Calcium channels in lymphocytes (2001)

On B and T lymphocytes, ligation of the antigen receptor (AgR) induces a biphasic Ca2+ response. In the initial phase there is a large elevation in the intracellular Ca2+ concentration as a consequence of Ca2+ release from intracellular stores. This is followed by a lower, but prolonged elevation that is dependent on extracellular Ca2+.1,2 This simple description belies the complexity of the response. The initial phase may involve as many as three different intracellular Ca2+ channels, while the second phase depends not only on plasma membrane Ca2+ channels, but also on at least two different intracellular channels. The complexity of the signal, and the many opportunities for regulation of individual components of the signalling mechanism, lead to a tremendous flexibility in outcome, ranging from single, brief elevated Ca2+ transients, through a range of oscillatory responses, each of which can be decoded by the cell into a differing outcome.2 In this review we concentrate on the Ca2+ channels involved in the AgR-mediated Ca2+ signal, but we briefly discuss other Ca2+ channels present in lymphocytes. Figure 1 shows two possible schemes for the involvement of Ca2+ channels in TCR signalling, and Fig. 2 shows possible roles for Ca2+ channels in B cells. A possible scheme for the involvement of Ca2+ channels in TCR signalling (a) depicts the simplest possible scheme for the role of Ca2+ channels in TCR-induced Ca2+ signalling. TCR-induced Ins(1,4,5)P3 production causes Ca2+ release from intracellular stores, which in turn relays a signal to the plasma membrane store-operated Ca2+ channel (ICRAC channel), causing it to open. In an alternative scheme (b) intracellular Ca2+ flux results from the TCR-induced production of Ins(1,4,5)P3, cADPR and possibly NAADP, in concert with the activation of the ICRAC channel in the plasma membrane. The Ca2+ signal is sustained by the activity of mitochondria (shown in yellow), KCa channels, and cADPR. Note that the localization of the NAADP receptor is unknown, and that RyR3 and InsP3R may not be present on the same intracellular stores. The roles of plasma membrane InsP3Rs and the l-type Ca2+ channel are unknown – the possibility that they may mediate Ca2+ influx is indicated by dotted lines. The identification of the l-type Ca2+ channel as an NAADP receptor is speculative. Intracellular stores are depicted in blue, and activation steps are shown by red arrows. Possible roles for Ca2+ channels in B cells. The BCR-induced Ca2+ signal involves the production of Ins(1,4,5)P3 and the release of Ca2+ from intracellular stores gated by InsP3Rs and RyR1. This is followed by an influx of Ca2+ through an unidentified store-operated channel (SOC). The mechanism of activation of RyR1 is unknown. Note that RyR1 and InsP3R are unlikely to be present on the same intracellular stores. The possible involvement of NAADP receptors in BCR signalling is highly speculative. The roles of plasma membrane InsP3Rs and the l-type Ca2+ channel are unknown – the possibility that they may mediate Ca2+ influx is indicated by dotted lines. The identification of the l-type Ca2+ channel as an NAADP receptor is speculative. CD20 and annexin V are shown as possible Ca2+ channels. Intracellular stores are depicted in blue, and activation steps are shown by red arrows. A plethora of studies of AgR signalling have highlighted the role of inositol trisphosphate [Ins(1,4,5)P3]-mediated release of Ca2+ from internal stores (reviewed in refs 1–3). However, it is becoming apparent that there is more to the regulated release of intracellular Ca2+ in lymphocytes than inositol trisphosphate receptors (InsP3Rs). Recent studies are beginning to unravel roles for ryanodine receptors (RyRs) and the newly described and little understood NAADP receptor. Three types of InsP3R are known, and they vary in their sensitivities to Ins(1,4,5)P3 and in the properties of their activation by Ca2+. InsP3Rs must bind Ins(1,4,5)P3 for Ca2+ release to occur. The response of the InsP3R can be regulated by phosphorylation, by various accessory proteins and by ATP, but by far the most important regulator is Ca2+. The exact mechanism is disputed4–6 but it is apparent that the differing sensitivities of the InsP3R isoforms to regulation by Ca2+ allow cells to fine-tune the temporal and spatial aspects of the Ca2+ signal.5 Much recent work has been directed towards determining the roles of the various isoforms. B and T cells express all three types of InsP3R to varying degrees depending on their stage of differentiation.7–11 It is not clear why lymphocytes simultaneously express all three isoforms, particularly since Sugawara et al.8 clearly demonstrated the redundancy of InsP3R expression. In a series of knockouts in DT40 B lymphocytes the BCR-induced Ca2+ signal could not be ablated until all three InsP3R isoforms were simultaneously knocked out.8 InsP3R3 was the first isoform to have a specific role ascribed to it. In both T and B cells, InsP3R3 is up-regulated in cells undergoing apoptosis.10 Inhibition of InsP3R3 expression using antisense RNA prevents TCR-induced apoptosis.10 However, Sugawara et al.8 provided convincing evidence against a specific role for InsP3R3 in apoptosis when they showed that knockout of any two isoforms in DT40 cells inhibited BCR-induced apoptosis. InsP3R3 can be expressed on the external surface of the plasma membrane of T and B cells and in T cells it can cocap with the TCR.9,10 The validity of this observation has been questioned, but support was provided by Tanimura et al.12 who demonstrated InsP3R3 expression on the external surface of Jurkat T cells. They showed that this pattern of expression was not limited to InsP3R3, and in fact the predominant isoforms in the plasma membrane were InsP3R1 and InsP3R2.12 The role of plasma membrane InsP3Rs is unknown, but Putney13 has suggested that, in some cell types, InsP3R3 may be expressed as an integral plasma membrane protein and function as all or part of a store-operated Ca2+ channel. However, the properties of InsP3R, in particular its Ca2+ selectivity profile, do not accord with those of known store-operated Ca2+ channels and the prevailing evidence strongly indicates that these channels are formed by distinct molecules from InsP3Rs (discussed below). In an attempt to explore the role of InsP3R1, Jayaraman et al.11 used antisense RNA to show that TCR-induced Ca2+ signals are exclusively transduced through this subtype. However, this is a controversial finding since the construct they used could cross-react with the type 2 and type 3 receptors. Hirota et al.14 subsequently demonstrated that T lymphocytes could develop normally in InsP3R1 knockout mice and showed normal Ca2+ fluxes in response to anti-CD3 stimulation, contradicting the claim that InsP3R1 was absolutely required for TCR signalling. In the most convincing study to ascribe roles for the various isotypes, Miyakawa et al.15 showed that InsP3R3 was involved in the generation of monophasic single Ca2+ transients following BCR ligation, whereas InsP3R1 and InsP3R2 were involved in the generation of Ca2+ oscillations with differing frequencies. The knockouts studied by Sugawara et al.8 suggest that inhibition of downstream events may be achieved simply by reducing the overall levels of InsP3Rs, rather than the specific levels of one particular isotype. This raises the possibility that cells do not express homotetramers of InsP3Rs; rather they may express heterotetramers of varying amounts of each isoform, allowing the cell to express a graded array of hybrid receptors with a wide variety of subtly different properties made up of combinations of the properties of the ‘pure’ homotetramers. Clearly much remains to be done to unravel the role of InsP3Rs in AgR signalling. RyRs are large (∼560 kDa) homotetrameric receptors that mediate Ca2+ release from the endoplasmic reticulum stores. Three isoforms, encoded by separate genes, have been described. RyRs can be gated by allosteric coupling to plasma membrane voltage-gated Ca2+ channels (in the case of RyR1) and by Ca2+ (all isoforms). All three isoforms can be activated by cyclic ADP ribose (cADPR). cADPR can initiate Ca2+ release and sensitise the RyR to further activation by Ca2+. This feed-forward mechanism, known as Ca2+-induced Ca2+ release, can lead to the prolonged propagation of Ca2+ signals.16,17 In contrast to InsP3Rs, only one isotype of RyR is expressed in lymphocytes, RyR1 in B lymphocytes and RyR3 in T lymphocytes, therefore there is no question of heterotetramer formation.18–22 The role of RyRs in AgR signalling is beginning to be addressed and appears to differ between B and T cells. In T cells the initial peak of Ca2+ release following TCR engagement is due to Ins(1,4,5)P3-mediated Ca2+ release from intracellular stores. This peak is followed by a sustained Ca2+ flux that requires both the influx of extracellular Ca2+ and the production of cADPR. Guse et al.22 showed that cADPR is produced following TCR ligation and that antagonists of cADPR inhibited TCR-induced proliferation and the expression of early and late activation markers. They showed that the long-lasting Ca2+ influx, rather than the initial Ca2+ peak, depended on cADPR production. In contrast, in B cells depletion of RyR1-gated stores significantly inhibits the BCR-induced Ca2+ peak, suggesting that in B cells the RyR is activated at the same time as Ins(1,4,5)P3-induced Ca2+ release.18 The mechanism that activates RyRs in B cells is unknown, but in T cells Guse et al.22 showed that it is TCR-induced cADPR production. The source of TCR-induced cADPR is controversial. cADPR is produced from β-NAD by the action of ADP-ribosyl cyclase. This enzyme was first isolated from Aplysia, and its mammalian homologues include CD38. CD38 is an ecto-enzyme having both ADP-ribosyl cyclase and cADPR-hydrolase activities. The extracellular location of CD38 has proved puzzling since the site of action of its product (cADPR) is intracellular. One study suggests that cADPR produced by CD38 on human haemopoietic precursors can raise intracellular Ca2+ levels via a specific plasma membrane cADPR transporter.23 However, da Silva et al.24 have shown that the TCR-induced production of cADPR is intracellular and independent of extracellular production by CD38. Supporting the intracellular origin of TCR-induced cADPR, Guse et al.22 demonstrated the presence of an ADP ribosyl cyclase activity in the cytosol of T cells. The presence of RyR in lymphocytes is an interesting recent discovery. The Ca2+-induced Ca2+ release properties of these receptors suggest a role in maintaining a long-term Ca2+ signal. In turn, this may suggest that InsP3Rs are more concerned with triggering of the Ca2+ signal than with its long-term propagation. Nicotinic acid adenine dinucleotide phosphate (NAADP) is a recently described intracellular Ca2+ mobilizing agent. NAADP is synthesized from NADP by the actions of ADP-ribosyl cyclase. Its actions are poorly understood, but it is increasingly evident that it mobilizes Ca2+ via a specific receptor, distinct from InsP3Rs and RyRs, and which is a channel in its own right.25 The actions of NAADP appear to vary depending on cell type (reviewed in refs 25 and 26). In Jurkat T lymphocytes, recent evidence suggests that mobilization of NAADP is an absolute requirement for signalling through the TCR.27 NAADP appears to act before Ins(1,4,5)P3 and cADPR to provide sufficient Ca2+ to sensitize the InsP3R and RyR to Ins(1,4,5)P3 and cADPR, respectively. The nature of the NAADP receptor is unknown, but recent data suggest that it is an intracellular Ca2+ channel. Its pharmacology is beginning to be explored. It is known to be inhibited by high concentrations of l-type voltage-gated Ca2+ channel inhibitors, and curiously also by activators of these channels. It is also sensitive to inhibitors of some classes of voltage-gated K+ channels.28 There are currently no reports of NAADP involvement in B-cell signalling, but the persistent reports of the effects of inhibitors of l-type Ca2+ channels on BCR signalling (discussed below) suggest the possibility of this system also being involved in the BCR-induced Ca2+ flux. Perhaps the most interesting feature of the NAADP receptor is its inactivation properties. Prior exposure to high concentrations of NAADP, such as might be expected if the TCR had previously been cross-linked, render the receptor inactive and unable to respond to further stimulation. In effect it allows the cell to retain a memory of a previous Ca2+ signal. This property led Berg et al.27 to speculate that the NAADP/Ca2+ system may provide a mechanism underlying anergy in T cells. Together, InsP3Rs, RyRs and the NAADP receptor form a complex web for priming, initiating and maintaining a signal, and then ‘remembering’ that is has been transduced. Much more is known about the AgR-activated Ca2+ channel in T cells than in B cells. In T cells there is strong evidence that the TCR-activated Ca2+ channel is identical to the store-operated ICRAC channel (reviewed in detail in ref 2). For example, both store depletion and TCR ligation activate a channel with identical properties29 and in a human primary immunodeficiency caused by an absence of ICRAC, there is no TCR-activated Ca2+ influx.30 Ca2+ influx occurs following BCR ligation or pharmacological depletion of intracellular stores. However, to date, there have been no electrophysiological studies demonstrating the presence of the ICRAC channel in B lymphocytes and it is not known whether B cells express this channel. While the majority of studies on transmembrane Ca2+ flux have been aimed at identifying the AgR-activated Ca2+ channel, it is unlikely to be the only plasma membrane Ca2+ channel present in lymphocytes. A number of other potential Ca2+ channels have been identified but their unambiguous identification as bona fide Ca2+ channels is still lacking. AgR ligation leads to a release of Ca2+ from intracellular stores. The decrease in store Ca2+ concentration causes the activation of Ca2+ channels (store-operated Ca2+ channel: SOC) in the plasma membrane by an as yet unresolved mechanism.31–33 Store-operated Ca2+ channels have been the subject of intensive research, but are still best described in terms of the current passing through the channel. The best characterized store-operated Ca2+ current in haemopoietic cells is the Ca2+-release-activated Ca2+ current (ICRAC), which was first identified in mast cells34 and subsequently in Jurkat T lymphocytes.29 The ICRAC channel appears to be widely distributed, but other types of store-operated current have been described, including the Ca2+-release activated non-selective cation current (ICRANC) in Jurkat T cells.35 There are several defining features of the ICRAC Ca2+ influx pathway. These include a high selectivity for Ca2+,36 a very small single channel conductance and blockade by divalent cations with a characteristic selectivity profile.29,37,38 The other notable feature of the ICRAC channel is Ca2+-dependent modulation. Ca2+ influx can be enhanced through the binding of Ca2+ to an extracellular site on the channel39 and the channel can be rapidly inactivated due to accumulation of Ca2+ close to its intracellular mouth.40 The properties of the ICRAC channel have been determined by electrophysiological means and obviously must be met by any candidate protein. In this context, it is important to note that, although these properties describe one current, they may represent the averaged properties of a number of individual different subunits. This in turn may explain why the molecular identity of the ICRAC channel has proved to be so hard to determine (discussed in more detail below). The importance of Ca2+ entry in normal lymphocyte function was highlighted when defects in this pathway were discovered. In 1994, Partesi et al.30 described a patient with an immunodeficiency associated with defective T lymphocyte proliferation, where the defect was shown to be in the Ca2+ entry pathway. This defect was sufficient to alter T lymphocyte expansion and function. More recently, two severe-combined immunodeficiency patients have been shown to suffer from defective Ca2+ influx in both T and B lymphocytes resulting in impaired lymphocyte activation.41 It is not surprising, then, that Ca2+ entry turns out to be a regulated event in lymphocyte activation. The entry of Ca2+ through ICRAC channels is ultimately driven by the membrane potential. lymphocytes are cells, both B and T lymphocytes express voltage-gated K+ channels and K+ channels function is to the membrane the in intracellular Ca2+ KCa are activated and the membrane. This the membrane potential more and Ca2+ influx by the for Ca2+ Ca2+ entry can be sustained by the action of mitochondria close to the of the ICRAC channel. They Ca2+ following Ca2+ entry the Ca2+-dependent inactivation of the on lymphocytes can also Ca2+ of following ligation on T lymphocytes results in the release of which is further to and can Ca2+ through ICRAC channels and this may be the mechanism and of lymphocyte It has been suggested that blockade of ICRAC channels could be for anergy in In B lymphocytes, engagement of is important in the regulation of Ca2+ a is to following with the and a role in the BCR-induced Ca2+ Ca2+ influx can be regulated by protein activated downstream of B lymphocytes activity are in Ca2+ influx following BCR in T cells there is defective TCR-induced Ca2+ These effects are due to a defect in the sustained production of Ins(1,4,5)P3, rather than a effect of the on the Ca2+ channel. DT40 B cells, et showed that BCR-induced Ca2+ influx, possibly through of the channel. is to have a role in the of Ca2+ entry in Jurkat T lymphocytes, although the exact mechanism is There are several for the number of ICRAC channels expressed on T lymphocytes. and that there are channels T whereas et that T cells have channels, to channels in activated T cells. et suggested that this in the number of channels T cell activation is for proliferation and for Ca2+ signalling T lymphocyte activation. It is clear that ICRAC can be regulated by a number of by intracellular Ca2+ and by lymphocyte allows lymphocytes the to fine-tune the Ca2+ signal in the In the activation can be sustained by of the number of channels expressed on the cell The molecular identity of the ICRAC channel is but the current is that it is formed by one or more of the proteins were in cells as channels required for sustained Ca2+ homologues of into three and and have all been to be expressed in Jurkat T et described a store-operated non-selective cation channel (ICRANC) in They suggest that and are to be of this channel when and are expressed they form a channel with very properties to It is possible that proteins are of Ca2+ are accessory in a channel or form the channel It may be for a number of different proteins to form in to channels. of the studies that have to roles to channels formed by proteins have on the of these This is to a channel that not represent the In turn, this may explain some of the data on the of For about it is possible to studies and its role as a store-operated Ca2+ the best candidate for the ICRAC channel is the which was from small and subsequently from Jurkat T et showed that expression of in cells results in a Ca2+ current with properties not to those of These include activation by store cation and of single channel determining some of the properties in cells levels of et the of and with other data provide evidence that may form all or a of the ICRAC channel. yet the expression of in B lymphocytes has not been Store-operated Ca2+ channels are unlikely to be the Ca2+ channels present in lymphocytes. There are reports suggesting the presence of other types of Ca2+ channel such as the channel (discussed annexin CD20 and channels to voltage-gated Ca2+ channels. form Ca2+ channels in lymphocytes. The are a large of Ca2+-dependent binding proteins that can form voltage-gated Ca2+ channels in V known as is expressed in B and T lymphocytes where its function is recent studies have addressed the involvement of annexin V in Ca2+ influx in lymphocytes. In T cells, the pharmacological extracellular of annexin V was associated with an intracellular Ca2+ which in turn was associated with an inhibition of However, the extracellular of large amounts of a protein that normally has an intracellular localization not represent a was the mechanism of and activation of the annexin V Ca2+ channel explored. In a more convincing et demonstrated annexin Ca2+ influx in B lymphocytes. DT40 B lymphocytes with in annexin they showed that and store-operated Ca2+ entry was normal in these cells but that Ca2+ by 2 were However, this study could not out a role of annexin V as an of a Ca2+ channel rather than an Ca2+ channel in its own the best known candidate for a Ca2+ channel is CD20 is a of B lymphocytes that is expressed on and activated B and on some T cell of CD20 into cells induces the expression of a Ca2+ conductance identical to that when CD20 is in T and B cells. This Ca2+ conductance is enhanced following the binding of to However, the of Ca2+ influx is not a In some where Ca2+ influx has been demonstrated there is evidence suggesting that it is a downstream consequence of in those studies that suggest that CD20 is a cation the possibility that CD20 could be a of a Ca2+ channel rather than a Ca2+ channel in its own be The role of CD20 in Ca2+ influx therefore remains and persistent observation is that lymphocytes express Ca2+ channels that are to voltage-gated Ca2+ channels. work on pharmacological the l-type Ca2+ channel antagonists such as and These were used at concentrations than required to voltage-gated Ca2+ channels and at concentrations far than l-type channel antagonists a number of studies showed effects on and B-cell activation and Ca2+ these pharmacological studies provide some evidence for the expression of Ca2+ channels to voltage-gated Ca2+ channels, they at the same time be with some due to the high concentrations of et demonstrated that Ca2+ influx in B lymphocytes through a channel with to the of l-type Ca2+ channels. l-type channel antagonists and an the Ca2+ response. The presence of and l-type Ca2+ channel in Jurkat T lymphocytes has been demonstrated by Recent studies show that a Ca2+ channel is involved in and TCR-induced in T et showed surface of T lymphocytes with an specific and the expression of an l-type Ca2+ channel in these cells. These studies the presence of an l-type channel and protein in lymphocytes, but there appears to be some which is These reports represent an finding in the of the pharmacology of the NAADP receptor. this channel be in some to the l-type channels so in This question turns it is clear that Ca2+ signals in lymphocytes a complex temporal and spatial The of as simple as a Ca2+ flux the lymphocyte to an extracellular signal into an to the and of the This is achieved via a web of intracellular and transmembrane Ca2+ channels in a complexity of that we are only beginning to is by the is by the of of are to for of this