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Ca2+-binding proteins (CaBPs) are expressed in a highly specific manner across many different cell types, yet the physiological basis underlying their selective distribution patterns remains unclear. We used confocal line-scan microscopy together with photo-release of IP3 in Xenopus oocytes to investigate the actions of mobile cytosolic CaBPs on the spatiotemporal properties of IP3-evoked Ca2+ signals. Parvalbumin (PV), a CaBP with slow Ca2+-binding kinetics, shortened the duration of IP3-evoked Ca2+ signals and ‘balkanized’ global responses into discrete localized events (puffs). In contrast, calretinin (CR), a presumed fast buffer, prolonged Ca2+ responses and promoted ‘globalization’ of spatially uniform Ca2+ signals at high [IP3]. Oocytes loaded with CR or PV showed Ca2+ puffs following photolysis flashes that were subthreshold in controls, and the spatiotemporal properties of these localized events were differentially modulated by PV and CR. In comparison to results we previously obtained with exogenous Ca2+ buffers, PV closely mimicked the actions of the slow buffer EGTA, whereas CR showed important differences from the fast buffer BAPTA. Most notably, puffs were never observed after loading BAPTA, and this exogenous buffer did not show the marked sensitization of IP3 action evident with CR. The ability of Ca2+ buffers and CaBPs with differing kinetics to fine-tune both global and local intracellular Ca2+ signals is likely to have significant physiological implications.
Cytosolic Ca2+ signals regulate cellular processes as diverse as fertilization, differentiation, synaptic plasticity and apoptosis. This versatility is possible because cells are equipped with a Ca2+ signalling ‘toolkit’ (Berridge et al. 2000), with many components (proteins) that can be selected to enable signalling over a wide range of different time and distance scales (Marchant & Parker, 2000). The most important components in the toolkit are the Ca2+ channels that generate intracellular Ca2+ signals; either by allowing Ca2+ influx across the plasma membrane, or by liberating Ca2+ from intracellular stores (through inositol 1,4,5-trisphosphate receptors (IP3Rs) or ryanodine receptors (RyRs)). The majority of Ca2+ ions entering the cytosol are rapidly captured both by mobile cytosolic Ca2+-binding proteins (CaBPs) and immobile buffers of unknown identity. In addition to simply reducing the availability of free cytosolic Ca2+ ions, immobile buffers reduce the effective diffusion coefficient for Ca2+, whereas mobile buffers can act as a ‘shuttle’ to speed Ca2+ diffusion in the presence of immobile buffers (Stern, 1992; Roberts, 1994). It is thus likely that cells utilize CaBPs to shape Ca2+ signals for their specific functions; a notion supported by observations that different cell types – particularly subpopulations of neurones – selectively express mobile CaBPs with differing properties (Andressen et al. 1993).
CaBPs vary significantly in functional versatility, and are classified accordingly. ‘Ca2+ sensors’, such as calmodulin (Cheung, 1980; Vetter & Leclerc, 2003), undergo conformational changes on Ca2+ binding which enable them to bind to and activate target proteins to translate changes in intracellular [Ca2+] into signalling cascades. On the other hand, ‘Ca2+ buffers’ such as parvalbumin (PV) and calretinin (CR) are thought to act solely to chelate Ca2+ ions – although this view may change as we learn more about their biology (Schwaller et al. 2002). Despite their apparent passive function, PV and CR have nevertheless generated great interest, mainly due to their exquisitely specific expression in certain subpopulations of nerve cells (Baimbridge et al. 1992; Andressen et al. 1993). In the cerebellum, for example, PV is present in Purkinje cells and a subpopulation of inhibitory interneurones (stellate and basket cells), whereas CR is mainly localized to granule cells and their parallel fibres (Schwaller et al. 2002). These selective distribution patterns provide an invaluable experimental tool for identifying subpopulations of neurones (antibodies against them are routinely used to stain for specific populations of nerve cells). However, the physiological basis underlying their specific expression patterns has remained largely elusive (Neher, 2000), although recent studies with knockout mice now point to specific roles for CaBPs in regulating Ca2+ pools essential for synaptic plasticity (Schwaller et al. 2002).
Most experimental and theoretical investigations regarding CaBPs have focused on their ability to modulate signals arising from Ca2+ influx through voltage-gated channels in the plasma membrane (Lee et al. 2000a,b; Meinrenken et al. 2003; Schmidt et al. 2003b). Actions of CaBPs on signals arising from Ca2+ release from intracellular stores (via IP3Rs or RyRs) are likely to reflect a more complex situation (Dargan & Parker, 2003) because these release channels are themselves regulated by cytosolic [Ca2+], such that small increases in cytosolic [Ca2+] promote channel opening whereas higher concentrations are inhibitory (Iino, 1990; Finch et al. 1991; Bezprozvanny et al. 1991; Mak et al. 1998; Fill & Copello, 2002). Moreover, IP3Rs are known to exist in clusters, comprising tens of channels, which act as functionally discrete Ca2+ release units (Callamaras et al. 1998a,b; Sun et al. 1998; Swillens et al. 1999). Clusters can operate autonomously to generate local signals (Ca2+ puffs) that arise because Ca2+-induced Ca2+ release (CICR) leads to the near-simultaneous opening of multiple channels within a cluster (Yao et al. 1995; Bootman et al. 1997), and their activity can be synchronized by successive cycles of Ca2+ diffusion and CICR to generate Ca2+ waves that propagate in a saltatory manner across multiple clusters (Lechleiter & Clapham, 1992; Bootman et al. 1997; Berridge, 1997; Callamaras et al. 1998a,b; Dawson et al. 1999). Therefore, in addition to influencing the fate of Ca2+ ions already released into the cytosol, CaBPs are also likely to interfere with the Ca2+ feedback loops that act on very different distance and time scales to generate local signals by interactions between individual IP3Rs, and on the cluster–cluster interactions responsible for transitioning from local to global modes of Ca2+ signalling.
We had previously studied these processes utilizing Xenopus oocytes as a model cell system in which to image perturbations of Ca2+ signalling resulting from intracellular injections of two synthetic buffers, EGTA and BAPTA (Dargan & Parker, 2003). The oocyte is a favourable system in which to study intracellular Ca2+ signals because Ca2+ liberation is mediated solely through type 1 IP3Rs (Parys et al. 1992), its large size greatly facilitates intracellular injections and it is among the best characterized cells for Ca2+ signalling. Moreover, EGTA and BAPTA were selected because the Ca2+-binding properties of these buffers are simple and well characterized and, while having similar affinities, they show very different binding kinetics. Our main findings were that the ‘slow’ buffer EGTA accelerates the time course of IP3-evoked Ca2+ signals and dissociates global Ca2+ waves into autonomous local release events, whereas the ‘fast’ buffer BAPTA results in ‘globalization’ of spatially diffuse, and slowly decaying Ca2+ signals. These actions were attributed to the differential effects of buffers with differing kinetics on Ca2+ interactions between individual IP3Rs within a cluster, and on interactions between neighbouring clusters.
In the present paper we extend these studies to the more complex CaBPs expressed endogenously within cells. PV and CR were selected for these experiments because they show cell-specific expression and have contrasting Ca2+-binding kinetics. Under physiological conditions PV acts as a slow buffer, because its binding sites are predominantly occupied by Mg2+ ions which must be displaced before Ca2+ can bind (Haiech et al. 1979; Eberhard & Erne, 1994). CR is less well characterized, but functions as a fast intracellular buffer (Edmonds et al. 2000) analogous to BAPTA. We show that the ‘slow’ CaBP (PV) closely mimics the actions of EGTA, by speeding IP3-evoked Ca2+ transients and ‘balkanizing’ global Ca2+ waves into local puffs. On the other hand, the ‘fast’ CaBP (CR) has more complex actions. High concentrations of CR result in spatially diffuse, slowly decaying Ca2+ signals similar to the action of BAPTA; but, different to BAPTA, CR sensitizes responses to IP3 and low concentrations of CR actually promote local puffs. The ability of CaBPs to specifically modulate both local and global intracellular Ca2+ signals is likely to have significant physiological implications.
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IP3Rs are distributed in clusters (comprising tens of channels) spaced a few micrometres apart in the cytoplasm of cells ranging from Xenopus oocytes (Callamaras et al. 1998a,b; Swillens et al. 1999; Shuai & Jung, 2002, 2003) to various mammalian cell lines (Bootman et al. 1997; Simpson et al. 1997). CICR between IP3Rs can thus act over two very different spatiotemporal scales: fast diffusion of Ca2+ over short (nm) distances within a release site to generate local Ca2+ puffs, and slower diffusion between neighbouring clusters (μm scale) to generate propagating saltatory waves (Yao et al. 1995; Bootman et al. 1997; Berridge, 1997; Callamaras et al. 1998a,b; Marchant & Parker, 2000). Intracellular Ca2+ buffers with differing kinetics are likely to exert complex actions on IP3-evoked Ca2+ signalling, by differentially modulating one, or both, of these CICR processes. To study these actions we measured the effects of injecting exogenous buffers into Xenopus oocytes. A complication of this approach is that little is known regarding the nature and properties of the endogenous buffers already present in the oocyte. However, the finding that the addition of small amounts of exogenous buffer (e.g. an intracellular concentration of 25 μm parvalbumin) produced large effects on Ca2+ signals suggests that only low levels of endogenous mobile buffer are present, and would not appreciably affect our results at higher concentrations of added buffers. We had previously investigated two exogenous buffers, EGTA and BAPTA, since they have simple and well-characterized binding properties and have similar affinities yet very different kinetics (Dargan & Parker, 2003). The results and working model that emerged from that study now provide a framework for our analysis of the two endogenous CaBPs PV and CR which, in contrast to EGTA and BAPTA, contain multiple Ca2+-binding sites and exhibit more complex Ca2+-binding properties in terms of both kinetics and binding affinities.
Spatiotemporal patterning of Ca2+ signals is differentially modulated by fast and slow exogenous buffers
The main findings from our previous study (Dargan & Parker, 2003) were that EGTA (a ‘slow’ Ca2+ buffer) caused IP3-evoked Ca2+ signals to become more transient, and ‘balkanized’ Ca2+ liberation such that individual release sites functioned autonomously to generate discrete puffs, whereas BAPTA (a ‘fast’ Ca2+ buffer) prolonged IP3-evoked Ca2+ responses and promoted ‘globalization’ of spatially uniform Ca2+ signals. These strikingly distinct actions were not due to chelation of Ca2+ subsequent to its liberation into the cytosol, changes in resting free [Ca2+] or alterations in Ca2+ store filling. Instead, EGTA and BAPTA are likely to act over different time and distance scales to modulate the processes of Ca2+ diffusion and CICR that shape the regenerative nature of IP3-evoked Ca2+ liberation. We previously proposed that slow Ca2+ buffers bind Ca2+ ions diffusing over the micrometer distances between neighbouring clustered release sites, render this Ca2+ unavailable for further CICR, and ‘shuttle’ it long distances before ‘dumping’ Ca2+ ions deep in the interior of the oocyte where release sites are absent (Dargan & Parker, 2003). The overall action of slow buffers is thus to disrupt intercluster Ca2+ communication, sharply restricting Ca2+ signals around individual release sites, whilst sparing short-range Ca2+ feedback. We additionally proposed that fast buffers may bind Ca2+ ions diffusing over nanometer distances to disrupt CICR between individual receptors within clusters. Ca2+ bound to fast buffers is rapidly (∼10 ms) ‘shuttled’ a few micrometres, a distance comparable to intercluster spacing, before it dissociates. In this manner, fast buffers may act to inhibit intracluster feedback by Ca2+ whilst simultaneously facilitating intercluster Ca2+ communication.
Ca2+-binding properties of PV and CR versus EGTA and BAPTA
Table 1 lists key factors determining the interactions of Ca2+ ions with PV and CR and, for comparison, with EGTA and BAPTA. Parameters include: (1) the mean time for which a Ca2+ ion will diffuse (τcapture) and the distance (dcapture) that it will diffuse before it becomes bound to a buffer molecule; (2) the mean time (dwell time; τdwell) for which a Ca2+ ion will remain bound to a buffer before dissociating, and the corresponding mean distance (‘shuttle’ distance; dshuttle) that the Ca2+–buffer complex will diffuse before dissociation. Further, diffusion of Ca2+ ions in the cytosol is slowed by binding to endogenous, immobile buffers. Less than 10% of the total Ca2+ ions in the cytosol are free at any given time, and the apparent diffusion coefficient for Ca2+ in the oocyte is thereby slowed about 10-fold as compared to free aqueous diffusion (20 μm2 s−1versus 200 μm2 s−1, respectively; Allbritton et al. 1992; Yao et al. 1995). PV is freely mobile in the cytosol (Schmidt et al. 2003a), and can therefore act to speed or facilitate Ca2+ transport by shuttling bound Ca2+ ions through this ‘forest’ of immobile buffers (Stern, 1992; Roberts, 1994). When calculating parameters for CR (Table 1) we assumed that CR is similarly mobile (Edmonds et al. 2000). However, other reports indicate that some CR molecules may bind in a Ca2+-dependent manner to membrane constituents (Winsky & Kuznicki, 1995; Hubbard & McHugh, 1995).
Table 1. Summary of kinetic parameters and diffusion distances for binding and unbinding of Ca2+ to EGTA, BAPTA, PV and CR
| ||‘Slow’ buffers||‘Fast’ buffers|
| EGTA|| PV|| BAPTA|| CR|
|Ca2+ sites (functional)||1 (1)||3 (2)||1 (1)||6 (5)|
|App. Kd (nm) (pH 7.2)||150||150 *||160||1500 **|
|kon (μm−1 s−1)||3–10||6 *||100–1000||100–1000 ***|
|DCabuffer (μm2 s−1)||200||43||200||< 40|
| ||([B]= 270 μm)||([B]= 540 μm)||([B]= 270 μm)||([B]= 1.35 mm)|
| ||([B]= 270 μm)||([B]= 540 μm)||([B]= 270 μm)||([B]= 1.35 mm)|
In comparison to PV, few quantitative data are available for CR. Based on observations that CR is sufficiently fast to influence presynaptic Ca2+ signalling (Edmonds et al. 2000) we assumed (Table 1) that its Ca2+-binding kinetics would be comparable to those of BAPTA. However, this is likely to be an oversimplification. In contrast to the single binding site of BAPTA, CR contains five Ca2+-binding sites which – by analogy with a closely related protein calbindin D-28k (Nagerl et al. 2000) – are likely to possess different binding properties and may display cooperative rather than independent binding (G. Faas, unpublished observation).
Actions of CaBP and exogenous buffers on IP3-evoked Ca2+ signalling in oocytes
To facilitate comparison between the actions of endogenous CaBP and those of exogenous synthetic Ca2+ buffers, we present in Fig. 7 a summary of the present results overlaid with results from Dargan & Parker (2003) obtained using EGTA and BAPTA. Data are grouped comparing slow (EGTA and PV) and fast (BAPTA and CR) buffers. Moreover, concentrations of buffers are normalized by expressing them as the equivalent concentration of functional binding sites. For example, 100 μm EGTA, which has a single binding site, corresponds to 100 μm binding sites; whereas 100 μm CR, with five functional sites (Stevens & Rogers, 1997), is equivalent to 500 μm.
Figure 7. Comparison of actions of synthetic Ca2+ buffers and CaBPs on IP3-evoked Ca2+ signals Graphs summarize data obtained using PV (light blue) and CR (green), together with data taken from Dargan & Parker (2003) obtained previously using EGTA (red) and BAPTA (dark blue). In order to facilitate comparison, buffer concentrations are expressed as the equivalent concentration of Ca2+ binding sites (see text for further explanation). Further, all fluorescence data are scaled relative to the maximum of the peak signal obtained at high [IP3] before loading buffer. Control measurements (before loading buffer) are indicated in black. Different batches of control oocytes were used for each buffer, but the inset plot in A shows that normalized control data from these four groups matched closely. For clarity, control data points are shown only in the lower panels of A and B, and the fitted control Hill curves are replicated in the other panels. A, plots show the peak amplitude of Ca2+ signals as a function of normalized photolysis flash duration for three different concentrations of slow buffers (EGTA and PV). B, corresponding concentration–response relationships for the ‘fast’ buffers BAPTA and CR. C, data derived from the Hill curves in A and B showing changes in apparent cooperativity of IP3 action, Vmax and EC50 as a result of increasing concentrations of buffers. Horizontal black lines mark control values in the absence of added buffer. Hill coefficients varied between about 4.5 and 10.5 among the different batches of control oocytes, and the cooperativity is therefore expressed as a percentage of that in each respective control group. Similarly, values of Vmax are scaled as a percentage of each control group.
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Considering first the slow buffers, PV has actions that are both qualitatively and quantitatively almost identical to EGTA. Both caused IP3-evoked Ca2+ signals to become short-lived, and ‘balkanize’ Ca2+ liberation, such that individual release sites function autonomously to generate discrete puffs. Furthermore, equivalent concentrations of each molecule result in closely similar changes in the concentration–response relationship for IP3 (Fig. 7A), and in parameters derived from Hill fits to these relationships (Fig. 7C).
The case with fast buffers is more complex. The effects of CR in some respects resemble those of BAPTA, in that both promote spatially diffuse and slowly decaying Ca2+ signals at high [IP3], reduce the apparent cooperativity for IP3 action, and diminish the amplitude of responses to maximal [IP3] (Fig. 7C). In other regards, however, CR and BAPTA differ considerably. CR uniquely potentiates responses to low [IP3], causing a leftward shift in the concentration–response relationship (Fig. 7B) and a corresponding decrease in EC50 (Fig. 7C). Moreover, these responses at low [IP3] are evident as localized Ca2+ puffs, whereas we never observed local signals in the presence of BAPTA (Dargan & Parker, 2003).
Mechanisms of CaBP action on IP3/Ca2+ signalling
Under physiological conditions, PV has an on-rate (kon) comparable to EGTA: thus, a Ca2+ ion can diffuse about 1 μm in the presence of 100 μm PV before it is captured (Dargan & Parker, 2003). PV should therefore be capable of disrupting Ca2+ communication whilst sparing short-range intracluster Ca2+ feedback (Roberts, 1994; Horne & Meyer, 1997; Song et al. 1998; Callamaras et al. 1998a,b; Kidd et al. 1999). By virtue of its slow binding kinetics, PV is expected to render captured Ca2+ ions unavailable for CICR for prolonged periods (Falcke, 2003) (tdwell∼1 s), during which time it can shuttle them long distances (dshuttle= 16 μm) before ‘dumping’ them deep in the interior of the oocyte where release sites are absent (Callamaras & Parker, 1999). The overall effect of PV would therefore be to functionally uncouple neighbouring clusters by reducing free [Ca2+] between them, thus sharply restricting Ca2+ signals around individual release sites. In agreement, PV strongly inhibited Ca2+ waves, dissociating global IP3-evoked Ca2+ signals into discrete, localized Ca2+ release events (John et al. 2001; Figs 5 and 6). Associated with this, the apparent cooperativity for IP3-evoked Ca2+ liberation decreased markedly – with a Hill coefficient reducing from ∼7 to ∼2 at high [PV]– suggesting that CICR between clusters contributes markedly to the cooperativity under normal conditions, and that the binding of two (or possibly one) molecule of IP3 to the tetrameric IP3R is sufficient for channel opening (Dargan & Parker, 2003).
As with EGTA, PV caused a marked acceleration in decay of global Ca2+ signals – an effect that appears to arise because both of these slow buffers inhibit a slow tail of Ca2+ liberation that lingers during the falling phase of a wave. We had previously proposed that this slow tail component of Ca2+ liberation is maintained by cluster–cluster interactions, precisely because it was inhibited by a slow buffer that was expected to disrupt such interactions. However, the present finding (Fig. 6) that puffs normally show a biphasic decay in the absence of added buffer, and that the slow tail component is abolished by PV (but not CR), suggest that the prolonged phase of Ca2+ liberation may also involve the properties of the IP3 receptors themselves, or their interactions within a cluster.
Interpretation of the actions of CR is more difficult, both because this ‘fast’ CaBP did not simply mimic the actions of the stereotypical fast buffer BAPTA (Dargan & Parker, 2003), and because of the complex and poorly characterized Ca2+-binding kinetics and other properties of CR. In the case of BAPTA we had proposed that it binds Ca2+ ions diffusing over nanometer distances within an IP3R cluster, thereby disrupting CICR between individual IP3Rs, and subsequently facilitates Ca2+ communication between clusters by rapidly shuttling Ca2+ ions over distances of a few micrometres (Dargan & Parker, 2003). However, this scheme would not account for the specific abilities of CR to balkanize Ca2+ signals as individual puffs at low [IP3] as discussed above, nor to sensitize responses to low [IP3]. Even though the fast site(s) of CR are believed to have roughly comparable on-rate(s) (kon) for Ca2+ binding to BAPTA (Edmonds et al. 2000; Table 1), other sites are probably much slower and may at least partly account for these differences. Moreover, the actions of CR may be further complicated if a fraction of this CaBP is immobilized (Winsky & Kuznicki, 1995; Hubbard & McHugh, 1995) and if CR shows cooperative Ca2+ binding. Finally, we cannot exclude the possibility that CR may interact directly with IP3Rs to modulate their functioning, as described for calmodulin-like neuronal Ca2+-binding proteins (Yang et al. 2002; Kasri et al. 2003). Clearly, a detailed kinetic characterization of CR and other complex CaBPs will be needed before we can hope to elucidate the mechanistic basis of their varying actions on IP3-evoked Ca2+ signals.
Our findings highlight the importance of CaBPs in shaping the spatiotemporal properties of IP3-evoked Ca2+ signals – effects that are more complex than for signals arising from a fixed ‘pulse’ of Ca2+ as with Ca2+ entry through voltage-gated channels (e.g. Lee et al. 2000a). CaBPs (such as PV and CR) are found in mammalian cells at concentrations ranging from 50 μm to 2 mm (Plogmann & Celio, 1993; Schwaller et al. 2002). We show that PV and CR, even at concentrations (25–250 μm) at the lower end of this range, strongly influence IP3-mediated Ca2+ signalling in the Xenopus oocyte model cell system. Most importantly, PV and CR produce specific and strikingly different effects that may arise largely because differences in their binding kinetics confer differential actions on CICR within and between clusters of IP3Rs. Our results suggest that the concentration and buffering kinetics of CaBPs expressed by a cell are important for tuning the spatiotemporal properties of both local and global IP3-evoked Ca2+ signals, as well as determining the sensitivity and cooperativity of IP3 action and for conferring a threshold for the ability of the cell to transition from a local to a global mode of Ca2+ signalling. It is therefore highly likely that cell-specific expression of CaBPs may serve to shape intracellular Ca2+ signals for specific physiological roles. Moreover, although our results concern only IP3-evoked signals, it should be noted that ryanodine receptors (RyRs), the other major type of intracellular Ca2+ release channel, also communicate via CICR (Berridge, 1997), and may therefore be susceptible to similar ‘shaping’ by CaBPs.