the total cellular InsP₃Rs that are found on the inner NE (or other putative nuclear stores; see below) is probably very small (<1%) (Laflamme et al., 2002). However, their privileged access to the nucleoplasm might confer them with an essential local signalling function.

It has also been proposed that InsP₃Rs are present within heterochromatin and euchromatin, where they are expressed on small vesicular Ca²⁺ stores that also contain high-capacity Ca²⁺-binding chromogranin proteins (Yoo et al., 2007). The presence of small InsP₃-sensitive vesicular stores could help to explain the curious spotted distribution of nuclear InsP₃R staining that is sometimes observed when using InsP₃R-specific antibodies (particularly type-2-InsP₃R-specific antibodies) (Garcia et al., 2004; Laflamme et al., 2002; Leite et al., 2003). If this is the case, these vesicular structures could represent nuclear InsP₃-sensitive Ca²⁺ stores that might operate independently of cytosolic Ca²⁺ channels. Exactly how these vesicles are formed and how chromogranins and InsP₃Rs are trafficked to them is unclear. It is known that chromogranins can interact with InsP₃Rs and potentiate their opening (Thrower et al., 2003), and it has been proposed that the colocalisation of chromogranins with InsP₃Rs on nuclear vesicles underlies the increased sensitivity of nuclei to InsP₃-evoked Ca²⁺ release (Huh et al., 2007). However, more work is needed to verify the existence and function of these nucleoplasmic Ca²⁺ stores.

In addition to InsP₃Rs, it has been demonstrated that ryanodine receptors (RyRs) and nicotinic acid adenine dinucleotide phosphate receptors (NAADPRs) are expressed inside, or on the NE, of nuclei in various cell types, and that they regulate nuclear Ca²⁺ signalling. For example, cyclic ADP ribose or NAADP have been shown to cause the release of Ca²⁺ from the NE and to evoke Ca²⁺ transients in nuclei isolated from Aplysia neurons (Bezin et al., 2008b), osteoblasts (Adebanjo et al., 2000) and pancreatic acinar cells (Gerasimenko et al., 2003).

Ca²⁺-release channels have been found on nucleoplasm-facing invaginations of the NE, which are known as the nucleoplasmic reticulum (or nuclear tubules) (Echevarria et al., 2003; Lee et al., 2006; Lui et al., 2003; Schermelleh et al., 2008). Although such structures have been observed in a wide range of cell types (Fricker et al., 1997), they might not be universal (Bezin et al., 2008b). For reasons that are unknown, the structure of the nucleoplasmic reticulum seems to vary from being made up of simple solitary projections (Fig. 2) to a complex branched network, and it is interesting that the tubules often colocalise with nucleoli (Fricker et al., 1997). The lumen in the centre of the nucleoplasmic reticulum tubules is contiguous with the cytosol and presumably allows cytosolic messengers to gain increased access to the deeper parts of the nucleus. There is evidence that functional Ca²⁺-release channels localise on the nucleoplasmic reticulum (Echevarria et al., 2003; Marius et al., 2006). For example, photorelease of caged InsP₃ (Echevarria et al., 2003) or caged Ca²⁺ (Marius et al., 2006) in the vicinity of the nucleoplasmic reticulum initiated regenerative nuclear Ca²⁺ signals. These data indicate that InsP₃Rs and RyRs are present on the nucleoplasmic reticulum. It has been suggested that the presence of a nucleoplasmic reticulum increases the surface-to-volume ratio within the nucleus, thereby facilitating both the entry and exit of Ca²⁺.

As indicated above, cytosolic and nuclear activities can be coordinated by the spread of Ca²⁺ between these compartments. In addition, the Ca²⁺-dependent movement of enzymes (such as calcineurin) or other signalling mediators can convey signals into and out of the nucleus. One such moiety is the ubiquitous Ca²⁺-binding protein calmodulin, which has been proposed to translocate into the nucleus following cellular stimulation. Although translocation of calmodulin from the cytosol into the nucleus has been observed in numerous cell types following elevation of cytosolic Ca²⁺ (Mermelstein et al., 2001; Thorogate and Torok, 2004; Wu and Bers, 2007), the mechanism by which calmodulin enters the nucleus is not clear. Calmodulin does not contain an obvious motif for nuclear localisation, and might therefore `piggy-back' on other proteins that are translocated into the nucleus (Thorogate and Torok, 2007).

Although calmodulin is constitutively present in the nucleus, translocation of additional calmodulin, together with an increase in Ca²⁺ levels, could be crucial for nuclear signalling pathways. For example, calmodulin translocation in depolarised neurons occurred over the same time-course as CREB phosphorylation (Deisseroth et al., 1998). Buffering nuclear calmodulin, or inhibiting the nuclear influx of calmodulin, prevented the depolarisation-induced phosphorylation of CREB on Ser133. Furthermore, transgenic mice expressing a calmodulin-binding protein that was specifically expressed in the nuclei of forebrain neurons had a decreased level of CREB phosphorylation and impaired long-term memory consolidation (Limback-Stokin et al., 2004). Calmodulin is one example of many proteins that probably move into the nucleus to allow Ca²⁺ signals to function. Ca²⁺ causes its many functions by binding to proteins and altering their configurations to make them active or inactive. Therefore, the movement of signalling proteins concomitant with nucleoplasmic Ca²⁺ changes is important for Ca²⁺ to trigger downstream events.

Nuclear pore complexes (NPCs) are the major gateway for ions and macromolecules to pass between the cytosol and nucleoplasm (Box 3). Measurements of the size of the NPC central channel indicate that particles smaller than 9 nm should pass through without restriction. In particular, Ca²⁺ ions, which have a hydrated radius of ∼4 Å, should pass through easily. Although the central NPC channel can become partially occluded during the transport of various cargos, which could plausibly affect Ca²⁺ movement, ions and small molecules can pass unhindered through other channels that are thought to exist at the periphery of NPCs (Kramer et al., 2007). From these simple considerations, it would be expected that NPCs provide a constitutive conduit for Ca²⁺ moving between the nucleoplasm and the cytosol.

However, it has been suggested that NPCs can attain a conformation in which they do not permit ion transport. Using patch-clamp techniques, it is possible to form giga-ohm seals on the outer NE for periods of many minutes without any evident ion flux (Bustamante et al., 2000). This is a striking finding, as even a conservative estimate of NPC density suggests that a typical patch-clamp experiment should encompass 10-100 NPCs. As the pores have a conductance of ∼1 nanosiemen each, there should be a considerable current whenever a patch electrode is attached (Danker et al., 1997). However, electrophysiological recordings often detect only a few (about six per patch) operative channels (Bustamante et al., 2000). These observations have led to the suggestion that NPCs mainly exist in a closed conformation in which they are not ion conductive.

An alternative viewpoint has arisen from studies carried out using a different electrophysiological approach to monitor NPC activity. Instead of using membrane-attached patches, Oberleithner and colleagues employed an `hourglass' technique in which an isolated nucleus was sandwiched in a thin capillary tube. Using this method, it was observed that NPCs are constitutively open under physiological conditions (Danker et al., 2001). It was possible to modulate the NPC current recorded using the hourglass technique. For example, the addition of ATP or the depletion of the NE Ca²⁺ store increased the conductance of the NPC, but the NPCs always permitted cation flux (Shahin et al., 2001).

These latter findings arguably convey the physiological properties of NPCs most accurately. It is likely that patch-clamping of the NE distorts the membrane and somehow causes the NPCs to become non-conductive, whereas, under native conditions that are maintained using the hourglass technique, NPCs allow the constitutive passage of Ca²⁺ and other ions. Therefore, NPCs cannot be considered a complete diffusion break for Ca²⁺. Rather, ions in the nucleoplasm or cytosol have permanent access to the other compartment (Eder and Bading, 2007). At most, NPCs might act as a diffusion filter and introduce a kinetic delay in the equilibration of nuclear-cytosolic Ca²⁺ concentrations. The extent of the kinetic delay might be subject to modulation: although NPCs do not close, their conductance can change in response to factors such as Ca²⁺ and ATP. Furthermore, the extent of NPC expression on the NE can vary from 1-50 NPCs per μm², depending on cell type (Wang and Clapham, 1999). A greater expression of NPCs would allow a more rapid equilibration of cytosolic and nuclear Ca²⁺ concentrations.

Numerous studies have proposed that NPCs are regulated by changes in Ca²⁺ concentration either within the lumen of the NE, or at the cytosolic-nucleoplasmic face. However, there is little consistency in the nature of the observed effect. A widely cited example is the demonstration that altering the Ca²⁺ concentration in the lumen of the NE regulates both passive diffusion and NLS-mediated protein transport through NPCs (Greber and Gerace, 1995): depletion of the NE Ca²⁺ store was found to attenuate the nuclear influx of proteins bearing a NLS and of a non-specific fluorescent dextran molecule. It was suggested that the Ca²⁺ sensor in the NE was the integral membrane protein gp210, a nucleoporin of 210 kDa that functions to anchor NPCs. gp210 interacts with NPCs via its cytosolic C-terminus, whereas the bulk of the protein projects into the lumen of the NE, where it can sense Ca²⁺ levels and thereby mediate changes in NPC structure (Greber et al., 1990). In support of this idea, an antibody specific for the lumenal domain of gp210 was found to inhibit both passive diffusion and signal-mediated transport into the nucleus (Greber and Gerace, 1992).

A similar finding was reported when the nuclear import of histone H1 (a ∼21 kDa constitutively expressed nuclear protein) and two smaller fluorescently tagged dextran molecules (of 3 and 10 kDa) was compared in intact cardiomyocytes (Perez-Terzic et al., 1999). Reducing the Ca²⁺ concentration in the NE lumen by treating cells with a Ca²⁺ chelator, a Ca²⁺-pump inhibitor or ionomycin prevented the energy-dependent import of the histone. The same treatments prevented the passive influx of 10 kDa dextran, but not the 3 kDa dextran species. These data indicate that NPC function is sensitive to the concentration of Ca²⁺ within the NE, and that altering lumenal Ca²⁺ concentration can regulate both active and passive flux of large, but not small, molecules. However, a similar study examining the diffusion of GFP between the nucleoplasm and cytosol of intact Xenopus oocytes reached the opposite conclusion; the NE reduced diffusion of GFP by ∼100-fold, but depletion of Ca²⁺ from the lumen of the NE made no difference (Wei et al., 2003). Yet a different finding was reported in a study of the movement of photo-activated GFP across the NE in hepatocytes (O'Brien et al., 2007). In this case, a rise in cytosolic Ca²⁺ levels, and not changes in NE Ca²⁺ content, increased the influx (but not the efflux) of photo-activated GFP across the NE. In this latter study, it was also demonstrated that Ca²⁺ signals evoked by InsP₃Rs regulated the influx of GFP into the nucleus, suggesting a putative link between cell activation by InsP₃-producing stimuli and the control of nuclear trafficking.

These discrepant studies highlight some of the controversy surrounding how NPC opening is regulated. As yet, there is little consensus about what effects cytosolic Ca²⁺, or Ca²⁺ within the NE, have on NPC structure. However, although the results of the various studies are not entirely consistent, they suggest that Ca²⁺ is somehow important in regulating nuclear traffic. The mechanism by which Ca²⁺ might regulate NPC permeability is unclear. One of the most commonly proposed theories is that a central `plug' moves and blocks the channel in response to changes in the Ca²⁺ concentration in either the cysotol or the lumen of the NE (see Box 3). Consistent with this idea, several studies reported Ca²⁺-dependent changes in the shape of NPCs (Stoffler et al., 1999) and, in particular, alterations in the number of NPCs bearing a central plug. A key technique in these investigations was atomic force microscopy (AFM), which can be used to scan the three-dimensional topography of the cytosolic face of NPCs. AFM can resolve changes in the surface of large macromolecules, and has been used to show that the depletion of Ca²⁺ from the lumen of the NE significantly and reversibly increased the proportion of NPCs with a central plug (Wang and Clapham, 1999). The observed change in NPC structure that is caused by lumenal Ca²⁺ depletion is in line with the Ca²⁺-dependent alterations in NPC transport described above (Perez-Terzic et al., 1999), and supports the concept of allosteric regulation of NPC gating by Ca²⁺. However, the reported changes in NPC structure are not the same in different studies (Erickson et al., 2006), and alteration in NPC structure following Ca²⁺ release from the NE is not universally observed (Stoffler et al., 2006).

Even if changes in NPC permeability do indeed occur under physiological conditions, it is not clear what difference this makes to cellular growth or activity. It has been argued that simply altering the permeability of the nucleus to species of different molecular masses is a rather crude mechanism for regulating nuclear-to-cytosolic flux (Torok, 2007). Much greater specificity for NPC transit can be achieved by cellular signalling mechanisms that either promote or diminish the exposure of molecular tags (such as the NLS and NES) that promote NPC transport.

It has been established that Ca²⁺ signals occur in the nucleus of intact cells. For example, confocal imaging studies have shown that as Ca²⁺ waves `sweep' through the cytosol of hormone-stimulated cells, the nucleus becomes engulfed by the signal (Lipp et al., 1997). However, a major controversial issue concerning nuclear Ca²⁺ signalling is whether Ca²⁺ transients are generated autonomously in the nucleoplasm, and whether they can be distinct from cytosolic Ca²⁺ changes. Numerous studies have examined Ca²⁺ signalling in isolated nuclei (Adebanjo et al., 2000; Bezin et al., 2008b; Gerasimenko et al., 1995). The advantage of studying nuclei in isolation is that changes in nucleoplasmic Ca²⁺ only occur if Ca²⁺ is released from channels within the inner NE. Although the NPCs are present in this type of preparation, it is unlikely that Ca²⁺ released from channels on the outer NE (that face away from the nucleus), or from potential remnants of the ER, can diffuse into the nucleoplasm. Rather, any Ca²⁺ that is released outside of the NE is rapidly dissipated and diluted. Isolated nuclei therefore represent a system in which nucleoplasm-directed Ca²⁺-release channels can be examined. However, although these studies are useful for examining the potential of nuclei to generate their own Ca²⁺ signals, they do not determine whether these signals are at all significant compared with the larger fluxes of Ca²⁺ that occur in the cytosol of intact cells. The key issue, therefore, is to establish how nuclear Ca²⁺ signals arise within the physiological context of intact cells, and whether Ca²⁺ channels facing the nucleoplasm have a significant role.

The idea that nuclear Ca²⁺ levels are regulated independently from those in the cytosol emerged as soon as imaging technology advanced to allow monitoring of Ca²⁺ changes simultaneously in different cellular compartments (Bkaily et al., 1997). Using both wide-field and confocal fluorescence imaging of living cells, several groups began to report that nuclear Ca²⁺ concentration differed from that in the cytosol. The extent to which the Ca²⁺ signals in these two compartments differed was highly variable between studies. In some cases, the nuclear and cytosolic Ca²⁺ levels were found to be entirely independent, whereas others reported modest differences in the kinetics or amplitude of the nucleoplasmic and cytosolic responses (Leite et al., 2003; Santella et al., 2003). Although the examples of differential Ca²⁺ concentration in the nucleus and cytosol are too numerous to mention in detail, some are discussed below, and Table 1 lists some of the reported findings.

Owing to their brightness, Ca²⁺-sensitive fluorescent indicators have been used in most studies that compared the properties of nuclear and cytosolic Ca²⁺ signals. However, it is certain that many of these studies are invalid as they failed to recognise that organic fluorochromes have different characteristics in the nuclear and cytosolic compartments (Birch et al., 1992; Bkaily et al., 2006). Indeed, one of the most prominent early studies of nuclear Ca²⁺ regulation – which suggested that Ca²⁺ concentration in the nucleus tracked that in the cytosol up to ∼300 nM, after which they were insulated against further rises (al-Mohanna et al., 1994) – is compromised by the lack of in situ calibration (O'Malley et al., 1999). We (Thomas et al., 2000b) and others (O'Malley et al., 1999; Perez-Terzic et al., 1997) have demonstrated that commonly used fluorescent Ca²⁺ indicators are much brighter in the nucleus than in the cytosol; they differ with respect to both their affinity for Ca²⁺ and their dynamic range. Therefore, to obtain absolute values for Ca²⁺ concentration in different cellular compartments, it is crucial to perform independent calibrations with the indicator in exactly the same environment in which it will be used (Thomas et al., 2000b). Typically, when quiescent cells are loaded with a Ca²⁺ indicator such as fura2 or fluo3, it is evident that the nucleus has a greater fluorescence than the cytosol. The difference in the intensity of the indicator fluorescence between the nucleus and cytosol is maintained or even exaggerated as the Ca²⁺ concentration globally increases. This exaggerated nuclear fluorescence has led some studies to erroneously conclude that nuclei amplify cellular Ca²⁺ signals, and even that there are intranuclear Ca²⁺ gradients (Birch et al., 1992). It is not known why fluorescent Ca²⁺ indicators behave differently in the nucleus, although it has been established that factors such as ionic strength, viscosity and pH can alter Ca²⁺ binding to the indicator. In addition, it appears that Ca²⁺ indicators interact with nuclear contents; for example, in Xenopus oocyte nuclei that had been loaded with fluo3, it was found that the indicator did not diffuse freely because it became somehow `trapped' in chromatin and/or DNA (Perez-Terzic et al., 1997). These technical artefacts are one of the main reasons why nuclear Ca²⁺ signalling has such a controversial history.

The observation of a nucleoplasmic Ca²⁺ signal does not necessarily indicate that the nuclei themselves are the actual source of Ca²⁺. It is evident that Ca²⁺ diffuses more slowly in the cytosol than in the nucleoplasm because of the much greater buffering and sequestration capacity in the cytosolic compartment (Fox et al., 1997). This means that Ca²⁺ signals that arise immediately outside of the nucleus have a greater potential to diffuse through NPCs and into the nucleus than to permeate across the cytosol. Consistent with this idea, it was observed that mobilisation of Ca²⁺ from intracellular stores surrounding the nucleus of various cell types caused simultaneous Ca²⁺ elevations within the cytosol and nucleoplasm (Chamero et al., 2002), whereas distal Ca²⁺ signals that arose from voltage-operated Ca²⁺ channels at the plasma membrane induced cytosolic Ca²⁺ transients, but only modest and delayed increases of nucleoplasmic Ca²⁺.

An emerging concept in Ca²⁺ signalling is that localised perinuclear Ca²⁺ release – that is, microscopic Ca²⁺ release events that occur next to the nucleus – can affect nuclear activities (Fig. 3A). It has been shown that brief openings of InsP₃Rs or RyRs induce microscopic Ca²⁺ events that do not diffuse farther than a few micrometres. If such events occur in the cytosol at sites that are distant from the nucleus, they probably have only a modest impact on cell behaviour, but if they occur next to the nucleus, then they can have a long-lasting and integrating effect on nucleoplasmic Ca²⁺. Such observations have been made using both excitable and non-excitable cell types (Lipp et al., 1997). Indeed, many of the studies that proposed the existence of independent nuclear Ca²⁺ signals could be explained by such a perinuclear cytosolic Ca²⁺ release, followed by anisotropic diffusion of Ca²⁺ through NPCs into the nucleoplasm. In particular, this probably applies to studies in which the nuclear Ca²⁺ signal lags behind the cytosolic Ca²⁺ response (Huh et al., 2007).

Perinuclear Ca²⁺ release, and the consequent elevation in nuclear Ca²⁺ that it induces, has recently become a particular focus in studies of cardiac myocytes (Higazi et al., 2009; Kockskamper et al., 2008b; Wu et al., 2006). Ca²⁺ signals occur rhythmically in cardiac myocytes and promote the interaction of protein filaments that make the cells contract and allow the heart to pump blood. This process of excitation-contraction (EC) coupling in the heart relies on the interplay between voltage-gated Ca²⁺ channels on the plasma membrane and RyRs on the sarcoplasmic reticulum (the major internal Ca²⁺ store). The voltage-gated Ca²⁺ channels respond to the depolarizing action potential that sweeps over the heart with each beat by opening and thereby permitting the influx of a small amount of Ca²⁺. This Ca²⁺ signal is greatly amplified by RyRs that are close to the voltage-gated Ca²⁺ channels. Ca²⁺ diffuses from the RyRs to the actin/myosin filaments and initiates cell contraction. RyRs are therefore considered to be the main Ca²⁺-release channel in the heart. However, cardiac myocytes also express InsP₃Rs (Lipp et al., 2000; Perez et al., 1997), and recent studies of neonatal (Garcia et al., 2004; Guatimosim et al., 2008; Luo et al., 2008b; Luo et al., 2006), atrial (Bootman et al., 2007; Kockskamper et al., 2008a; Zima et al., 2007) and ventricular myocytes (Higazi et al., 2009; Wu et al., 2006) demonstrated that InsP₃-generating agonists, or InsP₃ itself, have specific effects on nucleoplasmic Ca²⁺ signals. Indeed, it appears that a substantial proportion of InsP₃Rs in neonatal and adult cardiomyocytes is expressed on membranes close to the nucleus or on the NE (Bare et al., 2005; Higazi et al., 2009; Liu et al., 2001) (Fig. 3B). This strategic positioning of InsP₃Rs might allow the generation of nucleus-specific Ca²⁺ signals (even though they originate in the cytosolic compartment), which could have a significant role in regulating cardiac gene transcription, and thereby be involved in controlling processes such as cardiac hypertrophy. In support of this, it has been demonstrated that the release of perinuclear InsP₃-sensitive Ca²⁺ stores promotes the activation of calcineurin and nuclear transport of NFAT into the nucleus to trigger a hypertrophic gene programme (Higazi et al., 2009). In addition, perinuclear InsP₃-mediated Ca²⁺ release promotes the phosphorylation of histone deacetylase 5 via Ca²⁺-calmodulin-dependent kinase II, causing it to be exported out of the nucleus (Wu et al., 2006) and thereby de-repressing genes that underlie the hypertrophic growth of myocytes.

It is well known that the stimulation of myocytes with hormones that activate InsP₃ production can promote hypertrophy (Sugden and Clerk, 2005), but it is not clear how hormones induce subtle changes in Ca²⁺-dependent gene transcription within cells that already experience periodic surges of Ca²⁺ during EC-coupling (Roderick et al., 2007). Perinuclear InsP₃Rs might explain how this occurs, as they can provide a source of Ca²⁺ that is spatially and temporally distinct from the Ca²⁺ signals that are involved in EC-coupling (Molkentin, 2006). Consistent with this model, stimulation of rat neonatal myocytes with phenylephrine evoked InsP₃R-mediated perinuclear Ca²⁺-release events that entered the nucleoplasm (Luo et al., 2008). Together with the finding that inhibiting InsP₃R opening prevented hypertrophic growth in response to phenylephrine stimulation (Luo et al., 2006), it is logical to conclude that phenylephrine promotes hypertrophy by activating perinuclear InsP₃Rs and, consequently, causing nucleoplasmic Ca²⁺ signals. Comparable observations have been made in cardiac cells stimulated with another InsP₃-generating agonist, endothelin-1; InsP₃Rs in close proximity to the NE triggered nucleoplasmic Ca²⁺ signals and hypertrophic gene transcription, whereas EC-coupling did not (Garcia et al., 2004; Higazi et al., 2009). Although InsP₃Rs conduct significantly less Ca²⁺ and are expressed at levels that are ∼100-fold lower than RyRs, the activation of InsP₃Rs can clearly have a dramatic effect on gene transcription in cardiac myocytes owing to their specific impact on nuclear Ca²⁺. It remains to be determined whether this model of perinuclear Ca²⁺ release is involved in regulating gene transcription and other nuclear activities in other cell types. However, there are numerous studies that have examined InsP₃R distribution in various cell types and shown that these Ca²⁺ channels are often most densely concentrated around the nucleus (Bootman et al., 2001; Shirakawa and Miyazaki, 1996; Thomas et al., 2000a; Vermassen et al., 2003). It therefore seems probable that modulation of nucleoplasmic Ca²⁺ concentration by perinuclear Ca²⁺ release is a generic signalling paradigm.

The studies described above substantiate the idea that perinuclear Ca²⁺ release significantly affects nucleoplasmic Ca²⁺ concentration. However, the observations also raise questions about the function of Ca²⁺ channels located on the inner NE and in the nucleoplasmic reticulum. In particular, why don't the Ca²⁺ channels on the inner NE, or other putative nuclear Ca²⁺ stores, generate a Ca²⁺ signal that is independent from the cytosol? Or do Ca²⁺ signals that arise in the cytosol always dominate what happens in the nucleus? As mentioned earlier, it has been shown that isolated nuclei have the machinery to release Ca²⁺, but the majority of intact cell studies indicate that nucleoplasmic Ca²⁺ signals originate outside the nucleus (Lipp et al., 1997; Luo et al., 2008). Indeed, in one notable study, InsP₃ was introduced specifically to the nucleus or cytosol to examine the relative ability of this messenger to trigger Ca²⁺ signals in each compartment. As expected, when released in the cytosol, InsP₃ evoked a cytosolic Ca²⁺ signal that invaded the nucleus via diffusion. However, when InsP₃ was released in the nucleus, the resulting Ca²⁺ signal also initiated in the cytosol, and then invaded the nucleus (Shirakawa and Miyazaki, 1996). This indicates that even when InsP₃ is given immediate access to the InsP₃Rs on the NE, the messenger diffuses out of the nucleus and preferentially triggers cytosolic Ca²⁺ release. A similar conclusion was reached when an InsP₃R antagonist was introduced in the cytosol of cells during stimulation with an InsP₃-generating agonist. The antagonist, which was too large to enter the nucleus, blocked both cytosolic and nucleoplasmic Ca²⁺ transients (Allbritton et al., 1994). The obvious conclusion from such studies is that, although nuclei have the capacity to generate Ca²⁺ transients, their contribution is `swamped' by the larger cytosolic Ca²⁺ release [but see Lui et al. (Lui et al., 1998) for a contradictory observation].

Perhaps it is only under specific stimulation conditions that substantial Ca²⁺ responses are evoked from nuclear InsP₃Rs. For example, a comparison between the Ca²⁺ signals that are evoked by vasopressin and hepatocyte growth factor (HGF) in hepatic carcinoma cells revealed that vasopressin triggered Ca²⁺ release in the cytosol, whereas HGF caused nuclear Ca²⁺ mobilisation (Gomes et al., 2008). In the latter case, it was suggested that the HGF receptor was internalised and translocated to the nucleus, where it triggered local InsP₃ production. Vasopressin, on the other hand, stimulated InsP₃ production in the cytosol. It is therefore plausible that different stimuli activate InsP₃Rs in alternative cellular compartments, depending on where in the cell the InsP₃ is generated.

An example of nuclear-specific Ca²⁺ signalling is presented in Fig. 4A, which shows the spatial profile of spontaneous Ca²⁺ transients that occur in a neonatal cardiac myocyte. Such Ca²⁺ signals are largely generated by the stochastic activation of InsP₃Rs and RyRs (Luo et al., 2008). The black trace shows substantial nuclear Ca²⁺ elevations, but a corresponding increase in cytosolic Ca²⁺ on either side of the nucleus is not observed. It was not determined whether the nuclear Ca²⁺ signals were due to activation of Ca²⁺ channels on the inner NE, or to perinuclear Ca²⁺ release. However, the figure presents an example in which substantial Ca²⁺ changes can be clearly observed solely in the nucleus.

If nuclei are truly independent of cytosolic signalling, they must have the ability to generate Ca²⁺-mobilising messengers that then act locally on nucleoplasmic InsP₃Rs, RyRs and NAADPRs. Numerous studies have demonstrated that the biochemical machinery that is required for the generation of Ca²⁺-mobilising messengers is present in the nucleus. For example, the ADP-ribosyl cyclase enzyme that is responsible for the production of cyclic ADP ribose (cADPR) and/or NAADP is localised in the nucleus (Adebanjo et al., 1999; Bezin et al., 2008a; Trubiani et al., 2008). In addition, it has been well established that nuclei possess phosphoinositide signalling mechanisms that are similar to, but distinct from, those that occur at the plasma membrane, and which can lead to InsP₃ production (Visnjic and Banfic, 2007; Ye and Ahn, 2008). Indeed, nuclei have substantial amounts of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] and phosphoinositide-specific phospholipase C (PtdIns-PLC) (Downes et al., 2005). Interestingly, it has been reported that PtdIns(4,5)P₂ and PtdIns-PLC localise in particles known as nuclear speckles (see Fig. 1). These small nuclear substructures are believed to be to be the sites of storage and/or assembly of pre-mRNA splicing factors. The exact function of PtdIns(4,5)P₂ and PtdIns-PLC in nuclear speckles is currently under investigation, but they are involved in linking the production of diacylglycerol (DAG) and InsP₃ (and their metabolites) to the control of RNA processing (Alcazar-Roman and Wente, 2008).

Mammalian cells express at least 13 different PtdIns-PLC isozymes that can be grouped into six subfamilies (β, γ, δ, ϵ, η and ζ) (Cockcroft, 2006; Suh et al., 2008), of which PtdIns-PLCβ₁ is considered to be the predominant nuclear form (Faenza et al., 2008; Ye and Ahn, 2008). In some cell types, PtdIns-PLCβ₁ is constitutively present in nucleus, whereas in others, growth-factor stimulation causes the enzyme to translocate from the cytosol to the nucleus. This is facilitated by C-terminal sequences in PtdIns-PLCβ₁ that act either as a NLS or cause the retention of the protein in the nucleus (Kim et al., 1996). It is known that PtdIns-PLCβ isoforms are activated at the plasma membrane by G-protein-coupled receptors and are responsible for rapid InsP₃ production and acute Ca²⁺ signals (see Box 2). In the nucleus, however, PtdIns-PLCβ₁ plays an important role in signalling events that are evoked by growth-factor-receptor stimulation (Visnjic and Banfic, 2007). For example, PtdIns-PLCβ₁ was found to be essential during the differentiation of skeletal muscle myoblasts (Ramazzotti et al., 2008). The expression of the enzyme increases during myogenesis, and is required for the induction of cyclin D3 expression and the formation of the retinoblastoma-cyclin D3 complex that underlies differentiation (De Santa et al., 2007). The nuclear localisation of PtdIns-PLCβ₁ is essential in this context, as a cytosolically targeted form does not support myoblast differentiation (Faenza et al., 2007).

These studies highlight the importance of nuclear PtdIns-PLCs in growth factor responses. However, they mainly describe the roles of nuclear DAG, protein kinase C translocation or inositol polyphosphate formation that results from PtdIns-PLC activation (Divecha et al., 1991). Considerably less attention has been focussed on whether changes in the levels of nuclear Ca²⁺ participate in growth-factor-mediated responses. However, several recent reports have suggested a crucial role for Ca²⁺ downstream of nuclear PtdIns-PLC activation. For example, insulin receptors are found in the nucleus following insulin stimulation of cells, where they promote PtdIns(4,5)P₂ turnover and nuclear Ca²⁺ signalling independently of cytosolic Ca²⁺ signalling (Rodrigues et al., 2008). The specific action of insulin on nuclear InsP₃ production was confirmed by expressing the InsP₃-binding portion of the InsP₃R (known as an InsP₃ `sponge') to buffer changes in InsP₃ concentration. To affect its subcellular location, the InsP₃ sponge was fused to either a NLS or a NES. The expression of the nucleus-targeted InsP₃ sponge significantly attenuated responses to insulin, whereas the cytosol-targeted InsP₃ sponge did not (Rodrigues et al., 2008). The opposite effect was observed if the cells were stimulated with vasopressin, which acts on the plasma membrane pool of PtdIns(4,5)P₂ via cytosolic PtdIns-PLC isozymes (Gomes et al., 2008).

In addition to growth factor receptors being present in the nucleus, a number of studies have demonstrated that functional hormone receptors are also internalised and move across the NE. For example, a C-terminal NLS directs the nuclear import of the bradykinin B2 receptor (Savard et al., 2008). Specific bradykinin-binding sites are present in hepatocyte nuclear extracts, and the addition of bradykinin to isolated nuclei was found to evoke a rapid, transient nucleoplasmic Ca²⁺ signal. Furthermore, it has been demonstrated that endogenous and ectopically expressed mGluR5 metabotropic glutamate receptors are present on the nuclear membranes of primary neurons and of cultured cells, respectively (Kumar et al., 2008). Studies of isolated nuclei indicate that these receptors activate PtdIns-PLCs in the nucleus, leading to nucleoplasmic InsP₃ generation and Ca²⁺ signals.

Exactly how G-protein-coupled receptors in the nucleus become activated is unclear. The lumen of the NE is topologically the same as the extracellular space, so the ligand-binding domain of nuclear hormone receptors would be exposed within the NE lumen. It has been proposed that specific transporters allow the uptake of neurotransmitters such as glutamate into the NE, where they can bind to their cognate receptors and activate signalling (Jong et al., 2005). Similarly, receptors for endothelin, angiotensin and prostaglandin, the activation of which can also stimulate Ca²⁺-mobilising pathways, are reportedly expressed on nuclear membranes (Bkaily et al., 2006; Coffey et al., 1997).

Although many studies have focussed on investigating the nuclear functions of PtdIns-PLCβ₁, other PtdIns-PLC isoforms are also found in the nucleus, in particular PLCδ₁ (Okada et al., 2005) and PtdIns-PLCγ₁ (Gomes et al., 2008). In quiescent cells, PtdIns-PLCδ₁ is localised at the plasma membrane and in the cytosol, but following stimulation, it translocates to the nucleus in a Ca²⁺-dependent manner (Okada et al., 2005), where it plays a role in regulating PtdIns(4,5)P₂ levels, DNA synthesis and cell proliferation (Stallings et al., 2008). Growth-factor-induced Ca²⁺ signals, as well as non-specific Ca²⁺ transients caused by ionophores, promote nuclear PtdIns-PLCδ₁ translocation, thereby acting as positive feedback for further nuclear Ca²⁺ signalling (Yagisawa, 2006). Although data regarding the functions of different PtdIns-PLC isozymes in the nucleus are still emerging, current evidence suggests that they all play roles in cell survival, division and differentiation.

PtdIns-PLCζ is also known to accumulate in the nucleus owing to an NLS, but in this case, the translocation of the enzyme prevents Ca²⁺ signalling (Ito et al., 2008). Repetitive Ca²⁺ oscillations occur immediately following fertilisation in mammalian oocytes, and are believed to be driven by InsP₃ production that is caused by the introduction of PtdIns-PLCζ into the oocyte from the fused sperm. The Ca²⁺ oscillations cease around the time when the pronucleus is formed, owing to the sequestration of PtdIns-PLCζ in the nucleus (Larman et al., 2004). Unlike the other PtdIns-PLC isozymes described above, when PtdIns-PLCζ is in the nucleus, it does not evoke InsP₃ production and Ca²⁺ signalling.

The Ca²⁺-mobilising pathways described above have well-characterised effects on nuclear Ca²⁺ signalling. However, there are numerous additional messengers and signalling pathways by which cellular Ca²⁺ signals can be evoked (Bootman et al., 2002), and some of these have been shown to function in the nucleus. Another example of a putative nuclear messenger is arachidonic acid: this lipid is known to increase cytosolic Ca²⁺ concentration. Although little is known about the effect of arachidonic acid on nucleoplasmic Ca²⁺, it is plausibly an additional regulator of nuclear Ca²⁺ concentration. Indeed, phospholipase A2, an enzyme involved in the production of aachidonic acid, translocates into the nucleus in a Ca²⁺-dependent manner (Schievella et al., 1995). Furthermore, arachidonic acid can be metabolised into further Ca²⁺ signalling moieties by the action of cyclooxygenase-2, which translocates into the nucleus in response to growth-factor-stimulation (Coffey et al., 1997). Although the bulk of studies have focussed on InsP₃-mediated Ca²⁺ release in the nucleus, it is probable that nuclear Ca²⁺ signalling utilises the same diverse range of messengers and channels as the cytosol.

It has been noted in several studies that nuclear Ca²⁺ signals persist considerably longer than equivalent cytosolic Ca²⁺ transients (Bootman et al., 2007; Kockskamper et al., 2008a; Lipp et al., 1997). The reason for this is the lack of Ca²⁺ ATPases on the inner NE (Humbert et al., 1996). Because of the slow dissipation of nuclear Ca²⁺, it is plausible that rapid cellular stimulation could cause a progressive accumulation of Ca²⁺ in the nucleoplasm. Fig. 4B,C shows an example of the kinetic delay in both the rise and dissipation of nuclear Ca²⁺. The recordings were obtained by imaging fluo4 fluorescence from a single atrial cardiac myocyte that was activated by regular electrical depolarisation. In addition to the kinetic delay in recovery of the nuclear Ca²⁺ transient, it is evident that the nuclear fluo4 fluorescence was consistently higher than that in the cytosol, even when both signals had recovered. This is because fluo4 is brighter in the nucleus than in the cytosol (as described above), not because there was a persistent gradient of Ca²⁺ between the cytosol and nucleus. The calibrated diastolic Ca²⁺ levels are actually very similar in the cytosol and nucleus. The differences in the kinetics of the nuclear and cytosolic Ca²⁺ signals are illustrated in the line-scan image shown in Fig. 4C; it is evident that the nucleus takes longer to fill with Ca²⁺, and considerably longer for the Ca²⁺ signal to dissipate.

A major mechanism for the dissipation of nuclear Ca²⁺ signals is via simple diffusion through NPCs. To leave the nucleus, a Ca²⁺ ion has to pass through the nucleoplasm and then encounter and traverse a NPC. Once outside the nucleus, Ca²⁺ can be quickly sequestered by Ca²⁺ ATPases on the outer NE, ER and plasma membrane, or by other buffers such as mitochondria. The NE can be regarded as a diffusion barrier for both Ca²⁺ entry and exit. Although Ca²⁺ ATPases are not expressed on the inner NE, there is growing evidence that a splice variant of type 1 sodium-calcium exchanger (NCX) is expressed on the inner NE (Ledeen and Wu, 2007). It is suggested that the nuclear NCX uniquely associates with a ganglioside known as GM1, which potentiates Ca²⁺ transport and prevents Ca²⁺ overloading within the nucleus (Ledeen and Wu, 2008). NCX is a mechanism of Ca²⁺ transport that is usually associated with electrically excitable cells in which large and rapid fluxes of Ca²⁺ occur. However, many non-excitable cells also express NCX and, in some cells, expression of the transporters appears to be restricted to the nucleus (Xie et al., 2004). The configuration of the NCX on the inner NE is the same as on the plasma membrane, but the interaction with GM1 is atypical. In the presence of a Na⁺ gradient, nuclei sequester Ca²⁺, verifying the functionality of NCX (Xie et al., 2002). The Na⁺ gradient might be driven by a Na⁺/K⁺ ATPase or Na⁺H⁺ exchanger, all of which are also present on the NE (Bkaily et al., 2006; Garner, 2002). Therefore, in addition to passive diffusion out of the nucleus, nucleoplasmic Ca²⁺ signals can be reversed by active uptake processes.

Replenishment of Ca²⁺ in the NE lumen is probably mediated by ER-localised Ca²⁺ ATPases. Although these enzymes sequester cytosolic Ca²⁺ into the ER, the fact that the lumen of the NE and ER are connected means that Ca²⁺ can diffuse from one membrane system to the other (Wu and Bers, 2006). In addition, it has been reported that Ca²⁺ accumulates in the NE via a curious mechanism that is driven solely by InsP₄ (Hsu et al., 1998; Malviya and Klein, 2006). It has been reported that specific InsP₄ receptors are present on the outer NE (Malviya, 1994), but exactly how these binding sites activate Ca²⁺ uptake is unknown. A major problem with this proposed uptake pathway is that the ER and NE possess a substantially higher Ca²⁺ concentration than the cytosol, and, unless the Ca²⁺ content of these stores was completely emptied, the flux of Ca²⁺ would always be towards the cytosol. Complete depletion of ER Ca²⁺ is a pathological state that culminates in cell death.

In the process of signal transduction, the reversal of a signalling event is as important as its induction. Exactly how nucleoplasmic Ca²⁺ signals decline back to pre-stimulation levels is not entirely clear, as the majority of studies have focussed solely on the generation of nucleoplasmic Ca²⁺ transients. Currently, passive diffusion through NPCs and uptake into the NE by NCX appear to be the main mechanisms by which nuclear Ca²⁺ transients are limited and reversed.

Over the past decade there has been a tremendous increase in the knowledge of Ca²⁺ signalling systems in the nucleus, and progress has been made in unravelling the impact of changes in nuclear Ca²⁺ on cellular activity. Recent studies have clearly highlighted the idea that gene transcription and cell-cycle progression can be specifically influenced by nuclear Ca²⁺ signals. It is probable that many more cellular processes will be shown to be dependent on nucleoplasmic Ca²⁺ changes. Despite this understanding of the functions of nuclear Ca²⁺ signals, there is still some uncertainty concerning how nuclear Ca²⁺ signals are generated and whether Ca²⁺ signalling in the nucleus is really autonomous. This is a surprisingly contentious issue that has been debated for many years. It has been shown that NPCs do not prevent Ca²⁺ fluxes from crossing the NE, which would imply that Ca²⁺ signalling in the nucleus must follow cytosolic Ca²⁺ changes (providing that cytosolic Ca²⁺ changes persist for sufficient time). Yet, several studies have shown that Ca²⁺ signals initiate within, and are restricted to, the nucleus. Exactly how Ca²⁺ signals within the nucleus or cytosol are prevented from equilibrating with the other compartment remains unclear.

In future studies, it will be important to work towards defining unified views on several topics, including nuclear Ca²⁺-release mechanisms and the regulation of NPC structure and function by Ca²⁺. As some of the controversy in nuclear Ca²⁺ signalling might be owing to the use of different cell types in different laboratories, it will also be necessary to consider cell-type-specific versus generic Ca²⁺ signalling paradigms. Finally, another goal of future research will be to link putative nucleoplasmic Ca²⁺ signals with cellular processes.