Calcium Channels in Lymphocytes Franz Fenninger and Wilfred A

What's Bred in the Bone: Calcium Channels in Lymphocytes Franz Fenninger and Wilfred A. Jefferies This information is current as J Immunol 2019; 202:1021-1030; ; of September 24, 2021. doi: 10.4049/jimmunol.1800837 http://www.jimmunol.org/content/202/4/1021 Downloaded from References This article cites 112 articles, 47 of which you can access for free at: http://www.jimmunol.org/content/202/4/1021.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. What’s Bred in the Bone: Calcium Channels in Lymphocytes x { ‖ Franz Fenninger*,† and Wilfred A. Jefferies*,†,‡, , , ,# Calcium (Ca2+) is an important second messenger calcineurin, resulting in the dephosphorylation and nuclear in lymphocytes and is essential in regulating various translocation of NFAT proteins (Fig. 1). The NFAT tran- intracellular pathways that control critical cell functions. scription factor family includes NFAT1–5, of which Ca2+ channels are located in the plasma membrane and NFAT1, 2, and 4 have so far been described in lymphocytes, intracellular membranes, facilitating Ca2+ entry into the where they can have redundant and specific roles (1). cytoplasm. Upon Ag receptor stimulation, Ca2+ can en- Interestingly, although NFAT1 seems to be the main tran- 2+ 2+ scription factor in naive T cells, anergic T cells, which ter the lymphocyte via the Ca release-activated Ca 2+ exhibit reduced Ca signaling, preferentially activated Downloaded from channel found in the plasma membrane. The increase of 2+ 2+ NFAT2 (2). The Ca –calmodulin complex can also activate cytosolic Ca modulates signaling pathways, resulting 2+ in the transcription of target genes implicated in differ- transcription factor CREB via Ca /calmodulin-dependent entiation, activation, proliferation, survival, and apopto- protein kinase II (CaMKII) as well as myocyte enhancer factor 2 (MEF2) and NF-kB (3). The transcription factors sis of lymphocytes. Along with Ca2+ release-activated then induce gene transcription involved in cell proliferation, Ca2+ channels, several other channels have been found cytokine production, survival, and cell death. http://www.jimmunol.org/ in the membranes of T and B lymphocytes contribut- Over the last 20 years, there have been significant ad- ing to key cellular events. Among them are the tran- vancements in the identification and characterization of plasma sient receptor potential channels, the P2X receptors, 2+ membrane channels. We have become increasingly aware that voltage-dependent Ca channels, and the inositol the expression of different Ca2+ channels can vary based on the 1,4,5-trisphosphate receptor as well as the N-methyl- subset of lymphocyte and the stage of lymphocyte maturation. D-aspartate receptors. In this article, we review the Also, it is not uncommon that Ca2+ channels are expressed as 2+ contributions of these channels to mediating Ca cur- novel splice variants, often modifying their gating character-

rents that drive specific lymphocyte functions. The istics (4). Another phenomenon that adds to the complexity by guest on September 24, 2021 Journal of Immunology, 2019, 202: 1021–1030. of Ca2+ signaling is the formation of so-called Ca2+ microdomains, which refer to the spatiotemporal organization of alcium (Ca2+) signaling is a vital event in lymphocytes, Ca2+ currents (5). For example, the translocation of NFAT1 as it regulates various fundamental processes including into the nucleus is dependent on local Ca2+ increases in differentiation, activation, proliferation, survival, proximity to open Ca2+ release-activated Ca2+ (CRAC) C 2+ and apoptosis. The mechanisms that govern the levels of channels instead of a global increase of Ca levels in the intracellular Ca2+ involve membrane receptors, signaling entire cytoplasm (6). Similarly, elevated Ca2+ levels close to 2+ 2+ 2+ molecules, and a diverse array of Ca channels. The Ca voltage-dependent Ca (CaV) 1.1 channels are more efficient signaling cascade is commonly initiated by the stimulation in inducing CREB phosphorylation than increased Ca2+ close 2+ of Ag receptors like the TCR or BCR, resulting in Ca to CaV1.2 channels (7). Finally, different Ag affinities induce release from intracellular stores into the cytoplasm. After different Ca2+ dynamics that turn on different transcriptional depletion of these intracellular stores, Ca2+ enters the cell regulators (8). For example, large transient cytosolic increases fromtheextracellularspacethroughCa2+ channels in the in Ca2+ concentration selectively activate NF-kB and JNK plasma membrane. Ca2+ can then bind to the cytoplasmic signaling pathways, whereas NFAT translocation requires a adaptor molecule calmodulin, which forms a complex with smaller but sustained Ca2+ plateau (4). Importantly, this

*Michael Smith Laboratories, University of British Columbia, Vancouver V6T 1Z4, Address correspondence and reprint requests to Dr. Wilfred A. Jefferies, University of British Columbia, Canada; †Department of Microbiology and Immunology, University British Columbia, 2185 East Mall, Vancouver, BC V6T 1Z4, Canada. E-mail address: of British Columbia, Vancouver V6T 1Z3, British Columbia, Canada; ‡Vancouver [email protected] Prostate Centre, University of British Columbia, Vancouver V6H 3Z6, British Colum- 2+ x Abbreviations used in this article: Ca , calcium; cADPR, cyclic ADP-ribose; CaV, bia, Canada; Centre for Blood Research, University of British Columbia, Vancouver 2+ { voltage-dependent Ca ; CFS/ME, chronic fatigue syndrome/myalgic encephalomyelitis; V6T 1Z4, British Columbia, Canada; The Djavad Mowafaghian Centre for Brain CRAC, Ca2+ release-activated Ca2+; EAE, experimental autoimmune encephalomyelitis; Health, University of British Columbia, Vancouver V6T 1Z3, British Columbia, Canada; + ‖ ER, endoplasmic reticulum; IP3, inositol 1,4,5-trisphosphate; KO, knockout; Na , so- Department of Medical Genetics, University of British Columbia, Vancouver V6T 1Z4, dium; NMDA, N-methyl-D-aspartate; RyR, ryanodine receptor; SOCE, store-operated British Columbia, Canada; and #Department of Zoology, University of British Columbia, Ca2+ entry; STIM, stromal interaction molecule; T , T effector; Treg, regulatory T cell; Vancouver V6T 1Z4, British Columbia, Canada Eff TRP, transient receptor potential. Received for publication June 14, 2018. Accepted for publication August 22, 2018. Ó F.F. was supported by a DOC Fellowship of the Austrian Academy of Sciences. W.A.J. Copyright 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 was supported by grants from the Canadian Institutes of Health Research (IPR-139079 and MOP-102698) and a grant from Pascal Biosciences, Inc. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800837 1022 BRIEF REVIEWS: CALCIUM CHANNELS IN LYMPHOCYTES Downloaded from http://www.jimmunol.org/

FIGURE 1. Ca2+ channels in lymphocytes. Binding of Ag to the TCR/BCR initiates the signaling cascade that activates PLCg, which cleaves PIP2, producing IP3. IP3 subsequently binds to the Ca2+ channel IP3R in the endoplasmic reticulum (ER) membrane, triggering a release of Ca2+ from the ER stores into the cytoplasm. The depletion of Ca2+ in the ER can be picked up on by the Ca2+ sensor STIM1, which subsequently accumulates in regions of the ER close to the plasma membrane. Here, it can directly interact with the pore-forming units of the CRAC channel, Orai1/2, and trigger the opening of the channel. CaV1 channels, in contrast, are thought to be open in resting lymphocytes, possibly to maintain basal Ca2+ levels and therefore ensure T cell homeostasis. STIM1 can also interact with members of 2+ 2+ 2+ the CaV1 family but takes on a reciprocal role and inhibits CaV1uponCa depletion of the ER. Other Ca channels in the plasma membrane also lead to Ca inﬂux following TCR stimulation. These include plasma membrane IP3Rs, TRP channels, P2X receptors, and NMDARs. The exact mechanisms of how these channels are regulated, however, remain poorly understood. The P2X receptors are gated by ATP that is released from the cell through the hemichannel pannexin-1. by guest on September 24, 2021 The increase of cytosolic Ca2+ concentration activates calmodulin-calcineurin, which dephosphorylates NFAT and thereby induces its nuclear translocation. NFAT then prompts the transcription of target genes important in lymphocyte development, homeostasis, activation, proliferation, and apoptosis. Reprinted in adapted form from Smart Servier Medical Art, available at smart.servier.com. Licensed under a Creative Commons Attribution 3.0 Unported License.

2+ plethora of Ca channel regulation comes together to channels, such as transient receptor potential (TRP) and CaV control lymphocyte development and effector functions, channels, are thought to also participate in SOCE and will be which will be explored further in this review. discussed later (9, 10). The existence of the CRAC channel in T cells was recognized CRAC channels (Orais and stromal interaction molecules) well before its constituents Orai1 and STIM1 were discovered The best-described mechanism of extracellular Ca2+ entering (11). Interestingly, the importance of CRAC channels was lymphocytes is through the CRAC channel. The CRAC first demonstrated in patients with SOCE and CRAC channel channel is comprised of two components that act together: deficiencies, which presented with SCID (12–14). Although Orai1, the pore-forming subunit, which is found in the the T cells of these patients underwent normal development, plasma membrane, and stromal interaction molecule (STIM) 1, in mature cells, TCR-induced Ca2+ flux was impaired along the regulatory subunit located in the endoplasmic reticu- with effector functions, resulting in life-threatening recurrent lum (ER) membrane. TCR/BCR engagement triggers a infections, such as pneumonia and severe CMV infections signaling cascade that leads to the transport of Ca2+ from (12–14). A decade after the initial pathogenic characterization the ER into the cytoplasm via the Ca2+ channel inositol of these patients, genomic linkage analysis of more CRAC 1,4,5-trisphosphate (IP3) receptor upon binding of its ligand channel–deficient patients (15–17), as well as RNA interfer- IP3. The Ca2+ levels in the ER are monitored by the Ca2+ ence screens in Drosophila cells (18, 19), identified Orai1 sensing protein STIM1, the regulatory subunit of the CRAC and STIM1 as the main components of the CRAC channel. channel. STIM1 proteins, which are usually readily spread out The CRAC channel–deficient patients harbored mutations in in the ER membrane, oligomerize in certain puncta during either the Orai1- or STIM1-encoding genes that led to an low levels of Ca2+. These areas are in close proximity to the abrogation of their T cell Ca2+ influx and CRAC channel plasma membrane so that STIM1 can interact with and ac- function. Soon after, it was confirmed in a mouse model that tivate Orai1, the pore-forming unit of the CRAC channel, Orai1 and STIM1 were components of the CRAC channel and trigger a Ca2+ influx from the extracellular space. The and that their deficiency led to a reduced SOCE (20, 21). In process is termed store-operated Ca2+ entry (SOCE), and mice, Orai1 deficiency led to a less drastic phenotype, as although the CRAC channel is the prime example of it, other T cells exhibited residual SOCE, which was attributed to the The Journal of Immunology 1023 upregulation of the Orai1 paralog Orai2, partially compen- attenuated following adoptive transfer of STIM1-deficient sating for Orai1 deficiency (22). Along with this compensa- CD4 T cells into MHC-mismatched recipient mice (24). tory role in the absence of Orai1, an interesting function for Finally, a functional CRAC channel was also necessary for Orai2 was recently described by Vaeth et al. (23). Although T cell homing. T cell homing involves the migration Orai1/Orai2 double-deficient T cells were completely devoid of lymphocytes from the thymus into the secondary lymphoid of SOCE, the deletion of the paralog Orai2 alone surprisingly organs such as spleen and lymph nodes, where they encounter increased SOCE into mouse cells. This demonstrates an dendritic cells that present Ag for their activation (32). In inhibitory role of Orai2 in Ca2+ flux, which is thought to fine- transgenic mice expressing a dominant-negative Orai1 mutant tune immune responses. The authors suggest that Orai2 can (E106A), T cell migration into the peripheral organs was confer its inhibitory role by forming a heteromeric complex impaired (33). However, another study found no defects in with Orai1 (23). homing when examining T cells expressing a similar Orai1 STIM2, in contrast, was found to have a similar activating mutant (E106Q) (34), leaving this aspect controversial. role to that of its paralog STIM1. However, its absence did Along with T lymphocytes, STIM1/2 and Orai1/2 also play not cause severe defects equal to the STIM1 deficiency, and it a role in B cells. For example, it was previously demonstrated interfered mostly with sustained Ca2+ influx (21). In STIM1- that Orai1-deficient B cells exhibited an impaired BCR- deficient T cells, STIM2 could also partially compensate for induced Ca2+ flux, as well as defects in cell proliferation the absence of STIM1, and on a molecular level, STIM1 and (20). In addition, STIM1/2 double deficiency led to a com- 2+ STIM2 deficiency both impaired the downstream NFAT plete abrogation of Ca flux and severe proliferative defects Downloaded from pathway (21). (35). Furthermore, upon BCR cross-linking, this impairment In Orai1- or STIM1/STIM2-deficient T cells, the reduced in Ca2+ flux dampened NFAT signaling, resulting in the SOCE led to decreased effector functions such as impaired reduced production of anti-inflammatory cytokine IL-10 cytokine production and proliferation (20, 21, 24, 25). (35). As IL-10 is a negative regulator of autoimmunity, its Because of these deficits in cytokine production, particularly absence resulted in the heightened severity of EAE in mice lower production of TNF-a and IFN-g along with impaired (35). Intriguingly, despite these cellular and regulatory de- http://www.jimmunol.org/ degranulation of cytotoxic T cells, STIM1/2 was required fects, the Ab responses of these B cells were normal, suggesting for the suppression of invasive tumors. As such, STIM1/2- the mechanisms controlled by STIM1/2 do not regulate this decifient mice succumbed to induced melanoma and adeno- process (35). carcinoma (26). Similarly, STIM1/2-dependent production Analogous to T cells, B cells developed normally in the of IL-2 and IFN-g was critical during early antiviral responses absence of STIM1 (36). However, STIM1 overexpression of CD8 T cells (27). Also, during acute secondary viral in- increased the Ca2+ entry into maturing B cells and was suf- fection, STIM1/2 was required for memory responses and ficient to activate a newly discovered proapoptotic ERK sig-

specifically CD4 T cell helper function, which reactivates naling pathway predisposing the cells to negative selection by guest on September 24, 2021 cytotoxic CD8 T cells upon re-exposure (27). Although Orai1 (36, 37). Despite these findings, T cells are more dependent and STIM1 were important for proper effector functions of on CRAC channels than B cells, as the mediated Ca2+ flux is T cells, they were dispensable for their maturation, as dem- not required for Ab production and other B cell related im- onstrated in traditional murine TCRab+ T cells (20). Con- mune responses (38). versely, agonist-selected T cells, which include regulatory Although the CRAC channel is the best-studied contributor T cells (Tregs) and invariant NKT cells, exhibited a defective to the TCR/BCR-induced Ca2+ flux, several additional Ca2+ development in the absence of STIM1 (21, 28). It is believed channels have been detected in the plasma membrane that this occurred because of reduced NFAT translocation, of lymphocytes (39). which is necessary for the induction of FOXP3, a transcription factor required for differentiation and function of Tregs TRPs (29). Because Tregs are important for suppressing T effector The TRP channels are permeable for Ca2+ and sodium (Na+) (TEff) function and potentially deleterious immune responses, and are best known for their role as pain receptors in sensory STIM1 and STIM2 double deficiency provoked the devel- neurons (40). They contain six transmembrane domains, of opment of a type of lymphoproliferative disorder, a condition whichthetwomostC-terminalonesencompassthepore- characterized by the uncontrolled expansion of T cells (21). forming domain (41). Currently, 28 mammalian TRP Orai1 was also indispensable for differentiation of naive channel homologs have been described and can be divided CD4 T cells to TH17 cells because of impaired NFAT sig- into six subfamilies based on their amino acid sequence: naling, which would normally activate the TH17 lineage TRPC (canonical), TRPM (melastatin), TRPV (vanilloid), transcription factor retinoic-acid-receptor-related-orphan- TRPA (ankyrin), TRPML (mucolipin), and TRPP (poly- receptor (ROR) (30). TH1 and TH2 cell differentiation, in cystin). Of these TRP channels, TRPC, TRPM, TRPV, and contrast, was not affected by the absence of Orai1 (30). Given TRPA have previously been shown to play roles in the proinflammatory role of TH17 cells, in the absence of lymphocyte development or function. Orai1, the suppression of TH17 differentiation resulted in the TRPV1, for example, is known to contribute to the TCR- reduced severity of experimental autoimmune encephalomy- induced Ca2+ flux as a non-SOCE channel in CD4 T cells elitis (EAE), a murine model of multiple sclerosis. Similar to and is gated by phosphorylation, which is dependent on the phenotype exhibited by Orai1 deficiency, T cell–specific the lymphocyte-specific protein tyrosine kinase (LCK). Using 2 2 STIM1 and STIM2 deficiencies dampened EAE severity by a TRPV1 / mouse model, Bertin et al. showed that the 2+ impairing TH1 and TH17 proinflammatory cytokine pro- reduced Ca flux translated to impaired TCR signaling, duction (31). Intriguingly, graft-versus-host disease was also resulting in reduced NFAT and NF-kB translocation and 1024 BRIEF REVIEWS: CALCIUM CHANNELS IN LYMPHOCYTES subsequent defects in CD4 T cell activation and proin- Interestingly, multiple single-nucleotide polymorphisms in flammatory cytokine production (42). The role of TRPV1 in the TRPM3 gene from PBMCs appear to correlate with the cytokine production was also confirmed using TRPV1 an- disease, and although TRPM3 is expressed on B as well as tagonists in murine splenic T cells (43). Because of lower NK cells of healthy donors, its expression is reduced on those proinflammatory mediator production, TRPV1 deficiency of CFS/ME patients (57). Additionally, the Ca2+ influx upon was also shown to be protective in a mouse model of T cell– BCR stimulation or thapsigargin treatment (which induces mediated colitis (42). SOCE) was significantly reduced in the patients’ B cells TRP channels can also inhibit Ca2+ flux by modulating the compared with healthy controls, suggesting that TRPM3 is activity of one another. An example of this is TRPA1, an a store-operated Ca2+ channel (57). Although immunological ankyrin TRP channel that was found to inhibit the activity of dysfunctions have been reported in CFS/ME patients, it is TRPV1. It is thought that this inhibition is mediated by the unknown if they are causative of the disease. formation of heteromeric complex between TRPA1 and Different types of Ca2+ channels can influence one another, TRPV1, similar to what was suggested for Orai1 and Orai2 as a recent study linking a TRP and the CRAC channel earlier. In a mouse model of colitis, TRPA1 deficiency suggests (58). The authors demonstrated that SOCE was re- resulted in increased TRPV1 activation and therefore ampli- duced in the absence of TRPM7. Interestingly, TRPM7 is not fied TCR-induced Ca2+ flux and subsequent hyperactivation a store-operated Ca2+ channel itself but rather activates SOCE of inflammatory mediators, exacerbating the disease (44). by phosphorylation of CRAC components. One can hy-

TRPC channels (specifically TRPC5) have been found to be pothesize that this mechanism also applies to the previously Downloaded from important in mediating Treg-influenced inhibition of TEff mentioned TRPM3 channel. cells. Specifically, once bound to a Treg, TRPC5 becomes 2+ P2X receptors overexpressed in the interacting TEff cell, triggering a Ca flux and inhibiting the proliferation of that cell (45). Purinergic P2X receptors include seven different channels, of 2+ The exact mechanism of how this Ca flux can inhibit which the foremost receptors, P2X7 as well as P2X1, P2X4, TCR-induced proliferation, however, remains elusive. In and P2X5, were shown to be functional in T lymphocytes. http://www.jimmunol.org/ addition to TRPC5, other TRPC channels like TRPC3 These receptors are cation channels that can flux Ca2+ and 2+ and TRPC6 are also involved in T cell Ca flux, and in Na+ into the cell and potassium (K+) out of the cell. They do thecaseofTRPC3,thisCa2+ flux modulated cell prolif- so upon the binding of the energy metabolite and nucleic acid eration (46–48). building block ATP, giving it a role as a signaling molecule Another TRP channel important for T cell effector function (59). ATP is usually released from the mitochondria through is TRPM2 (49). A mouse model deficient in TRPM2 membrane channels, such as pannexin-1, by stressed or apo- exhibited reduced T cell proliferation, proinflammatory cy- ptotic cells that are sheared or under osmotic pressure. Con- tokine secretion, and thus reduced EAE severity after TCR sequently, ATP release is amplified and spread in an by guest on September 24, 2021 stimulation (49). In another study using Jurkat cells, it was autocrine/paracrine fashion by binding to P2X receptors on shown that Ca2+ flux by the TRPM2 channel could be ac- the cells from which the ATP was released, as well as neigh- tivated with the second messenger molecules cyclic ADP- boring cells, to induce apoptosis (60). This ATP-mediated ribose (cADPR) and nicotinic acid adenine dinucleotide apoptosis was found to play an important role in negative phosphate (NAADP) (50). Upon TCR stimulation and Ca2+ selection of thymocytes. Negative selection takes place in the entry, cADPR is released from the ER (51), from which it thymus during T cell maturation and is a critical event as it potentially binds to TRPM2, resulting in additional and results in the deletion of thymocytes with a self-reactive TCR. sustained Ca2+ release. Another member of the TRPM Apoptosis of cells due to negative selection causes the release family, TRPM4 is a monovalent channel that can flux Na+ of ATP and, in a paracrine fashion, induces apoptosis of into T cells. This, however, has profound impacts on Ca2+ neighboring thymocytes (60). Importantly, the P2X7 receptor signaling because the entry of Na+ depolarizes the plasma was found to be essential to this ATP-mediated induction of membrane, thereby reducing the driving force for Ca2+ in- apoptosis (61) Additionally, in mature T cells, P2X7 was flux during SOCE (52). In a TRPM4 Jurkat cell mutant, crucial for induction of cell death not only by ATP but also PHA treatment induced a prolonged Ca2+ influx and led to NAD (62). increased IL-2 production (52). A similar effect was seen in Despite this clear role of ATP signaling in apoptosis, a mouse TH2 cells, in which small interfering RNA-mediated baseline mitochondrial ATP production and signaling are knockdown of TRPM4 amplified Ca2+ fluxaswellasNFAT necessary for T cell homeostasis and for the cells’ ability to translocation and IL-2 production (53). TRPM4 knock- recognize Ag. This basal purinergic signaling is transmitted down in TH1-polarized cells, however, showed the opposite via P2X1 (63). Upon activation, T cells upregulate their effect (53). It is hypothesized that the differences in the two mitochondrial activity and therefore ATP secretion, which TH subsets are due to different expression levels of TRPM4 causes further autocrine activation via P2X receptors. The as well as distinct Ca2+ clearance dynamics. increased metabolism of T cells requires augmented cytosolic In B cells, very little is known about the role of TRP Ca2+ that is transported into the mitochondria to fuel ATP channels. Although TRPC1, 3, and 7 were all shown to be production. This Ca2+ is provided via the CRAC channel involved in BCR-induced signaling, these observations have so discussed earlier (64) and therefore demonstrates an inter- far been limited to the DT40 chicken B cell line (54–56). esting link between CRAC and P2X channels. The concen- Recently, however, a clinical study of patients with chronic tration of ATP seems to play a role in whether a T cell is fatigue syndrome/myalgic encephalomyelitis (CFS/ME) shed resting, becomes activated, or induces apoptosis (65). P2X some light into the role of TRPM3 in lymphocytes (57). activation by ATP was found to be essential for sustained The Journal of Immunology 1025

ERK signaling in murine T cells, and antagonizing P2X re- forms the pore in the membrane, which consists of four ceptors resulted in decreased IL-2 expression and T cell pro- homologous repeated domains (I–IV), each containing six liferation (66). Furthermore, inhibiting P2X receptors also led transmembrane segments (S1–S6). S5 and S6 are separated by to a reduced TCR-mediated Ca2+ flux and induced T cell a pore-forming loop containing an ion-selectivity filter, anergy (66). Similarly, in Jurkat cells the use of ATP scav- whereas the S4 region contains the voltage sensor. The aux- engers or P2X7 inhibitors caused a reduction of TCR- iliary subunits a2d and b do not take part in pore formation mediated Ca2+ flux and impaired NFAT translocation and but instead modulate the expression and biophysical prop- IL-2 production (67). Furthermore, P2X7 was essential for erties of the channel. The g subunit was found to constitute ATP-induced shedding of CD62L, CD27, and CD23, as well the L-type channel complex, but little is known about its as IL-6R, from T cells via the activation of metalloproteases. function (77). Several studies have now demonstrated that This shedding is usually induced upon T cell activation and the pore-forming CaV1 a1aswellastheb regulatory sub- converts the membrane proteins into soluble effector proteins units are, along with neuronal and muscle cells, expressed in (68–71). lymphocytes (78–80). Although P2X7 receptors were always uniformly distributed The first CaV channel that was found to be expressed on the cell surface, P2X1 and P2X4 receptors have also been in lymphocytes was CaV1.4, whose a1 subunit is encoded detected in human peripheral blood CD4 T cells but were by the Ca2+ voltage-gated channel subunit a1 F gene shown, together with pannexin-1, to translocate to the immune (CACNA1F ). CaV1.4 is best known for its role in the retina, 2+ synapse upon T cell activation (72). This might be important where it mediates Ca entry into photoreceptors. Mutations Downloaded from for the formation of Ca2+ microdomains and the increase of in CACNA1F have been linked to congenital stationary night local Ca2+ levels for TCR signaling next to the immunolog- blindness (81). Interestingly, the splice variants first found in ical synapse. Similar to P2X7, P2X1 and P2X4 were also the Jurkat T cell leukemia line and human peripheral blood important during T cell activation, as their pharmacological T lymphocytes differ from those in the retina (78). One of antagonism and genetic mutation reduced TCR-mediated them, called CaV1.4a, misses exons 31–34 and 37, which 2+ Ca flux as well as NFAT translocation and IL-2 synthesis translates to a deletion of the transmembrane segments S3, http://www.jimmunol.org/ (72). Given this generally inflammatory role of P2X receptors, S4, S5, and half of S6 of motif IV. The affected region in- it was also shown that P2X antagonism had a protective role cludes the voltage-sensing domain and might impact voltage in a diabetes and inflammatory bowel syndrome mouse model gating and reaction to depolarization (78). Additionally, a due to the suppression of cytokine production and T cell frameshift caused a change in the amino acid sequence of the proliferation. C terminus, resulting in a 40% homology with CaV1.1. Apart from autocrine signaling during T cell activa- CaV1.4b, another novel splice variant, is missing the exons 32 tion, ATP signaling can have paracrine effects on surrounding and 37, resulting in a deletion of the extracellular loop be- 2+

lymphocytes, particularly on their motility by inducing Ca tween S3 and S4 and part of the transmembrane segment S6 by guest on September 24, 2021 flux through P2X4 and P2X7. During T cell priming, T cells in motif IV. Although the voltage sensor is still present in this reduce their velocity, allowing them to better interact with splice variant, loss of the S3–S4 extracellular loop might still cognate dendritic cells. Interestingly, ATP released from these have an impact on the voltage gating characteristics, as it is in T cells can reduce the motility of bystander T cells, allowing close proximity (78). for the creation of lymphocyte clusters that can effectively Also, CaV1.1 was recently shown to exist as a splice variant tackle infection (73). in activated T cells. In this spliceoform, exon 29 was excised, Another study demonstrated that P2X signaling can also and the initial two N-terminal exons that are expressed in convert T cells into different subsets. In Tregs, P2X7 activation muscle cells were replaced with five new exons. Paralleling the inhibited the immunosuppressive role of the cells and instead earlier study describing the novel CaV1.4b splice variant promoted their conversion to TH17 cells in vivo. P2X7 inhi- reported by Kotturi et al. (78), skipping exon 29 of CaV1.1 bition, in contrast, promoted the differentiation of CD4 T cells resulted in the deletion of the linker region between S3 and into Tregs (74). Finally, a novel P2X5 transcript was discovered S4 next to the voltage sensor in domain IV. In human em- in human CD4 T cells, and its expression was upregulated upon bryonic kidney cells, the transfection with this splice variant T cell activation (75). Its small interfering RNA-mediated increased the basal Ca2+ levels, which was not the case in cells knockdown led to an increased production of IL-10 (75). transfected with a variant in which exon 29 was restored (82). Various splice variants of CaV1.4 were also found during the CaV channels extensive examination of retinal tissues, likely contaminated The CaV channels are expressed in neuronal and muscle cells, with blood cells like B and T lymphocytes. The discovery of 2+ where they flux Ca in response to membrane depolarization 19 splice variants of CaV1.4 suggests that splicing has a very 2+ (76). They are grouped into three major families, which are significant role in tuning CaV-dependent Ca currents (83). further divided into different subtypes based on their amino As already mentioned, novel splice variants have been shown acid sequence: the CaV1 family (CaV1.1–CaV1.4) contains L to possess altered gating characteristics (79) and are some- (long-lasting and large)-type channels; the CaV2 family con- times completely insensitive to membrane depolarization sists of P/Q (Purkinje)-type (CaV2.1), N (neuronal)-type (84). It therefore has been suggested that CaV channels (CaV2.2), and R (toxin-resistant)-type (CaV2.3) channels; in lymphocytes can be gated by Ag receptor signaling. Phar- and the CaV3 family (CaV3.1–CaV3.3) is also referred to as macological studies by Kotturi et al. (85) have shown that this the T (transient and tiny)-type channels (77). is indeed the case. Treating Jurkat cells as well as human The fully assembled CaV channels are structurally com- peripheral blood T cells with the CaV1 channel antagonist prised of the a1, a2d, b, and g subunits. The a1 subunit nifedipine severely reduced their TCR-induced Ca2+ flux, 1026 BRIEF REVIEWS: CALCIUM CHANNELS IN LYMPHOCYTES

2+ ERK phosphorylation, and IL-2 production. In Jurkat cells, This suggests that Ca currents mediated by CaV1.2 are the transcriptional activity of NFAT was reduced by nifedi- important in resting T cells and crucial for T cell survival, pine after TCR cross-linking. The CaV1 agonist Bay K8644, whereas the CRAC channel is important for T cell activation 2+ in contrast, increased intracellular Ca levels and phosphor- and effector function (80). As the role of CaV1.2 is similar to ylation of ERK. Later, the role of CaV1 channels in T cells the previously proposed function of CaV1.4, the question was also confirmed using genetically engineered mouse arises whether STIM1 can also inhibit CaV1.4. It remains models (79). Badou et al. (79) showed that mice lacking the to be seen if other CaV channels can be gated by STIM1 b3orb4 regulatory subunit exhibit an impaired TCR- interaction. mediated Ca2+ response. This further resulted in reduced Apart from the different subunits that are essential to form a NFAT translocation and compromised cytokine production CaV channel, it was found that the protein AHNAK1 acts as a of CD4 T cells. Additionally, the absence of the b regulatory scaffold protein for the channel. It is thought to physically subunits also led to a decreased expression of the pore- interact with the b regulatory subunits and thereby stabilize forming unit CaV1.1, which suggests that it might assemble the channel complex in the plasma membrane. Consequently, with the regulatory subunits and likely has a role in T cells AHNAK1 was also required for CaV1.1 a1 subunit expres- 2+ (79). Eventually, CaV1.1 was shown to be required for TCR- sion, and its deficiency caused a reduced TCR-induced Ca induced Ca2+ entry by the same group in knockdown ex- flux. This furthermore led to improper effector function of periments using lentiviral short hairpin RNA (82). CD4 T cells as well as cytolysis of target cells by cytotoxic

Whereas the b3 subunit is important for effector functions T cells (90, 91). Downloaded from of CD4 T cells, CD8 T cells require it for survival. The Additional studies highlight the role of CaV channels in number of CD8 T cells was significantly reduced in b3- leukocyte biology. CaV1.2 is expressed in human TH2 cells, deficient mice because of spontaneous apoptosis induced by where its antisense and pharmacological inhibition decreased high expression of the apoptosis-inducing cell surface receptor Ca2+ and cytokine responses in a protein kinase C–dependent Fas (first apoptosis signal receptor). This suggests that CaV1 manner (92). Recently, it has also been demonstrated that the channels provide a tonic survival signal that prevents CD8 inhibition of the a2d2 auxiliary subunit in CaV channels in http://www.jimmunol.org/ 2+ T cells from apoptosing (80). The remaining CD8 T cells TH2 cells is also sufficient to disrupt TCR-induced Ca flux exhibited an activated memory T cell phenotype along with and cytokine production (93). defects in TCR-induced Ca2+ flux and NFAT translocation Finally, T-type channels also play role in lymphocyte and proliferation (80). The authors also showed that the physiology. Recently, CaV3.1 was shown to be expressed in b3 subunit formed a complex with CaV1.4 in naive CD8 murine T lymphocytes (94). Interestingly, it retained its T cells and suggested that the observed phenotype in the b3- voltage-gated characteristics and conducted a substantial deficient mice can be attributed to impaired CaV1.4–b3 current at resting membrane potential in CD4 T cells but did

channel formation (80). not contribute to SOCE. The authors went on to demonstrate by guest on September 24, 2021 By using a CaV1.4–a1-deficient mouse model, our labo- that CaV3.1 deficiency had a protective role in an EAE mouse ratory has previously shown that CaV1.4 is essential for TCR- model, most likely because of reduced GM-CSF production induced SOCE into naive CD4 and CD8 T cells and by TH1 and TH17 cells (94). subsequent activation of the ERK and NFAT pathways (86). Other channels: IP3Rs, ryanodine receptors, The T cells in CaV1.4-deficient mice also displayed a memory T cell phenotype and upregulated activation markers, sug- and N-methyl-D-aspartate receptors gesting that CaV1.4 is necessary for naive T cell maintenance. IP3Rs are well described in the ER membrane, where they 2+ Upon Listeria monocytogenes infection, the CaV1.4 knockout release Ca from the ER stores into the cytoplasm upon (KO) mice exhibited severe immune deficiencies, as reflected binding of their cytosolic ligand IP3 (95). There have also in a reduced number of functional Ag-specific CD4 and CD8 been a few reports of IP3R presence in the plasma membrane T cells (86). Interestingly, CaV1.4-deficient T cells were still of T cells, although their role is not quite clear (96, 97). In sensitive to membrane depolarization. Because of the low DT40 chicken and mouse B cells, it was reported that only activation threshold of CaV1.4 and its relatively small current two functional IP3Rs are located in the plasma membrane, (87), it is possible that the channel is active in resting cells and where they conduct, despite their small number, a substantial contributes to tonic filling of intracellular Ca2+ stores. In BCR-induced Ca2+ current (98, 99). If true, these two high- addition to its role in the murine immune system, our labo- conductance IP3Rs can flux a similar amount of Ca2+ upon ratory has recently demonstrated that CaV1.4 deficiency can BCR stimulation as thousands of low-conductance Orai also lead to a new form of X-linked immunodeficiency in proteins (100). It would be interesting to identify the exact humans, thereby establishing its important function in the sites of influx of these plasma membrane IP3Rs to see what human immune system (F. Fenninger and W.A. Jefferies, proteins and signaling pathways are modulated by it. manuscript submitted for publication). In addition to IP3Rs, ryanodine receptors (RyRs) are located Although it has become clear that Ag receptor signaling can in the ER membrane. In cardiac and muscle cells, they release 2+ regulate CaV channels, an exact signaling pathway remains to Ca from intracellular stores upon excitation (101). The be elucidated. However, a very intriguing regulatory mech- pharmacogenetic disorder malignant hypothermia is caused 2+ anism for CaV1.2 and potentially other L-type Ca channels by RyR1 gain-of-function mutations that lead to an increased in general was described by Wang et al. (88) and Park et al. release of Ca2+ in myotubes of muscle. For easier diagnosis of (89). Apart from the activation of the CRAC channel de- the disease, which usually involves the surgical excision of scribed earlier, the Ca2+ sensor STIM1 was also found to muscle, studies investigated whether the increased Ca2+ release inhibit CaV1.2 by internalization upon TCR stimulation. can also be observed in B cells of affected individuals. Indeed, The Journal of Immunology 1027 stimulating B cells with a RyR1 agonist showed a greater Ca2+ consideration when evaluating results using this drug as an response in B cells of malignant hypothermia patients, which NMDAR inhibitor. harbor a RyR1 mutation, compared with controls and also correlated with a greater metabolic activity (102, 103). An- Conclusions other study demonstrated that caffeine, which is also used to Although the best-known contributor to Ag receptor– activate RyRs, increased intracellular Ca2+ concentrations in mediated Ca2+ flux in lymphocytes is the CRAC channel, the naive splenic lymphocytes from mice (104), further sup- role of many other Ca2+ channels that are found in the plasma porting the notion that RyRs transport Ca2+ in lymphocytes. membrane remains poorly understood. In fact, not long ago, Similar to the aforementioned TRPM2 channel, RyR there was doubt that additional plasma membrane Ca2+ activity was found to be ligand-modulated by the second channels resided in the membranes of lymphocytes. Studies messenger molecule cADPR (105). In T cells, cADPR has on CaV channels in lymphocytes eventually settled this de- Ca2+-mobilizing properties, and pharmacological studies in bate, and the literature has been expanding since this time to Jurkat T cells have confirmed that these properties can also include a constellation of leukocyte Ca2+ channels. be conferred through RyRs (106). As another study showed The expression of many of these channels may be restricted that the flux of Ca2+ from RyRs was induced upon SOCE to specific developmental stages and subsets of lymphocytes, (107), it is likely that cADPR release after SOCE activates giving rise to the hypothesis that they all control very specific RyRs to sustain the induced Ca2+ influx. mechanisms. Indeed, we have seen that Ca2+ channels play key 2+ The functional role that RyR-mediated Ca flux plays roles during lymphocyte development, particularly negative Downloaded from in lymphocytes is not very well explored, but one recent re- selection, homeostasis, and TH cell polarization. However, the 2+ port claims that RyR inhibitors reduced the amount of TH1 most prominent role of Ca channels appears to be in T cell and TH17 cells in cultured primary human T cells (108). The inflammation, in which we have seen the involvement of authors further reported that a gain-of-function mutation of many different channels, including the CRAC channel, TRP RyR1 in an EAE mouse model exacerbated the disease and a channels, P2X receptors, and CaV channels. Although some

RyR1 inhibitor reduced CNS inflammation and neurologic channels might act in parallel and have to some extent re- http://www.jimmunol.org/ symptoms (108). Although exact signaling mechanisms have dundant roles, it is perplexing how the KO of a single channel not been explored, this suggests a proinflammatory role for sometimes almost completely abrogates the TCR/BCR- RyR-mediated Ca2+ currents. induced Ca2+ flux and effector functions and raises the 2+ N-Methyl-D-aspartate (NMDA) receptors are ionotropic question of what role is left for other Ca channels in these glutamate receptors that permeate Ca2+ upon binding of the processes. One possible explanation for this phenomenon is neurotransmitter L-glutamate. They are best described in the formation of heteromeric channels, as we have seen with neurobiology, where their dysfunction has been associated Orai1/Orai2 and TRPV1/TRPA1. Although in these specific

with several neurologic disorders such as stroke, epilepsy, examples one subunit has an inhibitory role, it would also be by guest on September 24, 2021 and Alzheimer disease (109). However, NMDARs have also possible that both subunits have activating properties and the been identified in lymphocytes, and there is some evidence absence of either one will disrupt the Ca2+ signaling. We have that they regulate lymphocyte function (9). Pharmacological also seen that STIM1 not only regulates Ca2+ flux through studies, for example, demonstrated that NMDAR antago- Orais but it also regulates CaV1.2. It is likely that STIM1/2 nists can inhibit thapsigargin-induced Ca2+ flux in T cells, also control other channels, which would be a reason why a suggesting a role for NMDARs during SOCE (110). An- STIM1/2 double-KO B cell exhibits an almost complete ab- other study also found that NMDAR antagonists reduced sence of BCR-induced flux. Furthermore, ligand-gated Ca2+ TCR-mediated Ca2+ mobilization, resulting in an impair- channels like the P2X receptors are activated by second ment of NFAT and ERK signaling as well as reduced effector messenger molecules to amplify or sustain Ca2+ currents. In functions like cytokine production and proliferation (111). many cases, the activating ligands, however, are only secreted Lastly, by using pharmacological blockers in thymocytes, upon lymphocyte activation so that the P2X receptors are Affaticati et al. (112) showed that NMDARs were required only activated downstream of an initial Ca2+ flux. Therefore, for caspase-3 activation and, thus, induction of apoptosis CRAC channel or other SOCE deficiencies will most likely during negative selection. During this process, NMDARs also lead to a reduced flux in these downstream channels. migrate to the immunological synapse where the thymocytes Additionally, the spliceoforms that are expressed in and Ag-pulsed dendritic cells interact with each other (112). lymphocytes are different from those found in neuronal and The authors suggest that glutamate released by the dendritic muscle cells, where these channels were originally identified. cells binds to the NMDARs found on the thymocytes, Often the lymphocyte-specific splicing mechanisms alter the 2+ triggering the Ca signaling pathway and subsequent gating characteristics of the channels, particularly that of CaV caspase-3–induced cell death. channels. Although most channels directly or indirectly are All of the mentioned publications investigating the role of responsive to Ag receptor cross-linking, the mechanisms that NMDARs used the pharmacological drug MK801, which, lead to the opening/closing of the channels remain for the apart from NMDARs, was also found to impair the con- most part elusive. ductivity of the K+ channels Kv1.3 and KCa3.1 (111). These Despite many unanswered questions, because of the mostly channels also play a crucial role during T cell activation, and inflammatory role of Ca2+ channels, blocking them is a importantly, the role of NMDARs during T cell activation promising avenue for therapeutic drug intervention for au- demonstrated using MK801 could not be replicated when toimmune disorders and complications due to transplant re- using a NMDAR-deficient mouse model (111). Therefore, jection. By using small molecules or Abs that bind to the the effect of MK801 on K+ channels should be taken into extracellular portion of a channel, it should be possible to 1028 BRIEF REVIEWS: CALCIUM CHANNELS IN LYMPHOCYTES modulate their activity and, for example, block the produc- 19. Zhang, S. L., A. V. Yeromin, X. H.-F. Zhang, Y. Yu, O. Safrina, A. Penna, J. Roos, K. A. Stauderman, and M. D. Cahalan. 2006. Genome-wide RNAi screen of Ca tion of harmful cytokines. Particularly Orais, P2X receptors, (2+) influx identifies genes that regulate Ca(2+) release-activated Ca(2+) channel and CaV channels, whose ablation decreases lymphocyte ef- activity. Proc. Natl. Acad. Sci. USA 103: 9357–9362. 20. Gwack, Y., S. Srikanth, M. Oh-Hora, P. G. Hogan, E. D. Lamperti, fector function, have therapeutic potential. The fact that the M. Yamashita, C. Gelinas, D. S. Neems, Y. Sasaki, S. Feske, et al. 2008. Hair loss inhibition or genetic deletion of many of these channels is and defective T- and B-cell function in mice lacking ORAI1. Mol. Cell. Biol. 28: protective during autoimmune CNS inflammation, inflam- 5209–5222. 21. Oh-Hora, M., M. Yamashita, P. G. Hogan, S. Sharma, E. Lamperti, W. Chung, matory bowel syndrome, colitis, and graft-versus-host disease M. Prakriya, S. Feske, and A. Rao. 2008. Dual functions for the endoplasmic highlights their suitability as druggable targets. Finally, if reticulum calcium sensors STIM1 and STIM2 in T cell activation and tolerance. Nat. Immunol. 9: 432–443. it becomes possible to inhibit the specific splice variants 22. Vig, M., W. I. DeHaven, G. S. Bird, J. M. Billingsley, H. Wang, P. E. Rao, that exist in lymphocytes, therapies could target lymphocyte A. B. Hutchings, M.-H. Jouvin, J. W. Putney, and J.-P. Kinet. 2008. Defective subset-specific isoforms while leaving other cells untouched mast cell effector functions in mice lacking the CRACM1 pore subunit of store- operated calcium release-activated calcium channels. Nat. Immunol. 9: 89–96. and thereby prevent adverse side effects. 23. Vaeth, M., J. Yang, M. Yamashita, I. Zee, M. Eckstein, C. Knosp, U. Kaufmann, P. Karoly Jani, R. S. Lacruz, V. Flockerzi, et al. 2017. ORAI2 modulates store- operated calcium entry and T cell-mediated immunity. Nat. Commun. 8: 14714. Acknowledgments 24. Beyersdorf, N., A. Braun, T. Vo¨gtle, D. Varga-Szabo, R. R. Galdos, S. Kissler, T. Kerkau, and B. Nieswandt. 2009. STIM1-independent T cell development and We thank C.G. Pfeifer for editorial assistance as well as S.R. Stanwood and effector function in vivo. J. Immunol. 182: 3390–3397. C.J. Lu for critical proofreading. 25. McCarl, C.-A., S. Khalil, J. Ma, M. Oh-hora, M. Yamashita, J. Roether, T. Kawasaki, A. Jairaman, Y. Sasaki, M. Prakriya, and S. Feske. 2010. 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