Ion Channels and Transporters in Lymphocyte Function and Immunity

REVIEWS

Ion channels and transporters in lymphocyte function and immunity

Stefan Feske1, Edward Y. Skolnik2 and Murali Prakriya3 Abstract | Lymphocyte function is regulated by a network of ion channels and transporters in the plasma membrane of B and T cells. These proteins modulate the cytoplasmic concentrations of diverse cations, such as calcium, magnesium and zinc ions, which function as second messengers to regulate crucial lymphocyte effector functions, including cytokine production, differentiation and cytotoxicity. The repertoire of ion-conducting proteins includes calcium release-activated calcium (CRAC) channels, P2X receptors, transient receptor potential (TRP) channels, potassium channels, chloride channels and magnesium and zinc transporters. This Review discusses the roles of ion conduction pathways in lymphocyte function and immunity.

Ion channels and ion transporters function as gateways Store-operated calcium channels Ion channels 2+ Pore-forming transmembrane for charged ions that cannot freely diffuse across lipid Ca is a well-established second messenger in lympho proteins that enable the flow of membrane barriers. They regulate the intracellular cytes that regulates proliferation, gene expression, motil- ions down an electrochemical concentration of various ions, such as calcium (Ca2+), ity and other functions. Similarly to in other mammalian gradient. magnesium (Mg2+) and zinc (Zn2+). The movement of cell types, the intracellular Ca2+ concentration in unstim-

Ion transporters these cations across the plasma membrane depends on ulated B and T cells is maintained at ~50–100 nM, which 4 2+ Pore-forming transmembrane electrical gradients that are maintained in turn by potas- is ~10 -fold lower than the Ca concentration in the proteins that carry ions sium (K+), sodium (Na+) and chloride (Cl−) channels. serum. Following antigen binding to the T cell receptor against a concentration In the past couple of years, fundamental progress has (TCR) or B cell receptor (BCR), the intracellular Ca2+ gradient using energy, been made towards identifying the molecules that con- concentration can increase to ~1 μM1. Several ion chan- typically in the form of ATP. trol the function of Ca2+ release-activated Ca2+ channels nels have been identified in lymphocytes that mediate 2+ 1 (FIG. 1; TABLE 1) 1Department of Pathology, (CRAC channels) — which are the predominant antigen Ca influx . In the following sections, we 2+ New York University Langone receptor-activated Ca channels in lymphocytes — and discuss store-operated CRAC channels as well as P2X Medical Center, New York, channels that mediate Mg2+ and Zn2+ influx in T cells. purinoreceptor channels, transient receptor potential New York 10016, USA. We discuss the mechanisms that regulate the function of (TRP) channels and voltage-gated Ca2+ (Ca ) channels. 2Helen L. and Martin V S. Kimmel Center for Biology these ion channels in lymphocytes and review their roles and Medicine at the Skirball in immunity and their emerging potential for therapeutic CRAC channels. Antigen binding by the TCR or BCR Institute for Biomolecular immunomodulation. is coupled — via protein tyrosine kinases — to the Medicine; Division of Several other ion channels, pumps and organelles activation of phospholipase Cγ1 (PLCγ1) in T cells Nephrology, Department of are also required for the regulation of ion homeostasis and PLCγ2 in B cells and the generation of the lipid Medicine; and Department of 2+ Pharmacology, New York in lymphocytes. For example, transient increases in the metabolite InsP3. InsP3 promotes the release of Ca 2+ 2+ University Langone Medical intracellular Ca concentration are mediated by the from ER stores, and this leads to Ca influx across the Center, New York, release of Ca2+ from endoplasmic reticulum (ER) stores plasma membrane, a process termed store-operated Ca2+ New York 10016, USA. via Ca2+-permeable inositol‑1,4,5‑trisphosphate receptor entry (SOCE)2 (FIGS 1,2). The store-operated Ca2+ chan- 3Department of Molecular ryanodine receptor Pharmacology and Biological (InsP3 receptor) and (RYR) channels. nels of T cells, known as CRAC channels, have been 2+ 3,4 Chemistry, Northwestern Conversely, Ca is cleared from the cytoplasm by uptake extensively characterized and are distinguished by an University, Feinberg School of into mitochondria via the mitochondrial Ca2+ uniporter extremely high ion selectivity for Ca2+ and a low conduct- Medicine, Chicago, (MCU)190,191 and into the ER via sarcoplasmic/endoplas- ance5 (TABLE 1). CRAC channels are activated through Illinois 60611, USA. mic reticulum Ca2+ ATPases (SERCAs) and by Ca2+ export the binding of the ER Ca2+ sensors stromal interaction Correspondence to S.F. plasma membrane Ca2+ ATPases e-mail: [email protected] through (PMCAs). Owing molecule 1 (STIM1) and STIM2 to the CRAC channel doi:10.1038/nri3233 to space limitations, these intracellular ion channels proteins ORAI1, ORAI2 and ORAI3 (also known as Published online 15 June 2012 and transporters are not discussed here. CRACM1, CRACM2 and CRACM3)6.

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TCR BCR Ca2+ >> Mg2+, Na+ Ca2+, Na+, Mg2+ ORAI1 CRAC Ca2+ release-activated Ca2+ ORAI2? channel P2X 2+ –3 channels ORAI3? [Ca ]o ~10 M (CRAC channels). Highly 2+ –7 2+ [Ca ]i ~10 M (resting) Ca -selective ion channels ζ ζ 2+ –6 [Ca ]i ~10 M (activated) Negative located in the plasma Igα Igβ – membrane membrane that are encoded – potential by ORAI proteins. – (–60 mV) PLCγ1 PLCγ2 SOCE Inositol‑1,4,5‑trisphosphate K 3.1 receptor Ca2+ – Ca (InsP receptor). A – 3 InsP3 2+ Ca -permeable channel STIM1 or K+ located in the membrane of STIM2 K 1.3 the endoplasmic reticulum (ER) – V InsP R that mediates the release of 3 – 2+ Ca from ER stores following SERCA Ca2+ Calcineurin Na+ binding by the second Ca2+ ER TRPM4 messenger InsP . 3 2+ –3 [Ca ]ER ~0.5–1x10 M Ryanodine receptor P NFAT (RYR). A Ca2+-permeable channel located in the membrane of the sarcoplasmic NF-κB Cytokine expression reticulum (SR) and p50 p65 endoplasmic reticulum (ER) CREB MEF2 NFAT Diﬀerentiation Proliferation that mediates the release of Cytoplasm Nucleus Ca2+ from the SR or ER stores following binding by the second messenger cyclic ADP-ribose or Ca2+ itself. Figure 1 | Ion channels regulating calcium signalling in lymphocytes. Ca2+ release-activated Ca2+ (CRAC) channels are activated following the engagement of antigen receptors (that is, T cell receptors (TCRs)Nature or B cell Reviews receptors | Immunology (BCRs)). Sarcoplasmic/endoplasmic This is mediated through the activation of phospholipase Cγ (PLCγ), the production of inositol‑1,4,5‑trisphosphate reticulum Ca2+ ATPases (InsP ) and the release of Ca2+ from endoplasmic reticulum (ER) Ca2+ stores1,6,17. The ensuing activation of stromal 2+ 3 (SERCAs). Ca pumps located interaction molecule 1 (STIM1) and STIM2 results in the opening of ORAI1 CRAC channels and store-operated Ca2+ entry in the membrane of the (SOCE) (for details, see FIGS 2,3). Sustained Ca2+ influx through CRAC channels leads to the activation of Ca2+-dependent endoplasmic reticulum (ER) that move Ca2+ from the enzymes and transcription factors, including calcineurin and nuclear factor of activated T cells (NFAT). P2X receptors, 2+ 2+ cytoplasm into the ER through such as P2X4 and P2X7, are non-selective Ca channels activated by extracellular ATP. Ca influx in lymphocytes the hydrolysis of ATP. depends on the gradient between the extracellular Ca2+ concentration (~1 mM) and the intracellular Ca2+ concentration + + (~0.1 μM) and on an electrical gradient established by two K channels (namely, KV1.3 and KCa3.1) and the Na -permeable Plasma membrane Ca2+ channel TRPM4 (transient receptor potential cation channel M4)76,92. CREB, cAMP-responsive-element-binding protein; ATPases InsP3R, InsP3 receptor; MEF2, myocyte-specific enhancer factor 2; NF-κB, nuclear factor-κB; SERCA, sarcoplasmic/ (PMCAs). A family of ion endoplasmic reticulum Ca2+ ATPase. transport ATPases located in the plasma membrane that export Ca2+ from the cytoplasm. Identification of ORAI1. An important milestone in the channel protein and appears to be the predominant iso- 6,17 Store-operated Ca2+ entry identification of ORAI1 as the prototypical CRAC chan- form mediating SOCE in lymphocytes . By contrast, (SOCE). A Ca2+-influx process nel was the discovery that human patients with a severe there is no direct functional or genetic evidence for a role triggered by the depletion of form of combined immunodeficiency (CID) lack func- of ORAI2 or ORAI3 channels in immune cells as yet. 2+ endoplasmic reticulum Ca 7–11 stores and activation of plasma tional CRAC channels and SOCE in T cells . ORAI1 membrane ORAI Ca2+ channels was identified nearly simultaneously by three laborato- Activation of CRAC channels. The activation of ORAI by STIM proteins. ries as the gene encoding this CRAC channel by linkage CRAC channels involves a complex series of coordinated analysis in patients with CID and using RNA interfer- steps, during which STIM proteins fulfil two crucial roles. Ion selectivity ence (RNAi) screens for regulators of SOCE and nuclear First, they sense the depletion of ER Ca2+ stores, and sec- The specificity of an ion factor of activated T cells 12–14 channel for a particular species (NFAT) function . ORAI1 is ond, they communicate store depletion to the CRAC 18–20 2+ of ion, for example Ca2+, Mg2+, a widely expressed surface glycoprotein with four pre- channels (FIG. 2). In resting cells with replete Ca Na+ or K+. Non-selective dicted transmembrane domains, intracellular amino and stores, STIM proteins are diffusely distributed through- channels do not discriminate carboxyl termini (BOX 1; FIG. 2) and no sequence homol- out the ER membrane18,21. Following the depletion of between different types of ion. ogy to other ion channels except its homologues ORAI2 Ca2+ stores, STIM proteins are activated, oligomerize Conductance and ORAI3. CID arises from a single amino acid substi- and redistribute into discrete puncta located in junc- A measure of the ability of an tution (R91W) in ORAI1 that abrogates CRAC channel tional ER sites that are in close proximity to the plasma ion channel to carry electrical activity12. All three ORAI isoforms form Ca2+ channels membrane22–25. In these puncta, STIM1 colocalizes with charge. The conductance is with broadly similar functional properties when ectopi- and interacts directly with ORAI1 to activate the CRAC determined by dividing the 26 electrical current by the cally expressed, although they differ in their inactivation channel . The formation of overlapping STIM1–ORAI1 potential difference (voltage) characteristics, pharmacological properties and tissue puncta involves direct binding of a cytoplasmic domain and is measured in siemens. expression15,16. ORAI1 remains the best-studied CRAC of STIM1 to the N and C termini of ORAI1 (REFS 27–29)

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Table 1 | Properties and functions of ion channels and transporters in lymphocytes Channel Selectivity Activation Function in lymphocytes Associated channelopathies Calcium channels ORAI1 Ca2+ Antigen receptor stimulation and B, T and NK cell proliferation, cytokine CRAC channelopathy 2+ depletion of ER Ca stores by InsP3, production and/or cytotoxicity in vitro; with immunodeficiency, resulting in activation by STIM1 and STIM2 immunity to infection, T cell-mediated autoimmunity, muscular autoimmunity and inflammation, and hypotonia and ectodermal

allogeneic T cell responses in vivo; TReg dysplasia caused by mutations cell development in STIM1 and ORAI1 ORAI2, Ca2+ ND ND ND ORAI3 TRPC Ca2+, Na+ ND ND ND

2+ + TRPM2 Ca , Na ADP-ribose, cADP-ribose, H2O2, NAADP ND ND TRPM4 Na+ Intracellular Ca2+ Depolarization of the membrane ND potential; cytokine production

2+ + CaV1.2, Ca CaV currents in T cells are not well Cytokine production; CD8 T cell ND 2+ + CaV1.3, documented; CaV-dependent Ca influx is survival; CD8 T cell-mediated immunity CaV1.4 activated by an unknown mechanism (not to infection; TH2 cell function in asthma depolarization) following TCR stimulation;

CaV function is inhibited by STIM1 P2X7 Ca2+, Na+, Extracellular ATP T cell proliferation; cytokine production; ND

other promotes TH17 cell and inhibits TReg cell cations differentiation P2X1, P2X4 Ca2+, Na+ Extracellular ATP T cell proliferation; cytokine production; ND thymocyte apoptosis Magnesium channels and transporters TRPM7 Ni2+ > Zn2+ > Upstream cellular activation mechanism Thymocyte development; production of ND Mg2+, Ca2+ unknown; regulators include intracellular thymocyte growth factors; proliferation 2+ Mg , PtdInsP2 and extracellular pH and survival of DT40 B cells MAGT1 Mg2+ TCR stimulation; activation mechanism CD4+ T cell development and activation; XMEN syndrome caused by unknown immunity to infection (with EBV) X‑linked mutations in MAGT1 Zinc transporters ZIP3, ZIP6, Zn2+ Activation mechanism unknown; requires T cell activation (ZIP6); T cell Acrodermatitis enteropathica ZIP8 TCR stimulation (ZIP6) development (ZIP3)? with immunodeficiency is caused by mutations in the intestinal transporter ZIP4 ZNT Zn2+ ND ND ND Potassium channels

+ Kv1.3 K Membrane depolarization Regulation of the membrane potential; ND T cell activation (in TH17 and TEM cells); cytokine production; T cell-mediated autoimmunity and inflammation

+ 2+ KCa3.1 K Intracellular Ca Hyperpolarization of the membrane ND potential; T cell activation (in TH1, TH2 and TCM cells); cytokine production; autoimmune colitis Chloride channels

− − − Clswell Cl (I , Br ) Molecular identity of the channel is unknown; Apoptosis in T cells ND cell swelling activates Clswell currents CFTR Cl− cAMP Cytokine production by T cells? ND

− GABAA Cl Extracellular GABA Inhibition of T cell proliferation, ND cytokine production, cytotoxicity and T cell-mediated autoimmunity This table includes most of the ion channels and transporters reported to be functional or expressed in lymphocytes. Some molecules, such as CRAC channels and K+ channels, are well studied and widely recognized to have important roles in lymphocyte function. By contrast, our understanding of the properties and roles of − 2+ 2+ other channels (including TRPC, CaV and Cl channels as well as Zn transporters) is still in its infancy and requires further clarification. CaV, voltage-gated Ca channel; CFTR, cystic fibrosis transmembrane conductance regulator; EBV, Epstein–Barr virus; ER, endoplasmic reticulum; CRAC, Ca2+ release activated Ca2+; + 2+ + 2+ GABA, γ‑aminobutyric acid; InsP3, inositol‑1,4,5‑trisphosphate; KV, voltage-gated K channel; KCa, Ca -activated K channel; MAGT1, Mg transporter protein 1; NAADP, nicotinic acid adenine dinucleotide phosphate; ND, not determined; NK, natural killer; PLC, phospholipase C; STIM, stromal interaction molecule; TCM, central memory T; TCR, T cell receptor; TEM, effector memory T; TH,T helper; TReg, regulatory T; TRP, transient receptor potential; XMEN, X‑linked immunodeficiency with Mg2+ defect and EBV infection and neoplasia; ZIP, ZRT/IRT-like protein; ZNT, zinc transporter.

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2+ ORAI1 Ca ORAI1 CD4 TCR (closed) Recruitment (open) of ORAI1 Plasma into puncta LAT membrane

ζζ PLCγ1 LCK PtdInsP2 ZAP70 SLP76 CC C C C C

InsP3

STIM1 CC2 Ca2+ CAD K

ER CC1 InsP R membrane CC3 3 Translocation to ER–plasma membrane junctions ER lumen SAM and into puncta Conformational

2+ change and EF hand Ca2+ Ca oligomerization

Ca2+ dissociation from EF hand EF hand–SAM unfolding

Figure 2 | The molecular choreography of CRAC channel activation. In resting lymphocytes, the endoplasmic reticulum 2+ 2+ 2+ Combined (ER) Ca stores are full, and Ca is bound to the EF hand Ca -binding domain in the amino terminusNature ofReviews stromal | interactionImmunology immunodeficiency molecule 1 (STIM1) and STIM2 (not shown). The stimulation of T cell receptors (TCRs) or B cell receptors (BCRs; not shown) (CID). CID is caused by causes the activation of antigen receptor-proximal signalling cascades and the production of inositol‑1,4,5‑trisphosphate inherited defects in T cell 2+ (InsP3), resulting in the release of Ca from the ER through InsP3 receptors (InsP3Rs), which are non-selective ion channels. function (but not T cell The fall in ER Ca2+ concentration leads to the dissociation of Ca2+ from the EF hand domain in STIM1, the unfolding of the development). By contrast, STIM1 N terminus and the multimerization of STIM1 proteins6. STIM1 multimers translocate to junctional ER sites at which severe CID (SCID) is caused by inherited defects in T cell the ER membrane is juxtaposed with the plasma membrane. STIM1 multimers form large clusters, into which they recruit 2+ 2+ (and in some cases B cell) ORAI1 tetramers, which are the functional unit of Ca release-activated Ca (CRAC) channels. A minimal CRAC channel development. SCID and CID activation domain (CAD) in the carboxyl terminus of STIM1 is necessary and sufficient for ORAI1 binding, CRAC channel result in severe (often lethal) activation and store-operated Ca2+ entry29,184,186,187. This domain contains two coiled-coil (CC) domains, which interact infections in early infancy. with a CC domain in the C terminus and additional domains in the N terminus (not shown) of ORAI1(REF. 27) . LAT, linker for activation of T cells; PLCγ1, phospholipase Cγ1; SAM, sterile alpha motif; ZAP70, ζ-chain-associated protein kinase of 70 kDa. Nuclear factor of activated T cells (NFAT). A family of Ca2+-dependent transcription (BOX 1). Lymphocytes express two closely related STIM other immune cells (reviewed in REF. 34). Autosomal factors that are activated via dephosphorylation by the isoforms, STIM1 and STIM2, and both mediate SOCE recessive mutations in the human genes ORAI1 (which is 30,31 phosphatase calcineurin. in B and T cells . Like STIM1, STIM2 also binds to located at 12q24) and STIM1 (which is located at 11p15) They mediate the expression and activates ORAI1 CRAC channels, but it does so fol- abolish CRAC channel function and Ca2+ influx in of many cytokine genes in lowing smaller decreases in the ER Ca2+ concentration B cells, T cells and natural killer (NK) cells. This results lymphocytes. and with slower kinetics than STIM1 (REFS 32,33). This, in CID with increased susceptibility to severe infections 35,36 CRAC channelopathy and the higher expression levels of STIM1 compared with viruses (especially herpesviruses ), bacteria and 36,37 CRAC channel dysfunction with STIM2 in naive mouse T cells, may explain why fungal pathogens (such as Candida albicans ) (BOX 1; caused by autosomal recessive STIM2‑deficient T cells have initially normal Ca2+ levels TABLE 1). The combination of CID with autoimmunity mutations in ORAI1 and after TCR stimulation but fail to sustain Ca2+ influx. By and associated non-immunological clinical symptoms STIM1 that results in a CRAC channelopathy38,39 pathognomonic clinical contrast, STIM1‑deficient T cells display a near-complete is referred to as . 31 + + combination of lack of SOCE . CD4 and CD8 T cells from ORAI1- and immunodeficiency, STIM1‑deficient patients and mice show defective autoimmunity, congenital Control of lymphocyte function by CRAC channels. production of many cytokines, including interleukin‑2 muscular hypotonia and Genetic studies in patients with mutations in ORAI1 or (IL‑2), IL‑4, IL‑17, interferon‑γ (IFNγ) and tumour ectodermal dysplasia with 7,40 impaired dental enamel STIM1 genes and in mice lacking functional Orai1, Stim1 necrosis factor (TNF) . This is partly due to impaired 2+ calcification and sweat gland and/or Stim2 genes have established important and non- activation of the Ca -dependent transcription factor dysfunction. redundant roles for CRAC channels in lymphocytes and NFAT 7,31. SOCE-deficient human T cells also fail to

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Box 1 | Molecular structure of the CRAC channel components ORAI1 and STIM1 ORAI1 is localized in the plasma membrane and constitutes the pore-forming subunit of ORAI1 L194P the Ca2+ release-activated Ca2+ (CRAC) Plasma E106 channel. The channel is formed by the membrane A103E assembly of four ORAI1 subunits6, of which the first transmembrane (TM1) domains TM2 TM4 TM1 TM3 line the channel pore178,179. The selectivity Cytoplasm filter of the CRAC channel is formed by a K quartet of glutamate‑106 (E106) residues R91W R429C that form a high-affinity Ca2+-binding site to A88SfsX25 S/P provide the CRAC channel with high Ca2+ CC CC3 selectivity180–183. Analyses of other pore-lining residues in TM1 indicate that the CRAC CC2 CAD channel pore is narrow178,179, potentially explaining its low conductance (that is, the C 2+ small number of Ca ions passing through it), N STIM1 which limits the increase in intracellular Ca2+ CC1 concentration following channel opening. ER membrane 1538–1G>A The intracellular carboxyl terminus of ORAI1 features a coiled-coil (CC) domain that TM comprises a binding site for stromal interaction molecule 1 (STIM1). STIM1 and ER lumen SAM STIM2 (not shown) are single-pass membrane proteins in the endoplasmic reticulum (ER). E128RfsX9 (E128X) Their N termini are located in the lumen of Ca2+ EF hand the ER and contain an EF hand Ca2+-binding domain that allows them to sense the ER Ca2+ concentration. Mutations in the EF hand domain of STIM1 result in impaired Ca2+ binding and constitutive activation of CRAC channelsNature independently Reviews | Immunology of ER Ca2+ store depletion18,21. The second and third coiled-coil domains (CC2 and CC3) in the C terminus of STIM1 are part of a minimal CRAC channel activation domain (CAD; also known as SOAR, OASF and CCb9)29,184,186,187, which binds directly to ORAI1 to activate CRAC channels. A lysine (K)-rich domain at the end of the C terminus of STIM1 facilitates its recruitment to the plasma membrane. Autosomal recessive mutations in ORAI1 and STIM1 that have been identified in patients with CRAC channelopathy are indicated by stars12,35–38. These mutations abolish CRAC channel function and store-operated Ca2+ entry, either by eliminating channel function (in the case of the R91W substitution) or by abolishing ORAI1 or STIM1 protein expression (all other mutations shown). SAM, sterile alpha motif.

proliferate in response to TCR or mitogen-mediated and T cell-independent antibody responses follow- stimulation8,10,36,37. This dependence of lymphocyte ing immunization were normal in Stim1−/− mice43 and effector functions on SOCE is not limited to T cells. Stim1flox/floxStim2flox/flox Mb1–Cre mice30. It is noteworthy, NK cells from an ORAI1‑deficient patient showed however, that in some ORAI1- and STIM1‑deficient impaired production of IFNγ, TNF and CC‑chemokine patients, antibodies specific for the recall antigens ligand 2 (CCL2) and failed to degranulate and kill tar- diphtheria and tetanus toxoid were absent44. get tumour cells41. Moreover, B cells from mice lacking In humans, immunodeficiency in STIM1‑deficient ORAI1, or STIM1 and STIM2, exhibit a decrease in (and to a lesser extent ORAI-deficient) patients is asso- BCR-induced proliferative responses (but not in pro- ciated with autoimmunity characterized by haemolytic liferative responses that are dependent on CD40 liga- anaemia, thrombocytopenia and lymphoproliferative dis- tion or lipopolysaccharide)30,42. SOCE is also required ease. This autoimmunity is probably due to the reduced hi + + + for the production of IL‑10, especially by CD1d CD5 numbers of CD25 FOXP3 regulatory T (TReg) cells found 37 regulatory B cells. The impaired expression of this anti- in these patients . A more profound reduction in TReg inflammatory cytokine in mice with a B cell-specific cell numbers is observed in the thymus and secondary deletion of Stim1 and Stim2 was associated with exac- lymphoid organs of mice with a combined T cell- erbated autoimmune inflammation in the central nerv- specific deletion of Stim1 and Stim2 (REF. 31). In addi-

ous system (CNS) in the experimental autoimmune tion, TReg cells deficient in both STIM1 and STIM2 show encephalomyelitis (EAE) model of multiple sclero- severely impaired suppressive function31. Accordingly, sis30. However, despite the profound defect in SOCE these mice develop massive splenomegaly, lymph in B cells from ORAI1- and STIM1‑deficient patients adenopathy and pulmonary inflammation. The depend-

and mice, CRAC channels do not have a major role in ence of TReg cell development and function on SOCE is antibody production. Serum immunoglobulin levels probably explained by the Ca2+-dependent activation in these patients are not reduced, and T cell-dependent of NFAT and the role of this transcription factor in the

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Ca2+, Na+, Mg2+ K+ rejection and autoimmune inflammation. CD4+ T cell- dependent skin contact hypersensitivity responses are ATP (paracrine, abolished in ORAI1‑deficient mice, and these mice TCR ATP cell death) CRAC also fail to efficiently reject MHC-mismatched skin 45 + −/− ORAI1 channel Pannexin 1 P2X1, P2X4 allografts . Likewise, CD4 T cells from Stim1 mice or P2X7 mediate slower and attenuated acute graft-versus-host disease (GVHD) compared with wild-type T cells when ζζ PLCγ1 Plasma transferred to fully allogeneic mice43. When investigated membrane ? for their ability to mediate autoimmunity in animal ATP models of multiple sclerosis and inflammatory bowel Ca2+ Ca2+ K+ disease (IBD), CRAC channel-deficient T cells from ORAI1-, STIM1- and STIM2‑deficient mice failed to STIM1 45–47 Ca2+ ATP induce disease . T helper 1 (TH1) and TH17 cells are crucial mediators of inflammation in these models as Calcineurin in their human disease counterparts48,49. Accordingly, Mitochondrion IκBα ERK1 or IFNγ and IL‑17 production by CRAC channel-deficient ERK2 P NFAT p50 p65 T cells isolated from the CNS-draining lymph nodes and mesenteric lymph nodes of mice with EAE and IBD, NF-κB respectively, was severely impaired, indicating that SOCE 45–47 is required for the function of TH1 and TH17 cells . Disease severity correlated with residual SOCE in T cells, Nucleus FOXP3 as STIM1‑deficient mice were completely protected NF-κB JUN or from developing EAE, whereas mice lacking STIM2 p50 p65 47 46 NFAT FOS RORγt showed either delayed onset or reduced severity of disease. Collectively, these studies emphasize the crucial importance of CRAC channels for T cell immunity.

Figure 3 | P2X receptors are non-selective calcium channels mediating T cell Other calcium-permeable ion channels activation. P2X receptors are homotrimeric ion channels locatedNature in Reviews the plasma | Immunology membrane P2X purinoreceptor channels. P2X receptors are a family of lymphocytes. They form non-selective ion channels that allow the influx of Ca2+, Na+ of non-selective ion channels (FIG. 3) that are activated and other cations52,55. P2X1, P2X4 and P2X7 are activated by extracellular ATP, for which by extracellular ATP and allow the influx of Na+, Ca2+ they have distinct affinities52. P2X7 is unusual among P2X receptors, as it functions as a REF. 52 non-selective cation channel at low extracellular ATP concentrations, but forms large and other cations (reviewed in ). At least three 2+ pores following prolonged exposure to high extracellular ATP concentrations. In different P2X receptors have been implicated in Ca addition, it was reported to mediate the K+ efflux required for NLRP3 (NOD-, LRR- and influx in human T cells: P2X1, P2X4 (REF. 53) and P2X7 pyrin domain-containing 3) inflammasome activation in innate immune cells188. The ATP (REF. 54). Their opening, especially that of P2X7, causes required for P2X receptor opening in T cells originates from dying cells, ATP-secreting cells Ca2+ influx and the activation of downstream signalling (a paracrine source) or the T cells themselves (an autocrine source). T cells were shown to molecules such as calcineurin, resulting in the prolifera- release ATP through pannexin 1 hemichannels following T cell receptor (TCR) stimulation tion of B and T cells55,56 and IL‑2 production53,57. RNAi- 58 2+ and mitochondrial ATP production . The opening of P2X receptors results in Ca influx, mediated depletion of P2X1, P2X4 and P2X7 or their 2+ which has been suggested to synergize with store-operated Ca entry to activate pharmacological inhibition with P2X receptor antago- Ca2+-dependent signalling molecules and transcription factors, resulting in enhanced nists decreases Ca2+ influx, NFAT activation and IL‑2 cytokine expression. P2X7‑dependent activation of extracellular signal-regulated kinase 1 (ERK1) or ERK2 was shown to repress the transcription of forkhead box P3 (FOXP3) in production following TCR stimulation in Jurkat T cells + 53,54 favour of retinoic acid receptor-related orphan receptor‑γt (RORγt) expression, thereby and human CD4 T cells . Potential sources of the promoting the differentiation of CD4+ T cells into T helper 17 cells59. IκBα, NF-κB inhibitor-α; ATP required for P2X receptor activation include the NFAT, nuclear factor of activated T cells; NF-κB, nuclear factor-κB; PLCγ1, phospholipase Cγ1. T cells themselves, which are reported to release ATP in an autocrine manner through pannexin 1 hemichannels that colocalize with P2X7 at the immunological syn- expression of forkhead box P3 (FOXP3)50,51. By contrast, apse53,58 (FIG. 3). It has been suggested that autocrine ATP

the development and suppressive function of TReg cells signalling in T cells via P2X receptors serves to amplify from ORAI1- or STIM1‑deficient mice were only mod- weak TCR signals, gene expression and T cell effector erately impaired43,45, indicating that residual Ca2+ influx functions52. — which is probably mediated by ORAI2 or ORAI3, Although the biophysical properties of P2X chan-

and STIM2, respectively — is sufficient for TReg cell nels in lymphocytes remain poorly characterized, sev- development and function. Taking these data together, eral lines of evidence suggest that P2X receptors regulate CRAC channels emerge as important regulators of T cell responses in vivo. The inhibition of all P2X recep- T cell-dependent self-tolerance and autoimmunity. tors with oxidized ATP protects mice from diabetes following the adoptive transfer of T cells specific for CRAC channels mediate autoimmunity and inflam- pancreatic β‑cells and from colitis in an adoptive T cell- mation. CRAC channels in T cells are crucial not only transfer model of IBD58. Protection was associated with

for host defence to infection and TReg cell function, but impaired production of IL‑17, IFNγ and TNF, suggesting also for T cell-mediated hypersensitivity, allotransplant that P2X receptor signalling is required for the function

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of pro-inflammatory T cells58. Further analysis revealed most investigators. Although it is theoretically possible

that P2X7 controls the differentiation of TH17 cells and that CaV channels in T cells are activated by other path- + + CD4 CD25 TReg cells, as stimulation of TReg cells with the ways that are depolarization independent, this remains P2X7 agonist BzATP inhibited the expression of FOXP3 speculative. Complicating the picture further are recent

but enhanced the levels of the TH17 cell-specific tran- studies that report the inhibition of CaV channels by scription factor retinoic acid receptor-related orphan STIM1 (REFS 72,73), which raises the possibility that the 59 receptor‑γt (RORγt) . A similar ATP-dependent con- functions of CRAC and CaV channels might be recip-

version into TH17 cells was not observed in TReg cells rocally regulated. Evidence against a significant role of −/− from P2x7 mice. P2X7 signalling in T cells therefore CaV channels in T cells comes from human CaV channel appears to be pro-inflammatory by mediating the dif- genes with loss-of-function mutations, which are not 74 ferentiation and function of TH17 cells and by inhibit- associated with an overt immunological phenotype , 59 ing the stability of TReg cells . However, the effects of and from pharmacological inhibitors of L‑type CaV P2X7 on adaptive immune responses are not always this channels (such as nifedipine), which are in wide clini- unambiguous, as several studies using P2x7−/− mice have cal use for cardiovascular diseases but have no reported alternatively shown an increased60 or decreased61 suscep- effects on immune function. In the absence of a thor-

tibility to autoimmune CNS inflammation in EAE. The ough validation of CaV channel currents by patch-clamp

cause of these discrepancies is not known. Future studies measurements and a molecular mechanism for CaV will need to carefully address which P2X receptors con- channel activation in T cells, the effects of genetic dele- 2+ tribute to the influx of Ca and other cations in T cells at tion of CaV channel components on T cell function and physiological ATP concentrations and which P2X recep- immune responses remain difficult to interpret. tors regulate adaptive immune responses in vivo, taking advantage of the various P2X receptor knock-out mice Channels controlling membrane potential that have been generated62–64. Ca2+ influx in lymphocytes depends on a negative membrane potential that provides the electrical driving force 2+ 75 Voltage-gated calcium channels. CaV channels are for Ca entry . Two classes of channels regulate the highly Ca2+-selective channels that mediate Ca2+ influx membrane potential in lymphocytes: K+ channels and in response to the depolarization of excitable cells, TRPM4 channels. such as myocytes, cardiomyocytes and neurons65. All + members of the L‑type family of CaV channels (CaV1.1, Potassium channels. K channels protect against mem- + CaV1.2, CaV1.3 and CaV1.4) and their regulatory β3 and brane depolarization by mediating the efflux of K β4 subunits were found to be expressed in human and to hyperpolarize the plasma membrane76. The best- mouse T cells, and several studies have reported the studied K+ channels that predominately regulate mem-

presence of truncated or alternatively spliced CaV iso- brane potential in lymphocytes are the voltage-gated 66–68 + 2+ + forms . Recent genetic studies in mice have impli- K channel KV1.3 and the Ca -activated K channel + + 2+ cated CaV channels in T cell function. CD4 and CD8 KCa3.1 (also known as KCNN4, IKCa or SK4). KV1.3 T cells from mice with mutations in the genes encoding is a homotetramer of four α‑subunits, each composed of the β3 and β4 subunits had a partial reduction in Ca2+ six transmembrane segments (S1–S6), and is activated influx in response to TCR stimulation and impaired by membrane depolarization77. Depolarization of the IL‑4, IFNγ and TNF production66,69. The impaired Ca2+ membrane is sensed by four arginine residues that are influx in β3‑deficient CD8+ T cells was associated with localized in the S4 segment, and this results in a con- 69 78 a lack of CaV1.4 protein expression . Likewise, naive formational change that causes channel opening . By + + 2+ + CD4 and CD8 T cells from CaV1.4‑deficient mice had contrast, KCa3.1 is a Ca -activated K channel, but it has 2+ impaired TCR-induced Ca influx. CaV1.4‑deficient a similar membrane topology and pore architecture to

mice failed to mount an effective T cell response to KV1.3. However, rather than containing a voltage sensor, 70 infection with Listeria monocytogenes , and this was the C terminus of KCa3.1 is constitutively bound to cal associated with reduced cytotoxic function of CD8+ modulin, and channel opening occurs after Ca2+ binds to 70 79 T cells . CaV1.4‑deficient T cells also had increased calmodulin . KCa3.1 channels powerfully hyperpolarize rates of cell death70, which is consistent with the previ- the membrane following elevations in the intracellular ously reported role of the β3 subunit in CD8+ T cell sur- Ca2+ concentration and help to sustain the driving 69 2+ vival . RNAi-mediated depletion of CaV1.2 and CaV1.3 force for Ca entry. In addition to its requirement 2+ 2+ expression in T cells reduced TCR-induced Ca influx in for Ca , KCa3.1 channel activity depends on a class II

TH2 cells, attenuated IL‑4 production and reduced airway phosphoinositide 3‑kinase (PI3K), PI3K‑C2β, which inflammation in a mouse model of allergic asthma71. increases the concentration of phosphatidylinositol-

Despite these intriguing findings, the role of CaV 3‑phosphate (PtdIns(3)P) in the plasma membrane. channels in lymphocytes remains highly controver- This allows the histidine kinase nucleoside diphosphate sial. A major gap in our understanding of the role of kinase B (NDKB; also known as NM23H2) to activate

L‑type CaV channels in lymphocytes is whether they KCa3.1 by phosphorylating histidine‑358 in the C termi- Membrane potential function as Ca2+ channels or facilitate Ca2+ influx by nus of K 3.1 (REFS 80,81). In agreement with the finding The difference between the Ca electrical potential inside and other mechanisms. Cell depolarization — which is the that both PtdIns(3)P and histidine phosphorylation of outside a cell. It is typically canonical mechanism for activating CaV channels — fails KCa3.1 are crucial for KCa3.1 activation, the PtdIns(3)P 60 to 80 mV in resting cells. − − to evoke typical CaV channel currents in the hands of phosphatase myotubularin-related protein 6 and the

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histidine phosphatase phosphohistidine phosphatase 1 from patients with multiple sclerosis99. Similar studies 2+ inhibit KCa3.1, TCR-stimulated Ca influx and the have shown an increase in disease-associated TEM cells proliferation of activated naive human CD4+ T cells in patients with type 1 diabetes, rheumatoid arthritis

by dephosphorylating PtdIns(3)P and KCa3.1, respec- and psoriasis, and the treatment of these diseases with 80,82 tively . In addition, the E3 ubiquitin ligase tripartite a KV1.3 blocker (ShK or PAP‑1) led to the ameliora- 90,100–102 motif-containing protein 27 (TRIM27; also known as tion of disease . Consistent with a role for KV1.3 2+ 88 RFP) inhibits KCa3.1 and TCR-stimulated Ca influx in the activation of TH17 cells , KV1.3 blockers may have and cytokine production by ubiquitylating and inhibiting a therapeutic benefit in autoimmune diseases driven by 83 103,104 PI3K‑C2β . TH17 cells . By contrast, TH1 and TH2 cells depend 88 The relative contributions of KV1.3 and KCa3.1 in on KCa3.1 for their activation . Inhibition of KCa3.1 lymphocyte Ca2+ influx are determined primarily by protected mice from developing colitis in two mouse 88 their expression levels, which are dependent on the models of IBD , suggesting that KCa3.1 may be a novel lymphocyte subset and its state of activation. Under therapeutic target to treat patients with Crohn’s disease resting conditions, CCR7+CD45RA+ naive human or ulcerative colitis.

T cells predominantly express KV1.3 channels and 84 depend on KV1.3 for activation . Following activa- TRPM4 channels. TRPM4 channels are expressed by

tion, naive human T cells upregulate KCa3.1 expres- T cells and many other immune cells. Unlike most 85 sion , and inhibition of KCa3.1 in pre-activated T cells other TRP channels, their role in lymphocyte func- blocks TCR-stimulated Ca2+ influx and proliferation is well documented. TRPM4 channels mainly 86,87 + + tion . Furthermore, mouse TH1 and TH2 cells pre- conduct Na and K and, in contrast to other TRP 2+ (REF. 105) dominantly express KCa3.1 and depend on KCa3.1 for channels, are only weakly permeable to Ca . TCR-stimulated Ca2+ influx and cytokine produc- The activation of TRPM4 channels — which occurs 2+ tion, whereas TH17 cells mainly express KV1.3 and in response to increases in intracellular Ca concen- + require KV1.3 for their activation and production of tration — results in Na influx, membrane depolari- IL‑17 (REF. 88). Differential use of K+ channels is also zation and a reduction in the electrical driving force 2+ observed in effector memory T (TEM) and central for Ca influx. TRPM4 channels thus provide a nega- 76,80,89,90 memory T (TCM) cells . When activated at sites tive feedback mechanism for the regulation of SOCE 2+ of inflammation, TEM cells (which have the phenotype and were proposed to prevent cellular Ca overload. − low − CCR7 CD62L CD45RA ) produce various cytokines, Given that TRPM4 and KV channels elicit opposing including IFNγ, IL‑4 and IL‑5, and exclusively upregu- effects on membrane potential, it remains to be eluci-

late KV1.3 expression. By contrast, TCM cells (which are dated precisely how TRPM4 works together with KV1.3 + hi − CCR7 CD62L CD45RA ) upregulate their expression and KCa3.1 to regulate changes in membrane potential 2+ of KCa3.1 following activation in lymph nodes and and intracellular Ca concentration. Overexpression

mucosal lymphoid organs. As a result, KV1.3 block- of a dominant-negative mutant of TRPM4 or deple-

ers are effective inhibitors of TEM cells, whereas KCa3.1 tion of TRPM4 using RNAi in Jurkat T cells resulted 2+ blockers are effective at inhibiting TCM cells. in enhanced Ca signalling and increased IL‑2 pro- 2+ 106 Given their prominent role in regulating Ca signal- duction . Similar effects were observed in mouse TH2 2+ ling, KV1.3 and KCa3.1 have emerged as important drug cells, in which TRPM4 was shown to regulate Ca 91,92 targets . KV1.3 can be specifically inhibited by sev- levels, motility and the production of IL‑2 and IL‑4 eral potent peptide toxins (such as Stichodactyla toxin by controlling the nuclear translocation of NFAT107. (ShK), which is derived from sea anemone venom) and Mast cells from Trpm4−/− mice had higher levels of oral small-molecule inhibitors (such as Psora‑4 and Ca2+ influx, degranulation and histamine release than 93–95 PAP‑1) . Several specific inhibitors of KCa3.1 chan- wild-type mast cells following stimulation of the high- nels have also been developed and include TRAM34 affinity Fc receptor for IgE (FcεRI); accordingly, the (REFS 96,97) and ICA‑17043 . Inhibitors of KV1.3 and acute passive cutaneous anaphylactic response medi- 108 KCa3.1 have been very useful for studying the role of ated by IgE was more severe in these mice . The way K+ channels in immune responses in vivo, especially as in which the enhanced Ca2+ influx that occurs in the

KV1.3‑deficient mice have no overt T cell defect owing absence of TRPM4 affects lymphocyte-dependent to the upregulation of a Cl− channel that compensates for immune responses in vivo remains to be elucidated. (REF. 98) the loss of KV1.3 . TRP channels The finding that KV1.3 and KCa3.1 function to activate distinct lymphocyte subsets provides an oppor- In humans, TRP channels form a large superfamily of 28 tunity to more selectively target lymphocyte subsets cation channels, which can be divided into 7 subfami- for therapeutic purposes. Studies in a rat model of lies109. T cells predominantly express channels belong-

multiple sclerosis have revealed that KV1.3 expression ing to the TRPC and TRPM subfamilies, including is upregulated and required for the proliferation of TRPC1, TRPC3, TRPC5, TRPM2, TRPM4 and TRPM7 encephalitogenic T cells, and the treatment of rats with (REF. 110) (TABLE 1). Most TRP channels are non-selective 2+ + KV1.3 blockers in models of EAE markedly ameliorated and permeable to several cations, including Ca and Na disease89. The relevance of these findings to humans (REFS 111,112). We briefly discuss the role of TRPC and was demonstrated by the observation of high levels of TRPM2 channels; TRPM7 channels will be discussed 2+ KV1.3 expression by myelin-reactive T cells isolated further below in the context of Mg signalling.

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TRPC channels. Members of the TRPC subfamily RNA and ATP. Although >90% of all cellular Mg2+ is (TRPC1–TRPC7) form non-selective cation channels, in the form of Mg‑ATP123, ~5% is free and can poten- and their activation is generally linked to the stimula- tially function as a second messenger similar to Ca2+. tion of plasma membrane receptors that are coupled to Mg2+ is required for the proliferation of mitogen- PLCγ111,113. Before the identification of ORAI1 as the stimulated T cells124,125, and the stimulation of T cells main CRAC channel protein12–14, there was avid interest through the TCR results in a transient increase in the in the possibility that TRPC channels contribute to SOCE intracellular Mg2+ concentration126. Recent studies pro- in T cells. A recent study showed that RNAi-mediated vide evidence for an important role of TRPM7, a Mg2+- depletion of TRPC3 has a moderate effect on SOCE and permeable channel, and Mg2+ transporter protein 1 T cell proliferation110. Expression of TRPC5 was reported (MAGT1), a Mg2+ transporter, in T cell function and to increase following the activation of mouse CD4+ and development126,127. CD8+ T cells and to mediate Ca2+ influx in response to crosslinking of the ganglioside GM1 by the B subunit of TRPM7 channels. TRPM7 is a ubiquitously expressed cholera toxin114; whether TRPC5 mediates TCR-induced non-selective cation channel that exhibits nearly equal Ca2+ influx has not been examined. Although these stud- permeabilities for Mg2+ and Ca2+ (REF. 128) (FIG. 4). ies were generally interpreted as supporting a role for TRPM7 channels are thought to regulate cellular and TRPC channels in SOCE, recent evidence has questioned whole-body Mg2+ homeostasis because of their high whether TRPC channels are activated by store deple- Mg2+ permeability. This is supported by evidence tion115. Overall, the biophysical and immunological evi- indicating that mutations in the gene encoding the dence for a significant role of TRPC channels in SOCE, closely related TRPM6 channel cause hypomagne- lymphocyte function and adaptive immune responses is saemia owing to impaired renal and intestinal Mg2+ unclear and awaits further evaluation. absorption129–131. Direct evidence for a role of TRPM7 in immune function came from genetic deletion of TRPM2 channels. TRPM2 is a non-selective Ca2+- TRPM7 in DT40 chicken B cells; the mutant cells failed permeable channel that is activated by intracellular to proliferate, showed increased cell death and had ADP-ribose and regulated by several intracellular factors, reduced total cellular levels of Mg2+ (REF. 132). These including Ca2+, cyclic ADP-ribose (cADP-ribose), hydro- defects could partially be rescued by growing the cells

gen peroxide (H2O2), nicotinic acid adenine dinucleotide in a medium containing high (10 mM) extracellular phosphate (NAADP) and AMP116,117. TRPM2 channels levels of Mg2+ (REF. 132). mediate stress-induced Ca2+ signals in a diverse group In vivo, an important role for TRPM7 in T cell of immune cells116,117. In T cells, TRPM2 expression was development was identified using mice with a T cell- found to increase after TCR stimulation110, and endog- specific deletion of Trpm7. Trpm7flox/− Lck–Cre mice enous TRPM2 currents could be activated by cADP- had a severe block in T cell development at the double- ribose, ADP-ribose and NAADP118. Although there is no negative (CD4−CD8−) stage, resulting in reduced direct evidence that TRPM2 is required for Ca2+ influx in numbers of double-positive (CD4+CD8+) and single- lymphocytes or for T cell function, TCR stimulation has positive (CD4+) T cells in the thymus as well as been reported to evoke the release of cADP-ribose from decreased numbers of T cells in the spleen127. The lack the ER119, thus potentially activating TRPM2 in T cells. of TRPM7 in T cells was associated with impaired Studies in myeloid cells indicate that cADP-ribose and expression of growth factors such as fibroblast growth

H2O2 synergize in the activation of TRPM2 (reviewed in factor 7 (FGF7), FGF13 and midkine, and consequently REFS 116,117 ). As H2O2 is produced by several immune with a progressive loss of medullary thymic epithe- cell types under inflammatory conditions, Ca2+ influx lial cells (mTECs)127. A central question that remains through TRPM2 has been investigated as a potential unresolved is whether the primary role of TRPM7 in mediator of reactive oxygen species (ROS)-induced T cells is in Mg2+ influx and homeostasis. Given that pathologies120,121. TRPM2‑deficient mice are largely there is little or no Mg2+ gradient across the plasma resistant to colitis induced by dextran sulphate sodium membrane192 (FIG. 4), it is unclear how TRPM7 channels owing to defects in Ca2+ influx, in nuclear factor‑κB can induce Mg2+ influx. Although TRPM7 currents in activation and in the production of CXC-chemokine thymocytes from Trpm7flox/− Lck–Cre mice were mark- ligand 2 (CXCL2) by monocytes, as these defects result edly reduced, Mg2+ influx and total cellular Mg2+ con- in impaired neutrophil infiltration into the gut122. In addi- tent were normal, consistent with the idea that TRPM7 tion, TRPM2 inhibits ROS production in phagocytic cells is not required for Mg2+ influx in T cells127. So, how by attenuating the function of NADPH oxidase and pre- does TRPM7 control T cell development and func- vents endotoxin-induced lung inflammation in mice120. tion? A possible explanation is that the main function Whether TRPM2 channels are modulated by ROS in of TRPM7 in T cells is to promote Ca2+ not Mg2+ influx, T cells and regulate T cell responses during inflammation which is consistent with the documented Ca2+ perme- in vivo remains to be elucidated. ability of TRPM7 (REF. 133). Alternatively, it cannot be excluded that TRPM7 regulates T cell development Magnesium channels and transporters through its C-terminal kinase domain, although a Mg2+ is the most abundant divalent cation in eukary- recent study showed that the role of TRPM7 in apop- otic cells. It binds to and regulates the function of many tosis in T cells depends on its channel — but not kinase polyphosphate-containing molecules, such as DNA, — function193. Although the biophysical mechanisms of

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a Ni2+ > Zn2+> Mg2+ > Ca2+, Mn2+ b Mg2+ >> Ca2+, Zn2+, Ni2+ TRPM7 MAGT1 CD4 TCR ORAI1 CRAC Plasma 2+ LAT channel membrane [Mg ]o ~0.8mM

TRP 2+ PLCγ1 PtdInsP2 [Mg ]i ~0.5–1mM ζζ LCK ZAP70 Ca2+ SLP76 Kinase C ? InsP N domain 3 Mg2+ Mg2+ ? Ca2+ STIM1

2+ • Mg homeostasis? InsP3R • Lymphocyte survival • T cell activation and proliferation? • CD4+ T cell • T cell development development ER

Figure 4 | Magnesium channels and transporters in lymphocytes. a | TRPM7 (transient receptor potential cation 2+ channel M7) is a Mg -permeable channel that is known as a ‘chanzyme’ because it functionsNature both Reviews as an ion | channelImmunology and as an enzyme through its carboxy-terminal serine/threonine kinase domain. As with other TRP channels, its ion channel pore is located between the fifth and sixth transmembrane domains. TRPM7 is a non-selective cation channel and conducts Mg2+ and Ca2+ with near equal permeabilities. One of the defining features of the TRPM7 channel is its inhibition by intracellular Mg2+, but the mechanism of Mg2+ regulation is incompletely understood76,128. TRPM7 function further depends 133 2+ on phosphatidylinositol‑4,5‑bisphosphate (PtdInsP2) and is regulated by the extracellular pH . b | Mg transporter protein 1 (MAGT1) belongs to a family of recently identified Mg2+ transporters. It is highly selective for Mg2+ over Ca2+, Zn2+, Ni2+ and other divalent cations134. MAGT1 opening in response to T cell receptor (TCR) stimulation results in a global increase in the intracellular Mg2+ concentration, activation of phospholipase Cγ1 (PLCγ1) and Ca2+ influx, presumably via Ca2+ release-activated Ca2+ (CRAC) channels. The mechanisms by which TCR signalling causes MAGT1 to open and the way in which Mg2+ activates PLCγ1 are not understood. Two MAGT1 isoforms have been described: a short one (335 amino acids) with a confirmed tetraspanning membrane topology135 and a longer version (367 amino acids)126 that is predicted to contain five transmembrane domains and an intracellular amino terminus, which may facilitate the TCR-dependent

activation of MAGT1. ER, endoplasmic reticulum; InsP3R, inositol‑1,4,5‑trisphosphate receptor; LAT, linker for activation of T cells; STIM1, stromal interaction molecule 1; ZAP70, ζ-chain-associated protein kinase of 70 kDa.

TRPM7 function and regulation and its role in lympho- One of the main functions of Mg2+ influx through cyte function remain in debate, it is noteworthy that MAGT1 is the activation of PLCγ1 and Ca2+ influx, as TRPM7 is the only ion channel identified so far that is TCR crosslinking of MAGT1‑deficient T cells resulted required for lymphocyte development. in delayed activation of PLCγ1 and abolished SOCE126. By contrast, proximal TCR signalling events — such as MAGT1. MAGT1 is a Mg2+ transporter that is essential the phosphorylation of CD3ε, ζ‑chain-associated pro- for Mg2+ signalling in T cells (FIG. 4). It was discovered in tein kinase of 70 kDa (ZAP70) and linker for activation two independent screens and has little sequence similar- of T cells (LAT) — occurred normally126. The mecha- ity to other ion channels or transporters134,135. MAGT1 nisms by which Mg2+ influx through MAGT1 regulates is highly selective for Mg2+ and does not conduct Ca2+, PLCγ1 activation are not understood (FIG. 4). Another Zn2+, Ni2+ or other divalent cations when expressed in open question is how TCR stimulation activates MAGT1 xenopus oocytes134. It mediates Mg2+ influx in T cells, and thus Mg2+ influx, especially as MAGT1 has only two and RNAi-mediated depletion of MAGT1 in these cells short intracellular domains available to interact with resulted in a moderate decrease in cytoplasmic Mg2+ cytoplasmic signalling molecules135 (FIG. 4). Despite these concentrations135. The importance of MAGT1 and unresolved questions, the profound immunological Mg2+ signalling in T cells is emphasized by patients with effects of MAGT1 deficiency validate the important role inherited mutations in MAGT1 who suffer from a rare form of Mg2+ in T cell function. of immunodeficiency126 (TABLE 1). Patients with XMEN disease (X‑linked immunodeficiency with magnesium Zinc transporters defect and EBV infection and neoplasia) suffer from CD4+ Zinc is an essential trace element and a structural lymphocytopenia and increased susceptibility to viral component of numerous metalloproteins, such as infections, particularly with Epstein–Barr virus (EBV), zinc finger-containing transcription factors, through owing to a loss of MAGT1 protein expression and Mg2+ which it contributes to immune function (reviewed in influx following TCR stimulation. In contrast to T cells, REFS 136–138). In addition, emerging evidence suggests B cells develop and function normally in these patients, that Zn2+ regulates lymphocyte function directly as a sec- which is consistent with the lack of Mg2+ influx in control ond messenger. The free intracellular Zn2+ concentration B cells stimulated by IgM- or CD40‑specific antibodies. in lymphocytes is very low (~0.35 nM)139, whereas that

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in the serum is ~16 μM140, establishing a >104-fold gradi- lysosomal stores142, respectively. When primary human ent between extracellular and intracellular Zn2+ concen- CD4+ T cells were stimulated by incubation with DCs, 2+ trations that can potentially be exploited for signalling their intracellular Zn concentrations increased within purposes. Stimulation of human and mouse T cells by 1 minute of the formation of immunological synapses IL‑2 or incubation with DCs results in rapid increases between the T cells and DCs. This increase in the in the intracellular Zn2+ concentration accompanied by intracellular Zn2+ concentration and the subsequent T cell proliferation and cytokine production, suggest- expression of CD25 and CD69 depended on ZIP6. ing a potentially important role for Zn2+ in lymphocyte Interestingly, increases in the intracellular Zn2+ concen- signal transduction141–143. tration were spatially restricted to the immunological The role of Zn2+ in immunity is highlighted by the synapse143, potentially as a result of rapid sequestration inherited Zn2+-malabsorption syndrome acroderma- by Zn2+-binding proteins such as metallothionein. titis enteropathica, which is caused by impaired Zn2+ Another mechanism to limit increases in the uptake through the zinc transporter ZRT/IRT-like intracellular Zn2+ concentration is provided by ZNT protein 4 (ZIP4) in the intestinal epithelium144,145. transporters, which mediate the uptake of Zn2+ into The acrodermatitis enteropathica phenotype includes intracellular organelles or promote Zn2+ export across immunodeficiency with recurrent infections in ~30% the plasma membrane. Of the ten ZNT transporters in of patients. Immunodeficiency is associated with thy- mammalian cells, only a few are known to be functional mus atrophy and lymphopenia146,147, which have been in immune cells. ZNT5 is required for mast cell func- attributed to increased glucocorticoid production and tion157,158, but the ZNT molecules controlling intracel- the apoptosis of immature B and T cells147. Several lular Zn2+ concentrations in lymphocytes remain to be in vitro studies have shown that Zn2+ is required for elucidated. It is noteworthy that primary human B and T cell proliferation143,148 and for the production of T cells express ZNT1, ZNT4, ZNT6 and ZNT7 (REF. 157) cytokines such as IL‑2 and IFNγ149. At higher con- and that in T cells the expression of mRNA encoding centrations, however, Zn2+ was shown to inhibit the these transporters was strongly reduced following stim- proliferation of mouse T cells150 and the expression ulation by phytohaemagglutinin. Thus, downregulation of cytokines by Jurkat T cells151 and human CD4+ of ZNT levels may be a means to maintain elevated T cells142. The molecular mechanisms underlying these intracellular Zn2+ concentrations during T cell activa- concentration-dependent effects of Zn2+ are only par- tion. Despite these leads, the overall role of Zn2+ trans- tially understood. FIGURE 5 shows some of the signal- porters in immune function, immune cell development ling pathways in lymphocytes that are either activated and adaptive immunity remains poorly understood. or inhibited by Zn2+ (reviewed in REFS 136–138). For instance, increases in the intracellular Zn2+ concentra- Chloride channels tion were reported to enhance the activation of kinases Several Cl− channels that allow the flux of Cl− anions such as LCK and PKC152, but to inhibit the phosphatase across the plasma membrane were reported to be active calcineurin153,154 (FIG. 5). More recently, Zn2+ influx was in lymphocytes and to control the function of these reported to mediate T cell activation by enhancing cells. Volume-regulated Cl− channels (also known as

the phosphorylation of ZAP70 and by decreasing the Clswell channels) open following the swelling of T cells in recruitment of the tyrosine phosphatase SHP1 to the a hypotonic environment, resulting in the efflux of Cl−, TCR, thereby prolonging Ca2+ influx143. and ultimately water, from the cell, and thus a return The proteins that control Zn2+ levels in lymphocytes to normal cell volume76,159,160. The osmotic activation of and their molecular regulation are still poorly defined. Cl− channels in Jurkat T cells depends on the SRC family Two classes of Zn2+ transporters have been described to kinase LCK161. Interestingly, the induction of apoptosis regulate the intracellular Zn2+ concentration: ZIP trans- in T cells by crosslinking of FAS (also known as CD95) porters (also known as SLC39A family transporters) and induces Cl− currents in a LCK-dependent manner162, zinc transporters (ZNTs; also known as SLC30A family suggesting that Cl− channels may regulate apoptosis in transporters). ZIP and ZNT proteins are localized either T cells. A further analysis of the physiological role of in the plasma membrane or in the membranes of intra- volume-regulated Cl− channels in lymphocytes is ham- cellular organelles, where they mediate Zn2+ influx (in pered, however, by the fact that the molecular identity of the case of ZIP transporters) or Zn2+ efflux (in the case of these channels is unknown (reviewed in REF. 76). ZNTs) into or from the cytoplasm, respectively (reviewed Several studies have demonstrated the expression of in REF. 155) (FIG. 5). GABA (γ‑aminobutyric acid) receptors in human, mouse 163,164 Fourteen mammalian ZIP genes have been iden- and rat T cells . GABAA receptors are heteropenta- tified137. ZIP3 is highly expressed by CD34+ human meric ligand-gated Cl− channels, and their inhibitory role haematopoietic stem cells, and genetic deletion of Zip3 in neuronal function in the CNS is well established165. in mice resulted in the depletion of CD4+CD8+ T cells GABA-activated Cl− currents have been reported in in the thymus under Zn2+-limiting conditions. By con- mouse and rat T cells and macrophages164,166,167. GABA trast, the numbers of single-positive (CD4+ or CD8+) administration inhibited T cell proliferation, the produc- thymocytes were increased, which suggests accelerated tion of IL‑2 and IFNγ as well as the cytotoxic function of T cell maturation156. In T cells, the Zn2+ transporters CD8+ T cells in vitro163,164,168,169. In vivo, the administra- ZIP6 and ZIP8 were reported to mediate Zn2+ influx tion of GABA or GABAergic agents ameliorated disease across the plasma membrane143 and Zn2+ release from outcome in several animal models of autoimmunity, such

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a CD4 TCR CRAC IL-1Rα/β Plasma LAT channel membrane

ζζ PLCγ1 PKC LCK IRAK4 Zn2+ SLP76 LCK Stimulatory ZAP70 2+ Inhibitory InsP Ca 2+ PKC eﬀects of Zn2+ 3 Zn eﬀects IKK of Zn2+ STIM1 IKK Calcineurin

InsP3R p50 p65 P NFAT p50 p65 ER NF-κB NF-κB

Nucleus ↓ IL-2 and IFNγ with high Zn2+

b Zn2+ ZIP6 ZNT? Plasma 2+ [Zn ]o~11–22 µM membrane

2+ [Zn ]i~0.35 nM

Metallothionein Zn2+ Zn2+ Eﬀects on signal ZIP8 transduction

Lysosome

Figure 5 | Zinc signalling and zinc transporters in T cells. a | Zn2+ ions have activating and inhibitory effects on signal transduction in T cells136–138. Zn2+ mediates the recruitment of the SRC family kinase LCK to CD4Nature and CD8 Reviews and promotes | Immunology LCK dimerization, resulting in enhanced T cell receptor (TCR) signalling152. Zn2+ also promotes protein kinase C (PKC) signalling, probably by recruiting PKC to the plasma membrane. By contrast, Zn2+ inhibits the activity of the phosphatase calcineurin, thus preventing nuclear translocation of the transcription factor NFAT (nuclear factor of activated T cells)153,154. Furthermore, Zn2+ inhibits the function of IL‑1R‑associated kinase 4 (IRAK4), thereby restraining signalling through the interleukin‑1 receptor (IL‑1R) and the activation of nuclear factor-κB (NF‑κB). Inhibitory effects of Zn2+ on both NFAT and NF‑κB may explain the reduced production of cytokines such as IL‑2 and interferon-γ (IFNγ) in the presence of increasing extracellular Zn2+ concentrations. b | Increases in intracellular Zn2+ concentrations in lymphocytes are mediated by Zn2+ influx from the extracellular space or Zn2+ efflux from intracellular organelles mediated by ZRT/IRT-like proteins (ZIPs). These Zn2+ transporters contain eight transmembrane domains with an aqueous pore predicted to be formed by the fourth and fifth transmembrane domains189. Zn2+ is exported from the cytoplasm by zinc transporters (ZNTs), resulting in decreased intracellular Zn2+ concentrations. In T cells, the Zn2+ transporters ZIP3, ZIP6 and ZIP8 have been implicated in Zn2+ influx142,143,156, whereas the identities of the ZNT proteins mediating Zn2+ efflux in lymphocytes are presently unknown. In addition to being regulated by Zn2+ transport, intracellular Zn2+ levels are modulated by the binding of Zn2+ to metallothionein and other proteins. CRAC,Ca 2+ 2+ release-activated Ca ; ER, endoplasmic reticulum; IKK, IκB kinase; InsP3R, inositol‑1,4,5‑trisphosphate receptor; LAT, linker for activation of T cells; STIM1, stromal interaction molecule 1;ZAP70, ζ-chain-associated protein kinase of 70 kDa.

as type 1 diabetes163, rheumatoid arthritis170 and multi- Another Cl− channel that has been reported to regu- 166 ple sclerosis . This suggests that GABAA receptors may late T cell function is the cystic fibrosis transmembrane inhibit the activation of T cells to protect GABA-secreting conductance regulator (CFTR), mutations of which cells from T cell-mediated inflammatory tissue damage. cause cystic fibrosis. Cyclic AMP-activated Cl− currents However, the mechanism by which GABA receptor- were originally reported in Jurkat T cells, CD4+ T cell mediated Cl− influx inhibits T cell function has not been clones and EBV-transformed B cells and were shown to

elucidated. In excitable cells, GABA receptors inhibit CaV be defective in B and T cells from patients with cystic channels through membrane hyperpolarization, but this fibrosis171,172. The effects of the ΔF508 CFTR mutation mechanism is unlikely to account for the effects of GABA — the most common mutation in patients with cystic on T cells. fibrosis — on mouse and human T cells were, however,

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very different. Whereas T cell clones from patients with receptors53. It is tempting to speculate that different cystic fibrosis showed impaired production of IL‑5 and types of ion channel aggregate in signalling complexes IL‑10 after stimulation with CD3‑specific antibodies in lymphocytes, where they modulate each other’s func- and PMA (phorbol 12‑myristate 13‑acetate)171, CD4+ tion, but more in-depth studies are needed to investigate T cells from Cftr−/− mice showed increased IL‑4 and IL‑13 this possibility. production compared with wild-type T cells when stimu- Many of the ion channels discussed here contrib- lated with congenic monocytes and antigen173. The cause ute to T cell-mediated autoimmune and/or inflamma- of this discrepancy between human and mouse T cells is tory responses and therefore are attractive targets for not clear. Further studies are required to corroborate a pharmacological immune modulation. Whereas drugs role for CFTR in lymphocyte function and to provide acting on ion channels have successfully been used for a better mechanistic understanding of how CFTR may the treatment of neurological and cardiovascular dis- regulate T cell function. orders174, ion channels have not been systematically exploited as drug targets for immune therapy. Plasma Summary and perspectives membrane channels are readily accessible to small- Lymphocytes express an abundance of ion channels molecule compounds and biological reagents such as that are crucial for their development and function. blocking antibodies and peptides. Inhibitory antibodies Although the importance of individual ion channels specific for the TRPC5 and TRPM3 channels have been and transporters for T cell effector function is now well developed that target an extracellular loop in close prox- recognized, the ways in which different ion transport imity to the ion channel pore175,176. It is possible that mechanisms interact with each other to fine-tune over- these approaches could be extended to TRPM2 channels all cellular responses for the most optimal outcome still (given their pro-inflammatory role in monocytes122) and remain poorly understood. It seems likely that inter- ORAI Ca2+ channels. As described above, genetic dele- actions among the various ion transport mechanisms tion of Orai1 and Stim1 in mice abolishes the expression could help to generate complex signal transduction pat- of several pro-inflammatory cytokines7,46,47 and protects terns and generate specificity by enhancing the dynamic mice from autoimmune CNS inflammation, colitis, allo- range of the individual signalling pathways and by graft rejection and GVHD43,45–47. Inhibition of SOCE improving signal-to-noise ratios. Examples of crosstalk can be achieved directly by targeting ORAI1 CRAC include the regulation of Ca2+ influx by the MAGT1 channels, or indirectly by inhibiting the function of Mg2+ transporter126 and by Zn2+ influx143, as well as the K+ channels. As discussed above, considerable progress well-known modulation of Ca2+ influx by K+ channels has been made in developing K+ channel blockers92,177. through the control of membrane potential. Such cross- Similarly, P2X7 receptor antagonists may provide a talk could permit more finely tuned regulation of cell multi-pronged approach to anti-inflammatory therapy, signalling than may be possible through the action of given the role of these channels in the pro-inflammatory individual independent pathways. In most cases, the function of lymphocytes and innate immune cells58,59. It molecular foundations of crosstalk are unclear, but will therefore be an important long-term goal to develop possible explanations include the colocalization of ion safe, selective and potent inhibitors of ion channels for transport proteins, as suggested for ORAI CRAC chan- the treatment of inflammation, autoimmunity, allergy nels and K+ channels9 and for CRAC channels and P2X and transplant rejection.

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GABAA receptors mediate inhibition of T cell 182. Yamashita, M., Navarro-Borelly, L., McNally, B. A. & Acknowledgements responses. J. Neuroimmunol. 96, 21–28 (1999). Prakriya, M. Orai1 mutations alter ion permeation We thank H. Wulff, B. N. Desai and H. McBride for their criti- 169. Bergeret, M. et al. GABA modulates cytotoxicity of and Ca2+-dependent inactivation of CRAC channels: cal reading of the manuscript and their insightful comments.

immunocompetent cells expressing GABAA receptor evidence for coupling of permeation and gating. This work was supported in part by US National Institutes of subunits. Biomed. Pharmacother. 52, 214–219 J. Gen. Physiol. 130, 525–540 (2007). Health grants AI066128 (to S.F.), NS057499 (to M.P.) and (1998). 183. Yeromin, A. V. et al. Molecular identification of the GM084195 (to E.Y.S.). 170. Tian, J., Yong, J., Dang, H. & Kaufman, D. L. CRAC channel by altered ion selectivity in a mutant Oral GABA treatment downregulates inflammatory of Orai. Nature 443, 226–229 (2006). Competing interests statement responses in a mouse model of rheumatoid arthritis. References 180, 181 and 183 demonstrate that The authors declare competing financial interests: see Web Autoimmunity 44, 465–470 (2011). ORAI1 (also known as CRACM1) is the version for details. 171. Moss, R. B. et al. Reduced IL‑10 secretion by CD4+ pore-forming subunit of the CRAC channel by T lymphocytes expressing mutant cystic fibrosis identifying a glutamate residue in the first transmembrane conductance regulator (CFTR). transmembrane domain of ORAI1 as the selectivity FURTHER INFORMATION Clin. Exp. Immunol. 106, 374–388 (1996). filter of the CRAC channel. Stefan Feske’s homepage: 172. Chen, J. H., Schulman, H. & Gardner, P. 184. Kawasaki, T., Lange, I. & Feske, S. A minimal http://labs.pathology.med.nyu.edu/feske-lab A cAMP-regulated chloride channel in lymphocytes regulatory domain in the C terminus of STIM1 binds Edward Y. Skolnik’s homepage: that is affected in cystic fibrosis. Science 243, to and activates ORAI1 CRAC channels. Biochem. http://saturn.med.nyu.edu/research/mp/skolniklab 657–660 (1989). Biophys. Res. Commun. 385, 49–54 (2009). Murali Prakriya’s homepage: http://www.pharm. 173. Mueller, C. et al. Lack of cystic fibrosis transmembrane 185. Mullins, F. M., Park, C. Y., Dolmetsch, R. E. & Lewis, R. S. northwestern.edu/faculty/prakriya/PrakriyaLab/Home.html + conductance regulator in CD3 lymphocytes leads to STIM1 and calmodulin interact with Orai1 to induce ALL LINKS ARE ACTIVE IN THE ONLINE PDF aberrant cytokine secretion and hyperinflammatory Ca2+-dependent inactivation of CRAC channels.

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