Ion channelopathies of the Immune System

Abstract

Ion channels and transporters move ions across membrane barriers and are essential for a host of cell functions in many organs. They conduct K⁺, Na⁺ and Cl⁻, which are essential for regulating the membrane potential, H⁺ to control intra- and extracellular pH and divalent cations such as Ca²⁺, Mg²⁺ and Zn²⁺, which function as second messengers and cofactors for many proteins. Inherited channelopathies due to mutations in ion channels or their accessory proteins cause a variety of diseases in the nervous, cardiovascular and other tissues, but channelopathies that affect immune function are not as well studied. Mutations in ORAI1 and STIM1 genes that encode the Ca²⁺ release-activated Ca²⁺ (CRAC) channel in immune cells, the Mg²⁺ transporter MAGT1 and the Cl⁻ channel LRRC8A all cause immunodeficiency with increased susceptibility to infection. Mutations in the Zn²⁺ transporters SLC39A4 (ZIP4) and SLC30A2 (ZnT2) result in nutritional Zn²⁺ deficiency and immune dysfunction. These channels, however, only represent a fraction of ion channels that regulate immunity as demonstrated by immune dysregulation in channel knockout mice. The immune system itself can cause acquired channelopathies that are associated with a variety of diseases of nervous, cardiovascular and endocrine systems resulting from autoantibodies binding to ion channels. These autoantibodies highlight the therapeutic potential of functional anti-ion channel antibodies that are being developed for the treatment of autoimmune, inflammatory and other diseases.

Introduction

Ion channels and transporters (ICT) move ions across hydrophobic lipid membrane barriers including the plasma membrane (PM) and membranes of intracellular organelles such as mitochondria, the endoplasmic reticulum (ER) and vacuoles. The passive transport of ions through ion channels is driven by concentration and electrical gradients between two compartments (e.g. the intra- and extracellular space). By contrast, ion transporters, pumps and exchangers move ions actively with or against their gradient by using energy provided by the hydrolysis of ATP or coupling transport to the potential of other ion gradients. The mammalian genome encodes for more than 600 ICT and accessory proteins that conduct Ca²⁺, Mg²⁺, Zn²⁺, Fe³⁺, Fe²⁺, Cu²⁺, Mn²⁺, K⁺, Na⁺, H⁺, Cl⁻, HCO₃⁻ and trace elements, which play important roles in the regulation of cell function. Relatively few ICTs have known roles in the immune system based on genetic or solid pharmacological evidence (Tables 1 and 2)^{1, 2}, although it is likely that many more channels operate in immune cells and control their function.

Table 1. Ion channels and transporters (ICT) controlling immune responses in mice.

ICTs and their regulatory proteins included in this table were selected because their role in immune cell function and immunity in vivo is supported by gene targeted mice and human channelopathies (see Table 2). Abbreviations: Ae2, anion exchange protein 2; Ca_v, voltage-gated calcium channel; CRAC, calcium release-activated calcium; EBV, Epstein-Barr virus; EAE, experimental autoimmune encephalomyelitis; IBD, inflammatory bowel disease; IEL, intraepithelial lymphocyte; Fcγ R, Tc gamma receptor; GABA_A, γ amino butyric acid receptor A; GvHD, graft versus host disease; H_v, voltage-gated proton channel; IP₃, inositol 1,4,5 trisphosphate; IP₃R, IP3 receptor; K_Ca, calcium-activated potassium channel; K_v, voltage-gated potassium channel; LRRC8A, leucine-rich repeat containing 8 family member A; MAGT, magnesium transporter; Na_v, voltage-gated sodium channel; P2RX, purinergic receptor; PCA, passive cutaneous anaphylaxis; RA, rheumatoid arthritis; ROS, reactive oxygen species; STIM, stromal interaction molecule; TASK, WIK-related acid-sensitive potassium channel; Tfh, follicular T helper cell; Treg, regulatory T cell; TRPM, transient receptor potential melastatin; TRPV, transient receptor potential vanilloid; ZIP, zinc/iron-regulated transporter-like protein; ZnT, zinc transporter; FcεRI, Fc epsilon receptor 1.

ICT	Permeability	Activation	Expression in	Immune Function in vitro and in vivo	References
ORAI1	Ca2+	Antigen and G protein-coupled receptors, STIM1, STIM2	T, B, NK, mast cells, DC, ubiquitous	T cell function (proliferation, cytokine production, cytotoxicity, apoptosis, metabolism), T cell development (Treg, iNKT, IELs) and differentiation (Th17, iTreg, Th2, Tfh), immunity to viral, fungal and bacterial infection; T- dependent antibody responses; T cell-mediated auto- and alloimmunity (EAE, IBD, GvHD). NK cell function (degranulation, cytotoxicity). Mast cell function (degranulation, histamine release, cytokine production, PCA).	9, 11, 87, 88, 89, 90, 91
ORAI2	Ca2+	Antigen and G protein-coupled receptors, STIM1, STIM2	T cells, ubiquitous	Together with ORAI1 regulates murine T cell function (proliferation, cytokine production) and T cell immune responses in vivo (IBD, GvHD, antiviral immunity)	9
STIM1, STIM2	–	Antigen and G protein-coupled receptors, Ca2+ depletion from ER stores	T, B, NK, mast cells, DC, macrophages, neutrophils, ubiquitous	Immune phenotype is similar to ORAI1. Neutrophil function (ROS and cytokine production)	31, 33, 92, 93, 94, 95, 96
TRPM2	Ca2+, Na+	ADPR, cADRP, NAADP, oxidative stress	T cells, macrophages	Macrophages function (cytokine production, inflammasome activation), T cell activation and cytokine production; immunity to Listeria infection; DSS colitis, obesity-induced diabetes, EAE	97, 98, 99
TRPV1	Ca2+ > Na+, K+	Chemical ligands, toxins, temperature, low pH	T cells, DCs, ubiquitous	T cell function (cytokine production); IBD	100
TRPV2	Ca2+, Na+	FcγR	Macrophages	Chemotaxis and phagocytosis by macrophages. Trpv2−/− mice show increased susceptibility to L. monocytogenes.	101, 102
Cav 1.2, 1.3, 1.4, (β3, β4 subunits)	Ca2+	unknown	T cells, B cells, myeloid cells	T cell function; Positive selection of CD4+ T cells (CaV1.4), CD8+ T cell survival, cytokine production (CaV.1.3, CaV1.4), immunity to Listeria, Th2 cell function in asthma (CaV1.2).	103, 104
Cav 3.1	Ca2+	unknown	T cells	T cell function; autoimmunity (EAE)	105
P2RX7	Ca2+, Na+, K+, other	Extracellular ATP	Ubiquitous	T cell function (proliferation, cytokine production, Th17 and Tfh differentiation, inhibition of Treg differentiation); autoimmunity (IBD), allograft rejection, GvHD, antitumor immunity. Innate immune cell function (inflammasome activation; DC priming).	106
P2RX1,2,3,4,5	Ca2+, Na+	Extracellular ATP	T, B, mast cells	T cell function (proliferation, cytokine production, thymocyte apoptosis (P2RX1,4,5). Mast cell activation and cytokine production (P2RX1,3). IgE receptor shedding (P2RX2). Hypocellularity of bone marrow and thymus in P2rx2−/−/P2rx3−/− mice.	107
Piezo1	Ca2+, Na+	Mechanosensation	Ubiquitous	T cell function (activation, proliferation). Normal immune profiling in patients with PIEZO1 mutations and lymphedema.	108, 109
IP3R1,2,3	Ca2+	IP3	Ubiquitous	T cell activation, IP3Rs regulate CRAC channel function. Itpr1/2/3−/− mice show defective thymocyte and B cell development, defective antibody and IL-10 production	110, 111
TRPM7	Ni2+ > Zn2+ > Mg2+, Ca2+	Regulation by intracellular Mg2+, PIP2, extracellular pH	T, B, NK cells, macrophages	Thymocyte development and cytokine secreation. Defective cytokine production, gut homing and GvHD induction of TRPM7-kinase dead T cells. Macrophage function (IL-1β production), LPS-induced peritonits.	49, 50, 112
MAGT1	Mg2+	TCR	T, NK cells	CD4+ T cell homeostasis, NKG2D expression by NK cells, cytotoxicity, immunity to viral infection (EBV)	2, 51, 53
ZIP3, 4, 6, 8, 10, 14	Zn2+	TCR; unknown	T, B cells, Macrophages	T cell activation (ZIP6), IFNγ and perforin expression (ZIP8); T cell development (ZIP3). B cell development and B cell apoptosis, cytokine signaling (ZIP10). Macrophages: Inhibition of cytokine production (ZIP8, ZIP14). DC: Inhibition of expression of MHC class II and costimulatory molecules (ZIP6).	66, 67, 68, 69, 70, 71
ZnT5	Zn2+	Unknown; FcεRI ?	Mast cells	NF-κB activation, FcεRI-dependent delayed type hypersensitivity	113
Kv1.3	K+	Plasma membrane depolarization	T, B, plasma cells, DC, Macrophages	T cells function (Proliferation, cytokine production by Th17 cells). Autoimmunity (EAE, diabetes, RA), and TEM antitumor immunity. Macrophage function (proliferation, iNOS expression). DC function (expression of costimulatory receptors and cytokines; chemotaxis)	1, 114, 115

Table 2. Human immune channelopathies.

Abbreviations: AD, autosomal dominant; AR, autosomal recessive; CRAC, calcium release-activated calcium; EBV, Epstein-Barr virus; EVER, Epidermodysplasia verruciformis; ICT, ion channel and transporters; MAGT, magnesium transporter; OMIM, online mendelian inheritance in man; STIM, stromal interaction molecule; TMC, transmembrane channel-like; VRAC, volume regulated anion channel; LRRC8A, leucine rich repeat containing 8 family member A; XL, X linked; ZIP, zinc/iron-regulated transporter-like protein; ZnT, zinc transporter.

ICT	Gene	Chromo- some	Inheri- tance	Disease	OMIM	Symptoms
ORAI1	ORAI1	12q24.31	AR	CRAC channelopathy	612782	Combined Immunodeficiency (CID; autoimmunity (AIHA, ITP); muscular hypotonia; ectodermal dysplasia with anhidrosis (EDA)
STIM1	STIM1	11p15.4	AR	CRAC channelopathy	12783	(same as ORAI1)
MAGT1	MAGT1	Xq21.1	XL	XMEN (X-linked immunodeficiency with magnesium defect, EBV infection and neoplasia)	300853	Chronic EBV infections, B cell lymphoma, splenomegaly, dysgammagloblobulinemia, decreased CD4/CD8 ration, increased susceptibility to viral infections
ZIP4	SLC39A4	8q24.3	AR	Acrodermatitis enteropathica (AE)	201100	Periorificial dermatitis, alopecia, and diarrhea; recurrent infections
ZnT2	SLC30A2	1p34.11	AD	Transient neonatal zinc deficiency	608118	Dermatitis (Perioral, genital, neck, fingers), diarrhea, hair loss.
TMC6, TMC8	EVER1, EVER2	17q25.3	AR	Epidermodysplasia verruciformis (EV)	605828, 605829	Dermatosis with abnormal susceptibility to human papillomavirus (HPV) infection and high rate of progression to squamous cell carcinoma.
VRAC (SWELL1)	LRRC8A	9q34.11	AD	Agammaglobulinemia	613506	Congenital agammaglobulinemia with B cell deficiency (infections not reported)

Channelopathies are a heterogeneous group of diseases caused by the dysfunction of ICTs due to mutations in their pore-forming alpha subunits or accessory proteins that regulate their function. Channelopathies have been widely studied in many organ systems and linked to diseases such as epilepsy, ataxia or migraine in the nervous system, Brugada syndrome, long QT syndrome and atrial fibrillation in the heart, cystic fibrosis, neonatal forms of diabetes mellitus and polycystic kidney disease, to name a few³. In the immune system, only a few inherited ion channelopathies affecting ICTs conducting Ca²⁺, Mg²⁺, Zn²⁺ and Cl⁻ have been reported so far (Table 2). Their dysfunction is associated with immunodeficiency and in some cases autoimmunity and hematologic malignancies^{1, 2, 4}. Disease-causing autoantibodies binding to ICTs in variety of organs can be considered as secondary (auto)immune channelopathies and will be discussed briefly at the end of this review⁵.

ORAI1, STIM1 and CRAC channelopathy

The Ca²⁺ release-activated Ca²⁺ (CRAC) channel is the main Ca²⁺ influx channel in T cells and most other immune cells^{1, 6, 7, 8}. Besides its important role in immunity, the CRAC channel mediates Ca²⁺ influx in many other cell types and tissues owing to its ubiquitous expression. The highly Ca²⁺ selective CRAC channel is composed of a hexameric complex of ORAI1 proteins or its homologues ORAI2 and ORAI3 (Figure 1)^{8, 9}. ORAI proteins, named after the horae Eunomia, Dike, Eirene in Homer’s Iliad, who were the custodians of the gates of Olympus¹⁰, are highly conserved small tetraspanning plasma membrane proteins^{11, 12, 13}. They contain intracellular N- and C-termini, which allow them to bind to stromal interaction molecule (STIM) 1 and STIM2 and other accessory proteins that modulate CRAC channel function^{8, 14}. STIM1 and STIM2 are single-pass transmembrane proteins with an ER lumenal N terminus and cytoplasmic C terminus. They respond to a reduction in the Ca²⁺ concentration in the ER ([Ca²⁺]_ER) and the dissociation of Ca²⁺ from their EF hand Ca²⁺ binding domains with extensive conformational changes, which allow them to interact with ORAI channels and phospholipids in the plasma membrane within ER-PM junctions. The molecular choreography of STIM activation and the subsequent activation of ORAI channels has been extensively reviewed elsewhere^{8, 15, 16}.

Figure 1. Mutations interfering with the function or expression of ion channels (ORAI1, STIM1, LRRC8A) or transporters (MAGT1) in T cells cause channelopathies and immunodeficiency.

T cell receptor (TCR) stimulation activates the kinases Lck and ZAP-70 and phospholipase Cγ1, resulting in the hydrolysis of PIP₂ into the second messengers inositol-1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG). IP₃ binding to IP₃ receptor channels leads to Ca²⁺ release from the endoplasmic reticulum (ER). The decrease in the ER Ca²⁺ concentration ([Ca²⁺]_ER) activates stromal interaction molecules 1 (STIM1) and STIM2, which subsequently bind to and open Ca²⁺ release-activated Ca²⁺ (CRAC) channels formed by ORAI1 and ORAI2 proteins in the PM. The resulting influx of extracellular Ca²⁺ is called store-operated Ca²⁺ entry (SOCE). Free cytosolic Ca²⁺ is pumped back into ER Ca²⁺ stores by the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). LoF mutations in ORAI1 or STIM1 genes cause CRAC channelopathy with impaired T and NK cell function. TCR stimulation also activates the Mg²⁺ transporter (MAGT1) that mediates Mg²⁺ influx into T cells and promotes activation of PLCγ1, SOCE and expression of NKG2D. The mechanisms by which TCR stimulation activates MGAT1 and how Mg²⁺ causes PLCγ1 activation are not well understood (indicated by question marks). LoF mutations in the MAGT1 gene cause X-linked immunodeficiency with Mg²⁺ defect, EBV infection and neoplasia (XMEN) disease. LRRC8A is a volume-regulated anion channel (VRAC) that conducts Cl⁻ and is required for the regulatory volume decrease after cell swelling as well as T and B cell development. LoF mutations in LRRC8A cause agammaglobulinemia. For details see text.

The Ca²⁺ influx mediated by CRAC channels, called store-operated Ca²⁺ entry (SOCE) because of its regulation by [Ca²⁺]_ER, is initiated by engagement of immunoreceptors such as the TCR, BCR and FcR and subsequent activation of phospholipase Cγ (PLCγ) and IP₃ production, which results in Ca²⁺ release from the ER through inositol-1,4,5-trisphosphate (IP₃) receptor channels and activation of STIM1 and STIM2 (Figure 1). Ca²⁺ signaling following SOCE leads to the activation of many Ca²⁺ regulated enzymes and transcription factors among which the serine/threonine-phosphatase calcineurin and the nuclear factor of activated T cells (NFAT) have prominent roles in T cell function and immunity^{17, 18, 19, 20, 21}. An important function of the SOCE-calcineurin-NFAT signaling pathway is the transcriptional regulation of gene expression. SOCE controls the expression of cytokines, transcription factors, glycolytic enzymes and mitochondrial genes that control a variety of cellular processes including T cell differentiation, proliferation and metabolism^{22, 23, 24, 25}. Besides gene regulation, SOCE also controls a multitude of other immune cell functions such as the degranulation of cytotoxic vesicles by CD8⁺ T cells and NK cells or the production of reactive oxygen species (ROS) by neutrophils^{26, 27, 28, 29, 30, 31}. It therefore comes as no surprise that defects in CRAC channel function have profound effects on immunity.

Loss-of-function (LOF) mutations in ORAI1 or STIM1 genes that abolish CRAC channel function and SOCE in T cells, NK cells and other immune and non-immune cells cause a unique disease syndrome called CRAC channelopathy (Table 2)^{11, 32, 33, 34, 35, 36}. It is characterized by combined immunodeficiency (CID), autoimmunity and non-immune symptoms including congenital muscular hypotonia and ectodermal dysplasia with anhidrosis and amelogenesis imperfecta^{4, 37}. CID in most patients manifests with severe viral, bacterial and fungal infections in the first months or years of life and generally requires treatment by hematopoeitic stem cell transplantation. Common pathogens include cytomegalovirus (CMV), Epstein-Barr virus (EBV), Candida albicans, Streptococcus pneumonia. Uncontrolled viral infections with EBV and human herpes virus 8 have resulted in EBV⁺ B cell lymphoma and Kaposi sarcoma in a subset of patients with CRAC channelopathy^{32, 34, 38}. Unlike severe combined immunodeficiency (SCID), ORAI1 and STIM1 deficient patients have mostly normal T, B and NK cells counts and lymphocyte development seems to proceed normally despite the absence of SOCE^{4, 35}. However, some unconventional lymphocyte populations are altered in CRAC-deficient patients including iNKT cells, γδ T cells and in particular Foxp3⁺ Treg cells^{33, 34, 35}. Abnormal iNKT and γδ T cells may contribute to the CID phenotype. The main cause of CID in patients with LOF mutations in ORAI1 and STIM1, however, is impaired T cell function. In the absence of SOCE, CD4⁺ and CD8⁺ T cells fail to proliferate and do not produce a large variety of cytokines required for T cell effector function and differentiation. The proliferation defect is not due to lack of the growth cytokine IL-2 but caused by an impaired switch of SOCE-deficient T cells to aerobic glycolysis, which is required to support the anabolic metabolism of stimulated T cells that allows them to grow and proliferate³⁹. In the absence of SOCE and NFAT activation, T cells fail to express glucose transporters, glycolytic enzymes and transcription factors that control their expression and they do not activate the AKT-mTOR pathway²³. The role of SOCE in metabolism is not limited to glycolysis, but extents further to the regulation of mitochondrial function and lipid metabolism²⁵. CID in CRAC channelopathy is in part caused by impaired production of antigen-specific antibodies after vaccination or infection, despite overall normal or elevated serum immunoglobulin levels^{4, 33, 35}. Studies in CRAC channel-deficient mice show that defective seroconversion results from the attenuated differentiation and function of follicular T helper (Tfh) cells that promote B cell maturation in the germinal centers (GC) of secondary lymphoid organs²⁴. T cell-specific deletion of SOCE in mice strongly impaired antibody responses to immunization with T-dependent antigens and after viral infection²⁴.

Besides CID, many CRAC-deficient patients present with lymphoproliferation and autoimmunity characterized by autoimmune hemolytic anemia (AIHA) and thrombocytopenia associated with anti-erythrocyte, anti-platelet and other autoantibodies^{4, 35}. The seemingly paradoxical combination of CID and autoimmunity is not unique to patients with ORAI1 or STIM1 mutations but is common to many primary immunodeficiencies (PIDs)⁴⁰. Autoantibody production in CRAC channelopathy patients likely results from impaired development or function of follicular Treg (Tfr) cells that suppress the GC reaction and prevent the formation of autoantibodies^{41, 42}. Whereas Tfr cells are difficult to study in patients due to their paucity in the blood, mice with T cell- or Treg-specific deletion of Stim1 and Stim2 are almost completely devoid of CXCR5⁺ Tfr cells in their lymph nodes and showed spontaneous development of large GCs and autoantibody production²⁴. These findings indicate that SOCE is required for the differentiation of thymus-derived Treg cells into Tfr cells. A strong reduction of effector Treg (eTreg) cells was observed in ORAI1-deficient patients, likely accounting for their autoantibody production. Taken together, SOCE plays a dual role in controlling the function of Tfh cells that are required for the production of pathogen-specific antibodies and the function of Tfr cells that prevent spontaneous autoantibody production. Intriguingly, B cell-specific deletion of SOCE had no effect on antibody production after immunization⁴³, indicating that the humoral immunodeficiency in CRAC deficient patients is due to impaired T cell but not B cell function.

Non-immunological symptoms of CRAC channelopathy include ectodermal dysplasia and anhidrosis (EDA), constituting a new form of EDA with immunodeficiency (ID) that is distinct from EDA-ID due to mutations in IKBKG and NFKBIA genes in the nuclear factor kB (NF-kB) signaling pathway^{35, 44}. Anhidrosis in CRAC channel-deficient patients is caused by impaired sweat gland function because SOCE is required for the opening of the Ca²⁺ activated Cl⁻ channel TMEM16A (or Ano1), which mediates Cl⁻ secretion⁴⁵. Ectodermal dysplasia is mainly characterized by hypocalcified amelogenesis imperfecta due to impaired dental enamel formation and calcification⁴⁶. It is noteworthy that gain-of-function (GOF) mutations in ORAI1 and STIM1 have been reported that cause constitutive Ca²⁺ influx. They cause disease with overlapping phenotypes, Stormorken syndrome and tubular aggregate myopathy, but are not associated with an overt immune phenotype⁴.

MAGT1 and XMEN syndrome

Mg²⁺ is the most abundant divalent cation in immune cells and important for their proliferation and survival⁴⁷. Several Mg²⁺ channels and transporters are expressed in immune cells including SLC41A1, SLC41A2, TRPM6, TRPM7 and MAGT1, but only the last two have so far been shown to regulate lymphocyte development and function⁴⁷. TRPM7 is a non-selective, Mg²⁺-permeable channel that also conducts other divalent cations including Ca²⁺, Zn²⁺, and Ni²⁺. It is ubiquitously expressed and thought to regulate cellular Mg²⁺ homeostasis and thereby survival and proliferation⁴⁸. Deletion of TRPM7 in murine T cells profoundly impairs T cell development, which suggests that Mg²⁺ influx through TRPM7 regulates thymocyte survival and/or proliferation, an interpretation that is complicated, however, by the fact that Mg²⁺ influx and cellular Mg²⁺ content in thymocytes were normal⁴⁹. Since TRPM7 is also a functional serine threonine kinase, it remains possible that the function of TRPM7 in T cell development depends not on its channel but rather its kinase activity. A recent study using mice with macrophage specific deletion of TRPM7 showed that the channel mediates cytosolic Ca²⁺ elevations essential for LPS-induced macrophage activation and production of pro-inflammatory cytokines like IL-1β⁵⁰. Human diseases linked to TRPM7 have not been identified yet. In contrast to TRPM7, MAGT1 is a highly Mg²⁺ selective transporter with a tetraspanning PM topology (Figure 1). Together with MAGT2, which is much less selective for Mg²⁺ and can also conduct other bivalent cations such as Fe²⁺, Mn²⁺ and Cu²⁺, MAGT1 constitutes a new family of transporters without major similarities to other ICTs except the non-selective Mg²⁺ transporter TUSC3^{51, 52}.

Much of what we know about the physiological role of MAGT1 in immune function comes from patients with LoF mutations in MAGT1 who suffer from X-linked immunodeficiency with magnesium defect, EBV infection and neoplasia (XMEN) disease (Table 2)⁵¹ (reviewed in detail in ^{2, 53, 54, 55}). The disease is dominated by recurrent and persistent infections with EBV that often cause EBV-associated B cell lymphomas. Patients show CD4⁺ lymphopenia and an inverted CD4:CD8 ratio that is due to diminished thymic production of CD31⁺ naïve T cells^{54, 55}. The immunodeficiency in XMEN patients is mainly caused by impaired function of NK cells and cytotoxic CD8⁺ T cells^{54, 55}. NK cells of XMEN patients lack expression of NKG2D, an activating NK cell receptor that is required for their antiviral and antitumor immunity. TCR stimulation was shown to result in Mg²⁺ influx, which is abolished in T cells of XMEN patients. While MAGT1 deficiency did not alter the total intracellular Mg²⁺ content (95% of intracellular Mg²⁺ is bound to phosphates and protein molecules), it impaired T cell activation by reducing the activation of PLCγ1, the production of IP₃ and the subsequent SOCE via CRAC channels (Figure 1). Defective SOCE in XMEN patients was only observed in T but not NK cells, which may be due to the effects of Mg²⁺ on the activation of the T cell-specific Tec kinase ITK⁵⁶. Thus, it is likely that some defects in XMEN patients are a consequence of impaired SOCE, which is also required for T and NK cell function. The T cell activation defect in MAGT1-deficient patients can be overcome by strong and prolonged TCR stimulation⁴⁸, which may explain why XMEN patients do not develop other life-threatening infections besides EBV as seen in patients with CRAC channelopathy. Intriguingly, Mg²⁺ supplementation of T cells and NK cells from XMEN patients elevated their free intracellular Mg²⁺ levels, restored NKG2D expression on NK cells and significantly improved their cytotoxic function in vitro⁵¹. Furthermore, dietary supplementation of two XMEN patients with Mg²⁺ L-threonate was furthermore able to control of their EBV viremia⁵¹. Although XMEN patients provided clear evidence that MAGT1 and Mg²⁺ signaling are critical for NK and CD8⁺ T cell function, the molecular mechanisms by which TCR stimulation activates MAGT1 or the mechanism of Mg²⁺-dependent activation of PLCγ1 and other signaling pathways remains to be elucidated.

LRRC8A and Agammaglobulinemia

Leucine-rich repeat containing 8A (LRRC8A, also known as SWELL1) was identified by two siRNA screens as an essential component of the volume-regulated anion channel (VRAC). It mediates Cl⁻ secretion after cell swelling in response to changes in intracellular osmolytes or decreased extracellular osmolarity^{57, 58}. LRRC8A is a tetraspanning plasma membrane protein that together with one or more of its four homologues (LRRC8B-E) forms a channel complex (likely a hexamer) (Figure 1)⁵⁹. Mammalian LRRC8A contains 17 leucine-rich repeats at its C terminus that were shown to mediate signaling and activation of T cells^{60, 61, 62}. LRRC8A is expressed in T and B cells and has important roles in adaptive immunity.

LRRC8A was first identified as a novel gene mutated in a 17 year-old girl who was heterozygous for a balanced chromosomal translocation, t(9;20)(q33.2;q12), that replaced two-and-a-half LRR domains by 35 novel amino acids encoded by intronic nucleotides and likely acts as a dominant negative on VRAC function⁶¹. The girl presented congenital agammaglobulinemia and B cell deficiency (Table 2)⁶¹. Expression of the truncated form of LRRC8A in murine bone-marrow cells and Lrrc8a-deficient mice confirmed its important role in B cell development and antibody production^{61, 62}. Lrrc8a^−/− animals not only showed increased prenatal and postnatal mortality, growth retardation, multiple tissue abnormalities and severely reduced peripheral B cell numbers, but also revealed a role of LRRC8A in thymocyte development and survival not found in human patients⁶². A spontaneous insertion mutation of Lrrc8a in mice (ebo/ebo) that causes a frameshift results in the expression of a truncated LRRC8A protein with its 15 C-terminal LRR domains missing (Table 1)⁶⁰. T cells from ebo/ebo mice have abolished VRAC currents, suggesting that the LRR domains are required for channel function. However, T and B cell development, antibody responses after immunization as well as proliferation and cytokine production by T cells were normal⁶⁰. These data suggest that the channel function of LRRC8A is dispensable for lymphocyte development and function⁶⁰. The molecular mechanisms, including the relationship of the channel pore and LRR domains, by which LRRC8A regulates lymphocyte development and function require further study.

Zinc transporters, zinc deficiency and infections

Zn²⁺ is critical for normal immune function and nutritional Zn²⁺ deficiency in developing countries is a leading cause of immunodeficiency due to impaired innate and adaptive immune responses^{63, 64}. Zn²⁺ levels in cells are regulated by two families of Zn²⁺ transporters, SLC30 (ZnT) and SLC39 (ZIP) proteins⁶⁵. Two human diseases characterized by Zn²⁺ deficiency and impaired immune function have been linked to mutations in these gene families (Table 2). Acrodermatitis enteropathica (AE) is caused by LoF mutations in SLC39A4 encoding the intestinal Zn²⁺ transporter ZIP4 and resulting in an inability to resorb intestinal Zn²⁺. The disease resembles in many aspects systemic Zn²⁺ malnutrition and manifests with diarrhea, skin inflammation and blistering, alopecia, abnormal leukocyte populations and an increased susceptibility to infections⁶⁴. Conditional deletion of Slc39a4 in the intestinal epithelium of mice showed an important role of ZIP4 in Zn²⁺ uptake from the gut, maintenance of the intestinal stem cell niche and function of the gut mucosa, resulting in systemic Zn²⁺ deficiency but the effects on immune function were not tested⁶⁶.

Transient neonatal Zn²⁺ deficiency is due to heterozygous mutations in SLC30A2 that encodes ZnT2. It transports Zn²⁺ into lysosomes, and mutations result in decreased Zn²⁺ levels in breast milk and nutritional Zn²⁺ deficiency of babies. Thus, immunodeficiency caused by mutations in ZnT2, similar to ZIP4, is not due to its role in immune cell function but can be attributed to systemic Zn²⁺ deficiency. Nevertheless, genetic deletion of Zn²⁺ transporters such as ZIP3, ZIP6, ZIP8, ZIP10 and ZnT5 in mice indicates that ZIP and ZnT proteins have important roles in immune cell development and function. Mice lacking ZIP3 have reduced CD4⁺ CD8⁺ double-positive (DP) but increased CD4⁺ and CD8⁺ single positive thymocytes suggesting that ZIP3 regulates T cell development⁶⁷. However, deletion of ZIP3 does not alter intracellular levels of Zn²⁺ and trace metals nor did it affect the expression of two known Zn²⁺-responsive genes⁶⁷. Stimulation of mature T cells was shown to cause Zn²⁺ influx via ZIP6. The increase in intracellular Zn²⁺ concentration inhibits the recruitment of the phosphatase SHP1 and results in sustained Ca²⁺ influx and T cell activation⁶⁸. The lysosomal transporter ZIP8 is upregulated after T cell activation and its knockdown by RNAi attenuated IFNγ and perforin expression in human T cells⁶⁹. In B cells, genetic deletion of ZIP10 reduces intracellular Zn²⁺ levels, increases caspase activation and apoptosis, resulting in impaired B cell development⁷⁰. Residual ZIP10-deficient B cells proliferated poorly and had decreased GC formation and T cell-dependent and T cell-independent antibody production after immunization, suggesting that Zn²⁺ uptake via ZIP10 regulates humoral immunity⁷¹.

Two members of a family of transmembrane channel-like (TMC) proteins, TMC6 (EVER1) and TMC8 (EVER2) were shown to form a complex and interact with ZnT1 in the ER membrane and facilitating Zn²⁺ uptake into the ER (Table 2)⁷². TMC8 prevented the influx of free Zn²⁺ into the nucleus and inhibited the activation of the Zinc-regulated transcription factor MTF1⁷². Autosomal recessive mutations in TMC6 and TMC8 are associated with epidermodysplasia verruciformis (EV), a dermatosis characterized by abnormal susceptibility to human papillomaviruses (HPVs) and a high rate of progression to squamous cell carcinoma on sun-exposed skin. Given the role of TMC6 and TMC8 in Zn²⁺ uptake into the ER, their mutation has been suggested to result in increased cytosolic Zn²⁺ concentrations and enhanced transcription of Zn²⁺-dependent viral proteins in keratinocytes⁷². TMC8 is also expressed in T cells but is downregulated after TCR stimulation, resulting in increased concentrations of free cytosolic Zn²⁺ similar to primary T cells from EVER2-deficient patients⁷³. Increased Zn²⁺ impairs T cell activation and proliferation, but the impact of this T cell defect on EV pathogenesis remains to be elucidated. Despite these tantalizing findings, many questions about the role of ZIP, ZnT and TMC proteins in Zn²⁺ conductance and innate and adaptive immune function remain including the molecular mechanisms by which they are activated and how they regulate immune cell function.

Autoantibody-mediated ion channelopathies

Human LoF mutations in ion channels that impair immune cell function are relatively rare. However, the immune system can itself affect ion channel and cell function through the formation of autoantibodies (AA)³. AA binding to ion channels on the cell membrane causes disease by a number of mechanisms including blocking ion conductance, causing internalization of channel proteins or through complement fixation with subsequent inflammatory cell damage. The diseases associated with autoantibodies targeting ion channels predominantly affect the nervous system and skeletal muscle. Examples are diseases of neuromuscular transmission associated with muscle weakness such as myasthenia gravis due to AA against the nicotinic acetylcholine receptor (nAChR) or the receptor tyrosine kinase MuSK that facilitates nAChR clustering⁷⁴, Lambert-Eaton syndrome due to AA against P/Q-type voltage-gated Ca²⁺ channels⁷⁵, and Isaac and Morvan syndromes, which represent a form of neuromyotonia caused by AA against components of the voltage-gated K⁺ channel (VGKC) complex⁷⁶. AA against the neuronal N-methyl-D-aspartate (NMDA) receptor are responsible for the most common form of AA-mediated encephalitis that manifests with severe CNS and autonomic nervous system symptoms⁷⁷. Anti-NMDA receptor AA can also be found in patients with SLE, where they cause neurological and psychological symptoms. Apart from the CNS and skeletal muscle, examples of other organs affected by AA against ion channels are the heart in patients with congenital heart block (targeting L- and T-type voltage-gated Ca²⁺ channels)⁷⁸ and cardiomyopathy (L-type Ca²⁺ channels, K⁺ channels) and skin in pemphigus vulgaris patients (nAChR)⁷⁹. For more detailed reviews of AA-mediated channelopathies see^{3, 5}. It is noteworthy that the production of AA against ion channels is often the consequence of paraneoplastic syndrome caused by tumors including thymoma, small cell lung cancer or ovarian teratoma^{3, 80}.

Conclusions Outlook

Although the number of ion channels mutated in human patients with immune channelopathy syndromes are limited, genetic deletion of ion channels in mice has taught us that many more ion channels play important roles in regulating innate and adaptive immune responses than previously anticipated. Equally important, the fact that autoantibodies can bind to ion channels and interfere with their function indicates that the deliberate use of ion channel selective functional antibodies is a promising approach to the treatment of diseases in which ion channels contribute to pathophysiology. Indeed, functional monoclonal antibodies (mAb) are emerging as a promising new drug family for the therapeutic modulation of ion channels following on the heels of mAb against other protein classes that is continuing to revolutionize the treatment of cancer, autoimmunity and other diseases⁸¹. Generating inhibitory mAb against ion channels is generally more challenging compared to mAb against other protein classes due to the complex membrane topology and small extracellular loop domains of many channels⁸². mAb against about half a dozen ion channels have been generated that block channel function and are effective in preclinical studies⁸². Two of these channels, P2RX7 and ORAI1, have important roles in immune cell function and immunity (Tables 1 and 2). Two mAbs against human ORAI1 were shown to block CRAC currents, SOCE and cytokine production by PBMC and attenuate GvHD in a preclinical xenograft model^{83, 84}. Similarly, mAb and nanobodies antagonizing P2X7 function on mast cells, macrophages and T cells ameliorated immune pathology in mouse models of colitis, glomerulonephritis and contact hypersensitivity^{85, 86}. While no therapeutic mAb targeting ion channels have been approved for clinical use yet, preclinical studies with mAb are promising and a variety to technological platforms are available to efficiently produce functional mAb against ion channels that may be useful to treat autoimmune disease.

Highlights.

• Channels and transporters for Ca²⁺, Mg²⁺, Zn²⁺ and other ions regulate immunity

• Mutations in ORAI1 and STIM1 abolish Ca²⁺ influx and cause CRAC channelopathy

• XMEN disease results from mutations in the Mg²⁺ transporter MAGT1

• Loss of LRRC8A function blocks lymphocyte development and causes agammaglobulinemia

• Dual role of antibodies as the cause of channelopathy and a new class of therapeutics