Timing in Cellular Ca²⁺ signaling

Abstract

Calcium (Ca²⁺) signals are generated across a broad time range. Kinetic considerations impact how information is processed to encode and decode Ca²⁺ signals, the choreography of responses that ensure specific and efficient signaling and the overall temporal gearing such that ephemeral Ca²⁺ signals have lasting physiological value. The reciprocal importance of timing for Ca²⁺ signaling, and Ca²⁺ signaling for timing is exemplified by the altered kinetic profiles of Ca²⁺ signals in certain diseases and the likely role of basal Ca²⁺ fluctuations in the perception of time itself.

Introduction

Crudely, the business of Ca²⁺ signaling is one of information delivery. How do biological systems interpret environmental cues to choreograph the generation of Ca²⁺ signals and thereby execute appropriate physiological responses? Understanding how inputs are processed and relayed via changes in cytoplasmic Ca²⁺ concentration (‘encoding’) to impact the activity of only a desired subset of Ca²⁺-sensitive targets (‘decoding’) has proved to be a durable and mechanistically intriguing field of research, as well as one of increasing pathological pertinence.

Ca²⁺ signals display great spatiotemporal malleability. They are generated across wide spatial and temporal ranges (nanometer to centimeter, microsecond to hour). This broad scope disguises additional flexibility in the size, source, spread, persistence and rhythm of cytoplasmic Ca²⁺ changes coordinated through the specific organization and properties (the ‘functional architecture’) of Ca²⁺ channels, pumps, buffers and exchangers in any given cell type. Therefore, as spatial and temporal controls are inseparable orchestrators of Ca²⁺ signals, our focus here on issues of ‘timing’ is somewhat contrived. Consequently, we direct readers to broader reviews [1-4] and discuss here solely principles of timing in Ca²⁺ signaling and examples that showcase their application.

Timing pervades all aspects of Ca²⁺ signaling, affecting how environmental information is compiled, encrypted and deciphered (overview in Figure 1). Temporal considerations govern: (1) how incoming information is both processed at the cell surface and resolved by effectors (‘interpreting inputs’), (2) how reactions are set into motion with appropriate sequentiality and interdependence, cueing processes in an order determined by feedback from and dialog with other signaling pathways (‘choreographing responses’) and (3) how Ca²⁺-dependent effectors are differentially activated (‘targeting effectors’) by temporal aspects of cytoplasmic Ca²⁺ signals (notably their frequency or duration). These topics are discussed below.

Figure 1. Kinetic orchestration of Ca²⁺ signals.

Schematic overview of the impact of timing in cellular Ca²⁺ signaling. Aspects of timing impact how extracellular signals are interpreted and choreographed into specific profiles of cytoplasmic [Ca²⁺] signals (‘encoding’). Different Ca²⁺ signals result depending on how cells interpret dynamic environmental cues (‘integrating inputs’, triangles) to contextualize and temporally order specific signaling processes (‘choreographing responses’). Temporal aspects of the resultant cytoplasmic Ca²⁺ signal – notably, variability in duration (top) and periodicity (lower) – are sensed by subsets of Ca²⁺-sensitive effectors via Ca²⁺-binding sites of varied affinity, kinetics and interdependence, resulting in their selective activation (‘decoding: targeting effectors’) to yield discrete responses (stars).

Interpreting Inputs

Single Inputs: Context

A first example of time-dependent sensitivity in Ca²⁺ signaling is ‘context’: scenarios where responsiveness to a signal is state-dependent (Figure 2a). At one end of the spectrum, this represents gain-of-function scenarios where a signal is ineffective at evoking a response at one point in time, but not at another. More subtly, different outcomes may be associated with the same input when presented at different times (e.g different antigen-evoked Ca²⁺ signals in naïve and primed lymphocytes [4,5]). If time is ‘that great gift of nature which keeps everything from happening at once’, context is the timekeeper that paces change via external or autonomous cues. In short, such contextual cues distill specificity from pervasive signals.

Figure 2. Temporal integration empowers diverse outcomes from limited inputs.

Principles of timing impacting signal interpretation for single and multiple (dual) inputs. (a) ‘Context’ determines whether a single (‘1’) signal will be effective at evoking a response (▲ → no response/response). Bottom, Two inputs associated with specific individual responses can produce several different responses (right) when (b) the duration of their effects compound (‘summation’), (c) the order of their presentation matters (‘sequentiality’; ▲△→response ‘x’, △▲→ response ‘y’), (d) the relative timing, but not necessarily order, of the arrival of each input is critical (‘coincidence detection’, ▲...△/△...▲→ response ‘x’, △▲ or ▲△→ response ‘y’) or (e) an example of ‘associative’ memory where specific combinations of presented signals (e.g. one of two signals ▲ and △ presented ‘n’ times) can trigger a number of unique (≤2ⁿ) responses. Strategies for temporal ‘memory’ of Ca²⁺ signals (e.g. priming & persistence, are discussed under ‘duration’).

Clearcut examples of state-dependent responsiveness are found during the natural temporal progression of development and cellular differentiation, and during acute refractory states in excitable tissue. Sperm-evoked Ca²⁺ signaling is ineffective at fertilizing oocytes, until maturation (minutes to days) renders a competent egg. Depolarizing stimuli are ineffective at stimulating Ca²⁺ entry until the developmental timepoint when voltage-gated Ca²⁺ channels are expressed [6,7]. Defined periods of ligand sensitivity are seen with several Ca²⁺-modulating agonists [7,8]. Windows of ‘competency’ to inductive signaling during embryogenesis exist transiently and execute irreversibly on receiving appropriate inputs. Since the activity of many inducing factors modulate, and/or are modulated by local Ca²⁺ concentration [9-12], it is unsurprising that embryonic Ca²⁺ transients/gradients are implicated in patterning and specification [13-16]. Potential mechanisms delimiting these periods include temporal regulation of receptor transcription/translation, and restricted spatial expression of Ca²⁺ channels [17,18]. Alternatively, targets may be present but inhibited, and time must pass before attenuatory regulation is relieved [19,20]. Finally, it is important to point out that the functional readout of Ca²⁺ signaling networks may change over developmental time [13]: early non-canonical Wnt (Wnt-Ca²⁺) signaling is associated with specification events (ventral patterning, [15,16]) but later with morphogenesis (e.g. convergent extension, [21]); Ca²⁺ oscillations can either initiate development or terminate cellular viability, depending on the age of the egg when fertilized [22].

Ca²⁺ signals observed during cellular differentiation [6,23,24], reinforce differentiative events and possibly impact their intrinsic timing. One example is the ordered production of first neurons, then glia from multipotent neuronal stem cells in the developing vertebrate cortex. This timing mechanism is contextual: environmental cues bias outcomes, even though the intrinsic timing mechanism is hard-wired into isolated clones [25]. Spontaneous Ca²⁺ entry signals observed during neuronal progenitor differentiation [6,23,24] may impact neuro/gliogenic switching through global epigenetic control. Methyl-CpG-binding protein 2, a transcriptional repressor with affinity for glial specific promoters, is subject to a Ca²⁺ influx dependent phosphorylation that relieves transcriptional repression [12,25,26].

Multiple Inputs: Summation, Sequentiality, Coincidence & Memory

Timing provides further flexibility in information processing where multiple inputs are involved (Figure 2). Kinetic considerations ensure that two inputs do not invariantly produce only two outputs. Changes in the duration, sequentiality and temporal convergence of incoming signals ensure a multiplicity of biological outcomes to a pair of stimuli. Here, we have broadly corralled examples under the headings of ‘summation’ (the effects of two Ca²⁺ signals compound to ensure a discrete outcome, Figure 2b), ‘sequentiality’ (the order of presentation of two signals dictates different outcomes, Figure 2c), ‘coincidence detection’ (the arrival of two different signals within a set timeframe ensures a discrete outcome, Figure 2d) and ‘memory’ (certain combinations/sequences of repetitive signals associate with specific responses, Figure 2e). Although this categorization is fluid (for example, many coincidence detectors are also sensitive to the order inputs are presented), they provide a simple framework for discussion.

Summation is frequently the basis of ‘amplitude modulation’ [4] in Ca²⁺ signaling (Figure 2b). When different inputs converge to the same signaling currency (Ca²⁺) and their effects overlap, the resulting cytosolic Ca²⁺ increases compound to yield unique responses. Summation of Ca²⁺ signals is not only important for activating targets of progressively lower Ca²⁺ affinity, but also for triggering cellular Ca²⁺ signals. In the majority of cell types, initiation of a Ca²⁺ wave is regulated by local summation of short lasting Ca²⁺ fluxes that drive the ambient cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyt) toward a threshold at which regenerative Ca²⁺ release occurs [2,27,28]. As Ca²⁺ channels integrate diverse regulatory inputs, there can be considerable variability in the occurrence and duration of unitary Ca²⁺ release events that impact their summation. The threshold local [Ca²⁺]_cyt needed to evoke regenerative Ca²⁺ release is also variable, related to the global behaviour of the cytoplasm as an ‘excitable medium’, an integrative gauge of second messenger levels and the repleteness of cellular Ca²⁺ stores at any point in time. Therefore, temporal summation shapes both the generation of Ca²⁺ signals and the ensuing responses.

In paradigms of ‘sequentiality’ (Figure 2c), the order of presentation of inputs is important. Certain input combinations render one response, other combinations another or no response at all. A classic paradigm involving sequentiality (and co-incidence) is spike-timing dependent plasticity (STDP), where the same presynaptic and postsynaptic action potentials can strengthen or weaken synaptic efficiency depending on their order [29,30]. At the molecular level, sequentiality is encoded by mechanisms that endow interdependence to binding/regulatory sites including overlap [31], conformational masking [32-35], de novo generation of binding sites/functionality by intermolecular assembly [36,37] or spatial translocation [33,38]. An elegant example of a scenario where one signal is ineffective unless another has been presented first is the sequential activation of conventional PKC isoforms. DAG analogs do not cause translocation of full length PKC unless Ca²⁺ is elevated [33]. This is because the diacylglycerol (DAG) binding sites (C1₂ domain) of the kinase are rendered inaccessible by a pseudosubstrate clamp until Ca²⁺ binds (C2 domain) to effect plasma membrane translocation and a series of stabilizing interactions (including DAG binding) that must be maintained to relieve kinase inhibition. Therefore overlapping Ca²⁺ (first step, translocation) and DAG signals (second step, activity), but neither signal independently, produces maximal kinase activity.

Many processes involved in Ca²⁺ signaling integrate different signals by detecting ‘coincident’ inputs (Figure 2d). In some cases, simple overlap of different signals is sufficient to evoke a supralinear response. In more stringent examples, the arrival of an input may define a set time window in which the coincident input must be received, or the ordering of co-incident inputs is important. Most notably in STDP, the type and extent of synaptic modification is dependent on both the respective timing (millisecond discrimination, [29]), and ordering of pre- and post-synaptic action potentials [29,30,39]. Examples of molecular coincidence detectors include Ca²⁺ channels [30,34,39-41]), Ca²⁺-regulated enzymes [42,43] and transcription regulators [44], all of which sense pairings of extracellular signals (agonists, depolarization), G proteins or second messengers (IP₃, Ca²⁺, cAMP, DAG).

Finally, the involvement of Ca²⁺ signaling in ‘memory’ (Figure 2e) spans work on speculative mechanisms of memory deposition in individual Ca²⁺ sensors [38], through to intensely researched changes in local synaptic plasticity/connectivity that likely underpin neuronal ‘clique’ and network firing patterns involved in the deposition and consolidation of memories [30,45,46]. Ca²⁺ entry and Ca²⁺ release signals regulate short term (<hrs) activity-dependent changes in synaptic efficiency (of pre-existing synaptic components), as well as longer term changes (>days) in synaptic function and architecture [30,47-49]. Short term working memory (seconds), spatial memory (hours) and the reactivation of consolidated memories embedded via transcription/translational events harbor a common dependency on Ca²⁺ fluxes [48-50]. The increasing technical ability to monitor, and manipulate, the firing patterns of many neurons during mnemonic episodes [46,51] demonstrates how temporal precision (neuronal discharge frequency, latencies) is paramount for coupling clique dynamics and for preserving the fidelity of signals traversing neuronal networks.

Choreography

Signal transduction is an engagement of interlinked modules of reactions that adjust cellular behavior to environmental cues. Efficient signaling necessitates ordered temporal execution of these modules and their constituent reactions, each of which may regulate preceding or subsequent events. Kinetic considerations define the speed by which information is relayed between effectors and thereby the timeframe of the overall response, the oscillations in activity of individual components, as well as the impact of positive and negative feedback in determining the sensitivity, range and stability of output from the overall module.

Obviously, different signaling modules execute over different timeframes in different cells: synaptic [Ca²⁺] changes within microseconds, compared to latencies of up to tens of seconds with agonist-evoked Ca²⁺ signaling in non-excitable cells. The same module can however be customized to work over a malleable timeframe, as exemplified by considering the ubiquitous phospholipase C (PLCβ) Ca²⁺ signaling cascade. At one extreme, where this module underpins Drosophila visual transduction, responses are executed with minimal latency (<25ms), an obvious temporal adaptation to the demands of flight. A major contributing factor is the spatial compartmentalization and precoupling of signaling components by the scaffolding protein InaD in the rhabdomere, minimizing the need for amplification steps upstream of PLCβ [52]. The tight spatial coupling of this module permits the high temporal fidelity of phototransduction events. Indeed, the latency of the single photon response (time between activation of a rhodopsin molecule and ‘quantum bump’ depolarization), is increased ~6-fold in mutants lacking such spatial organization through InaD [52]. Scaffolding proteins impact signaling kinetics both generally - by increasing reaction speed and efficiency via compartmentalizing proteins within restricted surfaces that favor productive and privileged interactions - but also specifically, as is increasingly recognized (e.g. InaD [53], Homer-2 [54]) by regulating individual kinetic interactions within Ca²⁺ signaling modules. In contrast, the coupling of remotely synthesized hormones to phosphoinositide-coupled Ca²⁺ oscillations in the intact liver occurs over a timeframe >10,000-fold longer and more compatible with metabolic cycling [55]. Here, each protein component is separated by signal relay steps impacted by issues of messenger durability, such as agonist range, GTPase activity (e.g. RGS proteins [56]), IP₃ diffusion/metabolism and regionalized Ca²⁺ buffering.

Positive and negative feedback regulation of proteins in these modules is critical in shaping the periodicity and kinetics of cytoplasmic Ca²⁺ signals. Feedback regulation ensures that all the relay steps (G proteins, IP₃, Ca²⁺ [57]) as well as interlinked signaling outputs (e.g. phosphorylation, cAMP [43,58]) can display oscillatory, phase-locked changes in activity. The differential kinetic timecourse of individual feedback steps, even on the same component, is important in ordering reactions, sensing spatial distance and for switching reliably and stably between activity states [59]. For example, many intracellular Ca²⁺ channels exhibit time-dependent regulation by agonists, where the same ligand concentration may increase or decrease Ca²⁺ release depending on the duration of exposure [40,60]. The activity of distinct Ca²⁺ signaling modules is further choreographed by steps that interlink their ordered temporal execution, establishing complex regulatory circuits and crosstalk with other signaling pathways that orchestrate short term responses while maintaining longer term homeostatic balance.

Temporal decoding of Ca²⁺ signals

Cytoplasmic Ca²⁺ signals relay information to Ca²⁺-sensitive effectors. Issues of timing – notably the periodicity and duration of Ca²⁺ transients – are crucial in determining how information is decoded into appropriate cellular responses. These parameters are discussed separately below, prefaced with a reminder about their in vivo interdependence: the efficiency of frequency encoding of Ca²⁺ signals is impacted by the specific profile of Ca²⁺ spike duration and amplitude, as well as baseline level of Ca²⁺ over which they occur [33,61-65].

Frequency

Repetitive fluctuations in cytosolic Ca²⁺ occur over an exceptionally broad time range (Figure 3). These fluctuations encompass rhythmic changes in baseline Ca²⁺ observed in fungi, plants and animals [66-69], upon which are superimposed stimulation-evoked Ca²⁺ transients that occur episodically as bursts of oscillations or repetitive Ca²⁺ spikes with regulable intermittency [3,70] and cell-/agonist- specific profiles [71]. The duration of trains of Ca²⁺ spikes (which can be evoked for hours in experiments) is physiologically significant [72]. The ~10⁷-fold range in periodicity (likely constrained by our inability to resolve exceptionally slow, or fast signals without summation) is exemplified at the extremes by the rapid bursts of Ca²⁺ spikes generated within sound-producing muscles (e.g. ≥10ms spike duration in toadfish swimbladder muscle [73]) and the protracted and persisent (e.g. ≤24hr) oscillations in basal [Ca²⁺]_cyt associated with circadian rhythms [67-69]. In this latter case, Ca²⁺ signaling messengers may actually orchestrate the perception of time via entrainment and pacing of the oscillator itself [74]. Therefore the variable periodicity of Ca²⁺ signals coordinates responses from the cellular to the systems level.

Figure 3. Physiological harmonics: frequency encoding of Ca²⁺ signals.

The ~10,000,000-fold range in the periodicity (Hz, left) of repetitive Ca²⁺ changes observed in different cells/tissues, using data from non-excitable cells (orange, [112,113]), muscle (purple, [73,114,115]), embryos (green, [14,116]) and neurons (blue [67,117]). Numbers represent citations to examples delimiting each range. Right (red), estimates of optimal Ca²⁺ spiking frequencies for half-maximal activation of the indicated proteins from: Ras [75], NF-kB [64], NFAT [64], CamKII [63], PKC [33]. These obviously represent single point estimates from a broader in vivo range.

Irrespective of the absolute frequency of Ca²⁺ oscillations, the same key issues hold: what is the mechanistic basis of the oscillator and how is this oscillator read? This latter question may simply entail understanding ‘target selection’ (i.e. how the affinity, kinetics and localization of Ca²⁺ binding sites delimit which effectors respond) for outputs that directly track Ca²⁺ signals. More elaborately, many effectors have the ability to transduce different frequencies of Ca²⁺ transients into graded levels of activation (‘frequency-modulation’). Over the ~30 years since repetitive Ca²⁺ oscillations were resolved, considerable effort has been spent addressing these questions [2,4,57]. Several molecules (and many more processes) have been identified that are optimally regulated by specific Ca²⁺ spiking frequencies, including Ca²⁺-dependent transcription factors (NF-AT [64,65], NF-kB [64], Oct/OAP [64]), Ras [75], CamKII [61,63] and PKC [33]). Collectively these data show that different Ca²⁺ sensors are regulated, over distinct ranges (Figure 3) and between distinct limits, by the periodicity of Ca²⁺ transients [58,61,63,64,75].

Considerably less is known, however, about the molecular basis of frequency decoding. Non-linear activation mechanisms in most models are underpinned by elements of co-incidence and reinforcement (e.g. interdependent, cooperative or temporally geared reactions where active intermediates persist beyond the transient Ca²⁺ spike) to render integrative behavior. Seemingly subtle kinetic delays are critical in allowing activities to be summated at high Ca²⁺ spiking frequencies, as shown, for example, by the short delays (seconds) in PKCγ and DAG association and dissociation as Ca²⁺ rises and falls [33]. The strongest structural insight into frequency decoding is probably provided by CaMKII, and experimental observations that different CaMKII isoforms [63], and notably different splice variants of the same isoform (βCaMKII [61]), display unique in vitro sensitivities (~10-fold range) to the frequency of Ca²⁺ oscillations. Specific structural changes impacting initial autophosphorylation rates [61], or residues determining the dramatic decrease in calmodulin dissociation rate from the autophosphorylated holoenzyme are likely crucial [76]. Coupling such biochemical insight with crystallographic solutions [77] will ultimately reveal the conformational mechanics of frequency decoding.

It suffices to conclude that the diversity of responses regulated by Ca²⁺ oscillation frequency emphasizes the utility of frequency encoding as a strategy for relaying information [78]. Ca²⁺ spiking decreases the threshold [Ca²⁺]_cyt for activating responses, preserves signal fidelity and specificity while minimizing metabolic cost, effector desensitization and cytotoxic risk associated with sustained increases in [Ca²⁺]_cyt.

Duration

The duration of Ca²⁺ transients varies considerably: Ca²⁺ signals can be rapid (and likely localized), or more protracted (and likely pervasive). Their individualized duration is determined by the kinetic interplay of Ca²⁺ fluxes that increase, buffer and remove Ca²⁺ ions from the cytosol, the balance between which is regulated physiologically and disturbed pathologically through changes in the functional architecture of Ca²⁺ signaling molecules. The duration of a Ca²⁺ signal therefore depends on the precise molecular complement of Ca²⁺ transporters engaged during a response. Involvement of the endoplasmic reticulum (ER) is particularly crucial: Ca²⁺ signals that evoke regenerative Ca²⁺-induced Ca²⁺ release via IP₃Rs or RyRs trigger propagating Ca²⁺ waves that extend the duration and spatial reach of cytoplasmic Ca²⁺ changes. The consequent ER Ca²⁺ depletion stimulates a homeostatic ‘store-operated’ Ca²⁺ entry, which in conjunction with other ‘receptor-operated’ Ca²⁺ influx pathways, refill the ER with Ca²⁺ via delayed but enduring Ca²⁺ entry currents [79]. Therefore, the involvement of different families of intracellular Ca²⁺ channels in amplifying, and triggering the amplification of, Ca²⁺ signals is a key determinant of Ca²⁺ signal duration.

The timecourse of a Ca²⁺ signal is important as early and late phases of Ca²⁺ signals are often associated with different responses - sustained Ca²⁺ elevations will reach more targets, and occupy sites of appropriate affinity for longer. For example, transient Ca²⁺ signals are often insufficient to activate transcriptional responses [80] and the duration of more sustained Ca²⁺ signals is important in specifying which transcription factors are most efficiently activated [70,81]. Further examples of Ca²⁺ signals of different durations directing unique cellular responses are as diverse as stomatal opening/closing [62], exocrine function [82], the integrated initiation of Ca²⁺-dependent events during egg activation [83], positive and negative thymocyte selection [84] and the selective modulation of plasticity in neurons (LTD vs LTP, [29,30]).

In terms of decoding, one would underestimate the versatility of Ca²⁺ as messenger by assuming the effective duration of a Ca²⁺ signal was set only by the occupancy of Ca²⁺ binding sites on Ca²⁺ sensors with singular affinities. Ca²⁺-dependent effectors exhibit a variety of ‘temporal gearing’ strategies to ensure the consequences of transient Ca²⁺ signals persist beyond the duration of the Ca²⁺ signal itself. While cellular Ca²⁺ binding proteins exhibit a wide range of intrinsic Ca²⁺ binding affinities, even within specific subfamilies of Ca²⁺ sensors [85-87], these ‘basal’ affinities can change dramatically on regulatory modification (>25000-fold [76]), through cooperativity with other Ca²⁺ binding modules in the same proteins, and on forming interactions with substrate or additional partners. Such mechanisms ensure that ephemeral Ca²⁺ signals have lasting physiological value.

Many Ca²⁺-triggered interactions are enabled by Ca²⁺-dependent translocations to cellular membranes [88], such as those mediated by C2 domains (e.g. PKC [33,58], Ras regulators [89]), Ca²⁺/myristoyl switches [86], annexin repeats [87] or via exposure of binding sites for specific target proteins (e.g. CaMKII [90]). After these initial Ca²⁺-driven translocation, activities may become entirely Ca²⁺-independent (‘autonomous’) thereby extending the temporal reach of Ca²⁺ signals, exemplified by the importance of CaMK autonomy for transcription [91] and plasticity [92]. Localized autonomous CaMKII activity is maintained by autophosphorylation (e.g. on cardiac Ca²⁺ channels [93] and certain PSD targets [94]), or simply by immobilization on the target itself (e.g. for NR2B, [95] and certain K⁺ channels [96]). These varied routes to autonomy define whether temporal gearing is controlled by local phosphatase activity, target dissociation, or both. Therefore the extent of temporal amplification through translocation is likely unique for different subcellular compartments, as well as different targets.

Mistiming

When timing goes awry, pathological outcomes ensue. There are many examples of how the temporal profile of cytosolic Ca²⁺ signals is adversely remodeled by disease and specifically by mutations that impact the expression, activity and spatial organization of individual Ca²⁺ homeostatic genes (see reviews in [97]). In the heart, for example, abnormal Ca²⁺ cycling predisposes to arrhythmias [98] via decreased expression/functionality of Ca²⁺ buffers [99] or mutational dysfunction of voltage-operated Ca²⁺ channels [100], intracellular Ca²⁺ channels (e.g. ≤70 mutations in human RyR2) or proteins that compartmentalize Ca²⁺ channels and transporters [101]. Protracted Ca²⁺ transients diagnostic of diastolic dysfunction can result from decreased sarcoplasmic reticulum Ca²⁺ ATPase (SERCA) expression or mutational dysfunction of its regulators thereby impairing recovery from Ca²⁺ loads [102,103]. Timing of mutations is also critical for disease progression, by impairing [104] or persistently potentiating [105,106] Ca²⁺ signaling events at key points in the pathogenic timeline. For example, PKD1 is a gene mutated in a majority of patients with autosomal dominant polycystic kidney disease. It encodes polycystin-1 (TRPP1/polycystin-1), a cell surface protein which functions as part of a Ca²⁺-dependent mechanosensitive complex with PKD2 during renal development. Loss of polycystin-1 function results in differential pathological outcomes depending on when functionality is impaired. If deleted shortly after birth, kidney cysts form rapidly and mice die in a few weeks, whereas loss of function in adults is associated with a much milder phenotype [104].

Beyond the many ‘loss-of-function’ mutations that impact global Ca²⁺ cycling, rarer ‘gain-of-function’ mutations increase the duration of cellular Ca²⁺ fluxes. This is exemplified by mutants in several Ca²⁺ channelopathies where normal channel inactivation kinetics are delayed [107-109]. A dramatic example is the de novo Ca²⁺ channel mutation associated with Timothy syndrome (G406R, Ca_v1.2) where a near complete failure of voltage-dependent channel inactivation precipitates cardiac, neuronal and developmental defects [100]. Many kinetic changes are however much subtler, yet still cause disease. For instance, certain mutations (3 from 50+) identified in ATP2A2, which encodes the housekeeping SERCA2b isoform, confer relatively normal Ca²⁺ transport and ATP hydrolytic activities (>65% of wild type [110]). These mutants are none-the-less associated with the Darier disease pedigrees [111]. Modulation of SERCA2 affinities in the heart – either to increase or decrease the net Ca²⁺ affinity of SR Ca²⁺ sequestration – can both result in cardiomyopathies [103]. An important principle from such examples is that although different cell types show incredible versatility in their individualized ‘Ca²⁺ signatures’, there is considerably smaller tolerance for temporal deviation from differentiated Ca²⁺ profiles without pathological consequences.

Conclusions

The diversity of examples discussed above underscore the many ways in which timing impacts how information is relayed from external stimuli via Ca²⁺ signals into specific physiological outcomes. The broad community of Ca²⁺ handling proteins that increase, decrease and buffer cytoplasmic Ca²⁺ provide kinetic diversity in assembling signaling modules customized to cellular function at any point in time to generate Ca²⁺ signals across a remarkable range of durations and periodicities. The malleable role of Ca²⁺ as a messenger depends on precise temporal control of the activity of these individual molecules, as well as controlled regulation of the extent of feedback, diversification and gearing between them that rapidly set up and dissipate spatial gradients of activity within cells and tissues. Consequently, quantifying the tolerance of this kinetic architecture to pathological cues is key for delimiting how the temporal dynamics of specific modules within proteins contribute to Ca²⁺-related pathologies at a systems-level.