Beyond the CRAC: Diversification of ion signaling in B cells

Abstract Although calcium signaling and the important role of calcium release–activated calcium channels is well recognized in the context of immune cell signaling, there is a vast diversity of ion channels and transporters that regulate the entry of ions beyond calcium, including magnesium, zinc, potassium, sodium, and chloride. These ions play a critical role in numerous metabolic and cellular processes. The importance of ions in human health and disease is illustrated by the identification of primary immunodeficiencies in patients with mutations in genes encoding ion channels and transporters, as well as the immunological defects observed in individuals with nutritional ion deficiencies. Despite progress in identifying the important role of ions in immune cell development and activation, we are still in the early stages of exploring the diversity of ion channels and transporters and mechanistically understanding the role of these ions in immune cell biology. Here, we review the biology of ion signaling in B cells and the identification of critical ion channels and transporters in B‐cell development, activation, and differentiation into effector cells. Elucidating the role of ion channels and transporters in immune cell signaling is critical for expanding the repertoire of potential therapeutics for the treatment of immune disorders. Moreover, increased understanding of the role of ions in immune cell function will enhance our understanding of the potentially serious consequences of ion deficiencies in human health and disease.


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MAHTANI ANd TREANOR in various stages of B-cell life and function. Ions have been found to regulate B-cell development, activation, and downstream effector functions in vivo. Here, we describe genetic and pharmacological evidence of these ions and ion channels in B cells and their importance in various aspects of B-cell biology. This study is not meant to be a comprehensive review of the biophysical and structural properties of ion channels or their role in other cells of the immune system-where appropriate the reader is directed toward numerous excellent reviews on these topics.

| D IVER S IT Y OF I ON CHANNEL S/ TR ANS P ORTER S AND ION S IG NALING
Ions play a multitude of roles in cell development, maintenance, and activation. Anions, such as chloride (Cl − ), and monovalent cations, such as sodium (Na + ), potassium (K + ), and hydrogen (H + ), are known to regulate the membrane potential and their movement across the plasma membrane contributes to hyperpolarization and depolarization responses. Zinc (Zn 2+ ), magnesium (Mg 2+ ), and calcium (Ca 2+ ) are stored in various cellular organelles and are released into the cytoplasm, as well as influxed from the extracellular space to maintain/ replenish these stores and act as secondary messengers.
Ion channels and transporters are the predominant mechanisms of ion movement across the plasma membrane and the membranes of cell organelles. These proteins act as environmental sensors and can be activated by a variety of stimuli including stretch, osmotic pressure, temperature, and ligand or lipid binding. [1][2][3][4][5] Ion channels move ions across membranes by passive transport over a concentration or electrical gradient, whereas ion transporters actively transport ions against these gradients. These proteins are expressed in a vast range of tissues and in both excitable and non-excitable cells, indicative for their important role in regulating the membrane potential even in non-excitable cells. There are many types of ion channels and transporters, which vary in the ions they transport, their selectivity, the stimuli that regulate them, and the role they play in physiological processes. 6 Indeed, according to the HGNC database, there are over 320 ion channel genes in the human genome (www.genen ames.org).
These encode a diversity of ion channels and transporters categorized into a number of families including calcium-activated potassium channels, 7 voltage-gated potassium channels, 8 ryanodine receptors, 9 voltage-gated calcium channels, 10 voltage-gated sodium channels, 11 GABA receptors,12 P2X ion channels, 13 transient receptor potential (TRP) channels, [14][15][16][17] and volume-regulated anion channels (VRAC). 18,19 There are also one-member families including the voltage-gated proton channel. 20 Ion channels and transporters have been found to play a major role in regulating the development, maintenance, and activation of immune cells reviewed in [21][22][23][24][25][26][27][28][29][30] . However, we are still in the early stages of unraveling the diversity of ion channels and transporters expressed in immune cells during development, activation, and differentiation into effector cells and their crucial role in fine-tuning immune responses.
For instance, studies so far have only begun to explore about 10% of F I G U R E 1 Functional ion channels expressed in B cells. Genetic and pharmacological evidence of ion channel activity in B cells provides evidence for their expression. B cells express store-operated calcium entry channels on the ER (IP 3 R) and PM (CRAC, composed of ORAI and TRPC1, activated by STIM). Non-selective cation channels from the TRP family, TRPC3, TRPC7, TRPM3, TRPV4, contribute to calcium influx upon various stimuli. Voltage-gated calcium channels (Ca v 1.2/3) have also been found to contribute to calcium influx in B cells. TRPML1/2 is found on lysosomes and regulates calcium concentrations by effluxing these ions into the cytosol. NCLX is a mitochondrial sodium-calcium exchanger and contributes to cytosolic calcium levels from mitochondrial stores. Zinc stores are found in the Golgi and ER and zinc release into the cytosol is mediated by ZIP9 and ZIP7, respectively. Zinc is also influxed from extracellular media and ZIP10 on the plasma membrane contributes to this. TRPM7 and MAGT1 are both regulators of intracellular magnesium homeostasis and are found on the plasma membrane. TRPM7 has also been found to contribute to calcium store maintenance, as it is a non-selective ion channel. P2X7, a purinergic receptor, is a non-selective channel that has been found to influx sodium ions in B cells. Membrane potential changes upon B-cell activation activate voltage-gated potassium (K v 1.3) and proton channels (H v 1) to efflux these ions out of the cell. Additionally, anionic currents, namely chloride ions, have been identified in B cells potentially mediated by CFTR and LRRC8 the potential 300+ ion channels and transporters in B cells ( Figure 1).
Nonetheless, what is emerging is that ion channels and transporters work in concert to regulate effective immune cell activation, and at the same time, limit hyperactivation and autoimmune responses.
Elucidating the role of ion channels and transporters in immune cell signaling and identifying critical ion channels in the regulation of the immune response is critical for expanding the repertoire of potential therapeutics for the treatment of immune disorders, as ion channels are excellent pharmacological targets and there are already a number of ion channel and transporter specific inhibitors. In addition, increased understanding of the requirement for specific ions in immune cell function will enhance our understanding of the potentially serious consequences of ion deficiencies in human health and disease. Collectively, these developmental processes require the concerted action of cytokines, transcription factors, and cell-cell interactions, and consequently, the activation of numerous signaling pathways. Not surprisingly then, the regulation of intracellular ions plays an important, and in some cases essential, role in B-cell development ( Figure 2).

| Zinc transporters
Some of the earliest work to highlight a role for ions and ion channels in B-cell development came from studies examining nutritional deficiencies in human populations and the corresponding immunerelated phenotypes, many of which identified immune deficiencies, including lymphopenia in zinc deficient individuals. 32,33 To examine the role of zinc in immune cell development and function, rodent models of zinc deficiency were developed in which mice were fed a diet deficient in zinc to varying degrees. 34 These studies demonstrated that zinc deficiency led to a significant loss in the proportion of B-lineage cells in the bone marrow, with moderate zinc deficiency F I G U R E 2 Ion channels in B-cell development. Simplified schematic diagram of the stages of B-cell development in the bone marrow and fetal liver and periphery (separated by dashed line), with key regulators highlighted in red that block B-cell development or enhanced B-cell development in green. The degree of impact on B-cell development is indicated by the font size, with more severe impact on development in larger font and more modest impact on development represented by a smaller font reducing B220 + cells by approximately 50% and severe zinc deficiency associated with nearly complete (>90%) loss of B220 + cells.
Interestingly, early stages of B-cell development, including pro-and pre-B-cell stages, were most severely affected by zinc deficiency, whereas immature B cells (B220 + IgM + IgD − ) and mature B cells (IgM + IgD + ) were more resistant to zinc deficiency, albeit still grossly impacted by severe zinc deficiency.
Zinc is an essential trace element necessary for structural integrity of many proteins including enzymes and transcription factors. 35 Indeed, zinc is required for the activity of some 300 enzymes and 2000 transcription factors. Thus, the regulation of zinc is crucial for numerous cellular processes. Zinc homeostasis is regulated by 14 ZRT1-and IRT1-like protein (ZIP) importers and 10 zinc transporter (ZnT) exporters that control the movement of Zn 2+ between the cytosol and the extracellular space or cytoplasmic organelles reviewed in 36,37 . These transporters are differentially expressed in different immune cells, 38 and most have yet to be investigated in the context of B-cell development. Recently, however, two zinc transporters were identified as critical for B-cell development. 39,40 ZIP10 is a plasma membrane ion channel that regulates the influx of Zn 2+ from the extracellular space to the cytosol and was recently identified as important for the development of B cells. 40 To investi- and found that this induced caspase activation and apoptosis, which could be reversed by zinc supplementation, indicating that zinc transported by ZIP10 is critical for suppression of caspase-mediated apoptosis. In addition, the authors found that ZIP10 expression is altered by JAK-STAT-mediated cytokine signaling, suggesting a JAK-STAT-ZIP10-zinc signaling axis in B-cell development.
In a back-to-back accompanying paper, Fukada and colleagues deleted ZIP10 in mature B cells using Cre recombinase under control of the promoter of either the invariant chain (Ii; CD74), which is expressed in major histocompatibility class II (MHCII) expressing antigen-presenting cells (APCs), including B cells, or CD21, which is expressed in mature B cells. Consistent with their accompanying data described above, deletion of ZIP10 in mature B cells resulted in a significant reduction in the number of FO B cells in the spleen and blood, and recirculating mature B cells in the bone marrow. 42 In addition, the number of B1 B cells were also significantly reduced by deletion of ZIP10 using Ii-Cre. Transfer of mature splenic B cells from ZIP10-deficient mice into Rag1-KO mice revealed a gradual loss of transferred B cells over time, indicating a B-cell intrinsic defect, and suggesting that ZIP10 is required for persistence of mature B cells.
Although the molecular mechanism for loss of mature B cells was not identified, enhanced BCR signaling in ZIP10-deficient B cells (discussed below) was speculated to lead to B-cell anergy and deletion. Collectively, these studies identified an important role for Zn 2+ in B-cell development and identified two essential zinc import channels. The identification of critical ion channels regulating zinc import in murine models has helped reconcile observations of immune deficiencies in human patients deficient in zinc. Although the molecular mechanism for the requirement for zinc has not been fully elucidated, one important mechanism appears to involve zinc-mediated regulation of developmental checkpoints dependent on pre-BCR and BCR signaling.

| Transient receptor potential channels
The TRP family of ion channels is a large family of widely expressed ion channels that regulate the intracellular concentration of Ca 2+ , Mg 2+ , and trace metal ions, have diverse roles in cellular physiology, and respond to a variety of sensory stimuli such as taste, temperature, and pain reviewed in [15][16][17]43 . This superfamily of ion channels is classified into six subfamilies in vertebrates: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polysystin), TRPA (ankyrin), and TRPML (mucolipin), with different modes of activation and selectivity both within subfamilies and across the superfamily. Although many TRP channels are expressed in B cells, 44 to date, only TRPM7 has been demonstrated as critical for B-cell development. 45 TRPM7 is divalent cation channel with selectivity for Ca 2+ , Mg 2+ , and Zn 2+ . Notably, TRPM7 and its closest relative, TRPM6, are the only known ion channels that contain an associated kinase domain, suggesting it may play an additional role in regulating cell signaling.
The gene encoding TRPM7, Ltrpc7, was first cloned in an effort to identify novel Ca 2+ /cation channels expressed in hematopoietic cells. 46 To further investigate the role of TRPM7 in cell physiology, the authors made use of the chicken B cell line, DT40; a cell line widely used in gene disruption studies due to its high rate of homologous recombination and rapid doubling time. 47 Notably, targeted deletion of one allele was easily obtained; however, no clones were obtained with targeted disruption of both alleles, leading the authors to conclude that TRPM7 is required for cellular viability. 46 To overcome this challenge, the authors took an inducible approach using a tamoxifen-controlled Cre recombinase. The activation of Cre recombinase induced cell growth arrest and cell death within a 42-to 72-hour time frame, again supporting the notion that TRPM7 is essential for cell survival. Subsequent studies demonstrated that the cell growth arrest and apoptosis of DT40 cells could be rescued by supplementation of the culture media with high concentrations of extracellular Mg 2+ , providing the first evidence that TRPM7-mediated regulation of Mg 2+ homeostasis may be important for cell survival. 48 More recently, a murine model deficient in TRPM7 demonstrated that it is essential for embryonic development 49 ; however, lineagespecific deletion of TRPM7 does not necessarily lead to cell death in all cell types. For example, deletion of TRPM7 in T cells using Lck-Cre resulted in a defect in thymocyte development, but nearly normal numbers of peripheral T cells. 49 Moreover, Cre-mediated deletion of TRPM7 in megakaryocytes increased the number of these cells in the bone marrow and red pulp of the spleen, 50 and specific deletion in the myeloid lineage does not affect macrophage differentiation from bone marrow cells ex vivo. 51 To examine a role for TRPM7 in B cells, we generated mice that lacked TRPM7 in the B-cell lineage using TRPM7-flox mice expressing Cre recombinase under the pan B cell CD79a (Mb1) gene promoter. 45 We found a complete loss of B220 + CD19 + mature B cells in the spleen, lymph nodes, and peripheral blood, as well as nearly complete loss of B1 B cells in the peritoneum in TRPM7-deficient mice. Consistent with our findings of a lack of peripheral B cells, TRPM7-deficient mice completely lacked immature, transitional, and mature B cells in the bone marrow.
Indeed, we found a developmental block at the pre-pro-B-cell and pro-B-cell stages, coincident with promoter activity. 41 The kinase function of TRPM7, however, was not required for B-cell development, as knock-in mice expressing a kinase dead mutant of TRPM7 had normal numbers of all B-cell subsets. Notably, using an in vitro coculture system of HSCs that supports B-cell development, 52 we found a dose-dependent increase in the number of B220 + CD19 + cells derived from HSCs from TRPM7-deficient mice supplemented with increased concentrations of extracellular Mg 2+ . However, even 10 mmol/L Mg 2+ , the concentration that supported development of TRPM7-deficient DT40 B cells, 48 development of B220 + CD19 + cells from TRPM7-deficient HSCs was still less than from wildtype (WT) HSCs. Our findings demonstrate that TRPM7 is essential for B-cell development, and suggest that the Mg 2+ transport function is most important. This is consistent with the observation that supplementation with high concentrations of extracellular Mg 2+ is able to rescue the growth arrest seen in chicken DT40 B cells deficient in TRPM7. 48 The discrepancy in the ability of supplementary Mg 2+ to fully support primary murine B-cell development 45 44 It may also be that the Mg 2+ permeable channel, MagT1, has a more important role in T-cell development. 54 In light of the above discussion of the important role of zinc transporters in B-cell development, and the permeability of TRPM7 to zinc ions, one might speculate that the B-cell developmental defect we observe in TRPM7-deficient mice might be due to a possible role for TRPM7 in Zn 2+ transport. Although we did not test the ability of high concentrations of extracellular Zn 2+ to support B-cell development in our in vitro culture system of HSCs, the growth arrest observed in TRPM7-deficient DT40 cells could only be reversed by supplementation with Mg 2+ , but not by Ca 2+ , Zn 2+ , Mn 2+ , or Ni 2+ , 48 suggesting that lack of Zn 2+ transport by TRPM7 is unlikely to account for our findings.

| Volume-regulated anion channel
Volume-regulated anion channel is a heterooligomeric ion channel composed of leucine-rich repeat (LRR)-containing 8 (LRRC8) subunits (LRRC8A-E) that transports chloride ions and various organic osmolytes, such as taurine and glutamate, across the plasma membrane reviewed in 18,19 . In 2003, Sawada et al isolated the Lrrc8a gene, which was found to be mutated in a patient with congenital agammaglobulinemia and a lack of B cells. 55 To investigate the significance of the mutant LRRC8A, a vector encoding the mutant cDNA (resulting in a truncated protein) or control vector were used to infect bone marrow cells, which were then transferred into lethally irradiated mice to evaluate B-cell reconstitution. Three months post transfer, mice infected with mutant bone marrow had reduced percentage of B and T cells, but not granulocytes or macrophages. Analysis of developing B-cell subsets showed a developmental arrest at the pro-B-cell stage and a severe reduction in the proportion of pre-B cells. Interestingly, in the patient, the unaffected Lrrc8a allele was transcribed and intact LRRC8A expressed; however, B cells were totally absent from peripheral blood, suggesting that the truncated form has a dominant-negative effect. At the time of this finding, however, the role of LRRC8A in VRAC had not yet been identified so the molecular mechanism for loss of mature B cells was not investigated.
Subsequently, Kumar et al generated an Lrrc8a knockout mouse. 56 LRRC8A is the most important subunit for channel function as suppression of LRRC8A nearly eliminated the presence of VRAC in mammalian cells. 57,58 In contrast to the patient with mutated Lrrc8a, 55

| Calcium channels
Given the importance of calcium as a second messenger for many cell surface receptors, it seems likely that ion channels regulating Ca 2+ efflux from the ER into the cytoplasm, or Ca 2+ influx from the extracellular space into the cytosol would be important in B-cell development. It was surprising then, that B-cell development in the spleen, bone marrow, and peritoneal cavity was normal in mice deficient in the ER Ca 2+ sensors, stromal interaction molecule 1 (STIM1) and STIM2, using Cre-mediated deletion under control of CD79a. 60 Similarly, deletion of the calcium release-activated channel (CRAC) component ORAI1, 61 or knock-in of ORAI1 with a point mutation (R93W) that renders the pore-forming subunit non-functional, 62 had no effect on B-cell development. These findings are consistent with normal numbers of B cells in patients with mutations in ORAI1 and STIM1. 63,64 Taken together, these findings demonstrate that B-cell development does not require Ca 2+ mediated through store-operated calcium channels.
Recently, Tang and colleagues investigated the role of inositol 1,4,5-trisphosphate receptor (IP 3 R)-mediated Ca 2+ release in B-cell development. 65 They generated a triple knockout of IP 3 R (IP 3 R1, IP 3 R2, and IP 3 R3 encoded by Itpr1, Itpr2, and Itpr3) designated IP 3 R-TKO. They found total numbers of CD19 + B220 + in the bone marrow were not significantly different in IP 3 R-TKO mice compared to control mice. Consistent with this, numbers of pro-, pre-, and immature B cells were not altered. They did find, however, that the number of In addition to calcium permeable ion channels, Ca 2+ homeostasis is also controlled by kinases that regulate the intracellular concen-  67 Deletion of Itpkb had no effect on B-cell subsets in the bone marrow, except for recirculating mature B cells, which were reduced by approximately 50%. Consistent with this observation, splenic B-cell numbers were reduced 80%, with decreases in transitional, FO, and MZ subsets. In contrast, B1 B cells were increased approximately 50%. The authors also found Itpkb-deficient B cells had altered BCR signaling (discussed below) and therefore proposed that Itpkb and its product, IP 4 , are negative regulators of BCR signaling and deficiency in Itpkb leads to enhanced negative selection.
Taken together, the findings discussed here implicate Zn 2+ and Mg 2+ as necessary ions for B-cell development, and suggest that Ca 2+ has less of a role in B-cell development, albeit still being important for positive and negative selection of B cells through regulation of BCR and pre-BCR signaling.

| Store-operated calcium entry
Stimulation of a B cell through engagement of the BCR induces receptor clustering and initiation of signaling pathways. These pathways include the release of ion stores, changes in membrane potential, movement of ions across the membrane, and recovery of emptied ion stores. These ions act as secondary messengers and are important for effective BCR signaling ( Figure 3). The most classically profiled secondary messenger is calcium, as the activation of phospholipase C gamma 2 (PLCγ2) leads to hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP 2 ), a membrane localized lipid, and production of IP 3 and diacylglycerol (DAG). 68 IP 3 binds to IP 3 receptors on the ER and causes the release of calcium stores from the ER. 68 Once these stores have emptied, STIM1 and STIM2 are activated and can, in turn, activate CRAC channels composed of ORAI1 and ORAI2 subunits on the plasma membrane to replenish calcium stores. 68 This process, known as store-operated calcium entry (SOCE), is thought to govern the calcium response in B cells.
Indeed, knocking out all three IP 3 receptors (IP 3 R-TKO) in murine B cells leads to essentially no calcium flux upon BCR stimulation. 65 Both phases of calcium signaling, the initial peak from the emptying of ER stores, and the sustained flux in the presence of extracellular calcium were diminished in IP 3 R-TKO B cells. However, treating cells with thapsigargin, a drug that empties calcium stores by inhibiting the sarco/ER calcium ATPase (SERCA), showed no difference between IP 3 R-TKO and WT B cells. This suggests that IP 3 Rs are crucial for calcium signaling in response to BCR stimulation, but are dispensable for maintenance of calcium stores.
Similarly, a murine double knockout (DKO) of STIM1 and STIM2 also led to significantly reduced calcium flux upon BCR stimulation in both the peak and sustained phases. 60 Single gene knockouts demonstrated that STIM1 is more important than STIM2 for both maintenance of stores and calcium influx into cells, as STIM2-KO B cells do not show much of a defect in comparison to the DKO, whereas STIM1-KO B cells do. 60 The abrogation of calcium flux seen in the DKO signifies the key role of these proteins in regulating SOCE currents. Previous findings in DT40 B cells are in agreement, as overexpressing STIM1 markedly enhances calcium influx upon BCR stimulation. 69 However, in the murine STIM1/2-DKO, 60 unlike the IP 3 R-TKO, 65 treatment of cells with thapsigargin revealed a defect in ER store filling in DKO B cells, as Ca 2+ flux was greatly reduced. Therefore, STIM1/2 are critical for the maintenance of Ca 2+ stores and their absence results in reduced ER calcium release, as well as reduced calcium uptake upon BCR stimulation.
It was also found that CRAC channels behave similarly in chicken DT40 B cells, as knocking out ORAI1 had no effect on SOCE currents, whereas knocking out ORAI2 actually increased calcium entry. 70 Knocking out both of the ORAI subunits in DT40 cells completely abrogated the second phase of SOCE currents. 70 This F I G U R E 3 Ion channel activation and function during B-cell activation. B cells recognize antigen with their B-cell receptor (BCR) and signaling cascades are initiated that are fine-tuned for optimal B-cell activation. Genetic and pharmacological studies have demonstrated that the kinase domain of transient receptor potential melastatin 7 (TRPM7) phosphorylates phopholipase C gamma 2 (PLCγ2) and once activated, it can hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to produce inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol (DAG). IP 3 binds to IP 3 receptors (IP 3 Rs) on the endoplasmic reticulum (ER) and stimulates the release of calcium stores by IP 3 R and ryanodine receptors (RyR). IP 3 conversion to inositol 1,3,4,5-tetrakisphosphate (IP 4 ) by IP 3 kinase B (ITPKB) regulates the degree of calcium signal upon activation. Stromal interaction molecules (STIM) recognize a depletion of ER stores and activate calcium release activated calcium (CRAC) channels comprised of ORAI subunits and TRPC1. DAG also activates store-independent calcium influx channels TRPC3 and TRPC7. Additionally, TRPM3 and Ca v channels are activated and contribute to calcium influx. P2X7 receptors are activated by extracellular ATP and dampen calcium signals upon B-cell activation by influx of sodium ions. Zinc release from Golgi (ZIP9) and ER (ZIP7) stores into the cytoplasm inhibits phosphatases and dampens signaling, whereas ZIP10 positively regulate CD45 phosphatase activity. Proton (Hv1) and potassium efflux (K Ca 3.1, K v 1.3) upon membrane hyperpolarization are both regulated by the membrane potential changes or calcium entry into the cell suggests that CRAC channels may be composed of ORAI subunits together with other channel proteins to compensate for the lack of one subunit; however, loss of both subunits prevents the formation of CRAC channels.
As discussed above, IP 3 generated during the SOCE response can also be converted to IP 4 by ITPKB-mediated phosphorylation, and this conversion has been found to dampen SOCE in mast cells. 71 Deletion of Itpkb in a murine model enhanced calcium signaling upon BCR stimulation, especially in the presence of extracellular calcium. 66 This was specific for BCR stimulation, as there was no difference in Ca 2+ store release upon thapsigargin treatment. Therefore, ITPKB or its product, IP 4 , regulates SOCE channels to control the amount of calcium that enters B cells upon BCR stimulation.
In addition to the conventionally studied players in SOCE, TRP channels may also play a role in the composition of heterogeneous CRAC channels and maintenance of calcium stores. For example, deletion of TRPC1 in DT40 B cells impacted both the peak and sustained Ca 2+ flux upon BCR stimulation. 72 Treatment of cells with thapsigargin demonstrated that TRPC1 does not play a role in ER Ca 2+ release, but is involved in extracellular Ca 2+ entry upon ER store emptying. This finding demonstrated that, at least in B cells, other subunits may be incorporated as components of SOCE channels. In addition, TRPM7 has also been shown to be important for Ca 2+ flux upon BCR stimulation, as SOCE currents in the presence of extracellular calcium are reduced in TRPM7-KO DT40 B cells. 70 Interestingly, the kinase domain of TRPM7 was found to be important for refilling and maintaining calcium stores, as both store content at steadystate and rate of filling after release were impacted in DT40 B cells expressing a kinase dead mutant of TRPM7. 70 This finding may be related to the role of TRPM7 kinase in phosphorylation of PLCγ2, 73 although how this impacts PLCγ2 activity is not currently clear.

| Non-store-operated calcium channels
Transient receptor potential channels have also been found to play a role in store-independent calcium responses. These responses are characterized by the activation of plasma membrane calcium channels through direct interactions with secondary messengers or byproducts of PLCγ2 activation. For example, TRPC3 has been found to be important for DAG-mediated calcium responses upon BCR stimulation. [74][75][76] This was demonstrated both by the loss of DAG analog, 1-oleoyl-2-acetyl-sn-glycerol (OAG)-induced currents in TRPC3-mutant cells, as well as a reduction in Ca 2+ flux upon BCR stimulation. 74 Consistent with this, the overexpression of TRPC3 in DT40 B cells increases the second phase of Ca 2+ flux upon BCR stimulation and induces a current in the presence of OAG that is not present without TRPC3 expression. 75,76 OAG treatment can also induce TRPC3 currents in IP 3 R knockout DT40 B cells, suggesting a SOCE mechanism does not activate it. 75,76 However, it may be that TRPC3 is also important for SOCE currents, as Ca 2+ flux upon thapsigargin treatment was higher in both WT and IP 3 R-KO DT40 B cells expressing human TRPC3. 75 This study also showed TRPC3-dependent OAG-induced currents, and these were actually higher in IP 3 R-KO DT40 cells, suggesting the lack of IP 3 R allows for enhanced activation of TRPC3 and a potential dual role of this channel in B cells. 75 In addition to the role of TRPC3 in non-SOCE, deletion of TRPC7 in DT40 B cells demonstrated a lack of a clear, detectable non-store-operated calcium current in the presence of gadmium, a SOCE current inhibitor, upon BCR stimulation, indicating a role for this channel in this pathway as well. 77 Interestingly, the lack of TRPC7 also affected calcium stores, as they were enlarged in the absence of the channel. It has also been seen that chronic fatigue syndrome patients have reduced expression of TRPM3 on the surface of their B cells, and this leads to a significantly decreased calcium flux in response to treatment with thapsigargin and crosslinking of the BCR and CD21. 78 Interestingly, B cells have also been found to express calcium channels typically found on excitable cells, such as neurons or mus-  79 The use of RyR inhibitors, ruthenium red (RuR) and ryanodine at μmol/L concentrations, which completely shut the channel, on DT40 B cells reduced SOCE in response to ionomycin with and without extracellular calcium. 79 Additionally, RyR activators such as cyclic ADP ribose (cADPR) led to more rapid calcium currents compared to control EGTA treatment, whereas RyR inhibitors such as RuR and 8-N-cADPR led to decreased and slower calcium currents compared to control treatment. 79 Therefore, RyR receptors play a significant role in the release of calcium stores from the ER in B cells upon stimulation.
In addition to RyR receptors, a non-voltage-gated L-type calcium channel is expressed in primary rat B cells and human B lymphoma cell lines. 81,82 L-type calcium channels are a family of voltage-dependent calcium channels (the "L" standing for the long-lasting currents observed for these channels) composed of Ca v 1.1, Ca v 1.2, Ca v 1.3, and Ca v 1.4 subunits. Again, these channels are typically associated with excitable cells; however, a truncated form of L-type channels has been found in non-excitable cells, and is generally thought to be voltage-insensitive reviewed in 83 . In primary rat B cells, treatment with specific L-type calcium channel inhibitors, nicarpidine and calciseptine, completely abrogated the expected calcium peak response upon BCR stimulation, which was found to be Ca v 1.3 dependent. 82 Calcium responses upon BCR stimulation were also significantly reduced in human B cell lines upon treatment with the inhibitors of L-type calcium channels, diltiazem, verapamil, and nifedipine, and in at least one cell line, truncated Ca v 1.2 was found to be the L-type channel expressed. 81 Treatment of rat B cells with an L-type calcium channel agonist, Bay K 8644, induced a much longer peak calcium response, 82 whereas in human B-cell lymphoma lines, adding this agonist prior to BCR stimulation had no effect, but treatment post-BCR stimulation induced an abrupt change from peak to sustained phases of calcium signaling. 81 It was also found that the current induced by this L-type calcium channel is a result of increased cyclic guanosine monophosphate (cGMP) levels after BCR stimulation and that inhibiting guanylyl cyclase, which synthesizes cGMP from GTP, with LY83583 led to a greatly reduced BCR-induced calcium response. 82

| Unconventional ions
As outlined above, two other divalent cations, magnesium and zinc, both play very important roles in the development and maintenance of B cells. However, not much is known about the role these ions play during BCR stimulation. It has been shown that Mg 2+ does appear to increase in cytoplasmic concentration after BCR stimulation and is dependent on calcium influx; however, the kinetics are gradual and monophasic with no clear peak or drop-off. 84 Deletion of the Mg 2+ permeable channel, TRPM7, in DT40 B cells lowers intracellular Mg 2+ levels at resting state, and is associated with impaired growth of these cells, as high concentrations of extracellular Mg 2+ can rescue this defect. 48 Similarly, murine B cells deficient in the Mg 2+ channel, MAGT1, had reduced magnesium levels in both resting cell and upon activation. 85 Surprisingly, these cells also had increased calcium influx in both phases of SOCE upon BCR stimulation; however, there were no differences in calcium flux upon thapsigargin treatment, suggesting that regulation of Mg 2+ plays a key role during BCR signaling. As magnesium is important for the function of many enzymes, it is perhaps not surprising that store release and extracellular influx upon BCR stimulation would be crucial to downstream events and differentiation; however, further investigation of the role of Mg 2+ in BCR signaling is required to elucidate the specific requirements and downstream targets of Mg 2+ .
It has also been shown that cytoplasmic Zn 2+ levels increase over a 10-minute period upon BCR stimulation with no drop-off, 86 and activated CD69 + B cells have increased Zn 2+ levels. 87 Knocking out ZIP9, a zinc transporter found in the Golgi, in DT40 B cells impaired the intracellular rise in Zn 2+ upon BCR stimulation. 86 Similarly, ZIP10 has also been shown to be important for Zn 2+ influx in developing B cells and activation of mature naive B cells suggesting a crucial role for Zn 2+ flux upon BCR stimulation. 42 While these studies importantly identify transporters mediating Zn 2+ flux in B cells, further studies are required to understand the role of zinc in B-cell activation and its impact on downstream events.

| Monovalent ions
B cells are non-excitable cells, which do not exhibit activation upon membrane depolarizing conditions. However, upon BCR stimulation, they do undergo differential membrane potential and require tight regulation of membrane polarization. [88][89][90] Monovalent cation flux in B cells upon BCR stimulation is not well characterized; however, Na + , K + , and H + currents have been detected during BCR stimulation.  88 as this current is dependent on the influx of calcium. 90 Inversely, blocking potassium efflux, or supplementing extracellular media with potassium 88 was found to reduce calcium influx upon BCR stimulation. 90 These findings suggest a cyclical regulation of these two ions, whereby Ca 2+ influx activates K + channels dependent on this ion for their activation, thereby hyperpolarizing the cell and increasing the driving force for more Ca 2+ entry. Consistent with this idea, B cells transiently hyperpolarize upon BCR stimulation 88 ; however, agonist induced K Ca 3.1 current upon BCR stimulation led to prolonged hyperpolarization but unexpectedly, the authors did not see an increase in calcium currents. 97 Thus, further studies are needed to understand the codependence of Ca 2+ and K + currents on each other upon activation, as the activating cue for non-calciumdependent potassium channels is not well understood in B cells.

Sodium currents have not been well characterized in B cells;
however, a few studies suggest that Na + flux may also play a role in B-cell responses. The first evidence suggesting a role for Na + channels in B cells came from patch-clamp experiments that demonstrated Na + conductance in B cells upon stimulation in calcium-free media, 88 and the subsequent immunoprecipitation of a protein complex using an antibody specific for a bovine kidney Na + channel. 98 Additionally, P2X7, a purinergic receptor, has been found on B cells and was shown to regulate Na + influx upon BCR stimulation, and dampen calcium influx as a result. 89 Similar findings have been shown in T cells with TRPM4, as Na + influx depolarizes the cells upon activation and regulates calcium influx. 99 This depolarization from Na + influx is thought to dampen calcium currents and activate voltage-gated potassium channels, which would return the membrane to a hyperpolarized state and allow for more calcium influx. 99 This "see-saw" effect between depolarized and hyperpolarized membrane potential is what leads to the observed calcium oscillations. This suggests a crucial role for Na + in regulating Bcell activation and preventing enhanced calcium responses which can direct the cell toward apoptosis rather than proliferation and differentiation.
Anionic currents in B cells are also not well understood and B cells express very little of the ATP-gated anion channel, CFTR.
Indeed, antisense oligonucleotide blocking of gene transcription has been the only way to determine the presence and chloride current activity of the CFTR channel in B cells. 100 It has, however, been found that at resting state, B cells do express a large conductance anionic channel with preference for chloride ions, and electrophysiology demonstrated the channel preferentially transports Cl − over both K + and Na + when these ion were present on the intracellular side of the membrane. 101 However, when chloride was replaced by aspartate, channel reversal potential and conductance were not altered suggesting that this channel is permeable to larger anions. Interestingly, B cells have been found to not undergo regulatory volume decrease in hypotonic conditions, which is characteristic of cells that swell and reduce swelling by effluxing potassium and chloride ions. 102 This was found to be due to the lack of potassium efflux upon swelling 102 ; however, B cells did exhibit a chloride current comparable to other cells. 103 Additionally, this channel conductance can be blocked by inhibitors, stilbene derivatives disodium 4-acetamido-4′-isothiocyanato-stilben-2,2′-disulfonate (SITS) and 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), of chloride flux and the channel responsible was found to be voltage sensitive. 104 This suggests that chloride currents may play a role in regulating membrane potential after BCR stimulation, as other ion fluxes polarize the cell membrane.

| I ON CHANNEL S IN BCR AC TIVATI ON AND S IG NALING
Ion channels play a key role in fluxing secondary messengers and cofactors for protein activity and defects in ion concentrations during both steady-state and activating conditions can lead to abnormal signaling downstream of the mitogenic receptor. Ion dysregulation has been found to have impacts on signaling downstream of the BCR, which can lead to defects in processes that lead to humoral immunity. In the following sections, we discuss the evidence of ions and ion channels that play a key role in BCR signaling and B-cell activation ( Figure 3).

| Store-operated and non-store-operated calcium channels
Defects in SOCE currents in B cells lead to downstream signaling defects differentially depending on the targeted protein. In IP 3 R-TKO murine B cells, NFAT dephosphorylation is completely abrogated, but ERK and JNK signaling are increased compared to control B cells. 65  Consistent with these studies, knocking out TRPC1, which also plays a role in the SOCE currents, in DT40 B cells also led to decreased NFAT activity. 72 In contrast, ITPKB-KO B cells have increased calcium signaling and slightly reduced PLCγ2 phosphorylation despite normal levels of IP 3 generation. 66 These cells did not upregulate costimulatory molecules and activation markers CD86, MHC class II, and CD69, and failed to proliferate compared to control cells upon BCR stimulation.
They were also blocked from progressing to the G1 stage of the cell cycle post-BCR stimulation. This is in agreement with the finding that overexpressing STIM1 in DT40 B cells led to enhanced calcium signaling upon BCR ligation, which led to enhanced ERK phosphorylation and increased cell death. 69 This highlights the important finetuning of SOCE responses upon B-cell activation, as enhanced SOCE responses can lead to signaling defects and induction of apoptotic pathways.
Defective calcium influx from non-store-operated pathways can also impact B-cell activation. For example, expressing a truncated form of TRPC3 defective in channel function in DT40 B cells led to defective PLCγ2 and PKC translocation to the plasma membrane upon BCR stimulation, and ERK phosphorylation and NFAT activity were also reduced. 74 Similarly, BCR stimulation in the presence of ATP-activated P2X7 receptors and were found to influx Na + and dampen Ca 2+ currents, which led to defective NFAT activation. 89 Indeed, calcium ionophore treatment of B cells in the absence of any other stimulation elicits MAP kinase activation solely through Ca 2+ influx, indicating the key role for calcium signaling in distal B-cell signaling. 105 Similarly, human B cells require extracellular calcium for multiple signaling proteins involved in B-cell activation, as exposure to extracellular calcium without BCR stimulation increased expression of the activation marker, CD83, as well as phosphorylation levels of AKT, PKC, ERK, p38, CAMKII and NFκB. 106

| Mg 2+ permeable ion channels
TRPM7 is a non-selective cation channel that has been implicated in calcium influx and store recovery upon BCR stimulation, 70 as well as Mg 2+ homeostasis. 48 We recently identified a key role for both the channel and kinase functions of TRPM7 in BCR signaling and activation. 107  WT cells, demonstrating that the channel, even at lower expression levels, is sufficient to restore downstream processes in B-cell activation. Although BCR signaling was defective in cells expressing kinase dead TRPM7, these cells also internalized BCR comparable to WT cells, indicating that that kinase function of TRPM7 is not required for this specific process. We hypothesized that the requirement for the channel domain of TRPM7, but not the kinase function in BCR endocytosis may be linked to altered levels of PIP 2 , which are associated with impaired actin reorganization necessary for endocytosis, 109 as we found TRPM7-KO B cells had increased abundance of PIP 2 , but TRPM7-KD cells did not. Interestingly, we found that supplementation with extracellular Mg 2+ restored BCR signaling in TRPM7-KO cells to WT levels, but was unable to rescue the defect in BCR internalization. The reason for this difference in the ability of supplementary Mg 2+ to rescue these processes is not clear, but may be linked to the requirement of divalent cations for activity of DAG kinase ξ, which inhibits DAG signaling by phosphorylating DAG to produce phosphatidic acid and has been shown to regulate ERK signaling in mature FO B cells. 110 It should also be noted that although supplementary Mg 2+ was not sufficient to rescue the BCR internalization defect in TRPM7-KO cells this finding does not rule out a possible role for Mg 2+ in BCR endocytosis, as we cannot be sure that in the absence of TRPM7, uptake of Mg 2+ by other magnesium permeable channels is sufficient to restore physiological levels in intracellular Mg 2+ . Importantly, however, we found that this defect in BCR-antigen internalization was recapitulated in a B cell line treated with the TRPM7-specific inhibitor, NS8593, and that this im-

| Zn 2+ transporters
Zinc is not only crucial for immune cell development and specifically B-cell development, but also humoral immunity. Indeed, zinc deficiency leads to immunodeficiency, suggesting impaired B-cell activation and thus, a lack of antibody production. In addition, several gene deletion studies of Zn 2+ transporters have identified an important role for these channels in B-cell activation. For example, deficiency of ZIP9, a transporter of zinc from the Golgi to the cytosol, in DT40 B cells leads to reduced Akt and ERK phosphorylation upon BCR stimulation. 86 In contrast, a DT40-TKO of ZnT5/6/7, which transport Zn 2+ from the cytosol to the Golgi, did not show this same signaling defect. Decreased ERK and Akt phosphorylation were found to be due, in part, to enhanced phosphatase activity in ZIP9-KO B cells. Similarly, in mice expressing a ZIP7 (shuttles Zn 2+ from ER to cytoplasm) point mutant that renders the transporter non-functional, when crossed with mice expressing a transgenic hen egg lysozyme specific-BCR (SW HEL ) to bypass the developmental defect observed in ZIP7-KO, BCR signaling is also impaired. 39 Consistent with the ZIP9-KO, 86 decreased phosphorylation status of Syk, PLCγ2, and ERK upon BCR stimulation was due to decreased cytoplasmic zinc and consequently increased phosphatase activity in the ZIP7 mutant. 39 ZIP10 is a plasma membrane localized transporter than can flux zinc ions into the cytoplasm of cells. 40,42 A conditional knockout of this transporter in APCs allowed for the study of ZIP10 and its role in B-cell activation. 42 ZIP10-deficient B cells do not have lower resting state zinc levels, however zinc uptake is greatly impaired.
This lack of uptake of extracellular zinc upon BCR stimulation led to hyperactivation of B cells, as Lyn, Syk, ERK, Akt, and NFκB phosphorylation was greatly enhanced. Notably, in contrast to ZIP7 and ZIP9, it was found that ZIP10 positively regulates phosphatase activity; CD45 activity was significantly reduced in ZIP10-deficient B cells and supplementation with high levels of extracellular Zn 2+ restored Lyn phosphorylation to levels comparable to the control cells.
Interestingly, ZIP10-deficient B cells fail to proliferate, possibly due to induction of apoptotic or anergic pathways due to hyperresponsiveness to stimuli.
Taken together, these studies demonstrate the important role of zinc in regulating B-cell responses, primarily, it appears, through regulating phosphatase activity and thus BCR signaling. Further studies examining other zinc transporters expressed on B cells may help further delineate these roles and how dysregulation of zinc leads to immunodeficiency.

| Monovalent cations
Monovalent cations are also very important to B-cell activation, as the cell undergoes membrane potential changes rapidly upon BCR stimulation and efflux of these ions regulates membrane polarization and ROS production. [88][89][90][91] One group found that resting B cells preferably express voltage-gated potassium channels and there is a switch in expression to calcium-dependent channels in activated B cells. 93 However, it has also been shown that BCR stimulation increases the expression of both types of channels, whereas LPS only induces voltage-gated potassium channel expression. 94 Blocking both K Ca 3.1 and K v 1.3 in B cells decreased ERK activation and severely decreases their potential to proliferate upon BCR stimulation. 114 These findings may be correlated to reduced Ca 2+ influx due to lack of K + efflux and loss of the driving force needed for calcium to enter the cells.
Proton currents that are voltage dependent have been recorded in B cells 115 and knocking out HVCN1 demonstrated how important these currents are during B-cell activation. 91 This protein is found in the BCR cap upon stimulation and is internalized with the BCR. H V 1 deficiency led to diminished ROS production and reduced SHP-1 oxidation, which is crucial to inhibiting its negative regulatory effect during B-cell activation. Due to reduced SHP-1 oxidation, global tyrosine phosphorylation of proteins was decreased in H V 1-deficient B cells. Specifically, Syk and Akt phosphorylation were significantly reduced, however ERK and calcium signaling were comparable to WT cells if not slightly increased. In addition, H V 1-deficient cells produced less energy, had reduced metabolism and did not proliferate as well as WT B cells. This demonstrates the crucial role for proton efflux upon BCR stimulation, as reduced ROS production leads to impaired activation. In contrast, some B cell lines express a truncated H V 1 protein that is 20 amino acids shorter and is much more active. 116 Overexpressing this protein form in murine B cell lines resulted in increased ERK activation and surprisingly, increased proliferation upon BCR stimulation. Additionally, overexpressing full-length H V 1 in A20 murine B cells impaired antigen presentation due to reduced acidification of lysosomes, as this protein is endocytosed along with BCR-Ag complexes and causes proton efflux into the cytosol from the lysosome. 91 These findings demonstrate the importance of proton currents both at the plasma membrane and in MHC-class II positive lysosomes in B-cell activation. Loss of these currents, as well as enhanced currents both dysregulate B-cell activation and can have major impacts on humoral immunity.

| I ON CHANNEL S IN E ARLY B-CELL AC TIVATION AND SYNAPS E FORMATION
The first step in B-cell activation requires B cells to find and bind to antigen through the BCR. This is facilitated by the trafficking of B cells through secondary lymphoid organs such as the spleen and lymph nodes, scanning cells for the presence of antigen. B cells predominantly recognize antigen in membrane-bound form present on the surface of APCs including macrophages and dendritic cells. 117 Upon BCR recognition of membrane-bound antigen the B cell spreads rapidly over the antigen-bearing membrane to try and bind to as much antigen as possible, forming multiple discrete "microclusters" of BCR and antigen before contracting and gathering the antigen into a central cluster. 118 BCR-Ag microclusters that are formed are sites of signaling where tyrosine kinases act on their substrates to potentiate the signaling cascade. 119,120 Importantly, these early events not only dictate the propensity of the B cell being activated, but also the degree of antigen that is gathered, internalized, and presented to T cells and thus, the degree of T cell help the B cell receives. 118 In this section, we discuss the role of ions and ion channels important in these early processes of B-cell activation.

| Calcium channels
Calcium is known to be an important secondary messenger for motility of cells, including lymphocytes. 121,122 In the context of B cells, a sodium-calcium exchanger, NCLX, on mitochondrial membranes was found to play an important role to B-cell migration. 123 This channel effluxes Ca 2+ from stores located in the mitochondria in exchange for Na + in the cell. RNA silencing of this channel in both A20 murine B cells and chicken DT40 B cells increased random cell movement and migration which was not altered by addition of the chemokine, CXCL12. Interestingly, NCLX-silenced B cells had higher basal levels of cytosolic calcium but reduced Ca 2+ flux upon addition of CXCL12 in comparison to control cells. In addition, NCLX knockdown altered F-actin organization and Rac1 localization, consistent with lack of directional migration, which appeared to depend on mitochondrial polarization. Inhibition of NCLX in primary murine B cells recapitulated chemotaxis defects and reduced calcium response upon BCR stimulation due to ER calcium leak into the cytosol. Therefore, regulation of cytosolic Ca 2+ is crucial for normal cell motility and chemotaxis, which is critical for B cells to traffic to secondary lymphoid organs surveying for cognate antigen.
It has also been found that ion channels mediate inside-out integrin activation, which is crucial for B cells to traffic to lymphoid organs, 124 as well as mediate B-cell interactions with APCs. 117,125 Ligands that activate non-store-operated non-selective cation channels on B cells allowed for B-cell binding to VCAM-1 and ICAM- 1. 124 This was found to be dependent on their ability to regulate the membrane potential and not due to their ability to influx calcium. During mechanical stress, it was found that both NSCCs and SOCE currents were activated, suggesting an overlapping signaling pathway which involves PLCγ2. However, BCR stimulation attenuates NSCC currents, suggesting that currents from NSCCs such as TRPV4 are active during steady-state and may help B cells migrate, whereas integrin activation upon BCR stimulation to mediate attachment to APCs may rely on CRAC channel currents. These findings demonstrate a key role of calcium and ion channel currents in inside-out integrin activation in response to varied stimuli.
Calcium has also been found to be important in B-cell actin reorganization and B-cell spreading upon recognition of antigen. For example, inhibition of CRAC channels with IP 3 R antagonist 2-APB (2-aminoethoxydiphenyl borate) prevented A20 and primary murine B cells from adhering to anti-Ig-coated coverslips. 126 These cells do not produce radial lamellipodia and fail to spread in response to stimulation. Calcium ionophore treatment of these cells induces higher cytosolic calcium levels and remodeling of the actin cytoskeleton.
Inhibition of calcium influx with 2-APB, on the other hand, prevented dephosphorylation of the actin disassembling protein cofilin, which is necessary for B-cell spreading and microcluster formation. 127 Therefore, calcium influx by ion channels is crucial to the B-cell spreading response.

| TRP channels
We recently identified an important role for TRPM7 in regulating the actin cytoskeleton and consequently several early events in B-cell activation. 107 We demonstrated that deletion of TRPM7 in DT40 B cells led to cytoskeletal defects both at resting state and upon activation, as TRPM7-KO B cells exhibited elongated filopodia-like protrusions, but reduced lamellipodia when cells are adhered to glass.
However, in a planar lipid bilayer system with anti-Ig as surrogate antigen, TRPM7-KO B cells spread over a larger area and form an increased number of BCR-antigen microclusters compared to WT cells, but failed to centrally aggregate the antigen as efficiently as WT B cells. This finding was recapitulated in primary murine B cells expressing only one allele of TRPM7. Importantly, supplementation with high concentrations of extracellular Mg 2+ was not able to rescue the defect in B-cell contraction and centralization of antigen. We also investigated the role of the kinase function of TRPM7 and found that DT40 B cells expressing kinase dead TRPM7 (TRPM7-KD) also exhibited cytoskeletal defects, and also failed to centralize antigen.
Interestingly, however, these cells differed from TRPM7-KO cells as they were slower to spreading and had even higher antigen accumulation. The centralization of antigen was also more severely compromised in TRPM7-KD cells as BCR-antigen microclusters were largely immobilized where they formed and did not come together in one large aggregate as in TRPM7-KO B cells. Differences in microcluster aggregation may be in part due to the failure of actin clearance from the center of the contact with the antigen-containing bilayer in TRPM7-KD cells, whereas actin clearance did not appear to be affect in TRPM7-KO cells. Surprisingly, in contrast to TRPM7-KO cells, the defect in the centralization of antigen did not lead to increased total antigen intensity at the contact, suggesting internalization of antigen is not impaired when the channel is still active. We hypothesized that the phenotypic differences we observed between TRPM7-KO and TRPM7-KD cells may be due to channel functionality being intact in KD cells, but reduced phosphorylation of PLCγ2 as it is a target of TRPM7 kinase. 73 Therefore, magnesium seems to play a key role in antigen internalization, whereas the kinase domain is required for proper spreading and contraction.

| I ON CHANNEL S IN B-CELL EFFEC TOR FUN C TIONS
B-cell activation leads to differentiation into plasma cells that secrete antibodies and provide lasting humoral immunity, or memory cells that can be activated quicker upon re-exposure to the same antigen. B cells also produce and secrete cytokines that can govern local immune responses. Ion dysregulation can lead to improper antibody responses in vivo and defective humoral immunity, as well as impaired cytokine secretion that can impact immune cell responses to various pathogens ( Figure 4).

| Calcium channels
Calcium plays a key role in B-cell secretion of IL-10, an anti-inflammatory cytokine, as both the DKO of STIM1/2 and TKO of IP 3 Rs in mice led to defective IL-10 production after B-cell activation. 60,65 Regulatory B-cell numbers and IL-10 secretion were reduced in IP 3 R-TKO mice, which was attributed to poor induction of the calcineurin-NFAT pathway upon BCR stimulation. 65 In contrast, regulatory B-cell numbers were not impacted by STIM1/2 deficiency; however, IL-10 mRNA transcription was downregulated and IL-10 secretion by this B-cell subset was impaired as seen in IP 3 R-TKO cells. 60  However, by week 2 postimmunization, these mice had comparable levels of serum antibody titers as WT controls. IgM responses upon immunization with a T-independent antigen exhibited a similar trend, as week 1 titers were slightly less than WT, but recovered by week 2. Interestingly, IgM antibody titers in response to LPS were slightly higher than WT controls, but also dropped to being comparable to WT controls by week 2. week post immunization, 65 demonstrating that even minor differences in calcium signaling can lead to varying antibody production in initial stages.
In contrast to reduced calcium signaling, enhanced calcium signaling had strong impacts on antibody production in vivo. ITPKB-KO mice had significantly elevated basal serum titers of IgM, markedly reduced titers of IgG1 and IgA and no difference in IgG3 titers in comparison to WT mice. 66 These mice do have higher numbers of B1 B cells and lower numbers of T cells, which may reflect the differences in basal antibody titers. Interestingly, there was no difference in IgM titers 1 week F I G U R E 4 Ion channels regulate a multitude of B-cell functions. Naive circulating B cells can be activated by recognition and binding of specific antigen to their B-cell receptor (BCR). These B cells internalize this antigen and present it to provide T cell help (bottom). Knockout models of TRPM7 and ITPKB demonstrated impaired antigen presentation through reduced surface expression of costimulatory molecules and MHC class II. The overexpression of H v 1 was found to impair antigen presentation, as this channel is internalized with BCR-Ag complexes and prevents lysosomes from acidifying. TRPML1/2 mutants may impact antigen presentation due to associated defects in lysosomal. Activated B cells can differentiate into plasma cells (top), which secrete antibodies to tag pathogen for removal. IP 3 R knockout mice have impaired GC B-cell numbers and antibody production in the first week postimmunization, but recovered to normal levels by week 2. ITPKB knockout mice had altered antibody responses, as basal antibody levels were increased, but responses postimmunization were severely impaired. ZIP10 knockout mice also have impaired antibody responses, which mirror defects found in mice on zinc-deficient diets. H V 1 knockout mice also have impaired antibody responses and defective class switching. B cells can also regulate immune responses through secreting various cytokines (right). Dysregulated calcium influx in STIM1/2 and IP 3 R knockout B cells upon BCR stimulation led to decreased NFAT activity and thus, lower IL-10 secretion. TRPC1-and TRPC3-deficient B cells also have decreased NFAT activity, suggestive of decreased IL-10 secretion. P2X7 dampens calcium responses through sodium influx upon BCR stimulation in the presence of ATP and loss of this channel may lead to enhanced IL-10 secretion due to enhanced calcium and NFAT activity. B cells can differentiate into memory B cells after activation (left). Currently, it is unknown whether any ion channels play a role in memory B-cell generation and further research is needed to understand their role in regulating this process. Key regulators that inhibit B-cell functions are highlighted in red or enhance Bcell functions in green. The degree of impact on B-cell functions are indicated by font size, with more severe impact in larger font and more modest impact represented by a smaller font postimmunization with a T-independent antigen despite higher basal levels; however, there was a substantial reduction in IgG3 after 1 week.
This demonstrates the crucial role of fine-tuning calcium responses during B-cell activation, as hyperresponsiveness and increased calcium leads to dysregulated antibody production in some contexts.

| Unconventional ions
Magnesium homeostasis is essential for B-cell development, maintenance, and activation. Conditional knockout of TRPM7 in B cells results in a developmental block at the pro-B-cell stage, 45 which has made it difficult to study the importance of magnesium in humoral immune responses. It has been shown, however, that MAGT1-KO mice have impaired plasma cell numbers preimmunization. 128  Zinc has also been found to be very important in humoral immunity, as dysregulated homeostasis of this divalent cation leads to immunodeficiency. Patients with ZIP7 mutations and mouse models with a knock-in mutation of ZIP7 both have very low or non-detectable levels of basal serum titers of IgG and IgM, respectively. 39 Mice with knock-in ZIP7 mutations never survived long enough to determine basal serum IgG levels. A B-cell conditional knockout of ZIP10 also demonstrated that dysregulated zinc homeostasis effects humoral immunity. 42  immunization with a T-dependent antigen; however, IgM antibody titers were comparable to WT, suggesting lack of H V 1 impairs isotype switching. Interestingly, staining for antigen-specific plasma cells and GCs demonstrated that GC reactions proceeded normally in H V 1 deficient mice, which was supported by evidence of unaffected affinity maturation and normal GC sizes and cell numbers. Therefore, defects in isotype switching were due to plasma cell and plasmablast intrinsic defects. Immunization with a T-independent antigen also led to defective antibody production in HVCN1-KO cells, as titers of both IgM and IgG3 were reduced over This demonstrates the significance of potassium ion efflux upon reactivation of different populations of memory B cells and that dysregulation of these channels leads to abrogated proliferation, which may impact immune responses toward secondary infections.

| I ON CHANNEL S IN HE ALTH AND DISE A SE
Much of the progress in our understanding of ion channels stems from observations of human patients with ion deficiencies, either due to nutritional deficiencies or genetic mutations in ion channels.
Mutations in genes encoding ion channel components result in a wide range of "channelopathies," diseases that affect a variety of physiological processes and range from mild to life-threatening. 129 In the context of the immune response, the discovery of patients with severe combined immunodeficiencies due to mutations in CRAC channel components 63,64,130,131 and MAGT1 128 has facilitated and spurred the study of ion channels and ion signaling in the immune system. The study of these patients, as well as the generation of murine models with targeted deletion of genes encoding ion channels and transporters have helped identify many key roles of ions, the critical ion channels/transporters regulating these ions, and their important function in the immune response.
Although the primary immunodeficiencies associated with mutations in ion channel genes so far identified are largely associated with defects in T cells, the evidence gathered from murine models discussed here clearly demonstrate an important role for ion channels/transporters in B-cell biology. Notably, some of the first evidence for a role for ions in B-cell biology came from observations of patients not with genetic mutations in ion channels, but rather ion deficiencies due to malnutrition, 132 which is surprisingly common throughout the world. Indeed, it is estimated that 20%-30% of the global population are zinc deficient, 133 but for some subregions the estimates are >70%. Similarly, up to 75% are magnesium deficient. 134 These deficiencies resulting from malnutrition are rampant not only in developing countries, but also surprisingly common in developed countries; an estimated 50% of the US population do not meet the recommended daily allowance of magnesium. 135 In addition to dietary deficiencies, patients with malabsorption issues caused by intestinal or colon damage, such as Crohn's, irritable bowel syndrome, and celiac disease are frequently deficient in critical ions. 136 Moreover, many of the therapies used to treat a variety of diseases lead to ion deficiencies; for example, digoxin for the treatment of heart conditions, or the chemotherapeutic cisplatin, can lead to Mg 2+ deficiencies. [137][138][139] Thus, continued effort to elucidate the role of ion signaling, and the characterization of varied ion channels in immune cells, is fundamental in improving our understanding of human health and disease, and possibly the potential to improve human disease conditions through targeting ion channels with pharmacological inhibitors (for which several have already been identified) or nutritional ion deficiencies.

| FUTURE PER S PEC TIVE S
The study of ion channels and ion signaling in lymphocytes, and particularly in B cells, is in its infancy. Granted, we have learned much about Ca 2+ as a second messenger and the discovery of STIM and ORAI proteins was a key to elucidating how Ca 2+ fluxes are regulated. Yet, these proteins are found to be almost dispensable in B-cell development and activation. This suggests that other ion channels in B cells contribute to calcium regulation during these stages that have yet to be explored. In addition, new research into other ions, such as Mg 2+ and Zn 2+ , have led to surprising new discoveries of the critical role of these ions in B-cell development, activation, differentiation, and effector function, and the ion channels important in the regulation of these ions are being identified. However, B cells express a wealth of ion channels which remain completely unexplored in the context of B-cell biology. In addition, our knowledge of how these different ion channels work in concert to fine-tune B-cells responses is not well understood. We are also just beginning to unravel the impact of genetic mutations/SNPs and protein expression changes of ion channels in B cells and how these can contribute to disease. Understanding the complex network of ion signaling during the varying stages of B cell "life" could help in the development of therapeutics to treat B-cell diseases.

CO N FLI C T O F I NTE R E S T
The authors have no conflicting financial interests.