Decoding cytosolic Ca2+ oscillations

Review

Decoding cytosolic Ca2+ oscillations Anant B. Parekh Department of Physiology, Anatomy and Genetics, Sherrington Building, Parks Road, Oxford OX1 3PT, UK

A rise in cytosolic Ca2+ concentration is used as a universal signalling mechanism to control biological processes as diverse as exocytosis, contraction, cell growth and cell death. Ca2+ signals are often presented to cells in the form of Ca2+ oscillations, with signalling information encoded in both amplitude and frequency of the Ca2+ spikes. Recent studies have revealed that the sub-cellular spatial profile of the Ca2+ oscillation is also important in activating cellular responses, thereby suggesting a new mechanism for extracting information from the ubiquitous Ca2+ oscillation. The intracellular signalling paradox: combining specificity with universality Hundreds of hormones, neurotransmitters, paracrine, autocrine and physical stimuli bombard the plasma membrane (PM) of a cell each day and, if the appropriate surface receptors are expressed, elicit a cellular response. Many of these external signals alter cell behaviour by generating second messengers, which are small, diffusible molecules that activate intracellular effectors. Remarkably, only a small cohort of second messenger pathways are known to exist, leading to a fundamental question in cellular signalling: if so many different external stimuli can couple to the same second messenger cascade, how can a selective response be achieved? Research into several distinct physiological systems has provided valuable insight into how the same second messenger pathway can elicit outcomes specific to each stimulus. What is important is not the fact that the levels of the second messenger rise within the cytoplasm; rather, it is the precise spatial and temporal pattern of messenger generation that is crucial. These key principles have been extracted from studies devoted to understanding specificity to the pleiotropic universal intracellular messenger the calcium ion (Ca2+) and it is this system that I focus on here. A rise in cytosolic Ca2+ concentration ([Ca2+]c) is used as a signalling messenger in nearly all eukaryotic cells [1,2]. At rest, [Ca2+]c is maintained at values of 100 nM, but it can increase rapidly to levels in excess of 1 mM throughout the cytosol upon stimulation. The increase in [Ca2+] activates myriad responses, including neurotransmitter release, muscle contraction, mitochondrial metabolism, gene expression and cell growth and proliferation [2]. Ca2+, however, is like the sword of Damocles, in that it can lead to cell death through either apoptosis or the more indiscriminate process of necrosis [3]. A poignant example of the versatility of Ca2+ is found in vascular smooth muscle, where the same messenger can evoke physiologiCorresponding author: Parekh, A.B. ([email protected])

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cally opposing responses [4]. Ca2+ released from the subplasmalemmal sarcoplasmic reticulum opens plasma membrane Ca2+-activated K+ channels, hyperpolarizing the membrane potential and relaxing the muscle. Ca2+ influx through voltage-gated Ca2+ channels, by contrast, drives contraction. This example nicely encapsulates the concept that the spatial profile of the second messenger is crucial in determining which response will be activated. The fact that Ca2+ influences nearly all aspects of cellular life has served as a clarion call for researchers in the signalling field. Endeavours have been greatly facilitated by the synthesis of Ca2+-sensitive fluorescent dyes by Tsien and colleagues that can be loaded easily into the cytoplasm and, when combined with high-resolution microscopic imaging, permit the visualisation of changes in [Ca2+]c in living cells [5]. Researchers have observed a kaleidoscope of Ca2+ signals upon cell stimulation but perhaps most interest has been kindled by the finding that [Ca2+]c oscillates repetitively in response to low doses of agonist (Figure 1). Cytosolic Ca2+ oscillations Oscillations in [Ca2+]c are observed in all cell types, suggesting that they represent a universal signalling mode [6]. Their prevalence nevertheless belies complexity in the underlying mechanism. In excitable cells, such as nerve and muscle, rhythmic oscillations in [Ca2+]c are driven by changes in the plasma membrane potential that alter the activity of voltage-gated Ca2+ channels. This leads to bursts of Ca2+ entry, which can result directly in oscillations in [Ca2+]c or can trigger Ca2+-induced Ca2+ release from internal stores. The major way to evoke oscillations, however, is through activation of cell-surface receptors that activate the enzyme phospholipase C (PLC), which hydrolyses the minor membrane phospholipid phosphatidylyinositol-4,5bisphosphate (PIP2), thus forming diacylglycerol (DAG) and the Ca2+ mobilising second messenger inositol 1,4,5-trisphosphate (InsP3). These types of InsP3dependent oscillations in [Ca2+]c occur over a broad range of periodicities, from 10 s to >400 s. How receptors that stimulate InsP3 production evoke oscillations in [Ca2+]c is not entirely clear and the mechanisms might be both celltype and agonist-dependent. In some systems InsP3 levels oscillate owing to feedback pathways that uncouple the receptor from PLC [7,8]. InsP3 oscillations would therefore drive periodic release of Ca2+ from intracellular stores. In other cell types, oscillations in [Ca2+]c can develop in the presence of an elevated and stable background of InsP3. The oscillation is thought to arise from the fact that both InsP3 and [Ca2+]c regulate InsP3 receptors, with Ca2+ exerting a facilitatory effect at low concentrations but an inhibitory effect as its levels rise [6,9]. Regardless of the

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(Figure_1)TD$IG][ Review

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Figure 1. Oscillations in [Ca2+]c in response to an agonist that increases InsP3 arise from cyclical Ca2+ release from the stores and are maintained by store-operated Ca2+ entry. Here, RBL-1 cells (a mast cell line) have been stimulated with LTC4, an agonist of PLC-coupled cysteinyl leukotriene type I receptors. (a) A low dose of LTC4 (120 nM) evokes repetitive Ca2+ spikes when applied in the presence of external Ca2+. PMCA denotes plasma membrane Ca2+ATPase pump, a protein that removes Ca2+ from the cytosol and IP3R represents the InsP3 receptor. (b) Oscillations decrease in the absence of external Ca2+ (here, owing to the lack of Ca2+ entry through CRAC channels). (c) In the absence of external Ca2+, oscillations are maintained provided extrusion of Ca2+ from the cell is blocked with La3+. The cartoons above each graph depict the underlying mechanism. For simplicity, LTC4 receptors are omitted from the cartoons.

mechanism, oscillations in [Ca2+]c in most cell types can be supported for a few minutes in the absence of external Ca2+, revealing that Ca2+ recycling across the intracellular stores is the primary mechanism for driving them (Figure 1). Oscillations triggered in Ca2+-free solution nevertheless do decrease with time, because a fraction of the Ca2+ released during each cycle is transported out of the cell (Figure 1). Less Ca2+ is therefore available for resequestration into the stores to support the next oscillation. To sustain oscillations requires Ca2+ entry and this is generally accomplished through store-operated Ca2+ channels in the plasma membrane. These channels are opened by the loss of Ca2+ from the stores and the ensuing Ca2+ influx refills these stores and drives a plethora of other cellular activities [10,11]. The best understood store-operated Ca2+ channel is the CRAC channel [10,12,13]. The molecular basis of this Ca2+ entry pathway has now been teased apart with the discoveries of the stromal interaction molecule (STIM) proteins STIM1 and STIM2 and the Orai family of Ca2+-selective store-operated channels. STIM proteins are the Ca2+ sensors within the stores, and they form multimers upon store depletion [14–16]. These multimers then migrate within the endoplasmic reticulum (ER) membrane to reach specialised ER-PM junctions [17,18]. At these sites, they capture and then activate the plasma membrane CRAC channels [19], for which ORAI1 forms the pore [20–22]. Although STIM1 and STIM2 share many properties, they contrast in two important ways. First, STIM1 requires a substantial fall in store Ca2+ content before it translocates to ER–PM junctions whereas more modest

Ca2+ release is sufficient to mobilise STIM2 [23]. Second, after occupying ER–PM junctions, STIM1 is much more effective than STIM2 at opening CRAC channels [24]. Combined, these findings have led to the view that STIM2 is important in maintaining both basal Ca2+ entry and Ca2+ influx in response to weak levels of stimulation. By contrast, STIM1 plays an increasingly important role as stimulus intensity increases, reflecting the more extensive Ca2+ mobilisation from the stores. What happens during Ca2+ oscillations, which are widely believed to reflect physiological levels of receptor activation? A recent study clearly demonstrates a major role for STIM1 and not STIM2 in sustaining Ca2+ oscillations at low concentrations of agonist [25]. In addition, using total internal reflection microscopy to image events within 100 nm of the plasma membrane, the authors nicely demonstrated oscillatory movement of STIM1 to and from ER–PM junctions during each Ca2+ spike [25]. The dependence of CRAC channel activation on a substantial, albeit transient, fall in store Ca2+ content during each oscillation in [Ca2+]c points toward STIM1 acting as a digital decoder, translating essentially all-or-none Ca2+ release events into CRAC channel opening in the plasma membrane [25,26]. In the absence of Ca2+ influx, prevention of Ca2+ extrusion (e.g. by inhibition of the plasma membrane Ca2+ATPase pump with La3+) from the cell can support oscillations of [Ca2+]c for prolonged lengths of time (Figure 1) [26,27]. Under these conditions [28], the released Ca2+ is taken back into the stores by the sarco-endoplasmic reticulum calcium transport ATPase (SERCA) pumps in preparation for the next cyclical opening of InsP3 receptors. 79

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Table 1. Ca2+ oscillations and cellular functions Cell response

Cell type

Stimulus

Secretion

i) Gonadotrope

i) GnRH

Oscillatory Ca2+ signals i) Yes

ii) RBL-2H3 mast cell

ii) Antigen

ii) Yes

Mitochondrial metabolism (NAD(P)H fluorescence used as an indicator)

i) Adrenal glomerulosa ii) Hepatocyte iii) Pancreatic acinar cell

i) Angiotensin II ii) Vaspopressin iii) Cholecystokinin

i) Yes ii) Yes iii) Yes

Differentiation

Neurons

Gene expression

i) Jurkat T cells

Voltage-gated Ca2+ channels i) CRAC channel

ii) RBL-2H3

ii) InsP3

i) Yes i) Yes

ii) Yes

Comments

Ref

i) Oscillations are linked with secretion, but it is not known if they utilize FMa or AMb coding. ii) AM coding, but oscillations per se were not important. i) FM coding ii) FM coding iii) FM coding A threshold rise in mitochondrial matrix [Ca2+] (>1.5 mM) is needed to stimulate ATP production. Thus, FM coding probably occurs together with AM coding, to ensure that the threshold is reached. Stringent FM coding for neurite extension.

[73]

i) FM coding. Different transcription factors were sensitive to different oscillation frequencies. ii) FM coding

[80]

[74] [75] [76] [77] [78]

[79]

[81]

a

FM, frequency modulation. b AM, amplitude modulation.

Oscillations in [Ca2+]c have been implicated in the activation of a wide range of cellular responses (Table 1), ranging from exocytosis (active over seconds) to gene expression and cell differentiation (active over hours to days).

[(Figure_2)TD$IG]

Reading Ca2+ oscillations: the AM and FM catechism Electrical signals that have sinusoidal waveforms are common in electronics. For a simple harmonic oscillator, voltage output (V) can be described by: V ¼ A sin2p ft where A is the amplitude and f is the frequency (Hz). Hence sine waves carry information in their amplitude and frequency. Extrapolating this to oscillations of [Ca2+]c in biological systems, the conventional view is that information is encrypted in either the amplitude or frequency of the Ca2+ spikes (Table 1). What tools do cells have for extracting information from different patterns of Ca2+ signal? Amplitude modulation (AM) Signalling through amplitude modulation is conceptually straightforward because it is governed, at least in part, by the Ca2+ affinity of the decoder or sensor. If the decoder has a high-affinity Ca2+-binding site, then it will readily respond to small elevations in cytosolic Ca2+; by contrast, a low-affinity Ca2+ sensor would require a significantly larger increase in ambient Ca2+. Hence, Ca2+ signals of different amplitude would recruit sensors in a manner proportional to their respective Ca2+ affinities. Many Ca2+ sensors have more than one Ca2+-binding site. As the number of sites increases from one to four, and assuming affinity is the same for each site, an interesting change in the fractional Ca2+ occupancy of the sensor arises (Figure 2a). Calculations reveal a sharp rightwards shift in the relationship between [Ca2+]c and the fractional occupancy of the sensor as the number of independent binding sites increases. If one compares a one binding site 80

Figure 2. Ca2+ sensors with different numbers of identical Ca2+-binding sites or with an equivalent number of sites but with differing affinities are capable of detecting a wide range of Ca2+ spike amplitudes and impart apparent threshold phenomena. (a) The computed fractional occupancy of a sensor (KD 1 mM) is plotted against [Ca2+]. The number of identical Ca2+-binding sites (n) is varied from one to four. (b) The graphs compare fractional occupancy for Ca2+ sensors (four identical binding sites) with different KD values. In both graphs, note the emergence of a threshold (for higher n values in (a) and lower affinities in (b)), providing a mechanism for selective decoding of amplitude-based Ca2+ signals.

Review sensor (KD 1 mM) with a sensor harbouring four binding sites (each with the same affinity of 1 mM), what is striking is that negligible fractional occupancy is seen for the four site sensor at 1 mM Ca2+, whereas 50% saturation occurs for the one site sensor (Figure 2a). Hence, a threshold phenomenon develops; for <1 mM Ca2+, single Ca2+ binding site sensors would be potentially active whereas sensors with three or four sites would be largely ineffective. Fractional Ca2+ occupancy for sensors with different affinities for Ca2+ (each having four independent sites) is also shifted to higher concentrations of Ca2+ as affinity decreases (Figure 2b). Hence, increasing the number of binding sites provides a mechanism whereby different oscillatory amplitudes might activate different cellular responses in an almost digital manner. An interesting and hitherto unexplored concept is that agonists or certain conditions lead to the reversible formation of complexes that contain multiple Ca2+ sensor subunits. Varying the number of identical Ca2+-binding molecules in the complex gives rise to a range of Ca2+ sensitivities and, hence, provides a simple and dynamic means for obtaining amplitude-encoded selectivity. Further scope for diverse Ca2+ sensitivities is afforded by cooperative Ca2+ binding. However, the limitation of a simple amplitude-encoded system is that sensors with different Ca2+ affinities will be recruited sequentially, not independently. The bulk Ca2+ rise needed to recruit a low-affinity sensor would inadvertently activate almost all sensors with a higher affinity for Ca2+ (Figure 2). One potential bypass mechanism would be to restrict the location of sensors to discrete subcellular compartments, which is the concept of spatially restricted signalling (see below). Another way is to decipher the kinetics or time-course of the Ca2+ signal, which I now discuss. Frequency modulation (FM) In this form of decoding, the kinetic or temporal properties of the Ca2+ signal are detected by downstream Ca2+ sensors and translated into distinct cellular responses. Two such Ca2+ sensors are conventional (Ca2+-dependent) protein kinase C (PKCa, bI, bII, g) [29] and Ca2+/calmodulindependent protein kinase II (CaMKII)(30). Conventional PKC enzymes This family of serine/ threonine kinases requires both Ca2+ and DAG for activation, and these co-factors bind sequentially to C2 and C1 domains on the enzyme, respectively. Conventional PKC isozymes are cytosolic at rest but translocate to the plasma membrane upon an increase in [Ca2+]. Ca2+ binding to the C2 domain allows the kinase to attach to the plasma membrane, albeit with low affinity. If DAG is present, for example following receptor-driven hydrolysis of PLC, it binds to the C1 domain and stabilises PKC residency at the plasma membrane. As [Ca2+] falls, the enzyme migrates away from the plasma membrane and loses activity. Experiments using green fluorescent protein (GFP)-tagged PKCg showed that Ca2+ oscillations in response to low levels of agonist evoked oscillatory kinase movement to/from the plasma membrane that were phase locked with the Ca2+ signal [29,31]. However, in the presence of a DAG analogue, PKCg

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retention at the plasma membrane outlasted the Ca2+ oscillation by several seconds. A second Ca2+ spike during the time PKC is at the membrane would increase enzyme activity significantly. High-frequency oscillations of [Ca2+]c therefore ratchet up kinase activity, whereas enzyme activity closely mirrors low oscillation frequencies [29]. PKCg (and presumably other conventional PKC isoforms) could therefore act as a high-pass filter, converting highfrequency Ca2+ spikes into a response while filtering out slow signals. CaMKII This protein kinase requires Ca2+ and calmodulin for activation; autophosphorylation on a conserved threonine residue [32] results in increased and prolonged kinase activity even in the absence of the activating components. For autophosphorylation to occur, coincident binding of two calmodulin molecules onto two subunits of the holoenzyme is needed. The half time for calmodulin dissociation from the kinase is 0.17 s, but after autophosphorylation this increases to 3 s in the absence of Ca2+ and can be hundreds of seconds when [Ca2+] is maintained [33]. Autophosphorylation therefore prolongs CaMKII activity by ‘trapping’ calmodulin within the enzyme [33]. Low-frequency Ca2+ oscillations fail to evoke much autophosphorylation and kinase activity [30] because calmodulin dissociates from the kinase between Ca2+ spikes. By contrast, higher-frequency oscillations increase enzyme activity in a frequency-dependent manner through calmodulin trapping [30]. At a given frequency, an increased Ca2+ pulse duration also leads to increased autophosphorylation and kinase activity. Moreover, lowfrequency spikes of long duration are much more effective in triggering CaMKII autophosphorylation than higherfrequency spikes of shorter duration. CaMKinase II therefore converts both the number of oscillations and the duration of each oscillation into different levels of enzymatic activity [30]. Moreover, once autophosphorylated, kinase activity outlasts the duration of each oscillation, imparting a form of short-term memory. The oscillation frequencies that regulate CaMKII span a broad range, from 10 Hz, reflecting action potential-dependent spiking in neurons, to 0.1 Hz, typical of InsP3-driven oscillations in non-excitable cells [30]. There are four highly homologous isoforms of CaMKII (a, b, g and d), encoded by different genes, which themselves give rise to multiple splice variants. For the CaMKIIb isoform, the three splice variants require different Ca2+ oscillation frequencies to drive authophosphorylation and therefore autonomous activity [34]. Different CaMKII variants therefore differ in their ability to decode Ca2+ oscillation frequencies. This, coupled with their different patterns of expression within and between cells, provides a potential molecular basis for frequencydependent selectivity. Spatial profile: the third man The view that all the information within a series of Ca2+ oscillations is encoded by the amplitude and/or frequency of the spikes overlooks the potential involvement of spatially restricted Ca2+ signals. Oscillations in [Ca2+]c arise from the opening of Ca2+ channels that can be located 81

Review either in intracellular stores or in the plasma membrane. The diffusion of Ca2+ through an open Ca2+ channel in either of these locations results in the rapid build-up of a local microdomain of elevated Ca2+, which decays steeply with distance from the channel owing to cytosolic Ca2+ buffering [35,36]. Within a few nanometres of the open channel, buffers are too slow to capture the invading calcium ions and this, coupled with the tiny volume occupied by the microdomain, means that local [Ca2+] can reach tens of micromolar, several-fold higher than the rise in bulk [Ca2+]c [35,37]. An important element in the cellular arsenal for reading oscillations of Ca2+ could therefore be the subcellular spatial profile of the Ca2+ spikes. If this is indeed the case, then oscillations in [Ca2+]c that are identical in amplitude and frequency should differentially activate downstream targets depending on the spatial location of the underlying Ca2+ channels. To test this idea, endogenous cysteinyl leukotriene type I receptors (which are G protein-coupled receptors that activate PLC to produce InsP3) in a mast cell line were activated to generate two kinds of oscillation in [Ca2+]c with distinct spatial Ca2+ signatures [26]. One involved Ca2+ release from the ER but without any Ca2+ influx, whereas the other comprised Ca2+ release followed by Ca2+ influx through store-operated CRAC channels. Only the latter situation resulted in significant elevation of the subplasmalemmal [Ca2+]. Stimulation of these native receptors with the agonist leukotriene C4 (LTC4) evoked repetitive all-or-none oscillations in [Ca2+]c [26]. Over a range of agonist concentrations, the amplitude and frequency of the oscillations were indistinguishable between cells stimulated in the presence of external Ca2+ or in its absence (with the plasma membrane Ca2+-ATPase pump blocked to prevent loss of Ca2+ from the cell). Nevertheless, only those oscillations in which Ca2+ entry through CRAC channels occurred led to expression of the immediate early gene c-fos [26]. These findings therefore suggested a major role for the spatial profile of the [Ca2+]c oscillation, rather than amplitude or frequency, in excitation–transcription coupling in this system. Very close to a Ca2+ channel pore, intracellular Ca2+ chelators have little to no effect on the local [Ca2+], but do reduce the lateral spread of the microdomain in a manner dependent on the on-rate of the chelator for Ca2+ [35,37]. The chelator EGTA binds Ca2+ slowly (on-rate 106 M1 s1) and therefore has no impact on the build-up of local Ca2+ signals. 1,2-Bis(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA), in contrast, binds Ca2+ much more quickly (on-rate 5  108 M1 s1) and can reduce the extent of a microdomain [35]. Consistent with the role for local Ca2+ entry in excitation–transcription coupling driven by receptor activation, CRAC channel-driven gene expression was unaffected by EGTA, but was blocked by BAPTA [26]. A major determinant of the size of a Ca2+ microdomain is the single channel current, which depends on the prevailing electrochemical gradient for Ca2+ entry. Subsequent work revealed that manipulating either the electrical or chemical driving force for Ca2+ entry through CRAC channels strongly affected the expression of c-fos, despite having little impact on the bulk [Ca2+]c increase [38]. Collectively, these findings endorse the view that Ca2+ microdomains near 82

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CRAC channels and not global oscillations in [Ca2+]c per se effectively couple to gene transcription. Sensing local Ca2+ entry: what lies beneath The simplest mechanism whereby local Ca2+ signals can drive cellular responses is through co-localisation of the target with the source of Ca2+. In addition, local Ca2+ signals can influence events located at a considerable distance via the recruitment of intermediary Ca2+ sensors. Numerous examples exist that illustrate these different strategies (Figure 3 and Table 2). Calmodulin: a versatile Ca2+ sensor A crucial requirement for long-range signalling by spatially restricted Ca2+ signals is a Ca2+ sensor that is located close to the Ca2+ channel, and which then acts as an intermediary to recruit more distal targets. The ubiquitous Ca2+-binding protein calmodulin is commonly used as a

[(Figure_3)TD$IG]

Figure 3. Cellular strategies for coupling Ca2+ microdomains to molecular targets. (a) Ca2+ microdomain couples to (i) ion channels and (ii) plasma membrane enzymes. (b) Local Ca2+ entry (i) drives vesicular fusion, and Ca2+ release from InsP3 receptors (i) propagates into closely apposed mitochondria. Local Ca2+ entry (ii) is taken up into subplasmalemmal ER and tunnels through the ER to the opposite pole of the cell, where it is released to drive secretion. (c) Ca2+ microdomains activate intracellular enzymes, which generate paracrine signals that (i) sustain CRAC channel activity or(ii) regulate gene expression via Ca2+ sensors held close to the channel. See Table 2 for more details.

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Table 2. Mechanisms for sensing local Ca2+ Sensing mechanism Target adjacent to Ca2+ channel (Figure 3a)

Ca2+ channel co-localizes with organelle (Figure 3b) that itself expresses a Ca2+ sensor

Examples i) Ion channels: Ca2+-dependent K+ channels. ii) Enzymes: endothelial NO synthase, plasma membrane Ca2+ ATPase pump, adenylyl cyclase, Syk, CaMKII i) Secretory vesicle

i) Mitochondria

ii) ER Remote sensing (Figure 3C)

i) Paracrine signalling

ii) Gene expression

Comments These effectors are thought to sense the Ca2+ microdomain directly, resulting in rapid responses with high fidelity

Ref [82] [83–87]

i) Vesicles are held close to the Ca2+ channel through protein–protein interactions. Three or fewer channels can drive neurotransmitter release. i) Mitochondria are tethered to the ER via proteins including mitofusin-2, thus facilitating rapid propagation of Ca2+ microdomains from InsP3 receptors on the ER into the matrix, and stimulating ATP production. Many mitochondria are mobile, however, and can be recruited to the plasma membrane by Ca2+ microdomains near open CRAC channels, where they sustain channel activity by reducing Ca2+-dependent inactivation. ii) Local Ca2+ entry through CRAC channels rapidly refills the ER with Ca2+ i) Ca2+ microdomains near open CRAC channels lead to the synthesis and secretion of LTC4. Secreted LTC4 activates cysLT1 receptors on adjacent mast cells, driving further LTC4 production. A wave of excitation spreads through the cell population, arising from a feed-forward loop between CRAC channels and LTC4. ii) Local Ca2+ entry through either voltage-gated or CRAC channels is relayed to the nucleus by Ca2+ sensors, leading to gene expression

sensor by Ca2+ channels. The amino acid sequence of calmodulin, a versatile and highly conserved protein, is invariant among vertebrates. It has globular amino- and carboxy-terminal lobes, connected by a flexible hinge region [39]. Calmodulin contains four EF-hand Ca2+-binding motifs, two located in the amino-terminal lobe and two in the carboxy-terminal lobe. Calmodulin can bind up to four calcium ions, one on each EF hand. The protein can be either cytosolic or membrane-bound, the latter often involving a calmodulin-binding motif called the IQ motif, which has a consensus sequence of IQXXXRGXXX and is found in many calmodulin-binding proteins, including voltage-gated Ca2+ channels. The on-rate and the off-rate for Ca2+ binding to the EFhands of calmodulin are 70-fold and 170-fold faster in the amino-terminal lobe than that in the carboxy-terminal lobe [40], suggesting that the lobes might respond differently to distinct temporal patterns of Ca2+ signalling. Recently, new insight was gained regarding how calmodulin, tethered to a Ca2+ channel, can bifurcate the local Ca2+ signal in a lobe-specific manner into distinct responses. Ca2+-calmodulin has two opposing effects on voltage-gated P/Q-type (Cav2.1) Ca2+ channels: Ca2+-dependent facilitation (which increases the size of the Ca2+ current and is controlled by local [Ca2+]) and Ca2+-dependent inactivation (which reduces the current amplitude and is controlled by global [Ca2+]). Mutations in the Ca2+-binding sites in the amino-terminal lobe of calmodulin result in the loss of channel inactivation; facilitation, however, is unaffected. Conversely, mutations in the Ca2+-binding sites in the carboxy-terminal lobe impair facilitation but have no effect on inactivation [41]). Calmodulin tethered to the Ca2+ channel therefore enables lobe-specific regulation of the same target protein [41]. More generally, these findings

[35,90,91]

[36,88,92–95]

[89] [96–98]

[26,44,47]

reveal that intra-molecular segregation of Ca2+-binding sites renders a sensor able to elicit a range of responses. The two lobes of calmodulin are located within 6 nm of one another, and therefore both should be exposed to the same high local [Ca2+]. How can the amino-terminal lobe not sense this local Ca2+ but instead regulate P/Q-type Ca2+ channels through global [Ca2+]? The solution probably lies, at least partly, in the kinetics of Ca2+ unbinding within the lobes [42]. With its faster off-rate for Ca2+, the Ca2+ occupancy of the amino-terminal lobe tracks more closely the duration of the brief Ca2+ microdomain than the carboxy-terminal counterpart. Because Ca2+-dependent inactivation develops over hundreds of milliseconds whereas the build-up and collapse of microdomains occurs over hundreds of microseconds following channel opening and closing, Ca2+ would dissociate from the amino-terminal lobe long before Ca2+-dependent inactivation occurs. Amino-terminal lobe regulation therefore requires a sustained rise in [Ca2+] as afforded by a global signal rather than the transient local Ca2+ signals that accompany brief channel openings [42]. By contrast, the slow rate of Ca2+ dissociation from the carboxy-terminal lobe enables this lobe to respond faithfully to transient local Ca2+ entry through P/ Q-type Ca2+ channels. L-type (Cav1.2/1.3) Ca2+ channels are also controlled by the amino-terminal lobe of calmodulin but, unlike its interaction with P/Q-type channels, this lobe regulates channel activity by detecting local Ca2+ signals. How can the same lobe respond to such different types of Ca2+ signal? The answer lies in its physical interaction with the Ca2+ channels and the locus has been mapped to a Ca2+/ calmodulin-binding site (called the N-terminal spatial calcium-transforming element or NSCaTE) [43]. This site is found in L-type Ca2+ channels, but not in the P/Q-type 83

Review counterparts. Insertion of NSCaTE into P/Q-type channels renders them sensitive to calmodulin in a manner dependent on local, but not global, Ca2+ signals. Conversely, disruption of the NSCaTE site in L-type channels results in sensitivity to global rather than local Ca2+ [43]. The presence of NSCaTE domain therefore switches Ca2+-dependent regulation by calmodulin from the global level to the local level. Presumably, the binding of calmodulin to the NSCaTE site alters the kinetics of Ca2+ unbinding from the amino-terminal lobe. In addition to regulating Ca2+ channels, tethered calmodulin plays an important role in excitation–transcription coupling. Upon L-type Ca2+ channel opening in hippocampal neurons, tethered calmodulin dissociates from the channel and translocates into the nucleus where it leads to activation of the transcription factor CREB (cAMP response element-binding protein) [44]; activated CREB increases the transcription of genes associated with long-term memory formation [45,46]. In cortical neurons, opening of L-type channels activates CREB, whereas other Ca2+ channels do not, despite increasing [Ca2+]c to a similar extent [47]. These differences can be attributed to selective tethering of calmodulin to the L-type channel [47]. Calmodulin also couples CRAC channel activity to the nuclear factor of the activated T cells (NFAT) pathway [48]. Four members of the NFAT family (NFAT1–4) are Ca2+-activated transcription factors that localize to the cytoplasm in resting cells due to extensive phosphorylation in their regulatory domains. Dephosphorylation is accomplished by the Ca2+/calmodulin-dependent protein phosphatase calcineurin, and results in NFAT translocation into the nucleus where it upregulates genes involved in T cell proliferation and differentiation [49]. ORAI1, the poreforming subunit of the CRAC channel, binds calmodulin at the amino-terminal end [50], raising the possibility that Ca2+ microdomains near CRAC channels activate calcineurin and hence NFAT migration to the nucleus via tethered calmodulin. Other Ca2+-permeable channels also bind calmodulin. Several transient receptor potential (TRP) channels have a calmodulin-binding site on the carboxy terminus [51], which contributes to Ca2+-dependent regulation of channel activity [52–54]. Intracellular Ca2+ channels also bind to, and are controlled by, calmodulin [55,56]. Calmodulin also regulates mitochondrial Ca2+ uptake through the uniporter Ca2+ channel [57,58], but whether this involves tethered calmodulin awaits the molecular identification of this ubiquitous Ca2+ transporter. These findings indicate that tethering calmodulin to a range of distinct Ca2+-permeable ion channels in different subcellular locations holds the sensor at an optimal location for detecting local Ca2+ entry and can provide specificity in driving spatially distinct cellular responses. Other Ca2+ sensors Although much is now known about calmodulin, other Ca2+ sensors can reversibly associate with membranes in a Ca2+-dependent manner. Examples include proteins with C2 domains, such as the Ca2+ sensor for exocytosis, synaptotagmin and the annexin family of proteins. C2 domains comprise two sets of four-stranded b sheets with two 84

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protruding loops that form Ca2+-binding sites [59]. Ca2+ occupancy increases phospholipid binding, and thus reversibly localises the protein to membranes. Annexins bind to negatively charged phospholipids in a Ca2+-dependent manner and regulate several signalling pathways, (e.g. PKC, Ras and Ca2+) [60]. The neuronal Ca2+ sensor (NCS) group is a family of proteins that are distantly related to calmodulin and have very high affinity for Ca2+ [61]. NCS-1 protein, which regulates exocytosis, is likely to have its myristoyl tail exposed continuously and thus is embedded in membranes [62]. Other family members, including hippocalcin (which is thought to be antiapoptotic as well as a regulator of mitogen-activated protein kinase (MAPK) activity and AMPA receptor recycling) and recoverin (which is involved in light adaptation) expose the myristoyl tail upon binding Ca2+ and thus can reversibly associate with membranes, perhaps near sources of Ca2+ [62]. NCS proteins can interact directly with Ca2+-permeable TRPC5 ion channels [63] and regulate non-L-type Ca2+ channels in chromaffin cells [64]; however, it is not known whether this is a direct effect. Because of their high affinity for Ca2+, the dynamic range over which NCS proteins respond to cytosolic Ca2+ will be somewhat limited. Nevertheless, their differential distribution within and between cells, coupled with reversible Ca2+-dependent membrane attachment, adds a new element to spatial decoding of Ca2+ signals. Altering Ca2+ microdomains through dynamic changes in Ca2+ channels The amplitude and radial spread of a single Ca2+ channel microdomain depends on the single Ca2+ channel flux, which in turn is determined by the single channel conductance and the prevailing electrochemical gradient. The duration of the microdomain will also be influenced by the channel’s open probability. Consider an L-type Ca2+ channel. At hyperpolarised membrane potentials, channel open probability is very low but the electrical driving force is high. As the membrane potential depolarises, open probability increases, but electrical driving force falls. As the potential depolarises further, open probability approaches its maximum, but the driving force continues to fall. Hence, Ca2+ microdomains of different sizes and spatial extents can be generated, depending on the level of the membrane potential (Figure 4). Weak depolarisations would open a small number of channels with large Ca2+ microdomains whereas stronger stimuli would open many channels with smaller microdomains. It will be interesting to see if these different microdomains, determined by the prevailing membrane potential, recruit distinct downstream targets. A further dynamic can be provided by altering the duration of Ca2+ channel opening. Phosphorylation by CaMKII, which in turn depends on a preceding Ca2+ rise, shifts L-type channels into continuous bursts of activity [65–67]. Channels in this gating mode would generate longer-lasting Ca2+ microdomains with larger lateral spread. Microdomains also can be influenced by channel clustering. Co-localizing channels will allow for overlap of microdomains, greatly increasing both the amplitude and extent of the local Ca2+ signal. A dramatic example of this is seen with the CRAC channel, where store depletion

(Figure_4)TD$IG][ Review

Figure 4. The same population of Ca2+ channels can generate Ca2+ microdomain diversity. (a) Modest depolarisations open few voltage-gated Ca2+ channels but the driving force for Ca2+ entry is relatively large and results in a large Ca2+ microdomain. Strong depolarisations, to give the same amount of whole cell current, recruit many more channels, but the subsequent microdomains are smaller. Em denotes membrane potential and I represents whole cell Ca2+ current. (b) Clustering of plasmalemmal CRAC channels following store depletion by the ER Ca2+ sensor STIM1 produces hotspots of overlapping Ca2+ microdomains that will increase local [Ca2+] considerably in the restricted subplasmalemmal space.

leads to formation of Orai1 clusters or puncta in the plasma membrane opposite peripheral ER [13]. Although the number of endogenous CRAC channels in a cluster is unknown, over-expression studies have revealed 1300 channels in each cluster and, assuming each channel joins a cluster, there are 300 clusters [68]. Furthermore, the presence of peripheral ER within 20 nm of the CRAC channel clusters [18] will reduce the subplasmalemmal volume available for Ca2+ diffusion, further increasing the local Ca2+ concentration. It would be interesting to determine if variations in the number of Ca2+ channels within a cluster evokes distinct cellular responses and if spatially distinct clusters activate different targets. Concluding remarks Oscillations in [Ca2+]c are a universal signalling mechanism in eukaryotic cells. Oscillation can be triggered either by fluctuations in membrane potential, leading to the repetitive opening and closing of voltage-gated Ca2+ channels or, more commonly, through activation of cell-surface receptors that increase the levels of InsP3. An oscillatory Ca2+ signal confers numerous signalling advantages over a sustained increase in [Ca2+]. First, by increasing [Ca2+] to high levels only transiently, oscillations avoid the toxic effects that arise from a maintained Ca2+ signal [69,70]. Second, oscillatory signals support long-lasting cellular responses because they circumvent desensitization of Ca2+-dependent responses that can occur during a prolonged Ca2+ signal. Third, digital oscillations in [Ca2+] can increase the ability of low levels of stimulation to

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activate responses. One or two large Ca2+ spikes can be detected more easily than a sustained increase in [Ca2+] of the same average amplitude over the same length of time, increasing the signal-to-noise ratio. Finally, oscillatory Ca2+ signals impart information in both their amplitude and frequency and thus provide a mechanism for selective recruitment of downstream targets. In addition, there is now a growing appreciation that the spatial profile of the Ca2+ oscillation, reflecting the location of the underlying Ca2+ channels, also contributes information to the deciphering of Ca2+ signals. In a recent report [26], the loss of oscillatory Ca2+ signals failed to alter a downstream response provided local Ca2+ entry still occurred. Hence, some cellular activities might be better tuned to respond to Ca2+ microdomains than global oscillations in [Ca2+]. These local Ca2+ signals can activate a gamut of cellular processes, varying enormously in both distance from the Ca2+ microdomain and the time-course of development. Gene expression, for example, can occur hours after a brief burst of Ca2+ entry that lasts just a few seconds. Selective location of target systems or intermediary Ca2+ sensors near different sources of Ca2+ provides an effective mechanism for generating specific responses of high fidelity. Although much of our understanding of the impact of local Ca2+ signals on cellular responses is derived from plasma membrane Ca2+ channels, new Ca2+ release channels have recently been discovered and mapped to organelles not generally associated with Ca2+ signalling [70]. Hence, a multitude of Ca2+ channels expressed on separate membrane systems co-exist, providing enormous scope for spatially restricted Ca2+ signalling throughout the cell. Finally, several features of spatially restricted Ca2+ signalling are likely to be applicable to other second messenger pathways too. Visualisation of these latter gradients has been hampered by a dearth of suitable optical probes but the development of a FRET-based probe for cAMP has revealed the presence of cAMP gradients in cardiac myocytes in response to receptor stimulation [71]. Nitric oxide production, restricted to the apical pole, has been observed in epithelial cells [72]. Hence, compartmentalisation of second messengers, as epitomised by Ca2+, is probably a widespread mechanism for ensuring specific outputs to multifaceted intracellular signals. Acknowledgements Work in my laboratory is supported by the Medical Research Council. I am very grateful to Drs Daniel Bakowski and Joseph Di Capite for discussions on various aspects of this article.

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