Ion channels and transporters in lymphocyte function and immunity

Abstract

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

Figure 1. Ion channels regulating Ca²⁺ signalling in lymphocytes

CRAC channels are activated following antigen receptor (TCR, BCR) engagement, which results in the activation of phospholipase Cγ (PLCγ), production of inositol 1,4,5 triphosphate (InsP3) and release of Ca²⁺ from ER Ca²⁺ stores ^{, ,} . The ensuing activation of STIM1 and STIM2 results in the opening of ORAI1 CRAC channels and store-operated Ca²⁺ entry (SOCE) (for details see Fig. 2, 3). Sustained Ca²⁺ influx through CRAC channels leads to the activation of Ca²⁺-dependent enzymes and transcription factors, including calcineurin and NFAT. P2X receptors such as P2X4 and P2X7 are non-selective Ca²⁺ channels activated by extracellular ATP originating, for instance, from autocrine ATP release through pannexin hemichannels (Panx1) . Ca²⁺ influx in lymphocytes depends on the gradient between Ca²⁺ concentrations in the extracellular (~ 1 mM) and intracellular (~ 0.1 μM) compartments and on an electrical gradient established by two K⁺ channels, Kv1.3 and KCa3.1, and the Na⁺-permeable channel TRPM4 ^, . Abbreviations: SERCA, sarco/endoplasmic reticulum Ca²⁺ ATPase.

Figure 2. The molecular choreography of CRAC channel activation

In resting lymphocytes, ER Ca²⁺ stores are filled with Ca²⁺ bound to the EF hand Ca²⁺ binding domain in the N-terminus of STIM1. Antigen receptor stimulation causes the activation TCR/BCR-proximal signalling cascades and the production of InsP₃, resulting in the release of Ca²⁺ from the ER through InsP₃ receptors, which are non-selective ion channels. The fall in ER Ca²⁺ concentration leads to the dissociation of Ca²⁺ from the EF hand domain in STIM1, unfolding of the STIM1 N-terminus and the multimerization of STIM1 proteins . STIM1 multimers translocate to junctional ER sites in which the ER membrane is juxtaposed to the plasma membrane. STIM1 multimers form large clusters (or puncta) into which they recruit ORAI1 CRAC channels. A minimal CRAC channel activation domain (variously referred to as the CAD, SOAR, OASF or CCb9 domain) in the C terminus of STIM1 (green boxes) is necessary and sufficient for ORAI1 binding, CRAC channel activation, and SOCE ^{, , ,} . This domain contains two coiled (CC) domains, which interact with a CC domain in the C-terminus (red boxes) and additional domains in the N-terminus (not shown) of ORAI1 . Abbreviations: SAM, sterile-alpha motif.

Figure 3. P2X receptors are non-selective Ca²⁺ channels mediating T cell activation

P2X receptors are homotrimeric ion channels located in the plasma membrane of lymphocytes. They form non-selective ion channels that allow influx of Ca²⁺, Na⁺ and other cations ^, . P2X1, P2X4 and P2X7 are activated by extracellular ATP, for which they have distinct affinities . P2X7 is unusual among P2X receptors, as it functions as a non-selective cation channel at low extracellular [ATP], but forms large pores following prolonged exposure to high extracellular [ATP]. In addition, it was reported to mediate K⁺ efflux required for NLRP3 inflammasome activation in innate immune cells . The ATP required for P2X receptor opening in T cells originates from dying cells, ATP secreting cells (paracrine) or T cells themselves (autocrine). T cells were shown to release ATP through pannexin 1 hemichannels following TCR stimulation and mitochondrial ATP production . Opening of P2X receptors results in Ca²⁺ influx that has been suggested to synergizes with SOCE to activate Ca²⁺ dependent signalling molecules and transcription factors resulting in enhanced cytokine expression. P2X7 dependent ERK1/2 activation was shown to repress FOXP3 transcription in favor of RORγt expression, thereby promoting the differentiation of CD4 T cells into Th17 cells .

Figure 4. Mg²⁺ channels and transporters in lymphocytes

A, TRPM7 is a Mg²⁺-permeable channel that is a “chanzyme” because it functions as both an ion channel and an enzyme through its C-terminal serine/threonine kinase domain. As with other TRP channels, its ion channel pore is located between transmembrane (TM) domains 5 and 6. TRPM7 is a non-selective cation channel and conducts Mg²⁺ and Ca²⁺ with near equal permeabilities. One of the defining features of TRPM7 channels is inhibition by intracellular Mg²⁺ but the mechanism of Mg²⁺ regulation is incompletely understood . TRPM7 function further depends on PIP₂ and is regulated by extracellular pH . B, MagT1 belongs to a family of recently identified Mg²⁺ transporters. It is highly selective for Mg²⁺ compared to Ca²⁺, Zn²⁺, Ni²⁺ and other divalent cations . MagT1 opening in response to TCR stimulation results in a global increase in [Mg²⁺]_i, activation of PLCγ1 and Ca²⁺ influx, presumably via CRAC channels. The mechanisms by which TCR signalling causes MagT1 to open and how Mg²⁺ activates PLCγ1 are not understood. Two MagT1 isoforms have been described: a short one (335 aa) with a confirmed tetraspanning membrane topology (*) and a longer version (367 aa) predicted to contain five TM domains and an intracellular N terminus (**), which may facilitate TCR-dependent activation of MagT1.

Figure 5. Zinc signalling and Zinc transporters in T cells

A, Zn²⁺ ions have activating and inhibitory effects on signal transduction in T cells ^–. Zn²⁺ mediates the recruitment of the src kinase Lck to CD4 and CD8 and promotes Lck dimerization, resulting in enhanced TCR signalling . Zn²⁺ also promotes protein kinase C (PKC) signalling, likely by recruiting PKC to the plasma membrane. By contrast, Zn²⁺ inhibits the activity of the phosphatase calcineurin, thus preventing nuclear translocation of the transcription factor NFAT ^, . Furthermore, Zn²⁺ inhibits the function of interleukin-1 receptor-associated kinase (IRAK) 4, thereby restraining signalling through the IL-1R and activation of NF-kB. Inhibitory effects of Zn²⁺ on both NFAT and NF-kB may explain the reduced production of cytokines such as IL-2 and IFNg in the presence of increasing extracellular [Zn²⁺]. B, Increases in intracellular [Zn²⁺] in lymphocytes are mediated by Zinc influx from the extracellular space or efflux from intracellular organelles that are mediated by Zrt-Irt like proteins (ZIP). These Zn²⁺ transporters contain eight transmembrane domains (TM) with an aqueous pore predicted to be formed by TM4 and TM5 . Zn²⁺ is exported from the cytoplasm by ZnT transporters resulting in decreased intracellular [Zn²⁺]. In T cells, the Zinc transporters ZIP3, ZIP6 and ZIP8 have been implicated in Zn²⁺ influx ^{, ,} , whereas the nature of ZnT proteins mediating Zn²⁺ efflux in lymphocytes are presently unknown. In addition to Zn²⁺ transport, intracellular Zn²⁺ levels are modulated by Zn²⁺ binding to metallothionein and other proteins.