Intracellular calcium
endocrine cells, such as pituitary cells and pancreatic cells, where elevated intracellular Ca2 is also a trigger for secretion. Common characteristics
of Ca2 entry through VOCCs are the speed, brevity, and intensity of the observed Ca2 transients. The rather low affinity of the effectors is matched to the high concentrations of Ca2 achieved and the limited spatial volume in which it is confined. Rapid dissipation may then follow, ensuring that the subsequent effects are local and transient.
Receptor-operated channels
In some neuronal cells, low-specificity ion channels in the plasma membrane may open as a consequence of activation and allow Ca2 to enter. Glutamate receptors sensitive to NMDA are important postsynaptic ion channels that mediate excitatory transmission in the CNS. In order for these channels to open, two conditions must be satisfied: they must bind glutamate and the membrane in which they reside must already be depolarized in order to remove a blocking Mg2 ion.49 This may be achieved by the simultaneous effects of two different neurotransmitters, one of which depolarizes the cell in preparation for the action of glutamate itself. Alternatively, a sustained release of glutamate from the presynaptic cell could provide both signals, by first activating AMPA receptors to depolarize the postsynaptic cell. The ability of NMDA receptors to act as coincidence detectors, coupled with other processes, gives them a role in the long term potentiation of synaptic signalling.50,51
TRPM2 channels
TRPM2 is a plasma membrane cation channel that is Ca2 permeable. It is a member of the TRP superfamily, which is discussed below. It is activated from within cells by the adenine nucleotide, ADP-ribose (ADPR, not to be confused with cADPR, see Figure 7.14). The role of ADPR as a signalling molecule has yet to be fully established. Its principal function seems to be as a messenger that causes Ca2 influx in cells exposed to oxidative stress. It is not clear how its synthesis is initiated and it may take place at different locations, involving nuclear, mitochondrial or cytosolic enzymes, as well as the ectoenzyme CD38 acting on NAD .37 The ADPR-sensitivity of TRPM2 is Ca2 -dependent and probably involves calmodulin.52 TRPM2 does not interact with STIM1 (see below).
Replenishing depleted stores
The rapid transmission of signals between nerves and the fast responses of skeletal muscles, all of which depend on signalling through VOCCs, are
essential for survival. For other types of tissue, there is often less urgency. In hormone-secreting cells, the changes in Ca2 concentration may take the form of a series of oscillations that continue over a period of minutes (see
NMDA: N-methyl-d- aspartic acid, a glutamate analogue that activates a subclass of glutamate receptors.
209
Signal Transduction
Figure 7.6). In cells stimulated by growth factors or cytokines, there may be a need for a protracted period (hours) of Ca2 elevation in order to ensure full commitment to a proliferative response.53 Such demands present a problem since repetitive or sustained elevation of cytosol Ca2 must lead to depletion of the Ca2 stores. This is prevented by a mechanism that allows extracellular Ca2 to enter the cell through plasma membrane cation channels in response to store depletion. This is called store-operated Ca2 entry (SOCE). Originally proposed in 1986 and termed capacitative Ca2 entry,54,55 it took some 20 years before the sensor of ER/SR Ca2 depletion was identified. Although
the proteins likely to form the channel are now known, the details of the mechanism are still not clear.
Store-operated Ca2 channels
The first hint of a protein with potential SOCE activity was obtained from the photoreceptor cells of Drosophila. A spontaneous mutation caused the light response to decay to zero on continuous exposure to bright illumination. Patch clamp studies of isolated receptor cells revealed a reduced Ca2 current in the mutants,56, 57 and it was later concluded that lack of Ca2 in the stores could be responsible. In these cells, regions of the plasma membrane, close to elements of the ER, possess a Ca2 channel encoded by the trp gene (transient receptor potential).58 This finding stimulated a search for a vertebrate homologue.
The Drosophila trp gene product is a member of a superfamily of TRP proteins that are widely expressed across species. These transmembrane proteins
are predicted to assemble in tetramers to form Ca2 channels. There are six families and they have diverse functions. TRPV channels, which include the vanilloid receptors, and some TRPM channels are involved in sensory functions, such as sensitivity to temperature, osmolarity, odorants, and
mechanical stress. The so-called classical or canonical TRP channels, TRPC1–7, are activated following receptor-induced PI(4,5)P2 hydrolysis and seem most likely to be involved in SOCE. (Note that TRPC2 is a pseudogene in humans.)
Mammalian cells expressing cloned TRPCs have shown enhanced SOCE and, conversely, ablation of TRP genes or application of anti-TRPC antibodies reduces SOCE activity. Despite some conflicting data, there is evidence that TRPCs can contribute towards SOCE in a variety of cells, especially TRPC1.59 Drosophila Trp has 40% amino acid identity to TRPC1. However, there is no firm evidence of a homomeric TRPC1 channel activated by store depletion. Alternatively SOCE channels might be heteromers of different TRP subunits, which might account for the variation of channel properties that is observed.
There is, however, a more powerful contender for the role of store-operated channel that is not a TRP protein. First detected electrophysiologically in rodent mast cells, in which an inward current is activated within about 10 s of store depletion, the channel is of low conductance and specific for Ca2 . It was termed ICRAC (calcium release-activated calcium current).60 Ca2 entry
210
Intracellular calcium
through CRAC channels is a requirement for the activation of mast cells through the IgE receptor Fc RI, and for the expression of NFAT-controlled genes in lymphocytes. The channels are also present in other cells.
A gene encoding a transmembrane protein essential for SOCE was identified by genome-wide RNAi screening in Drosophila S2 cells (particularly suitable for RNAi studies). The assay used a fluorescent Ca2 -sensing dye to detect knockdown of SOCE and involved over 145 000 fluorescence measurements. The gene, called Orai, is highly conserved and has three mammalian homologues.61,62 A single mutation of a human homologue, Orai1, accompanies a rare form of severe combined immune deficiency (SCID) in which Icrac is absent from lymphocytes.63 Expression of wild type Orai1 in T cells from the SCID patients was found to restore CRAC channel activity.
Orai1 is a 33 kDa protein with four transmembrane stretches. It exists only on the plasma membrane and the tetramer has all the credentials of a CRAC channel (low conductance, Ca2 -selective, inwardly rectifying). For instance,
mutations of acidic residues in the putative pore region alter its ion selectivity and other channel characteristics, showing that it is able to act as a Ca2 selectivity filter. Over-expression of Orai1 together with its activator STIM1 (see below) produces very large Icrac responses.64 In consequence, Orai1 is considered to be an essential component of SOCE channels. Whether it forms homotetrameric channels or heteromeric channels with its homologues Orai2 and Orai3 is uncertain, but this could account for some of the observed variation in Ca2 -selectivity of CRAC responses.
The apparent and surprising lack of a requirement for any TRP protein raises many questions and has been the cause of some disagreement. It has been suggested that TRPC1 might provide a parallel or additional form of SOCE, or that it might function as a channel component together with Orai1 subunits, or it might be a channel regulator.59, 65 Furthermore, activation of Orai1 requires the clustering of its activator STIM1 (see below), but STIM1 clustering also proves to be necessary for agonist activation of all of the TRPC channels, except for TRPC7.66 (Note that TRPC7 is also known as TRPM2 and it is activated by ADP-ribose.)
The sensor that activates SOCE
The identification of Orai1 was made possible by RNAi screening. At about the same time, large-scale RNAi screening also led to the identification of a protein that senses the Ca2 level within the ER, and that activates the SOCE channels. This was achieved by screening Drosophila S2 cells with siRNA
from a panel of 170 genes known to encode proteins with potential roles in SOCE. These included the TRPs and other proteins with channel properties.67 In a similar, parallel study, human cells (HeLa) were screened against 2304 proteins with signalling motifs.68 These investigations identified Stim
Mast cells are associated with the immune system. They secrete inflammatory mediators in response to an antigen challenge. The antigen cross-links immunoglobulin E molecules (IgE) bound to Fc RI receptors. If the
antigen is also an allergen the response can present a particular problem for allergic individuals.
Orai1 is also known as CRACM. Orai is named after the keepers of the gates of heaven (in Greek mythology).
211
Signal Transduction
(stromal interaction molecule) as the only Ca2 sensor necessary for SOCE in Drosophila, and STIM1 as a key component of SOCE in mammalian cells. (The related molecule STIM2 was also identified, but despite early indications, it is now thought to function as a regulator of the resting level of cytosolic Ca2 .69)
STIM1 is widely expressed in vertebrates. It has a molar mass of 77 kDa and possesses a single transmembrane domain. It is predominantly present (75%) in the membrane of the ER, with its N-terminus within the lumen and C-terminus in the cytosol. The N-terminal chain contains a domain sterile motif (SAM) and a single Ca2 -sensing EF-hand motif (note: unpaired EFhands are unusual, see Chapter 8). The cytosolic chain has a coiled coil region and a number of phosphorylation sites. The protein has also been detected at the plasma membrane with its N-terminus outside the cell.
The evidence for STIM1’s function as a Ca2 sensor and activator of SOCE is strong. Knockdown of STIM1 abolishes SOCE and mutation of its EF-hand from high to low Ca2 affinity, causes activation of the Orai1 channel, even when the stores are replete. STIM1 is not only necessary for SOCE, it is sufficient if Orai1 is present in the plasma membrane. Also, as already mentioned, when STIM1 and Orai1 are co-over-expressed, a huge increase in SOCE activity ensues.
A number of mechanisms by which STIM1 might activate the plasma membrane channels have been proposed. These include (1) direct interaction, across the cytosol, of the C-terminal chains of ER-resident STIM1 with the intracellular chains of the channel, (2) movement of STIM1 from the ER membrane to the plasma membrane, where it can interact with the channels, and (3) the release from the ER of a soluble messenger that activates the channels.
Immunofluorescence studies show that the distribution of STIM1 changes upon store depletion. When the store is replete it is dispersed across the ER membrane. When depleted, it forms clusters (visible as punctuate
fluorescence) that are close to (or possibly even on) the plasma membrane. It has been proposed that the clustering enables the coiled coil regions of the cytoplasmic tail of STIM1 to interact directly with Orai1 channels at the regions where the ER juxtaposes the plasma membrane (Figure 7.16). This results in the opening of the channels which then admit Ca2 into a confined region of the cytosol, allowing it to be taken up into the ER through nearby SERCA pumps. This should happen before the ion can diffuse into the bulk cytosol where it would be buffered. This hypothesis accords with evidence that SOCE can occur without any change in the overall cytosol Ca2 concentration.70
A second proposal suggests that upon store depletion, STIM1 is transferred from the ER and inserted into the plasma membrane, presumably by vesicular trafficking. Once there, it activates the channels. A third idea concerns a rather mysterious substance called calcium influx factor (CIF), first discovered in 1993.71 CIF is a readily diffusible small molecule that is produced in cells when store
212
Intracellular calcium
Fig 7.16 Model showing how STIM1 may sense store depletion and activate plasma membrane Ca2 channels.
(a) When Ca2 stores in the ER are replete, the lumenal EF-hands of STIM1 bind Ca2 . The distribution of STIM1 (blue) across the ER membrane is diffuse. (b) When the store is depleted, the STIM1 molecules remain in the ER but extend hydrophobic chains into the plasma membrane. They cluster at the regions where
the ER is close to the cell surface and they interact with the SOC channels.
(c) Ca2 enters through the SOC channels and is taken up into the ER by sarcoplasmic/endoplasmic reticulum ATPase pumps (SERCA).
Modified from Wu et al.70
Ca2 is depleted. Its production ensues within 20–30 s of Ca2 depletion and it is detected in the ER fraction.72 Since its discovery, evidence has accumulated for CIF production in a wide variety of cell types.73 It is remarkable that in the 15 years following its discovery, its molecular identity remains unknown. CIF can only be obtained as a purified extract from cells with depleted stores and its activity is assayed by injecting it into Xenopus laevis oocytes and then measuring the rise in intracellular Ca2 . It has a molecular mass of 600 Da and seems most likely to be a phosphorylated sugar nucleotide (but not cADPR or NAADP, nor is
it S1P). The effect of CIF is to activate ICRAC and it is thought that this is mediated by a Ca2 -independent phospholipase A2 at the plasma membrane (iPLA2 ). It
has recently been shown that CIF production is closely linked to the expression of STIM1. Knockdown of STIM1 prevents the formation of CIF in vascular smooth muscle cells as well as inhibiting SOCE. Conversely, cells over-expressing STIM1 produce CIF extracts three times more potent than controls. If STIM1 is directly responsible for CIF production, the clustering of STIM1 upon store depletion would allow CIF to be delivered directly into the vicinity of the SOCE channels.
Clearly there have been major advances in identifying the molecules likely to be responsible for SOCE, but many questions remain.
213
Signal Transduction
Ca2 microdomains and global cellular signals
Digital imaging techniques and confocal microscopy have added considerable detail to our knowledge of Ca2 signals. Within cells, the initial increments
in Ca2 concentration tend to be confined to regions called microdomains. These are mostly close to locations where effector molecules are gathered and where action takes place, for example in the vicinity of the triad structures in skeletal muscle or in neurons near to neurotransmitter release sites or confined within individual dendritic spines. These localized increases in Ca2 may remain restricted and transient or they may be amplified by CICR and then spread further. They may eventually merge to generate a global increase in Ca2 concentration which permeates the whole cell, especially under conditions of strong stimulation, and may last for minutes or longer.
When these changes are visualized using fluorescent Ca2 indicators, a variety of optical phenomena may be observed. These range from ‘Ca2 sparks’,
the punctate flashes seen in cardiac muscle cells (10 m or more across and lasting tens of milliseconds), to broad waves that spread within seconds right across the cytoplasm. These have been observed in a wide range of cells that include pancreatic acinar cells, smooth muscle cells, megakaryocytes, and astrocytes (see Figure 7.7). Such Ca2 waves can also pass from cell to cell through gap junctions. In both electrically excitable and non-excitable cells, cytoplasm that is permeated by elements of the ER or SR bearing IP3Rs and RyRs may form an ‘excitable medium’74 in terms of its ability to release Ca2 . That is, once initiated, the process of Ca2 -induced Ca2 -release can produce
an expanding, regenerative response that progresses at a rate faster than that achievable by diffusion of Ca2 ions.
Ca2 signals in electrically excitable cells
Skeletal muscle
Ca2 release in skeletal muscle commences when dihydropyridine receptors (DHPR, see L-type channels above) in the plasma membrane sense a depolarization. The DHPRs are directly coupled to RyRs on the SR membrane in functional units of four DHPRs and one RyR1. Depolarization leads to allosteric activation of the RyRs and generates a localized release of Ca2 that spreads as neighbouring RyRs that are not coupled to DHPRs are activated by CICR. The DHPR/RyR complexes and surrounding RyRs are arranged so that Ca2 is released into a microdomain close to the contractile machinery. This is not only in the interests of efficiency, but also to avoid uncontrolled activation through further CICR. The Ca2 -sensitivity of RyRs, like that of IP3Rs, is bellshaped (see Figure 7.13), so that as the concentration of Ca2 approaches micromolar levels the rate of channel opening declines. This prevents the
214
Intracellular calcium
stores from emptying completely and it also sets a limit to the magnitude of the Ca2 signal. These factors shape the Ca2 transients that are seen in
skeletal muscle. They are less intense and less punctate than the spark events seen other cells such as cardiac muscle cells, and may last for hundreds of microseconds. They have been termed ‘embers’. Contraction of skeletal and smooth muscle is also discussed in Chapter 8.
Cardiac muscle
Ca2 signalling in cardiac muscle is different. In both atrial and ventricular cells the type 2 RyRs are not directly coupled to DHPR/L-type channels as they are in skeletal muscle. Instead they rely on CICR for activation. In ventricular cells, the T-tubule system that extends into the cell from the plasma membrane comes within a few nanometres of the SR at some 10 000 junctional zones. At each zone there are 10 L-type channels on the T-tubule and 100 RyR2s on the juxtaposed SR. A Ca2 spark occurs when one or more L-type channels open to activate 10–15 RyR2s 75. The high Ca2 -conductance and the longer open times of RyR2 channels, synchronized in all the zones by the plasma membrane depolarization, creates a rapid increase in Ca2 that is localized over the myofibrils.
In atrial cells there is no T-tubular system and the junctional zones are only present at the cell surface. However, there are many non-junctional RyR2s elsewhere on the SR. Depolarization causes sparks near the surface, but this excitation is prevented from propagating through the cell by a wall of mitochondria situated just under the plasma membrane. The mitochondria
begin to take up Ca2 when it rises to high levels, and together with the SERCA pumps, they have the effect of preventing the propagation of CICR beyond the surface. Thus, under normal conditions contractions are relatively weak. In the event of -adrenergic stimulation, more sparks are generated at the periphery and the Ca2 level becomes high enough to breach the wall. Then CICR can generate a global Ca2 signal that causes a strong contraction.76
Nerve cells
The morphology of nerve cells is complex and varied, and many preand postsynaptic interactions depend upon Ca2 signals. Postsynaptic signalling in the dendrites and dendritic spines that receive excitatory inputs involves VOCC or glutamate or AMPA receptors (see page 54). Brief openings of these channels produce Ca2 microdomains that are restricted to the immediate environment of the channel. Subsequent amplification can involve either RYRs or IP3Rs or both.77 Signals tend to be spatially restricted however, and this enables individual spines to process inputs independently.
At presynaptic nerve endings, Ca2 is the signal for exocytosis. VOCCs open transiently in response to depolarization. A microdomain of Ca2 is created that may be amplified by CICR, again through the opening of RyR and IP3R channels. Activation of neurotransmitter secretion is discussed in Chapter 8.
215