Ca²⁺-Activated K⁺ Channel K_Ca3.1 as a Therapeutic Target for Immune Disorders

Abstract

In lymphoid and myeloid cells, membrane hyperpolarization by the opening of K⁺ channels increases the activity of Ca²⁺ release-activated Ca²⁺ (CRAC) channels and transient receptor potential (TRP) Ca²⁺ channels. The intermediate-conductance Ca²⁺-activated K⁺ channel K_Ca3.1 plays an important role in cell proliferation, differentiation, migration, and cytokine production in innate and adaptive immune systems. K_Ca3.1 is therefore an attractive therapeutic target for allergic, inflammatory, and autoimmune disorders. In the past several years, studies have provided new insights into 1) K_Ca3.1 pharmacology and its auxiliary regulators; 2) post-transcriptional and proteasomal regulation of K_Ca3.1; 3) K_Ca3.1 as a regulator of immune cell migration, cytokine production, and phenotypic polarization; 4) the role of K_Ca3.1 in the phosphorylation and nuclear translocation of Smad2/3; and 5) K_Ca3.1 as a therapeutic target for cancer immunotherapy. In this review, we have assembled a comprehensive overview of current research on the physiological and pathophysiological significance of K_Ca3.1 in the immune system.

1. INTRODUCTION

More than 70 different K⁺ channel genes have been identified in the mammalian genome. These are classified as i) voltage-gated, ii) inward-rectifier, iii) Ca²⁺-activated, and iv) two-pore domain superfamilies. In non-excitable cells, including immune cells, the activation of K⁺ channels promotes Ca²⁺ signaling and transcriptional regulation by increasing the electrical driving force for Ca²⁺ entry from Ca²⁺ release-activated Ca²⁺ (CRAC) and transient receptor potential (TRP) Ca²⁺ channels. K⁺ channels therefore regulate proliferation, differentiation, migration, and cytokine/chemokine/chemical mediator expression in immune cells.^1,2) The auxiliary subunits of K⁺ channels influence gating kinetics, membrane trafficking, and the Ca²⁺ sensitivity of pore-forming α subunits.^3,4) A recent study has shown that the elevation of extracellular K⁺ concentration prevents T cell function and signaling, without changing membrane potential or Ca²⁺ entry; conversely, the reduction of intracellular K⁺ concentration by K⁺ channel activators improves T cell dysfunction.⁵⁾

The intermediate-conductance Ca²⁺-activated K⁺ channel K_Ca3.1 contributes to the control of Ca²⁺ signaling in the immune system.⁶⁾ Pharmacological blockade of K_Ca3.1 reduces the expression and secretion of pro-inflammatory cytokines and chemokines.^6–8) Therefore, K_Ca3.1 is an attractive therapeutic target for multiple sclerosis (MS), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), asthma, atherosclerosis, allergic rhinitis, and various tissue fibrosis.¹⁾ In addition, auxiliary subunits that positively or negatively control K_Ca3.1 activity, as well as transcriptional and post-transcriptional regulators that control K_Ca3.1 gene expression, have been identified: i) phosphoinositide-3-kinase, class 2, β polypeptide (PI3K-C2B), nucleoside diphosphate kinase-B (NDPK-B), phosphohistidine phosphatase 1 (PHPT-1), myotubularin related protein 6 (MTMR6), tripartite motif containing 27 (TRIM-27), and phosphoglycerate mutase family member 5 (PGAM5) as auxiliary subunits,^9–13) and ii) activating protein-1 (AP-1), repressor element 1-silencing transcription factor (REST/NRSF), histone deacetylase 2/3 (HDAC2/3), and microRNA-497-5p (miR-497-5p) as transcriptional and post-transcriptional regulators.^14–18) Here, we review current topics on the pathophysiological significance of K_Ca3.1 and its regulators in the immune system.

2. K_CA3.1 PHARMACOLOGY AND ITS AUXILIARY REGULATORS

Recent advances regarding the therapeutic potential of K_Ca3.1 blockers have been reviewed.^1,18) Gene silencing and the pharmacological blockade of K_Ca3.1 exhibit significant efficacy in treating RA, asthma, IBD, allergic rhinitis, Sickle cell disease, delayed type hypersensitivity, and immunoglobulin E (IgE)-mediated anaphylaxis.^1,6–8,18) Recently, Nguyen et al. visualized the mechanism of a small molecule K_Ca3.1 blocker (TRAM-34, PF-05416266, and NS6180) at the atomistic level using Rosetta ligand computational molecular modeling software.¹⁹⁾ This Rosetta K_Ca3.1 pore model enables the structure-based drug design of new K_Ca3.1 blockers belonging to different structural classes. Several studies on post-translational modifications of K_Ca3.1 have recently been reported. Oliván-Viguera et al. showed that ω3-fatty acids such as α-linolenic acid and docosahexiaenoic acid inhibited K_Ca3.1 by negatively modulating its expression in fibroblasts.²⁰⁾ These ω3-fatty acids decrease the disease activity of autoimmune diseases such as MS, RA, and IBD,²¹⁾ suggesting that they may down-regulate increased K_Ca3.1 expression in inflammatory helper T type 1 (Th1) and Th17 cells, in addition to suppressing pro-inflammatory cytokine production. The angiotensin II receptor type 1 (AT1) antagonist, telmisartan (TLM), also exerts an anti-inflammatory effect. Zhang et al. showed the inhibitory effect of TLM on K_Ca3.1 activity, suggesting that the TLM-induced reduction of pro-inflammatory cytokine production may be due, at least in part, to an inhibitory effect on K_Ca3.1 activity.²²⁾ Furthermore, the activation of protein kinase A reduced K_Ca3.1 gating and trafficking to the plasma membrane by phosphorylation of Ser332/334 in the calmodulin-binding C-terminus,²³⁾ and also led to the inhibition of K_Ca3.1 synthesis and down-regulation of PI3K-C2B.²⁴⁾

The following have been shown to be potent K_Ca3.1 activators: 1-ethylbenzimidazolin-2-one (1-EBIO), 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one (DCEBIO), 6,7-dichloro-1H-indole-2,3-dione 3-oxime (NS309), and naphtho[1,2-d]thiazol-2-ylamine (SKA-31), and they exhibit similar potency for small-conductance K_Ca2.x. The selective K_Ca3.1 activator SKA-121, a derivative of SKA-31, was developed using Rosetta modeling.²⁵⁾ Eil et al. showed that K_V1.3 and K_Ca3.1 activators are possible candidates for cancer immunotherapy,⁵⁾ suggesting that SKA-121 may have an effective therapeutic profile for cancer immunotherapy. In addition, K_Ca3.1 plays an important role in the fine-tuning of microglia activation and migration. In microglial cells, K_Ca3.1 was activated through protein kinase G (PKG)-dependent pathways,²⁶⁾ and was up-regulated by interleukin-4 (IL-4) receptor activation through Janus kinase 3 (JAK3) and Ras/MEK/extracellular signal-regulated kinase (ERK) signal pathways.²⁷⁾

The auxiliary regulators of K_Ca3.1 play a crucial role in T cell activation and quiescence, and are potential therapeutic targets in the treatment of K_Ca3.1-related immune disorders (Fig. 1). NDPK-B directly activates K_Ca3.1 by phosphorylating His358 in the C terminus,²⁸⁾ and PHPT1 directly inhibits K_Ca3.1 by dephosphorylation. PI3K-C2B acts upstream of NDPK-B to activate K_Ca3.1, and TRIM27 inhibits K_Ca3.1 activity by ubiquitinating PI3K-C2B. MTMR6 inactivates NDPK-B by dephosphorylating phosphatidylinositol 3-phosphate, which acts upstream of NDPK-B. Recently, a new K_Ca3.1 regulator, PGAM5, was identified as a negative regulator of CD4⁺ T cells.¹³⁾ PGAM5 negatively regulates NDPK-B to inactivate K_Ca3.1. It has also been shown that a reduction of K_Ca3.1 activity by gene silencing of PI3K-C2B decreased FcεRI-stimulated Ca²⁺ entry, cytokine production, and degranulation in mast cells.²⁹⁾ These positive and negative K_Ca3.1 regulators are both expected to be new pharmacologic targets for treating immune disorders, although their potent and selective inhibitors or activators have not yet been discovered.

Fig. 1. Positive and Negative Auxiliary Regulators of K_Ca3.1

(Color figure can be accessed in the online version.)

3. POST-TRANSCRIPTIONAL AND PROTEASOMAL REGULATION OF K_CA3.1

Our previous review summarized the transcriptional, epigenetic, spliceosomal, and proteasomal regulation of K_Ca3.1.³⁰⁾ The recycling endosome Rab8 contributes to the trafficking of K_Ca3.1 from the endoplasmic reticulum (ER) Golgi to the plasma membrane. Recent studies have identified the Rab8-dependent recycling pathway as an intracellular route for protein delivery to an immunological synapse (IS)^31,32) (Fig. 2). K_Ca3.1 is compartmentalized in the IS of T lymphocytes,³³⁾ suggesting that Rab8 may be an essential contributor to the localization of K_Ca3.1 and to the efficient coupling between K_Ca3.1 and CRAC channels in the IS. However, specific and potent promoting compounds of K_Ca3.1 degradation have not yet been developed.

Fig. 2. Transcriptional, Post-Transcriptional, and Post-Translational Regulation of K_Ca3.1

(Color figure can be accessed in the online version.)

Epigenetic mechanisms such as DNA methylation, histone modification, and RNA interference modulate gene expression. Histone deacetylase inhibitors are expected to be potentially therapeutic against autoimmune diseases such as type I diabetes, MS, pancreatitis, and IBD.^34–37) For instance, HDAC isoforms such as HDAC2, HDAC3, and HDAC6 are involved in chronic intestinal inflammation, and pan-HDAC inhibitors (HDACis) suppress pro-inflammatory cytokine production such as IL-6, tumor necrosis factor α (TNF-α), and interferon γ (IFN-γ).³⁷⁾ In K_Ca3.1-expressing breast and prostate cancer cells, pharmacological and small interfering RNA (siRNA)-mediated inhibition of HDAC2 and HDAC3 resulted in the down-regulation of K_Ca3.1.¹⁷⁾ In addition, K_Ca3.1 activity and expression were signficantly increased in CD4⁺ T cells of IBD model mice.⁸⁾ These suggest that K_Ca3.1 amplification in CD4⁺ T cells in IBD model mice may be induced by the up-regulation of HDAC2 and/or HDAC3, whereas the inhibition of K_Ca3.1 may, at least in part, be involved in HDACis-induced improvement in the pathogenesis of IBD (Fig. 2).

K_Ca3.1 is one of the putative target genes of the tumor suppressor miR-497-5p: miR-497-5p down-regulates K_Ca3.1 expression and contributes to the inhibition of angiosarcoma malignancy development.³⁸⁾ Additionally, miR-497-5p is up-regulated during myofibroblast differentiation in pulmonary fibrosis,³⁹⁾ and K_Ca3.1 has been associated with the induction of pulmonary fibrosis myofibroblasts.⁴⁰⁾ It remains to be determined whether immune disorder-induced changes in miR-497-5p are associated with the up-regulation of K_Ca3.1 in inflammatory T cells and macrophages (Fig. 2).

4. K_CA3.1 AS A REGULATOR OF IMMUNE CELL MIGRATION, CYTOKINE PRODUCTION, AND PHENOTYPIC POLARIZATION

K_Ca3.1 is related to immune cell migration. In activated T cells, K_Ca3.1 accumulates in the uropod rear portion, but not at the leading edge (predominant in K_v1.3^high T cells); together with TRPM7 Ca²⁺ channels it regulates T cell migration of IL-7Rα^low effector memory CD8⁺ cells^41,42) (Fig. 3). β1 integrins play important roles in the adhesion, migration, proliferation, and differentiation of T cells, and contribute to the efficient and functional assembly of ion channels and their regulators in lipid rafts. Recent studies have shown that the β1 integrin contributes to swelling activated K_Ca3.1 and its cellular localization in the rear.^43,44) The roles of ion channels in chemokine-induced migration are largely unknown. The blockade of K_Ca3.1 decreased chemokine-induced dendritic cell migration and monocyte chemotaxis by the down-regulation of migration markers (CCR5 and CCR7) and chemotaxis related factor CCL7, respectively.^45–47) In neutrophils, K_Ca3.1 is a key factor in the chemotactic response and migration capacity, without affecting Ca²⁺ entry.⁴⁸⁾

Fig. 3. Role of K_Ca3.1 in Immune Cell Migration

(Color figure can be accessed in the online version.)

Our previous study showed that pharmacological blockade of K_Ca3.1 reduced the expression of IFN-γ in CD4⁺ T cells and of IL-6 in inflammatory tissues from IBD model mice.⁸⁾ In mast cells and RA synovial fibroblasts, K_Ca3.1 blockade reduced the expression and secretion of pro-inflammatory cytokines IL-6 and IL-8.^7,49) Similarly, in activated brain microglia, K_Ca3.1 blockade suppressed pro-inflammatory cytokines and chemical mediators.^50,51) Inflammatory responses were regulated by the AKT/mammalian target of rapamycin (mTOR) signaling pathway, and the blockade of K_Ca3.1 inhibited the activation of the PI3K/AKT signaling pathway in immune cells.^50,52) A recent study showed that increased intracellular K⁺ by K⁺ channel blockade may reduce pro-inflammatory cytokine expression by inhibiting the AKT/mTOR signaling pathway.⁵⁾

K_Ca3.1-mediated activation of the AKT/mTOR signaling pathway is also responsible for an increase in tumor-infiltrating microglia/macrophages.⁵³⁾ Furthermore, K_Ca3.1-mediated activation of the signal transducer and activator of transcription 1 (STAT1) signaling pathway is responsible for macrophage polarization toward the M1 phenotype.⁵⁴⁾

5. ROLE OF K_CA3.1 IN THE PHOSPHORYLATION AND NUCLEAR TRANSLOCATION OF SMAD2/3

IL-10 is an anti-inflammatory cytokine produced by B cells, mast cells, macrophages, dendritic cells, and several T cell lineages (i.e., Th2 and regulatory T, T_reg). IL-10 induces immunosuppression and assists in an escape from tumor immune surveillance.^41,55) Smad2 and Smad3 are key regulators of the transforming growth factor-β (TGF-β)-mediated T_reg cell conversion of naïve T cells. Phosphorylation of Smad2/3 primarily mediates TGF-β-induced transcriptional regulation, and phosphorylated Smad2/3 translocate into the nucleus to regulate genes. In lung myofibroblasts, K_Ca3.1 constitutively promotes fibroblast to myofibroblast differentiation, mediation of the Smad2/3 signaling pathway, and the blockade of K_Ca3.1 prevented myofibroblast differentiation by attenuating the phosphorylation and nuclear translocation of Smad2/3.^56,57) Similarly, pharmacological blockade of K_Ca3.1 significantly inhibited the nuclear translocation of Smad2/3, and suppressed tissue fibrosis and vascular calcification.^58–60) Blockade of K_Ca3.1 led to a reduction of the phosphorylation of Smad2/3, and may be a novel target for therapeutic intervention in patients with tissue fibrosis. A recent study has shown that K_Ca3.1 blockade inhibits the TGF-β1 signaling pathway through PI3K/AKT/mTOR signaling pathways.⁶¹⁾ In an aggressive B cell lymphoma, the inhibition of PI3K/AKT/mTOR and STAT3 signaling pathways reduced the release of IL-10.⁶²⁾ Further studies will be needed to clarify the mechanisms underlying K_Ca3.1-mediated IL-10 expression in IL-10 producing immune cells.

6. K_CA3.1 AS A THERAPEUTIC TARGET OF CANCER IMMUNOTHERAPY

Recent studies have reported that ion channels, including K⁺ channels, are potential therapeutic targets for cancer immunotherapy.⁶³⁾ Koshy et al. concluded that K_Ca3.1, but not K_V1.3, is a beneficial target for cancer immunotherapy. Their study showed that K_Ca3.1 is preferentially up-regulated by adherent natural killer A-NK cells, and that a K_Ca3.1 blocker increases the ability of A-NK cells to reduce tumor growth.⁶⁴⁾ In addition, Eil et al. showed that K_Ca3.1 and K_V1.3 activators can maintain tumor-specific effector T cell function.⁵⁾ Within a tumor microenvironment, the elevation of extracellular K⁺ concentration suppresses the TCR-mediated Akt/mTOR signaling pathway. The reduction of intracellular K⁺ by K_Ca3.1 activators promotes pro-inflammatory cytokine expression through AKT/mTOR signaling, suggesting that K_Ca3.1 activators may increase the ability of tumor-specific effector T cells to reduce tumor growth.

7. CONCLUDING REMARKS

K⁺ channels play important roles in the regulation of immune and inflammatory processes, and are therefore attractive therapeutic targets for immune disorders.¹⁾ Previous studies have suggested the predominance of K_V1.3 on effector memory T (T_EM) cells, thus K_V1.3 inhibitors were developed to treat chronic immune disorders. However, there is emerging evidence that K_Ca3.1 is also a potential therapeutic target for immune and inflammatory disorders. Chiang et al. showed that K_V1.3 and K_Ca3.1 cooperatively and compensatorily regulate T_EM cell function.⁶⁵⁾ In addition, the physiological and pathophysiological roles of many molecules related to transcriptional, post-transcriptional, and post-translational modifications of K_Ca3.1 have been studied. Recently, K_Ca3.1 has attracted attention as a key player in cancer immune surveillance. Based on the recent advances in K_Ca3.1 research overviewed here, new therapeutic strategies may emerge for such immune disorders as autoimmune diseases, chronic inflammatory diseases and cancers.

Conflict of Interest

The authors declare no conflict of interest.

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