- Email: [email protected]
Calcium signalling, a spatiotemporal phenomenon
J. Krebs and M. Michalak (Editors) Calcium: A Matter of Life or Death 2007 Elsevier B.V. All rights reserved. ISSN: 0167-7306/DOI: 10.1016/S0167-7306(06)41019-X
CHAPTER 19
Calcium signalling, a spatiotemporal phenomenon Michael John Berridge The Babraham Institute, Babraham, Cambridge CB2 4AT, UK, Tel.: +44 (0) 1223 496621; Fax: +44 (0) 1223 496033; E-mail: [email protected]
Abstract A large number of cellular processes are controlled by Ca2+ signalling. The versatility of this signalling system depends on the existence of an extensive Ca2+ signalling toolkit from which cells can assemble cell-specific Ca2+ signalsomes that are precisely suited to deliver the signals to control their particular functions. The spatial and temporal properties of such cell-specific Ca2+ signals are a particularly important feature of this diversity. Ca2+ signalling systems are adapted to control cellular processes over a wide time domain for processes such as exocytosis (microseconds), muscle contraction (milliseconds), metabolism and gene transcription (seconds to minutes) and fertilization and cell proliferation (hours). Keywords: calcium, CRAC, cytoskeleton, endoplasmic reticulum, inositol 1,4,5trisphosphate, PMCA, puffs, ryanodine receptors, SERCA, sparklets, sparks, STIM, store-operated channels, TRP
One of the major control mechanisms used by cells is Ca2+ signalling [1–4]. It triggers new life at the time of fertilization and continues to control many processes during development. Once cells have differentiated into specialized cell types, it governs the activity of most cellular processes such as secretion, contraction, metabolism, gene transcription, cell proliferation and the synaptic plasticity responsible for learning and memory. There also is a darker side to its action because larger than normal elevations can cause cell death either in the controlled manner of programmed cell death (apoptosis) or in the more catastrophic necrotic change that occurs during processes such as stroke or cardiac ischemia.
1. Ca2+ signalling toolkit The basic mechanism of Ca2+ signalling is relatively simple in that it depends on an increase in the intracellular concentration of this ion. The Ca2+ concentration is low when cells are at rest, but when an appropriate stimulus arrives there is a sudden elevation, which is responsible for a change in cellular activity. However, there are multiple variations of this relatively simple theme. The versatility of Ca2+ signalling is achieved by having an extensive Ca2+ signalling toolkit (Fig. 1). Each cell type has
486
M. J. Berridge
Fig. 1. Summary of the major components of the Ca2+ signalling toolkit. Many of the individual components are present in multiple isoforms that further enhance the diversity of the Ca2+ signalling systems. The yellow arrows illustrate the ON reactions that introduce Ca2+ into the cell, and the blue arrows depict the OFF reactions that pump Ca2+ either out of the cell or back into the endoplasmic reticulum (ER). During its passage through the cytoplasm, Ca2+ resides temporarily on the Ca2+-binding proteins (CaBPs) that function as buffers, or it can pass through the mitochondria. To carry out its signalling function, Ca2+ binds to sensors that then employ a range of effectors to stimulate many different cellular processes. InsP3R, inositol 1,4,5-trisphosphate receptor; PLC, phospholipase C; NCX, Na+/Ca2+ exchanger; PMCA, plasma membrane Ca2+-ATPases; ROCs, receptor-operated channels; RYR, ryanodine receptor; SOCs, store OCs; SERCA, sarcoplasmic–ER Ca2+-ATPase; SMOCs, second-messenger OCs; VOCs, voltage OCs (See Color Plate 51, p. 546).
a clearly defined subset of toolkit components that will be referred to as a Ca2+ signalling signalsome [4]. These cell-specific signalsomes are put in place during development when a process of signalsome expression enables each differentiating cell to select out those signalling components it will require to control its particular functions. There are an enormous number of cell-specific Ca2+ signalsomes. The important point is that each signalsome generates a cell-specific Ca2+ signal with characteristic spatial and temporal properties. This large toolkit contains many different components that can be mixed and matched to create these different Ca2+ signalsomes. There are Ca2+ channels in the plasma membrane (PM), which control the entry of Ca2+ from the outside. There are Ca2+ release (CR) channels, which control the release of Ca2+ from internal stores (Fig. 1). The Ca2+ buffers ensure that the concentration of Ca2+ remains within its operation range and does not rise to levels that can induce cell death. Once Ca2+ has carried out its signalling function, there are Ca2+ pumps and exchangers that remove it from the cytoplasm by either extruding it from the cell or returning it to the internal stores. Ca2+ signalling functions are carried out by various Ca2+ sensors and Ca2+ effectors that are responsible for translating Ca2+ signals into a change in cellular activity.
Calcium signalling, a spatiotemporal phenomenon
487
2. Basic mechanism of Ca2+ signalling Cells at rest maintain a low intracellular concentration of Ca2+ (approximately 100 nM), but this increases rapidly into the micromolar range when cells are stimulated. This increase in intracellular Ca2+ can operate over a very wide time domain (e.g. microseconds to hours) to regulate many different cellular processes. An important feature of Ca2+ signalling is its dynamic nature as exemplified by the fact that Ca2+ signals invariably appear as a brief transient. The rising phase of each transient is produced by the ON reactions that introduce Ca2+ into the cytoplasm, whereas the falling phase depends on the OFF reactions that pump Ca2+ out of the cell or back into the internal stores (Fig. 1). External stimuli activate the ON reactions by stimulating both the entry and the release of Ca2+. Most cells make use of both sources, but there are examples of cells using either external or internal sources to control specific processes and this will depend on the type of signalsome they are using. Most of the Ca2+ that enters the cytoplasm is adsorbed onto buffers while a much smaller proportion activates the effectors to stimulate cellular processes. The OFF reactions remove Ca2+ from the cytoplasm using a combination of mitochondria and different pumping mechanisms. When cells are at rest, these OFF reactions keep the concentration low, but these are temporarily overwhelmed when external stimuli activate the ON reactions. Sequential activation of the ON and OFF reactions gives rise to Ca2+ transients, which are such a characteristic feature of Ca2+ signalling. At any moment in time, the level of Ca2+ is determined by the balance between these ON and OFF reactions. An important spatial aspect of the ON reactions is that they are often closely associated with the effector systems that respond to Ca2+. For example, voltageoperated channels (VOCs) in presynaptic endings are associated with the synaptic vesicles thus producing a highly localized puff of Ca2+ to trigger exocytosis. Similarly, the type 2 ryanodine receptors (RYR2s) of cardiac cells are lined up close to the contractile filaments to ensure that Ca2+ will rapidly stimulate contraction. In cases where cells need to be stimulated over a long time, these transients are repeated at set intervals to set up Ca2+ oscillations. These oscillations are part of the spatiotemporal aspects of Ca2+ signalling that will be described in Section 5.2. 2.1. Ca2+ ON reactions In response to external stimuli, channels in the PM or endoplasmic reticulum (ER) are opened, and Ca2+ flows into the cytoplasm to bring about the elevation of cytosolic Ca2+ responsible for cell activation (Fig. 1). During these ON reactions, the cell employs a variety of both Ca2+ entry channels and CR channels, which are organized into different modules (Fig. 2). The entry of Ca2+ across the PM is carried out by many different channels whose names indicate how they are opened. For example, there are receptor OCs (ROCs, see module 1 in Fig. 2), second-messenger OCs (SMOCs, see module 2 in Fig. 2), VOCs (see module 3 in Fig. 2) and store OCs (SOCs). The last have attracted a lot of interest recently because there have been some new developments regarding the nature of the SOC and how it might be regulated.
488
M. J. Berridge
Fig. 2. Summary of some of the main modules that are mixed and matched to create different Ca2+ signalling systems. (1) Agonists such as the neurotransmitters acetylcholine, glutamate and ATP act directly on receptoroperated channels (ROCs) in the plasma membrane (PM) to allow external Ca2+ to enter the cell. (2). Second messengers such as diacylglycerol, cyclic AMP, cyclic GMP and arachidonic acid acting from the cytoplasmic side open second-messenger OCs (SMOCs) in the PM. (3) Membrane depolarization (DV) activates voltage OCs (VOCs) in the PM to allow a rapid influx of external Ca2+. (4) Membrane depolarization (DV) activates the CaV1.1 L-type channel that then activates the type 1 ryanodine receptor (RYR1) in skeletal muscle through a direct conformational-coupling mechanism. (5) Membrane depolarization (DV) activates VOCs in the PM to allow a rapid influx of external Ca2+ to provide trigger Ca2+that then activates the RYR2 to release Ca2+ stored in the sarcoplasmic reticulum (SR). This mechanism is found in cardiac muscle and neurons. (6) Agonists acting on cell-surface receptors generate inositol 1,4,5-trisphosphate (InsP3) that then diffuses into the cell to activate the InsP3 receptor (InsP3R) to release Ca2+ from the endoplasmic reticulum (ER) (See Color Plate 52, p. 547).
Release of Ca2+ from internal stores is carried out by three main channel types: the RYRs, the inositol 1,4,5-trisphosphate receptors (InsP3Rs) and a putative nicotinic acid dinucleotide phosphate (NAADP)-sensitive channel. One of the major problems in Ca2+ signalling has been to determine how stimuli arriving at the cell
Calcium signalling, a spatiotemporal phenomenon
489
surface gain access to these internal stores. Two main mechanisms have been identified. Firstly, there is conformational-coupling through protein–protein interactions. This is a very fast mechanism that depends on a sensor in the PM interacting directly with an internal release channel. The receptor on the cell surface is the CaV1.1 L-type channel (a voltage sensor), which is coupled to the RYR1 (module 4 in Fig. 2). Information is transferred through a process of conformational-coupling. This mechanism is mainly found in skeletal muscle, but a similar mechanism may be present in certain neurons such as hypothalamic synaptic endings [5]. The second mechanism depends on the generation of diffusible second messengers. Activation of receptors or channels on the cell surface generates second messenger that then diffuses into the cell to activate release channels. One of the most significant Ca2+-mobilizing messengers is Ca2+ itself, which is a potent activator of the two main internal release channels, the RYRs and the InsP3Rs (Fig. 3). This Ca2+induced CR (CICR) mechanism has the unique property of being autocatalytic and plays a central role in generating those Ca2+ signals that appear as regenerative Ca2+ waves. Another important messenger for releasing internal Ca2+ is InsP3 (module 6 in Fig. 2) [1]. Other Ca2+-mobilizing messengers have been described such as cyclic adenine dinucleotide phosphate [6] and NAADP [7,8].
Fig. 3. The elementary events of Ca2+ signalling. Elementary events are the localized Ca2+ signals that arise from either individual or small groups of ion channels. The localized plumes of Ca2+ have been given different names depending on the channels that produce them. Voltage-operated channels (VOCs) in the plasma membrane (PM) produce sparklets and ryanodine receptors (RYRs) on the sarcoplasmic reticulum (SR) create sparks (and syntillas), whereas the inositol 1,4,5-trisphosphate receptors (InsP3Rs) produce puffs. Channels in the PM are linked to the internal release channels through a process of Ca2+-induced Ca2+ release (CICR). The intracellular channels have large conductances and gate so much Ca2+, which result in a local depletion of Ca2+ within the lumen immediately below the channel. This local emptying of the endoplasmic reticulum (ER) store by RYRs has been visualized and has been called a blink.
490
M. J. Berridge
The channels responsible for these Ca2+ ON reactions usually have powerful inactivation mechanisms that rapidly curtail the entry or release processes to prevent the cell being swamped with Ca2+, which can result in cell stress and apoptosis. Once the ON reactions have been curtailed, the Ca2+ OFF reactions rapidly take over to return the activated level of Ca2+ back to its resting level. 2.1.1. Store-operated Ca2+ entry Much of the Ca2+ used for signalling is released from internal stores that have a finite capacity. For signalling to continue, therefore, Ca2+ must be brought in from the outside to ensure that the store remains topped up. Much of this ER Ca2+ homeostasis depends on the sarcoplasmic–ER Ca2+-ATPase (SERCA) pumps that recycle the released Ca2+. However, there are always some losses to the outside resulting in store depletion, which will not only result in a decline in signalling capacity but also trigger the ER stress signalling pathways. To guard against the deleterious effects of Ca2+ store depletion, the cell employs SOCs that open in response to store emptying to ensure a constancy of its internal Ca2+ store [9]. Electrophysiological studies have revealed that there may be different types of SOCs that vary in their Ca2+ selectivity. The classical example of a SOC is the CR-activated Ca2+ (CRAC) channel found in lymphocytes, which has a very high selectivity for Ca2+ and a very low conductance [10]. One of the main functions of this entry mechanism, therefore, is to maintain the internal store of Ca2+. However, it can also function as a source of signal Ca2+ especially under conditions where Ca2+ signalling has to be maintained over a prolonged period as occurs during the stimulation of cell proliferation. The nature of the signal that emanates from the ER to activate SOCs is still a matter of some debate. One of the important questions to consider is whether the whole of the ER is involved in SOC activation. This seems unlikely because depletion of the ER Ca2+ can trigger stress signalling pathways by seriously interfering with the processes of protein synthesis and packaging. There is growing evidence that a small specialized region of the ER closely associated with the PM regulates the entry of Ca2+ (Fig. 4). The major unsolved problem concerns the mechanism whereby the empty store sends a message to open the SOC in the PM. Some of the proposed mechanisms include the generation of a calcium influx factor, exocytosis of vesicles containing SOCs or a conformational-coupling mechanism whereby the release channels in the ER interact directly with the SOCs in the PM (Fig. 5). The latter mechanism resembles that found in skeletal muscle where L-type VOCs in the PM interact directly with the RYR1 channels in the SR (module 4 in Fig. 2). A similar conformational-coupling mechanism has been proposed to explain how empty ER stores can stimulate entry through SOCs in the PM [11]. In this latter case, information is flowing in the opposite direction, i.e. the output signal travels from the ER to the PM. There are a number of possible molecular components of this putative coupling mechanism (Fig. 5), which are less well defined than those of the skeletal muscle system. One of the problems with trying to understand this entry process is that the identity of the entry channels is uncertain. An integral membrane protein called
Calcium signalling, a spatiotemporal phenomenon
491
Fig. 4. The hypothesis outlined in this figure suggests that store-operated Ca2+ entry occurs at localized regions where there is close apposition of the endoplasmic reticulum (ER) and plasma membrane (PM). The ER is an interconnected reticular network of tubules, and short arms of this ER network come into contact with the PM to form junctional zones. The narrow gap between the ER and the PM may be the site where the signal from the empty ER is transferred to the store-operated channels (SOCs). Agonists acting on cell-surface receptors stimulate phospholipase C (PLC) to produce a microdomain of inositol 1,4,5trisphosphate (InsP3) (*), which functions to deplete Ca2+ in the junctional zones. The latter then sends a message to the SOCs in the PM to promote entry (i.e. regions 1 and 2). This signal might be transmitted through a protein–protein interaction as outlined in the conformational-coupling hypothesis shown in Fig. 5. In some cases, the microdomain of InsP3 does not coincide with a junctional zone, and the local depletion of Ca2+ fails to trigger entry (see region 3).
Orai1 may be the CRAC channel [12]. There also are indications that there may be a number of other SOCs coded for by various members of the TRP ion channel family such as TRPC1, TRPC4 and TRPV6. Another area of uncertainty concerns the nature of the protein in the ER that senses store emptying and then relays information to the SOCs. Both the InsP3Rs and the RYRs, which are known to be able to sense the luminal level of Ca2+, have been implicated as signal initiators in the ER, which then convey information to the SOCs in the PM through a direct protein–protein interaction. There is some evidence to support the idea that either an InsP3R or an RYR may play a role in transmitting information from the ER to the channels in the PM (Fig. 5). Such an involvement of an RYR would mean that this release channel might play a pivotal role in both the release of internal Ca2+ (as occurs in skeletal muscle, see module 4 in Fig. 2) and the entry of external Ca2+ (Fig. 5). Indeed, there is some evidence to show that both mechanisms may coexist in skeletal muscle where the SOCs may function in longterm Ca2+ homeostasis to overcome fatigue during intensive exercise. The SOC may couple to the RYR1 through mitsugumin 29, which is a synaptophysin family-related protein positioned within the junctional space between the PM and the SR [13].
492
M. J. Berridge
Fig. 5. A conformational-coupling hypotheses for store-operated Ca2+ entry as might occur in lymphocytes. Entry of Ca2+ depends on activation of a store-operated channel (SOC) such as the Ca2+ releaseactivated Ca2+ (CRAC) channel. When an agonist activates phospholipase C (PLC), the increase in inositol 1,4,5-trisphosphate (InsP3) then releases Ca2+ from the endoplasmic reticulum (ER) and the emptying of the store opens the CRAC channel. Store emptying might be detected by the stromal interaction molecule 1 (STIM1), which has an N-terminal EF-hand domain that binds Ca2+ when the store is full. As the store empties, Ca2+ comes off STIM causing it to undergo a conformational change that is transmitted through some coupling protein [perhaps an InsP3 receptor (InsP3R) or a ryanodine receptor (RYR)] to open the CRAC channels. Formation of this coupling complex appears to depend on cytoskeletal remodelling driven by WAVE2 and controlled through the monomeric G proteins Vav and Rac (See Color Plate 53, p. 548).
Another candidate for the ER sensor is the stromal interaction molecule (STIM), which is located within the ER and has a single EF-hand that might function to sense Ca2+ (Fig. 5) [14]. If STIM is knocked out, there is a dramatic decline in storeoperated entry indicating that this protein plays some direct role in the coupling mechanism. Under resting conditions when the store is full of Ca2+, STIM appears to be distributed over the ER. Upon store emptying, STIM moves to the surface to aggregate at close contacts between the ER and the PM [15] and is thus ideally situated to act as a sensor for conformational coupling. However, STIM is a small molecule, and the problems remain as to how information is transferred across the 20-nm gap to activate CRAC or SOCs in the PM. In the model outlined in Fig. 5, the suggestion is that when STIM loses its Ca2+ and migrates towards the cell surface, it makes contact with some coupling protein (e.g. an InsP3R or an RYR) to induce the conformational change that results in channel opening.
Calcium signalling, a spatiotemporal phenomenon
493
The conformational-coupling hypothesis depends on the ER making close contact with the PM so that information can be relayed between proteins located in the two apposed membranes. Many of the components that function in store-operated entry are associated with the caveolae, which have been suggested as possible site for this entry processes. There are numerous examples of the ER making intimate contact with the caveolae as described in both smooth muscle [16] and cardiac cells [17]. Another important structural component for entry is the cytoskeleton, which may be particularly important in forming and/or maintaining the structural integrity of this complex (Fig. 5). Proteins that function in actin remodelling such as WAVE can profoundly influence store-operated Ca2+ entry. In the case of T cells, activation of the T-cell receptor relays information out to a number of signalling pathways. One of these is the activation of Vav, which seems to act through Rac and WAVE2 to remodel the actin cytoskeleton that has a role in regulating the entry of Ca2+ through the CRAC channel [18,19]. 2.1.2. CICR A process of CICR plays a central role in the way Ca2+ signals are generated. This positive feedback mechanism whereby Ca2+ triggers its own release has two important functions in cells. It enables Ca2+ entering across the PM to function as a messenger to release Ca2+ from the internal store (Fig. 3). This function of CICR was first described in cardiac cells where the CaV1.2 L-type channel provides an influx of trigger Ca2+ that then diffuses into the cell to activate the RYR2 (module 5 in Fig. 2). A similar interaction is particularly evident for neuronal Ca2+ entry and release channels. The other main function of CICR is to set up intracellular and intercellular waves where an elevated level of Ca2+ in one region of the cell (the initiation site) propagates throughout the rest of the cytoplasm as a regenerative Ca2+ wave (Fig. 6). Waves can progress by recruiting either RYRs or InsP3Rs [20]. These Ca2+ waves are made up of elementary Ca2+ events such as the sparks and puffs produced by the RYRs and InsP3Rs, respectively. It is these unitary events that are used to generate the regenerative waves that make up global Ca2+ signals.
3. Ca2+ OFF reactions Cells use a variety of mechanisms to remove Ca2+ from the cytoplasm (Fig. 1). The introduction of Ca2+ into the cell during the Ca2+ ON reactions usually occurs for a relatively brief period during which there is a rapid increase in the intracellular concentration of Ca2+. The increase in free Ca2+, which can be measured in the cytoplasm using aequorin or fluorescent indicators, is a very small proportion of the total amount of Ca2+ that enters during the ON reactions. Much of this Ca2+ is rapidly bound by cytosolic buffers or is taken up by the mitochondria. As the [Ca2+] returns to its resting level, Ca2+ leaves the buffers and the mitochondria and is returned to the ER or is pumped out of the cell resulting in a brief Ca2+ transient.
494
M. J. Berridge
Fig. 6. Intracellular waves depend on a process of Ca2+-induced Ca2+ release (CICR) whereby the Ca2+ being released from one channel diffuses across to neighbouring channels that are excited to release further Ca2+ thereby setting up a regenerative wave. When such waves meet a cell boundary, they can trigger waves in adjacent cells thus setting up an intercellular wave. There is still some debate about the way the wave travels between cells. (A) One mechanism proposes that when the intracellular wave reaches the cell boundary, some small molecular weight component, most likely to be Ca2+, diffuses across the gap junction to ignite another intracellular wave in the neighbouring cell. (B) An alternative mechanism suggests that the intracellular wave in one cell stimulates the release of ATP through hemichannels, which then diffuses across to ignite a wave in neighbouring cells by acting on P2Y receptors to produce inositol 1,4,5-trisphosphate (InsP3) (See Color Plate 54, p. 549).
The recovery process thus depends on a complex interplay between cytosolic Ca2+ buffers, mitochondria and Ca2+ pumps and exchangers on the internal stores and PM. These Ca2+ OFF reactions operate at different stages during the recovery phase of a typical Ca2+ spike. The buffers and mitochondria operate early, and the Ca2+ pumps and exchangers are responsible for restoring the status quo by pumping Ca2+ out of the cell or back into the ER. These pumps and exchangers operate at different times during the recovery process. The Na/Ca exchangers have low affinities for Ca2+ but have very high capacities, and this enables them to function at the beginning of the recovery process to rapidly remove large quantities of Ca2+. On the contrary, the PMCA and the SERCA pumps have lower capacities, but their higher affinities mean that they can complete the recovery process and can continue to pump at lower Ca2+ levels thus enabling them to maintain both the internal stores and the resting level of Ca2+ within the cytoplasm. Some of these OFF reactions interact with each other during the recovery period, and this is particularly evident in
Calcium signalling, a spatiotemporal phenomenon
495
the case of the ER/mitochondrial Ca2+ shuttle. When Ca2+ is released from the ER, Ca2+ is rapidly taken up by the mitochondria, and this is then released slowly back to the ER once Ca2+ has returned back to the resting level. Under pathological situations, the lack of oxygen reduces the supply of energy thus compromising the function of the OFF reactions resulting in the rise of Ca2+ that is so damaging during stroke or cardiac ischemia. Under normal circumstances, however, the fully energized OFF reactions can rapidly reduce the pulse of Ca2+ introduced by the ON mechanisms thus generating a brief Ca2+ transient. Such brief pulses of Ca2+ are a characteristic feature of the Ca2+ signalling pathway and contribute to the spatiotemporal aspects of Ca2+ signalling.
4. Ca2+ buffers Cells express a large number of Ca2+-binding proteins (CaBPs), which fall into two main groups, Ca2+ sensors and Ca2+ buffers. The Ca2+ sensors respond to changes in intracellular Ca2+ by activating the downstream effectors that control cellular responses (Fig. 1). In a sense, all proteins capable of binding Ca2+ will act as a buffer, and this also applies to the sensors. However, the concentration of these sensors is usually rather low so they have little buffering capacity. The role of Ca2+ buffering is carried out by another major group of CaBPs. The major cytosolic buffers in cells are parvalbumin (PV) and calbindin D-28k (CB). The major buffers that operate within the lumen of the ER are calsequestrin and calreticulin. The latter is unusual in that it functions both as a cytosolic and as a luminal buffer. The cytosolic buffers have subtly different Ca2+-binding properties and are expressed in cells in differing combinations and concentrations to create Ca2+ signals with kinetics that are tailored to carry out different functions. For example, neurons such as Purkinje cells express large amounts of PV and CB. As a consequence, Purkinje cells have a large endogenous Ca2+-buffering capacity, e.g. their buffers bind approximately 2000 Ca2+ ions for each free ion. Lower capacities of 50–100:1 are found in other cells. Motor neurons have a very low-buffering capacity and consequently have large Ca2+ signals in both the soma and the dendrites during normal physiological responses, and this makes them particularly susceptible to neurodegeneration. Buffer concentration is one of the important parameters in determining buffer capacity. The other key parameters include affinity for Ca2+ and other metal ions, the kinetics of Ca2+ binding and release and mobility. The sole function of these Ca2+ buffers is to bind Ca2+, and this has an important function in shaping both the spatial and the temporal properties of Ca2+ signals.
5. Spatiotemporal aspects of Ca2+ signalling The use of Ca2+ as a universal signal for cell regulation is somewhat paradoxical because this ion can be very toxic to cells if its level remains high for a prolonged period. Such toxicity is avoided by presenting Ca2+ signals in a pulsatile manner
496
M. J. Berridge
Fig. 7. In almost every example where Ca2+ is used as a signal, it is presented as a brief transient, which is the digital signal used to set up complex temporal patterns of Ca2+ signalling. In some cells, these pulses are produced on demand in that they are generated in response to periodic stimulation (arrows) as occurs in muscle contraction where a brief burst of Ca2+ activates the contractile machinery, which then recovers when the Ca2+ signal is removed. Likewise, the release of neurotransmitters from nerve terminals is triggered by brief localized pulse of Ca2+. In many other tissues, which receive a continuous stimulation over a prolonged period (bar), the Ca2+ signal is again presented as brief spikes that are produced rhythmically to give highly regular Ca2+ oscillations whose frequencies vary with the level of cell stimulation.
(Fig. 7). In addition to this temporal aspect, the Ca2+ signal is also highly organized in space. For example, muscle contraction is activated by a global elevation in Ca2+, whereas the release of neurotransmitters results from a minute punctate pulse of Ca2+ delivered directly to the docked vesicle by a Ca2+ channel tightly associated with the exocytotic machinery. In between these two extremes, there are many variations in the way the Ca2+ signal is presented to cells.
5.1. Temporal aspects of Ca2+ signalling The temporal aspect of signalling is based on the fact that most Ca2+ signals appear in cells as transients (Fig. 7). There are two main ways in which this digital Ca2+ signal is produced. Firstly, there are ‘on-demand’ Ca2+ transients such as the signals generated in muscle cells and neurons by membrane depolarization. Secondly, transients can appear as an oscillation when cells respond to stimuli that persist for long periods. Such oscillations, which can have a wide range of frequencies, are a characteristic feature of many cellular control systems. There are two main types of oscillatory activity: membrane oscillators and cytosolic oscillators. The output from membrane oscillators usually sets up a regular train of action potentials that
Calcium signalling, a spatiotemporal phenomenon
497
provide the rhythmical pacemaker activity responsible for driving cellular processes such as contraction, neuronal activity and secretion. These membrane potential oscillations can open VOCs that gate Ca2+ and can thus result in intracellular Ca2+ oscillations. Ca2+ oscillations can also be generated by cytosolic oscillators. The latter are of particular interest because oscillator frequency can vary with stimulus intensity, and such frequency modulation is used to encode information. There also is evidence that information carried as oscillations can greatly enhance the activation of gene transcription [21,22].
5.1.1. Membrane oscillators Membrane oscillators are usually generated through an interplay between outward currents, usually carried by K+, that induce membrane hyperpolarization that inhibits the inward currents (e.g. Na+ and Ca2+) that cause depolarization. The entry of Ca2+ during the depolarizing phase is responsible for the phasic elevation of Ca2+ that is returned to the cytoplasm by the PMCA during the OFF reaction. There are a number of cell types that set up pacemaking activity using such membrane oscillators. Sinoatrial node pacemaker cells establish the regular trains of action potential, which drive cardiac contraction. Thalamocortical neurons have a neuronal rhythmicity that displays oscillations at a frequency of 0.5–4 Hz using a combination of different inward and outward currents. Neurons that reside within the suprachiasmatic nucleus, which have the biological clock, also have a membrane Ca2+ oscillator that is responsible for generating the output signals from the biological clock. 5.1.2. Cytosolic Ca2+ oscillations Cytosolic Ca2+ oscillations are produced by the periodic release of internal Ca2+ through the operation of a cytosolic oscillator [1,3]. This Ca2+ oscillation mechanism depends on the release of Ca2+ from intracellular stores. In the case of agonistinduced oscillations, the InsP3/Ca2+ signalling cassette is primary responsible for initiating oscillatory activity. The information encoding/decoding of Ca2+ oscillations depends on the ability to modulate the different parameters of these Ca2+ oscillations. Such agonist-dependent cytosolic Ca2+ oscillations are a major feature of Ca2+ signalling in many cell types. Liver cells display Ca2+ oscillations with agonist concentration-dependent changes in frequency [23]. Ca2+ oscillations are evident during the acquisition of meiotic competence during development [24] and are responsible for oocyte activation during mammalian fertilization [25]. Such Ca2+ oscillations are also observed following artificial insemination by intracytoplasmic spermatozoa injection [26]. Smooth muscle cells surrounding cortical arterioles display spontaneous Ca2+ oscillations [27]. Astrocytes display spontaneous Ca2+ oscillations [28]. Airway epithelial cells respond to ATP by generating the Ca2+ oscillations that control ciliary beat frequency [29].
498
M. J. Berridge
5.2. Spatial aspects of Ca2+ signalling Components of the Ca2+ signalling pathways are often highly organized and localized to discrete cellular areas where Ca2+ microdomains are formed to regulate specific cellular processes [20,30,31]. Much attention has focused on the elementary and global aspects of Ca2+ signalling, which determine whether signalling occurs within highly localized regions or is spread more globally by the formation of both intracellular and intercellular Ca2+ waves. One of the exciting aspects of signalling microdomains is the way they are used by neurons to increase their capacity to process information. The fact that this information processing can be confined to very small volumes within the spines means that each neuron is capable of simultaneously processing large amounts of information. Such input-specific signalling is particularly relevant to the process of synaptic modifications during learning and memory. The high concentrations of neuronal Ca2+ buffers, such as CB, play a major role in restricting Ca2+ signals to individual spines, which are the smallest units of neuronal integration. The size of these signalling microdomains will depend on a number of aspects such as the rate of signal generation, the rate of signal diffusion, the rate of signal removal by the OFF mechanisms and the degree of buffering. Buffers thus play an important role in determining the volume of these signalling microdomains, and this may have been a particularly important feature for the miniaturization of Ca2+ signalling within the brain. 5.2.1. Elementary aspects of Ca2+ signalling The development of fluorescent indicators to visualize Ca2+ in real time in living cells has revealed a spatial dimension to its action that can account for both the universality and the versatility of Ca2+ signalling. The microdomains that have been recorded in cells are the result of elementary events produced by the brief opening of either entry channels in the PM or release channels in the ER. Elementary events are the basic building blocks of Ca2+ signalling. They can either perform highly localized signalling functions or can be recruited to generate global Ca2+ signals. Most of these elementary events are due to the brief opening of Ca2+ channels located either in the PM or in the ER and thus result in localized pulses of Ca2+ (Fig. 3). The presence of Ca2+ buffers helps to restrict these brief pulses to small microdomains within the cytoplasm. A number of different elementary events have now been described as outlined in the following sections. 5.2.2. Sparklet A sparklet is formed as a result of the brief opening VOCs (Fig. 3). Sparklets have been visualized in ventricular heart cells [32]. These sparklets play a critical role in ventricular cell E–C coupling because they provide the trigger Ca2+ that activates the RYR2s in the junctional zone. Another function for sparklets is to control exocytosis particularly at synaptic endings where a localized pulse of Ca2+ is responsible for transmitter release. An elementary event, which is equivalent to a sparklet, is formed in the stereocila of hair cells upon opening of the mechanosensitive channel [33].
Calcium signalling, a spatiotemporal phenomenon
499
5.2.3. Spark A Ca2+ spark is formed by the opening of a group of RYRs (Fig. 3). They were first described in cardiac cells where they are responsible for excitation–contraction coupling [34]. However, they have now been described in many other cell types where they play a variety of different functions. In ventricular cardiac cells, the spark is the unitary Ca2+ signal that is produced at each junctional zone. There are approximately 10 000 junctional zones in each cell, and to get a rapid contraction, the individual sparks must all be fired simultaneously. An electrical recruitment process is used for this synchronization. In atrial cells, the sparks have a different function from those in the ventricular cells. The initial sparks activated by membrane depolarization are restricted to the junctional zones at the cell surface where they provide a signal to ignite a Ca2+ wave that spreads into the cell by triggering a progressive series of sparks through the process of CICR. In mossy fibre presynaptic endings, spontaneous Ca2+ sparks can trigger transmitter release [35]. Spontaneous Ca2+ transients, which resemble sparks, have been recorded in cerebellar basket cell presynaptic endings [36]. Smooth muscle cell Ca2+ sparks function to control both contraction and relaxation. In the case of relaxation, the spark activates the large conductance (BK) channel to produce an outward current that hyperpolarizes the membrane [37]. 5.2.4. Syntilla A scintilla is an elementary event produced by RYRs and thus resembles a spark (Fig. 3). These syntillas function in hypothalamic neuronal presynaptic CR [5]. 5.2.5. Blink Blinks have been visualized within the SR of ventricular muscle cells [38]. As release channels such as the RYRs have a very high conductance, they can gate sufficient Ca2+ to cause a temporary depletion of Ca2+ within the lumen of the junctional SR (Fig. 3). These blinks are attracting considerable interest because they may play a role in the inactivation of ventricular RYR2s to curtail the cardiac Ca2+ transient. 5.2.6. Puff A puff is a unitary event that results from the release of Ca2+ from a small group of InsP3Rs (Fig. 3). These puffs, which are very similar to the Ca2+ sparks, are the building blocks of the intracellular Ca2+ waves in cells that result in global Ca2+ signals. Such localized Ca2+ signals form microdomains of Ca2+ to regulate localized cellular processes such as transmitter release from the astrocyte endings that form part of the tripartite synapse [39] and transmitter release from neocortical glutamatergic presynaptic endings [40]. Release of Ca2+ by InsP3Rs produces the microdomains of Ca2+, which are confined to individual spines in Purkinje neurons [41]. 5.3. Global Ca2+ signals Most of the global Ca2+ signals in cells are produced by the release of Ca2+ from internal stores. The intracellular release channels such as the InsP3Rs and RYRs can
500
M. J. Berridge
create such global signals if their release activity can be synchronized. There are two mechanisms of synchronization that is nicely illustrated by the way ventricular and atrial cardiac cells are controlled. In ventricular cells, electrical recruitment by the action potential is used to activate all the sparks in the junctional zones simultaneously. By contrast, atrial cells use a process of diffusional recruitment whereby Ca2+ sparks at the cell surface trigger Ca2+ waves that spread inwards through a process of CICR by recruiting RYRs located deeper within the cell. There also are examples where intracellular waves can spill across into neighbouring cells to set up intercellular Ca2+ waves, which thus provide a mechanism for coordinating the activity of a local population of cells. 5.3.1. Intracellular Ca2+ waves Intracellular Ca2+ waves can be generated by both InsP3Rs and RYRs, which are Ca2+-sensitive channels that contribute to the positive feedback process of CICR responsible for forming Ca2+ waves (Fig. 6A). The main condition that has to be met for Ca2+ waves to form is that their Ca2+ sensitivity must be increased such that they can respond to the local Ca2+ spark or puff produced by their neighbours. In both cases, the level of Ca2+ within the lumen of the ER appears to be a critical factor. This ER loading is achieved by entry of external Ca2+, and as the lumen loads up with Ca2+, the InsP3Rs and RYRs gradually increase their sensitivity such that they can participate in the regenerative processes that result in a Ca2+ wave. In effect, this increase in Ca2+ sensitivity of the release channels converts the cytoplasm into an ‘excitable medium’ capable of spawning these regenerative waves. These intracellular waves are an integral part of many cellular control processes. Intracellular Ca2+ waves provide the global Ca2+ signal that activates mammalian oocytes at fertilization [25]. Excitation–contraction coupling in atrial cardiac cells depends on Ca2+ waves that spread into the cell from the periphery [42]. Astrocyte excitability depends on an intracellular wave that spreads from the tripartite synapses down to the endfoot processes. In the pancreas, InsP3Rs in the apical region can trigger waves that then spread through the basal region through RYRs. 5.3.2. Intercellular Ca2+ waves There are a number of instances of Ca2+ waves travelling from one cell to the next. Such intercellular waves may act to coordinate the activity of a local population of cells. There is still some uncertainty concerning the way in which the wave is transmitted from one cell to the next (Fig. 6). One mechanism proposes that low molecular weight components such as InsP3 or Ca2+ spill through the gap junctions to ignite waves in neighbouring cells. In order for a cell to set up an intracellular wave, the internal release channels have to be sensitized, so it seems likely that all the cells in the population have to be in a similar state in order for an intercellular wave to pass from one cell to the next. In such a scenario, Ca2+ is the most likely candidate to be the stimulus that passes from one cell to the next. An alternative model proposes that the intracellular wave in one cell stimulates the release of ATP that then diffuses across to neighbouring cells where it acts on P2Y receptors to increase InsP3, which then acts to trigger a new wave (mechanism B in Fig. 6).
Calcium signalling, a spatiotemporal phenomenon
501
The function for such intercellular waves has not been clearly established. Much of the work on these waves has been done on cultured cells, but there are a number of reports showing that such intercellular waves do occur between cells in situ. Intercellular waves have been described in astrocytes, but their physiological function is unclear. As they only seem to appear following intense stimulation, they may be a manifestation of some pathological change. In this respect, the astrocyte wave moves at approximately the same rate as spreading depression that appears to be linked to the onset of migraines. Intercellular waves, which have been recorded in the intact perfused liver, appear to travel in a periportal to pericentral direction [43]. This directionality has led to the suggestion that the wave might function to regulate a peristaltic contraction wave to control the flow of bile. During development, there are pan-embryonic intercellular waves that sweep around the blastoderm margin in the late gastrula of zebra fish [44]. These spatiotemporal aspects greatly enhance the versatility of Ca2+ signalling thus providing the flexibility to regulate so many cellular processes.
6. Ca2+ signalling function The function of Ca2+ as an intracellular second messenger is carried out by a combination of Ca2+ sensors and Ca2+ effectors. The major sensors are the EFhand proteins troponin C, calmodulin (CaM), neuronal calcium sensor proteins and the S100 proteins. These sensors are then responsible for relaying information through a range of effectors such as Ca2+-sensitive K+ channels, Ca2+-sensitive Cl– channels, Ca2+–CaM-dependent protein kinases, calcineurin, phosphorylase kinase, myosin light chain kinase and Ca2+-promoted Ras inactivator. The activation of these different effectors is then responsible for stimulating the large number of Ca2+-sensitive cellular processes.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Berridge, M.J. (1993) Nature 361, 315–325. Clapham, D.E. (1995) Cell 80, 259–268. Berridge, M.J., Lipp, P. and Bootman, M.D. (2000) Nat. Rev. Mol. Cell Biol. 1, 11–21. Berridge, M.J., Bootman, M.D. and Roderick, H.L. (2003) Nat. Rev. Mol. Cell Biol. 4, 517–529. De Crescenzo, V., ZhuGe, R., Vela´zquez-Marrero, C., Lifshitz, L.M., Custer, E., Carmichael, J., Lai, F.A., Tuft, R.A., Fogarty, K.E., Lemos, J.R. and Walsh, J.V. (2004) J. Neurosci. 24, 1226–1235. Galione, A. and White, A. (1994) Trends Cell Biol. 4, 431–436. Patel, S., Churchill, G.C. and Galione, A. (2001) Trends Biochem. Sci. 26, 482–489. Lee, H.C. (2003) Curr. Biol. 13, R186–R188. Putney, J.W. (1986) Cell Calcium 7, 1–12. Hoth, M. and Penner, R. (1992) Nature (Lond) 355, 353–356. Berridge, M.J. (1995) Biochem. J. 312, 1–11. Feske, S., Gwack, Y., Prakriya, M., Srikanth, S., Puppel, S.-H., Tanasa, B., Hogan, P.G., Lewis, R.S., Daly, M. and Rao, A. (2006) Nature (Lond) 441, 179–185.
502
M. J. Berridge
13. Pan, Z., Yang, D., Nagaraj, R.Y., Nosek, T.A., Nishi, M., Takeshima, H., Cheng, H. and Ma, J. (2002) Nat. Cell Biol. 4, 379–383. 14. Roos, J., DiGregorio, P.J., Yeromin, A.V., Ohlsen, K., Lioudyno, M., Zhang, S., Safrina, O., Kozak, J.A., Wagner, S.L., Cahalan, M.D., Velic¸elebi, G. and Stauderman, K.A. (2005) J. Cell Biol. 169, 435–445. 15. Liou, J., Kim, M.L., Heo, W.D., Jones, J.T., Myers, J.W., Ferrell, J.E. and Meyer, T. (2005) Curr. Biol. 15, 1235–1241. 16. Forbes, M.S., Rennels, M.L. and Nelson, E. (1979) J. Ultrastruct. Res. 67, 325–339. 17. Gabella, G. (1978) J. Ultrastruct. Res. 65, 135–147. 18. Nolz, J.C., Gomez, T.S., Zhu, P., Li, S., Medeiros, R.B., Shimizu, Y., Burkhardt, J.K., Freedman, B.D. and Billadeau, D.D. (2006) Curr. Biol. 16, 24–34. 19. Zipfel, P.A., Bunnell, S.C., Witherow, D.S., Gu, J.J., Chislock, E.M., Ring, C. and Pendergast, A.M. (2006) Curr. Biol. 16, 35–46. 20. Berridge, M.J. (1997) J. Physiol. 499, 291–306. 21. Dolmetsch, R.E. Xu, K. and Lewis, R.S. (1998) Nature (Lond) 392, 933–936. 22. Li, W.-H., Liopis, J., Whitney, M., Xlokarnik, G. and Tsien, R.Y. (1998) Nature (Lond) 392, 936–941. 23. Robb-Gaspers, L.D., Rutter, G.A., Burnett, P., Hajno´czky, G., Denton, R.M. and Thomas, A.P. (1998) Biochim. Biophys. Acta 1366, 17–32. 24. Carroll, J., Swann, K., Whittingham, D. and Whitaker, M. (1994) Development 120, 3507–3517. 25. Miyazaki, S., Yuzaki, M., Nakada, H., Shirakawa, H., Nakanishi, S., Nakade, S. and Mikoshiba, M. (1992) Science 257, 251–255. 26. Nakano, Y., Shirakawa, H., Mitsuhashi, N., Kuwabara, Y. and Miyazaki, S. (2001) Mol. Hum. Reprod. 3, 1087–1093. 27. Filosa, J.A., Bonev, A.D. and Nelson, M.T. (2004) Circ. Res. 95, e73–e81. 28. Nett, W.J., Oloff, S.H. and McCarthy, K.D. (2002) J. Neurophysiol. 87, 528–537. 29. Zhang, L. and Sanderson, M.J. (2003) J. Physiol. 546, 733–749. 30. Bootman, M.D., Lipp, P. and Berridge, M.J. (2001) J. Cell Sci. 114, 2213–2222. 31. Rizzuto, R. and Pozzan, T. (2006) Physiol. Rev. 86, 369–408. 32. Wang, S.-Q., Song, L.-S., Lakatta, E.G. and Cheng, H. (2001) Nature 410, 592–596. 33. Lumpkin, E.A. and Hudspeth, A.J. (1998) J. Neurosci. 18, 6300–6318. 34. Cheng, H., Lederer, W.J. and Cannell, M.B. (1993) Science 262, 740–744. 35. Sharma, G. and Vijayaraghavan, S. (2003) Neuron 38, 929–939. 36. Conti, R., Tan, Y.P. and Llano, I. (2004) J. Neurosci. 24, 6946–6957. 37. Brenner, R., Pere´z, G.J., Bonev, A.D., Eckman, D.M., Kosek, J., Wiler, S.W., Patterson, A.J., Nelson, M.T. and Aldrich, R.W. (2000) Nature (Lond) 407, 870–876. 38. Brochet, D.X.P., Yang, D., Di Maio, A.D., Lederer, W.J., Franzini-Armstrong, C. and Cheng, H. (2004) Proc. Natl. Acad. Sci. U. S. A. 102, 3099–3104. 39. Grosche, J., Matyash, V., Mo¨ller, T., Verkhratsky, A., Reichenbach, A. and Kettermann, H. (1999) Nat. Neurosci. 2, 139–143. 40. Simkus, C.R.L. and Stricker, C. (2002) J. Physiol. (Lond) 545, 521–535. 41. Denk, W., Sugimori, M. and Llinas, R. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8279–8282. 42. Mackenzie, L., Bootman, M.D., Berridge, M.J. and Lipp, P. (2001) J. Physiol. 530, 417–429. 43. Robb-Gaspers, L.D. and Thomas, A.P. (1995) J. Biol. Chem. 270, 8102–8107. 44. Webb, S.E. and Miller, A.L. (2003) Nat. Rev. Mol. Cell Biol. 4, 539–551.
CHAPTER 19
Calcium signalling, a spatiotemporal phenomenon Michael John Berridge The Babraham Institute, Babraham, Cambridge CB2 4AT, UK, Tel.: +44 (0) 1223 496621; Fax: +44 (0) 1223 496033; E-mail: [email protected]
Abstract A large number of cellular processes are controlled by Ca2+ signalling. The versatility of this signalling system depends on the existence of an extensive Ca2+ signalling toolkit from which cells can assemble cell-specific Ca2+ signalsomes that are precisely suited to deliver the signals to control their particular functions. The spatial and temporal properties of such cell-specific Ca2+ signals are a particularly important feature of this diversity. Ca2+ signalling systems are adapted to control cellular processes over a wide time domain for processes such as exocytosis (microseconds), muscle contraction (milliseconds), metabolism and gene transcription (seconds to minutes) and fertilization and cell proliferation (hours). Keywords: calcium, CRAC, cytoskeleton, endoplasmic reticulum, inositol 1,4,5trisphosphate, PMCA, puffs, ryanodine receptors, SERCA, sparklets, sparks, STIM, store-operated channels, TRP
One of the major control mechanisms used by cells is Ca2+ signalling [1–4]. It triggers new life at the time of fertilization and continues to control many processes during development. Once cells have differentiated into specialized cell types, it governs the activity of most cellular processes such as secretion, contraction, metabolism, gene transcription, cell proliferation and the synaptic plasticity responsible for learning and memory. There also is a darker side to its action because larger than normal elevations can cause cell death either in the controlled manner of programmed cell death (apoptosis) or in the more catastrophic necrotic change that occurs during processes such as stroke or cardiac ischemia.
1. Ca2+ signalling toolkit The basic mechanism of Ca2+ signalling is relatively simple in that it depends on an increase in the intracellular concentration of this ion. The Ca2+ concentration is low when cells are at rest, but when an appropriate stimulus arrives there is a sudden elevation, which is responsible for a change in cellular activity. However, there are multiple variations of this relatively simple theme. The versatility of Ca2+ signalling is achieved by having an extensive Ca2+ signalling toolkit (Fig. 1). Each cell type has
486
M. J. Berridge
Fig. 1. Summary of the major components of the Ca2+ signalling toolkit. Many of the individual components are present in multiple isoforms that further enhance the diversity of the Ca2+ signalling systems. The yellow arrows illustrate the ON reactions that introduce Ca2+ into the cell, and the blue arrows depict the OFF reactions that pump Ca2+ either out of the cell or back into the endoplasmic reticulum (ER). During its passage through the cytoplasm, Ca2+ resides temporarily on the Ca2+-binding proteins (CaBPs) that function as buffers, or it can pass through the mitochondria. To carry out its signalling function, Ca2+ binds to sensors that then employ a range of effectors to stimulate many different cellular processes. InsP3R, inositol 1,4,5-trisphosphate receptor; PLC, phospholipase C; NCX, Na+/Ca2+ exchanger; PMCA, plasma membrane Ca2+-ATPases; ROCs, receptor-operated channels; RYR, ryanodine receptor; SOCs, store OCs; SERCA, sarcoplasmic–ER Ca2+-ATPase; SMOCs, second-messenger OCs; VOCs, voltage OCs (See Color Plate 51, p. 546).
a clearly defined subset of toolkit components that will be referred to as a Ca2+ signalling signalsome [4]. These cell-specific signalsomes are put in place during development when a process of signalsome expression enables each differentiating cell to select out those signalling components it will require to control its particular functions. There are an enormous number of cell-specific Ca2+ signalsomes. The important point is that each signalsome generates a cell-specific Ca2+ signal with characteristic spatial and temporal properties. This large toolkit contains many different components that can be mixed and matched to create these different Ca2+ signalsomes. There are Ca2+ channels in the plasma membrane (PM), which control the entry of Ca2+ from the outside. There are Ca2+ release (CR) channels, which control the release of Ca2+ from internal stores (Fig. 1). The Ca2+ buffers ensure that the concentration of Ca2+ remains within its operation range and does not rise to levels that can induce cell death. Once Ca2+ has carried out its signalling function, there are Ca2+ pumps and exchangers that remove it from the cytoplasm by either extruding it from the cell or returning it to the internal stores. Ca2+ signalling functions are carried out by various Ca2+ sensors and Ca2+ effectors that are responsible for translating Ca2+ signals into a change in cellular activity.
Calcium signalling, a spatiotemporal phenomenon
487
2. Basic mechanism of Ca2+ signalling Cells at rest maintain a low intracellular concentration of Ca2+ (approximately 100 nM), but this increases rapidly into the micromolar range when cells are stimulated. This increase in intracellular Ca2+ can operate over a very wide time domain (e.g. microseconds to hours) to regulate many different cellular processes. An important feature of Ca2+ signalling is its dynamic nature as exemplified by the fact that Ca2+ signals invariably appear as a brief transient. The rising phase of each transient is produced by the ON reactions that introduce Ca2+ into the cytoplasm, whereas the falling phase depends on the OFF reactions that pump Ca2+ out of the cell or back into the internal stores (Fig. 1). External stimuli activate the ON reactions by stimulating both the entry and the release of Ca2+. Most cells make use of both sources, but there are examples of cells using either external or internal sources to control specific processes and this will depend on the type of signalsome they are using. Most of the Ca2+ that enters the cytoplasm is adsorbed onto buffers while a much smaller proportion activates the effectors to stimulate cellular processes. The OFF reactions remove Ca2+ from the cytoplasm using a combination of mitochondria and different pumping mechanisms. When cells are at rest, these OFF reactions keep the concentration low, but these are temporarily overwhelmed when external stimuli activate the ON reactions. Sequential activation of the ON and OFF reactions gives rise to Ca2+ transients, which are such a characteristic feature of Ca2+ signalling. At any moment in time, the level of Ca2+ is determined by the balance between these ON and OFF reactions. An important spatial aspect of the ON reactions is that they are often closely associated with the effector systems that respond to Ca2+. For example, voltageoperated channels (VOCs) in presynaptic endings are associated with the synaptic vesicles thus producing a highly localized puff of Ca2+ to trigger exocytosis. Similarly, the type 2 ryanodine receptors (RYR2s) of cardiac cells are lined up close to the contractile filaments to ensure that Ca2+ will rapidly stimulate contraction. In cases where cells need to be stimulated over a long time, these transients are repeated at set intervals to set up Ca2+ oscillations. These oscillations are part of the spatiotemporal aspects of Ca2+ signalling that will be described in Section 5.2. 2.1. Ca2+ ON reactions In response to external stimuli, channels in the PM or endoplasmic reticulum (ER) are opened, and Ca2+ flows into the cytoplasm to bring about the elevation of cytosolic Ca2+ responsible for cell activation (Fig. 1). During these ON reactions, the cell employs a variety of both Ca2+ entry channels and CR channels, which are organized into different modules (Fig. 2). The entry of Ca2+ across the PM is carried out by many different channels whose names indicate how they are opened. For example, there are receptor OCs (ROCs, see module 1 in Fig. 2), second-messenger OCs (SMOCs, see module 2 in Fig. 2), VOCs (see module 3 in Fig. 2) and store OCs (SOCs). The last have attracted a lot of interest recently because there have been some new developments regarding the nature of the SOC and how it might be regulated.
488
M. J. Berridge
Fig. 2. Summary of some of the main modules that are mixed and matched to create different Ca2+ signalling systems. (1) Agonists such as the neurotransmitters acetylcholine, glutamate and ATP act directly on receptoroperated channels (ROCs) in the plasma membrane (PM) to allow external Ca2+ to enter the cell. (2). Second messengers such as diacylglycerol, cyclic AMP, cyclic GMP and arachidonic acid acting from the cytoplasmic side open second-messenger OCs (SMOCs) in the PM. (3) Membrane depolarization (DV) activates voltage OCs (VOCs) in the PM to allow a rapid influx of external Ca2+. (4) Membrane depolarization (DV) activates the CaV1.1 L-type channel that then activates the type 1 ryanodine receptor (RYR1) in skeletal muscle through a direct conformational-coupling mechanism. (5) Membrane depolarization (DV) activates VOCs in the PM to allow a rapid influx of external Ca2+ to provide trigger Ca2+that then activates the RYR2 to release Ca2+ stored in the sarcoplasmic reticulum (SR). This mechanism is found in cardiac muscle and neurons. (6) Agonists acting on cell-surface receptors generate inositol 1,4,5-trisphosphate (InsP3) that then diffuses into the cell to activate the InsP3 receptor (InsP3R) to release Ca2+ from the endoplasmic reticulum (ER) (See Color Plate 52, p. 547).
Release of Ca2+ from internal stores is carried out by three main channel types: the RYRs, the inositol 1,4,5-trisphosphate receptors (InsP3Rs) and a putative nicotinic acid dinucleotide phosphate (NAADP)-sensitive channel. One of the major problems in Ca2+ signalling has been to determine how stimuli arriving at the cell
Calcium signalling, a spatiotemporal phenomenon
489
surface gain access to these internal stores. Two main mechanisms have been identified. Firstly, there is conformational-coupling through protein–protein interactions. This is a very fast mechanism that depends on a sensor in the PM interacting directly with an internal release channel. The receptor on the cell surface is the CaV1.1 L-type channel (a voltage sensor), which is coupled to the RYR1 (module 4 in Fig. 2). Information is transferred through a process of conformational-coupling. This mechanism is mainly found in skeletal muscle, but a similar mechanism may be present in certain neurons such as hypothalamic synaptic endings [5]. The second mechanism depends on the generation of diffusible second messengers. Activation of receptors or channels on the cell surface generates second messenger that then diffuses into the cell to activate release channels. One of the most significant Ca2+-mobilizing messengers is Ca2+ itself, which is a potent activator of the two main internal release channels, the RYRs and the InsP3Rs (Fig. 3). This Ca2+induced CR (CICR) mechanism has the unique property of being autocatalytic and plays a central role in generating those Ca2+ signals that appear as regenerative Ca2+ waves. Another important messenger for releasing internal Ca2+ is InsP3 (module 6 in Fig. 2) [1]. Other Ca2+-mobilizing messengers have been described such as cyclic adenine dinucleotide phosphate [6] and NAADP [7,8].
Fig. 3. The elementary events of Ca2+ signalling. Elementary events are the localized Ca2+ signals that arise from either individual or small groups of ion channels. The localized plumes of Ca2+ have been given different names depending on the channels that produce them. Voltage-operated channels (VOCs) in the plasma membrane (PM) produce sparklets and ryanodine receptors (RYRs) on the sarcoplasmic reticulum (SR) create sparks (and syntillas), whereas the inositol 1,4,5-trisphosphate receptors (InsP3Rs) produce puffs. Channels in the PM are linked to the internal release channels through a process of Ca2+-induced Ca2+ release (CICR). The intracellular channels have large conductances and gate so much Ca2+, which result in a local depletion of Ca2+ within the lumen immediately below the channel. This local emptying of the endoplasmic reticulum (ER) store by RYRs has been visualized and has been called a blink.
490
M. J. Berridge
The channels responsible for these Ca2+ ON reactions usually have powerful inactivation mechanisms that rapidly curtail the entry or release processes to prevent the cell being swamped with Ca2+, which can result in cell stress and apoptosis. Once the ON reactions have been curtailed, the Ca2+ OFF reactions rapidly take over to return the activated level of Ca2+ back to its resting level. 2.1.1. Store-operated Ca2+ entry Much of the Ca2+ used for signalling is released from internal stores that have a finite capacity. For signalling to continue, therefore, Ca2+ must be brought in from the outside to ensure that the store remains topped up. Much of this ER Ca2+ homeostasis depends on the sarcoplasmic–ER Ca2+-ATPase (SERCA) pumps that recycle the released Ca2+. However, there are always some losses to the outside resulting in store depletion, which will not only result in a decline in signalling capacity but also trigger the ER stress signalling pathways. To guard against the deleterious effects of Ca2+ store depletion, the cell employs SOCs that open in response to store emptying to ensure a constancy of its internal Ca2+ store [9]. Electrophysiological studies have revealed that there may be different types of SOCs that vary in their Ca2+ selectivity. The classical example of a SOC is the CR-activated Ca2+ (CRAC) channel found in lymphocytes, which has a very high selectivity for Ca2+ and a very low conductance [10]. One of the main functions of this entry mechanism, therefore, is to maintain the internal store of Ca2+. However, it can also function as a source of signal Ca2+ especially under conditions where Ca2+ signalling has to be maintained over a prolonged period as occurs during the stimulation of cell proliferation. The nature of the signal that emanates from the ER to activate SOCs is still a matter of some debate. One of the important questions to consider is whether the whole of the ER is involved in SOC activation. This seems unlikely because depletion of the ER Ca2+ can trigger stress signalling pathways by seriously interfering with the processes of protein synthesis and packaging. There is growing evidence that a small specialized region of the ER closely associated with the PM regulates the entry of Ca2+ (Fig. 4). The major unsolved problem concerns the mechanism whereby the empty store sends a message to open the SOC in the PM. Some of the proposed mechanisms include the generation of a calcium influx factor, exocytosis of vesicles containing SOCs or a conformational-coupling mechanism whereby the release channels in the ER interact directly with the SOCs in the PM (Fig. 5). The latter mechanism resembles that found in skeletal muscle where L-type VOCs in the PM interact directly with the RYR1 channels in the SR (module 4 in Fig. 2). A similar conformational-coupling mechanism has been proposed to explain how empty ER stores can stimulate entry through SOCs in the PM [11]. In this latter case, information is flowing in the opposite direction, i.e. the output signal travels from the ER to the PM. There are a number of possible molecular components of this putative coupling mechanism (Fig. 5), which are less well defined than those of the skeletal muscle system. One of the problems with trying to understand this entry process is that the identity of the entry channels is uncertain. An integral membrane protein called
Calcium signalling, a spatiotemporal phenomenon
491
Fig. 4. The hypothesis outlined in this figure suggests that store-operated Ca2+ entry occurs at localized regions where there is close apposition of the endoplasmic reticulum (ER) and plasma membrane (PM). The ER is an interconnected reticular network of tubules, and short arms of this ER network come into contact with the PM to form junctional zones. The narrow gap between the ER and the PM may be the site where the signal from the empty ER is transferred to the store-operated channels (SOCs). Agonists acting on cell-surface receptors stimulate phospholipase C (PLC) to produce a microdomain of inositol 1,4,5trisphosphate (InsP3) (*), which functions to deplete Ca2+ in the junctional zones. The latter then sends a message to the SOCs in the PM to promote entry (i.e. regions 1 and 2). This signal might be transmitted through a protein–protein interaction as outlined in the conformational-coupling hypothesis shown in Fig. 5. In some cases, the microdomain of InsP3 does not coincide with a junctional zone, and the local depletion of Ca2+ fails to trigger entry (see region 3).
Orai1 may be the CRAC channel [12]. There also are indications that there may be a number of other SOCs coded for by various members of the TRP ion channel family such as TRPC1, TRPC4 and TRPV6. Another area of uncertainty concerns the nature of the protein in the ER that senses store emptying and then relays information to the SOCs. Both the InsP3Rs and the RYRs, which are known to be able to sense the luminal level of Ca2+, have been implicated as signal initiators in the ER, which then convey information to the SOCs in the PM through a direct protein–protein interaction. There is some evidence to support the idea that either an InsP3R or an RYR may play a role in transmitting information from the ER to the channels in the PM (Fig. 5). Such an involvement of an RYR would mean that this release channel might play a pivotal role in both the release of internal Ca2+ (as occurs in skeletal muscle, see module 4 in Fig. 2) and the entry of external Ca2+ (Fig. 5). Indeed, there is some evidence to show that both mechanisms may coexist in skeletal muscle where the SOCs may function in longterm Ca2+ homeostasis to overcome fatigue during intensive exercise. The SOC may couple to the RYR1 through mitsugumin 29, which is a synaptophysin family-related protein positioned within the junctional space between the PM and the SR [13].
492
M. J. Berridge
Fig. 5. A conformational-coupling hypotheses for store-operated Ca2+ entry as might occur in lymphocytes. Entry of Ca2+ depends on activation of a store-operated channel (SOC) such as the Ca2+ releaseactivated Ca2+ (CRAC) channel. When an agonist activates phospholipase C (PLC), the increase in inositol 1,4,5-trisphosphate (InsP3) then releases Ca2+ from the endoplasmic reticulum (ER) and the emptying of the store opens the CRAC channel. Store emptying might be detected by the stromal interaction molecule 1 (STIM1), which has an N-terminal EF-hand domain that binds Ca2+ when the store is full. As the store empties, Ca2+ comes off STIM causing it to undergo a conformational change that is transmitted through some coupling protein [perhaps an InsP3 receptor (InsP3R) or a ryanodine receptor (RYR)] to open the CRAC channels. Formation of this coupling complex appears to depend on cytoskeletal remodelling driven by WAVE2 and controlled through the monomeric G proteins Vav and Rac (See Color Plate 53, p. 548).
Another candidate for the ER sensor is the stromal interaction molecule (STIM), which is located within the ER and has a single EF-hand that might function to sense Ca2+ (Fig. 5) [14]. If STIM is knocked out, there is a dramatic decline in storeoperated entry indicating that this protein plays some direct role in the coupling mechanism. Under resting conditions when the store is full of Ca2+, STIM appears to be distributed over the ER. Upon store emptying, STIM moves to the surface to aggregate at close contacts between the ER and the PM [15] and is thus ideally situated to act as a sensor for conformational coupling. However, STIM is a small molecule, and the problems remain as to how information is transferred across the 20-nm gap to activate CRAC or SOCs in the PM. In the model outlined in Fig. 5, the suggestion is that when STIM loses its Ca2+ and migrates towards the cell surface, it makes contact with some coupling protein (e.g. an InsP3R or an RYR) to induce the conformational change that results in channel opening.
Calcium signalling, a spatiotemporal phenomenon
493
The conformational-coupling hypothesis depends on the ER making close contact with the PM so that information can be relayed between proteins located in the two apposed membranes. Many of the components that function in store-operated entry are associated with the caveolae, which have been suggested as possible site for this entry processes. There are numerous examples of the ER making intimate contact with the caveolae as described in both smooth muscle [16] and cardiac cells [17]. Another important structural component for entry is the cytoskeleton, which may be particularly important in forming and/or maintaining the structural integrity of this complex (Fig. 5). Proteins that function in actin remodelling such as WAVE can profoundly influence store-operated Ca2+ entry. In the case of T cells, activation of the T-cell receptor relays information out to a number of signalling pathways. One of these is the activation of Vav, which seems to act through Rac and WAVE2 to remodel the actin cytoskeleton that has a role in regulating the entry of Ca2+ through the CRAC channel [18,19]. 2.1.2. CICR A process of CICR plays a central role in the way Ca2+ signals are generated. This positive feedback mechanism whereby Ca2+ triggers its own release has two important functions in cells. It enables Ca2+ entering across the PM to function as a messenger to release Ca2+ from the internal store (Fig. 3). This function of CICR was first described in cardiac cells where the CaV1.2 L-type channel provides an influx of trigger Ca2+ that then diffuses into the cell to activate the RYR2 (module 5 in Fig. 2). A similar interaction is particularly evident for neuronal Ca2+ entry and release channels. The other main function of CICR is to set up intracellular and intercellular waves where an elevated level of Ca2+ in one region of the cell (the initiation site) propagates throughout the rest of the cytoplasm as a regenerative Ca2+ wave (Fig. 6). Waves can progress by recruiting either RYRs or InsP3Rs [20]. These Ca2+ waves are made up of elementary Ca2+ events such as the sparks and puffs produced by the RYRs and InsP3Rs, respectively. It is these unitary events that are used to generate the regenerative waves that make up global Ca2+ signals.
3. Ca2+ OFF reactions Cells use a variety of mechanisms to remove Ca2+ from the cytoplasm (Fig. 1). The introduction of Ca2+ into the cell during the Ca2+ ON reactions usually occurs for a relatively brief period during which there is a rapid increase in the intracellular concentration of Ca2+. The increase in free Ca2+, which can be measured in the cytoplasm using aequorin or fluorescent indicators, is a very small proportion of the total amount of Ca2+ that enters during the ON reactions. Much of this Ca2+ is rapidly bound by cytosolic buffers or is taken up by the mitochondria. As the [Ca2+] returns to its resting level, Ca2+ leaves the buffers and the mitochondria and is returned to the ER or is pumped out of the cell resulting in a brief Ca2+ transient.
494
M. J. Berridge
Fig. 6. Intracellular waves depend on a process of Ca2+-induced Ca2+ release (CICR) whereby the Ca2+ being released from one channel diffuses across to neighbouring channels that are excited to release further Ca2+ thereby setting up a regenerative wave. When such waves meet a cell boundary, they can trigger waves in adjacent cells thus setting up an intercellular wave. There is still some debate about the way the wave travels between cells. (A) One mechanism proposes that when the intracellular wave reaches the cell boundary, some small molecular weight component, most likely to be Ca2+, diffuses across the gap junction to ignite another intracellular wave in the neighbouring cell. (B) An alternative mechanism suggests that the intracellular wave in one cell stimulates the release of ATP through hemichannels, which then diffuses across to ignite a wave in neighbouring cells by acting on P2Y receptors to produce inositol 1,4,5-trisphosphate (InsP3) (See Color Plate 54, p. 549).
The recovery process thus depends on a complex interplay between cytosolic Ca2+ buffers, mitochondria and Ca2+ pumps and exchangers on the internal stores and PM. These Ca2+ OFF reactions operate at different stages during the recovery phase of a typical Ca2+ spike. The buffers and mitochondria operate early, and the Ca2+ pumps and exchangers are responsible for restoring the status quo by pumping Ca2+ out of the cell or back into the ER. These pumps and exchangers operate at different times during the recovery process. The Na/Ca exchangers have low affinities for Ca2+ but have very high capacities, and this enables them to function at the beginning of the recovery process to rapidly remove large quantities of Ca2+. On the contrary, the PMCA and the SERCA pumps have lower capacities, but their higher affinities mean that they can complete the recovery process and can continue to pump at lower Ca2+ levels thus enabling them to maintain both the internal stores and the resting level of Ca2+ within the cytoplasm. Some of these OFF reactions interact with each other during the recovery period, and this is particularly evident in
Calcium signalling, a spatiotemporal phenomenon
495
the case of the ER/mitochondrial Ca2+ shuttle. When Ca2+ is released from the ER, Ca2+ is rapidly taken up by the mitochondria, and this is then released slowly back to the ER once Ca2+ has returned back to the resting level. Under pathological situations, the lack of oxygen reduces the supply of energy thus compromising the function of the OFF reactions resulting in the rise of Ca2+ that is so damaging during stroke or cardiac ischemia. Under normal circumstances, however, the fully energized OFF reactions can rapidly reduce the pulse of Ca2+ introduced by the ON mechanisms thus generating a brief Ca2+ transient. Such brief pulses of Ca2+ are a characteristic feature of the Ca2+ signalling pathway and contribute to the spatiotemporal aspects of Ca2+ signalling.
4. Ca2+ buffers Cells express a large number of Ca2+-binding proteins (CaBPs), which fall into two main groups, Ca2+ sensors and Ca2+ buffers. The Ca2+ sensors respond to changes in intracellular Ca2+ by activating the downstream effectors that control cellular responses (Fig. 1). In a sense, all proteins capable of binding Ca2+ will act as a buffer, and this also applies to the sensors. However, the concentration of these sensors is usually rather low so they have little buffering capacity. The role of Ca2+ buffering is carried out by another major group of CaBPs. The major cytosolic buffers in cells are parvalbumin (PV) and calbindin D-28k (CB). The major buffers that operate within the lumen of the ER are calsequestrin and calreticulin. The latter is unusual in that it functions both as a cytosolic and as a luminal buffer. The cytosolic buffers have subtly different Ca2+-binding properties and are expressed in cells in differing combinations and concentrations to create Ca2+ signals with kinetics that are tailored to carry out different functions. For example, neurons such as Purkinje cells express large amounts of PV and CB. As a consequence, Purkinje cells have a large endogenous Ca2+-buffering capacity, e.g. their buffers bind approximately 2000 Ca2+ ions for each free ion. Lower capacities of 50–100:1 are found in other cells. Motor neurons have a very low-buffering capacity and consequently have large Ca2+ signals in both the soma and the dendrites during normal physiological responses, and this makes them particularly susceptible to neurodegeneration. Buffer concentration is one of the important parameters in determining buffer capacity. The other key parameters include affinity for Ca2+ and other metal ions, the kinetics of Ca2+ binding and release and mobility. The sole function of these Ca2+ buffers is to bind Ca2+, and this has an important function in shaping both the spatial and the temporal properties of Ca2+ signals.
5. Spatiotemporal aspects of Ca2+ signalling The use of Ca2+ as a universal signal for cell regulation is somewhat paradoxical because this ion can be very toxic to cells if its level remains high for a prolonged period. Such toxicity is avoided by presenting Ca2+ signals in a pulsatile manner
496
M. J. Berridge
Fig. 7. In almost every example where Ca2+ is used as a signal, it is presented as a brief transient, which is the digital signal used to set up complex temporal patterns of Ca2+ signalling. In some cells, these pulses are produced on demand in that they are generated in response to periodic stimulation (arrows) as occurs in muscle contraction where a brief burst of Ca2+ activates the contractile machinery, which then recovers when the Ca2+ signal is removed. Likewise, the release of neurotransmitters from nerve terminals is triggered by brief localized pulse of Ca2+. In many other tissues, which receive a continuous stimulation over a prolonged period (bar), the Ca2+ signal is again presented as brief spikes that are produced rhythmically to give highly regular Ca2+ oscillations whose frequencies vary with the level of cell stimulation.
(Fig. 7). In addition to this temporal aspect, the Ca2+ signal is also highly organized in space. For example, muscle contraction is activated by a global elevation in Ca2+, whereas the release of neurotransmitters results from a minute punctate pulse of Ca2+ delivered directly to the docked vesicle by a Ca2+ channel tightly associated with the exocytotic machinery. In between these two extremes, there are many variations in the way the Ca2+ signal is presented to cells.
5.1. Temporal aspects of Ca2+ signalling The temporal aspect of signalling is based on the fact that most Ca2+ signals appear in cells as transients (Fig. 7). There are two main ways in which this digital Ca2+ signal is produced. Firstly, there are ‘on-demand’ Ca2+ transients such as the signals generated in muscle cells and neurons by membrane depolarization. Secondly, transients can appear as an oscillation when cells respond to stimuli that persist for long periods. Such oscillations, which can have a wide range of frequencies, are a characteristic feature of many cellular control systems. There are two main types of oscillatory activity: membrane oscillators and cytosolic oscillators. The output from membrane oscillators usually sets up a regular train of action potentials that
Calcium signalling, a spatiotemporal phenomenon
497
provide the rhythmical pacemaker activity responsible for driving cellular processes such as contraction, neuronal activity and secretion. These membrane potential oscillations can open VOCs that gate Ca2+ and can thus result in intracellular Ca2+ oscillations. Ca2+ oscillations can also be generated by cytosolic oscillators. The latter are of particular interest because oscillator frequency can vary with stimulus intensity, and such frequency modulation is used to encode information. There also is evidence that information carried as oscillations can greatly enhance the activation of gene transcription [21,22].
5.1.1. Membrane oscillators Membrane oscillators are usually generated through an interplay between outward currents, usually carried by K+, that induce membrane hyperpolarization that inhibits the inward currents (e.g. Na+ and Ca2+) that cause depolarization. The entry of Ca2+ during the depolarizing phase is responsible for the phasic elevation of Ca2+ that is returned to the cytoplasm by the PMCA during the OFF reaction. There are a number of cell types that set up pacemaking activity using such membrane oscillators. Sinoatrial node pacemaker cells establish the regular trains of action potential, which drive cardiac contraction. Thalamocortical neurons have a neuronal rhythmicity that displays oscillations at a frequency of 0.5–4 Hz using a combination of different inward and outward currents. Neurons that reside within the suprachiasmatic nucleus, which have the biological clock, also have a membrane Ca2+ oscillator that is responsible for generating the output signals from the biological clock. 5.1.2. Cytosolic Ca2+ oscillations Cytosolic Ca2+ oscillations are produced by the periodic release of internal Ca2+ through the operation of a cytosolic oscillator [1,3]. This Ca2+ oscillation mechanism depends on the release of Ca2+ from intracellular stores. In the case of agonistinduced oscillations, the InsP3/Ca2+ signalling cassette is primary responsible for initiating oscillatory activity. The information encoding/decoding of Ca2+ oscillations depends on the ability to modulate the different parameters of these Ca2+ oscillations. Such agonist-dependent cytosolic Ca2+ oscillations are a major feature of Ca2+ signalling in many cell types. Liver cells display Ca2+ oscillations with agonist concentration-dependent changes in frequency [23]. Ca2+ oscillations are evident during the acquisition of meiotic competence during development [24] and are responsible for oocyte activation during mammalian fertilization [25]. Such Ca2+ oscillations are also observed following artificial insemination by intracytoplasmic spermatozoa injection [26]. Smooth muscle cells surrounding cortical arterioles display spontaneous Ca2+ oscillations [27]. Astrocytes display spontaneous Ca2+ oscillations [28]. Airway epithelial cells respond to ATP by generating the Ca2+ oscillations that control ciliary beat frequency [29].
498
M. J. Berridge
5.2. Spatial aspects of Ca2+ signalling Components of the Ca2+ signalling pathways are often highly organized and localized to discrete cellular areas where Ca2+ microdomains are formed to regulate specific cellular processes [20,30,31]. Much attention has focused on the elementary and global aspects of Ca2+ signalling, which determine whether signalling occurs within highly localized regions or is spread more globally by the formation of both intracellular and intercellular Ca2+ waves. One of the exciting aspects of signalling microdomains is the way they are used by neurons to increase their capacity to process information. The fact that this information processing can be confined to very small volumes within the spines means that each neuron is capable of simultaneously processing large amounts of information. Such input-specific signalling is particularly relevant to the process of synaptic modifications during learning and memory. The high concentrations of neuronal Ca2+ buffers, such as CB, play a major role in restricting Ca2+ signals to individual spines, which are the smallest units of neuronal integration. The size of these signalling microdomains will depend on a number of aspects such as the rate of signal generation, the rate of signal diffusion, the rate of signal removal by the OFF mechanisms and the degree of buffering. Buffers thus play an important role in determining the volume of these signalling microdomains, and this may have been a particularly important feature for the miniaturization of Ca2+ signalling within the brain. 5.2.1. Elementary aspects of Ca2+ signalling The development of fluorescent indicators to visualize Ca2+ in real time in living cells has revealed a spatial dimension to its action that can account for both the universality and the versatility of Ca2+ signalling. The microdomains that have been recorded in cells are the result of elementary events produced by the brief opening of either entry channels in the PM or release channels in the ER. Elementary events are the basic building blocks of Ca2+ signalling. They can either perform highly localized signalling functions or can be recruited to generate global Ca2+ signals. Most of these elementary events are due to the brief opening of Ca2+ channels located either in the PM or in the ER and thus result in localized pulses of Ca2+ (Fig. 3). The presence of Ca2+ buffers helps to restrict these brief pulses to small microdomains within the cytoplasm. A number of different elementary events have now been described as outlined in the following sections. 5.2.2. Sparklet A sparklet is formed as a result of the brief opening VOCs (Fig. 3). Sparklets have been visualized in ventricular heart cells [32]. These sparklets play a critical role in ventricular cell E–C coupling because they provide the trigger Ca2+ that activates the RYR2s in the junctional zone. Another function for sparklets is to control exocytosis particularly at synaptic endings where a localized pulse of Ca2+ is responsible for transmitter release. An elementary event, which is equivalent to a sparklet, is formed in the stereocila of hair cells upon opening of the mechanosensitive channel [33].
Calcium signalling, a spatiotemporal phenomenon
499
5.2.3. Spark A Ca2+ spark is formed by the opening of a group of RYRs (Fig. 3). They were first described in cardiac cells where they are responsible for excitation–contraction coupling [34]. However, they have now been described in many other cell types where they play a variety of different functions. In ventricular cardiac cells, the spark is the unitary Ca2+ signal that is produced at each junctional zone. There are approximately 10 000 junctional zones in each cell, and to get a rapid contraction, the individual sparks must all be fired simultaneously. An electrical recruitment process is used for this synchronization. In atrial cells, the sparks have a different function from those in the ventricular cells. The initial sparks activated by membrane depolarization are restricted to the junctional zones at the cell surface where they provide a signal to ignite a Ca2+ wave that spreads into the cell by triggering a progressive series of sparks through the process of CICR. In mossy fibre presynaptic endings, spontaneous Ca2+ sparks can trigger transmitter release [35]. Spontaneous Ca2+ transients, which resemble sparks, have been recorded in cerebellar basket cell presynaptic endings [36]. Smooth muscle cell Ca2+ sparks function to control both contraction and relaxation. In the case of relaxation, the spark activates the large conductance (BK) channel to produce an outward current that hyperpolarizes the membrane [37]. 5.2.4. Syntilla A scintilla is an elementary event produced by RYRs and thus resembles a spark (Fig. 3). These syntillas function in hypothalamic neuronal presynaptic CR [5]. 5.2.5. Blink Blinks have been visualized within the SR of ventricular muscle cells [38]. As release channels such as the RYRs have a very high conductance, they can gate sufficient Ca2+ to cause a temporary depletion of Ca2+ within the lumen of the junctional SR (Fig. 3). These blinks are attracting considerable interest because they may play a role in the inactivation of ventricular RYR2s to curtail the cardiac Ca2+ transient. 5.2.6. Puff A puff is a unitary event that results from the release of Ca2+ from a small group of InsP3Rs (Fig. 3). These puffs, which are very similar to the Ca2+ sparks, are the building blocks of the intracellular Ca2+ waves in cells that result in global Ca2+ signals. Such localized Ca2+ signals form microdomains of Ca2+ to regulate localized cellular processes such as transmitter release from the astrocyte endings that form part of the tripartite synapse [39] and transmitter release from neocortical glutamatergic presynaptic endings [40]. Release of Ca2+ by InsP3Rs produces the microdomains of Ca2+, which are confined to individual spines in Purkinje neurons [41]. 5.3. Global Ca2+ signals Most of the global Ca2+ signals in cells are produced by the release of Ca2+ from internal stores. The intracellular release channels such as the InsP3Rs and RYRs can
500
M. J. Berridge
create such global signals if their release activity can be synchronized. There are two mechanisms of synchronization that is nicely illustrated by the way ventricular and atrial cardiac cells are controlled. In ventricular cells, electrical recruitment by the action potential is used to activate all the sparks in the junctional zones simultaneously. By contrast, atrial cells use a process of diffusional recruitment whereby Ca2+ sparks at the cell surface trigger Ca2+ waves that spread inwards through a process of CICR by recruiting RYRs located deeper within the cell. There also are examples where intracellular waves can spill across into neighbouring cells to set up intercellular Ca2+ waves, which thus provide a mechanism for coordinating the activity of a local population of cells. 5.3.1. Intracellular Ca2+ waves Intracellular Ca2+ waves can be generated by both InsP3Rs and RYRs, which are Ca2+-sensitive channels that contribute to the positive feedback process of CICR responsible for forming Ca2+ waves (Fig. 6A). The main condition that has to be met for Ca2+ waves to form is that their Ca2+ sensitivity must be increased such that they can respond to the local Ca2+ spark or puff produced by their neighbours. In both cases, the level of Ca2+ within the lumen of the ER appears to be a critical factor. This ER loading is achieved by entry of external Ca2+, and as the lumen loads up with Ca2+, the InsP3Rs and RYRs gradually increase their sensitivity such that they can participate in the regenerative processes that result in a Ca2+ wave. In effect, this increase in Ca2+ sensitivity of the release channels converts the cytoplasm into an ‘excitable medium’ capable of spawning these regenerative waves. These intracellular waves are an integral part of many cellular control processes. Intracellular Ca2+ waves provide the global Ca2+ signal that activates mammalian oocytes at fertilization [25]. Excitation–contraction coupling in atrial cardiac cells depends on Ca2+ waves that spread into the cell from the periphery [42]. Astrocyte excitability depends on an intracellular wave that spreads from the tripartite synapses down to the endfoot processes. In the pancreas, InsP3Rs in the apical region can trigger waves that then spread through the basal region through RYRs. 5.3.2. Intercellular Ca2+ waves There are a number of instances of Ca2+ waves travelling from one cell to the next. Such intercellular waves may act to coordinate the activity of a local population of cells. There is still some uncertainty concerning the way in which the wave is transmitted from one cell to the next (Fig. 6). One mechanism proposes that low molecular weight components such as InsP3 or Ca2+ spill through the gap junctions to ignite waves in neighbouring cells. In order for a cell to set up an intracellular wave, the internal release channels have to be sensitized, so it seems likely that all the cells in the population have to be in a similar state in order for an intercellular wave to pass from one cell to the next. In such a scenario, Ca2+ is the most likely candidate to be the stimulus that passes from one cell to the next. An alternative model proposes that the intracellular wave in one cell stimulates the release of ATP that then diffuses across to neighbouring cells where it acts on P2Y receptors to increase InsP3, which then acts to trigger a new wave (mechanism B in Fig. 6).
Calcium signalling, a spatiotemporal phenomenon
501
The function for such intercellular waves has not been clearly established. Much of the work on these waves has been done on cultured cells, but there are a number of reports showing that such intercellular waves do occur between cells in situ. Intercellular waves have been described in astrocytes, but their physiological function is unclear. As they only seem to appear following intense stimulation, they may be a manifestation of some pathological change. In this respect, the astrocyte wave moves at approximately the same rate as spreading depression that appears to be linked to the onset of migraines. Intercellular waves, which have been recorded in the intact perfused liver, appear to travel in a periportal to pericentral direction [43]. This directionality has led to the suggestion that the wave might function to regulate a peristaltic contraction wave to control the flow of bile. During development, there are pan-embryonic intercellular waves that sweep around the blastoderm margin in the late gastrula of zebra fish [44]. These spatiotemporal aspects greatly enhance the versatility of Ca2+ signalling thus providing the flexibility to regulate so many cellular processes.
6. Ca2+ signalling function The function of Ca2+ as an intracellular second messenger is carried out by a combination of Ca2+ sensors and Ca2+ effectors. The major sensors are the EFhand proteins troponin C, calmodulin (CaM), neuronal calcium sensor proteins and the S100 proteins. These sensors are then responsible for relaying information through a range of effectors such as Ca2+-sensitive K+ channels, Ca2+-sensitive Cl– channels, Ca2+–CaM-dependent protein kinases, calcineurin, phosphorylase kinase, myosin light chain kinase and Ca2+-promoted Ras inactivator. The activation of these different effectors is then responsible for stimulating the large number of Ca2+-sensitive cellular processes.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Berridge, M.J. (1993) Nature 361, 315–325. Clapham, D.E. (1995) Cell 80, 259–268. Berridge, M.J., Lipp, P. and Bootman, M.D. (2000) Nat. Rev. Mol. Cell Biol. 1, 11–21. Berridge, M.J., Bootman, M.D. and Roderick, H.L. (2003) Nat. Rev. Mol. Cell Biol. 4, 517–529. De Crescenzo, V., ZhuGe, R., Vela´zquez-Marrero, C., Lifshitz, L.M., Custer, E., Carmichael, J., Lai, F.A., Tuft, R.A., Fogarty, K.E., Lemos, J.R. and Walsh, J.V. (2004) J. Neurosci. 24, 1226–1235. Galione, A. and White, A. (1994) Trends Cell Biol. 4, 431–436. Patel, S., Churchill, G.C. and Galione, A. (2001) Trends Biochem. Sci. 26, 482–489. Lee, H.C. (2003) Curr. Biol. 13, R186–R188. Putney, J.W. (1986) Cell Calcium 7, 1–12. Hoth, M. and Penner, R. (1992) Nature (Lond) 355, 353–356. Berridge, M.J. (1995) Biochem. J. 312, 1–11. Feske, S., Gwack, Y., Prakriya, M., Srikanth, S., Puppel, S.-H., Tanasa, B., Hogan, P.G., Lewis, R.S., Daly, M. and Rao, A. (2006) Nature (Lond) 441, 179–185.
502
M. J. Berridge
13. Pan, Z., Yang, D., Nagaraj, R.Y., Nosek, T.A., Nishi, M., Takeshima, H., Cheng, H. and Ma, J. (2002) Nat. Cell Biol. 4, 379–383. 14. Roos, J., DiGregorio, P.J., Yeromin, A.V., Ohlsen, K., Lioudyno, M., Zhang, S., Safrina, O., Kozak, J.A., Wagner, S.L., Cahalan, M.D., Velic¸elebi, G. and Stauderman, K.A. (2005) J. Cell Biol. 169, 435–445. 15. Liou, J., Kim, M.L., Heo, W.D., Jones, J.T., Myers, J.W., Ferrell, J.E. and Meyer, T. (2005) Curr. Biol. 15, 1235–1241. 16. Forbes, M.S., Rennels, M.L. and Nelson, E. (1979) J. Ultrastruct. Res. 67, 325–339. 17. Gabella, G. (1978) J. Ultrastruct. Res. 65, 135–147. 18. Nolz, J.C., Gomez, T.S., Zhu, P., Li, S., Medeiros, R.B., Shimizu, Y., Burkhardt, J.K., Freedman, B.D. and Billadeau, D.D. (2006) Curr. Biol. 16, 24–34. 19. Zipfel, P.A., Bunnell, S.C., Witherow, D.S., Gu, J.J., Chislock, E.M., Ring, C. and Pendergast, A.M. (2006) Curr. Biol. 16, 35–46. 20. Berridge, M.J. (1997) J. Physiol. 499, 291–306. 21. Dolmetsch, R.E. Xu, K. and Lewis, R.S. (1998) Nature (Lond) 392, 933–936. 22. Li, W.-H., Liopis, J., Whitney, M., Xlokarnik, G. and Tsien, R.Y. (1998) Nature (Lond) 392, 936–941. 23. Robb-Gaspers, L.D., Rutter, G.A., Burnett, P., Hajno´czky, G., Denton, R.M. and Thomas, A.P. (1998) Biochim. Biophys. Acta 1366, 17–32. 24. Carroll, J., Swann, K., Whittingham, D. and Whitaker, M. (1994) Development 120, 3507–3517. 25. Miyazaki, S., Yuzaki, M., Nakada, H., Shirakawa, H., Nakanishi, S., Nakade, S. and Mikoshiba, M. (1992) Science 257, 251–255. 26. Nakano, Y., Shirakawa, H., Mitsuhashi, N., Kuwabara, Y. and Miyazaki, S. (2001) Mol. Hum. Reprod. 3, 1087–1093. 27. Filosa, J.A., Bonev, A.D. and Nelson, M.T. (2004) Circ. Res. 95, e73–e81. 28. Nett, W.J., Oloff, S.H. and McCarthy, K.D. (2002) J. Neurophysiol. 87, 528–537. 29. Zhang, L. and Sanderson, M.J. (2003) J. Physiol. 546, 733–749. 30. Bootman, M.D., Lipp, P. and Berridge, M.J. (2001) J. Cell Sci. 114, 2213–2222. 31. Rizzuto, R. and Pozzan, T. (2006) Physiol. Rev. 86, 369–408. 32. Wang, S.-Q., Song, L.-S., Lakatta, E.G. and Cheng, H. (2001) Nature 410, 592–596. 33. Lumpkin, E.A. and Hudspeth, A.J. (1998) J. Neurosci. 18, 6300–6318. 34. Cheng, H., Lederer, W.J. and Cannell, M.B. (1993) Science 262, 740–744. 35. Sharma, G. and Vijayaraghavan, S. (2003) Neuron 38, 929–939. 36. Conti, R., Tan, Y.P. and Llano, I. (2004) J. Neurosci. 24, 6946–6957. 37. Brenner, R., Pere´z, G.J., Bonev, A.D., Eckman, D.M., Kosek, J., Wiler, S.W., Patterson, A.J., Nelson, M.T. and Aldrich, R.W. (2000) Nature (Lond) 407, 870–876. 38. Brochet, D.X.P., Yang, D., Di Maio, A.D., Lederer, W.J., Franzini-Armstrong, C. and Cheng, H. (2004) Proc. Natl. Acad. Sci. U. S. A. 102, 3099–3104. 39. Grosche, J., Matyash, V., Mo¨ller, T., Verkhratsky, A., Reichenbach, A. and Kettermann, H. (1999) Nat. Neurosci. 2, 139–143. 40. Simkus, C.R.L. and Stricker, C. (2002) J. Physiol. (Lond) 545, 521–535. 41. Denk, W., Sugimori, M. and Llinas, R. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8279–8282. 42. Mackenzie, L., Bootman, M.D., Berridge, M.J. and Lipp, P. (2001) J. Physiol. 530, 417–429. 43. Robb-Gaspers, L.D. and Thomas, A.P. (1995) J. Biol. Chem. 270, 8102–8107. 44. Webb, S.E. and Miller, A.L. (2003) Nat. Rev. Mol. Cell Biol. 4, 539–551.