Intermediate-conductance Ca 2 +-activated K + channel K Ca 3 . 1 and its related molecules in T-lymphocytes

The intermediate-conductance Ca-activated K channel KCa3.1 (also called IKCa, IK1 and KCNN4) plays an essential role for the positive-feedback mechanism required for the enhancement of Ca signaling in activated T-lymphocytes, and regulates the T cell activation, proliferation and differentiation. Recent reports have suggested that T-lymphocyte KCa3.1 K channel is an attractive target for the therapeutic strategies of inflammatory bowel disease (IBD). In addition, the potential KCa3.1 regulators also play critical roles in the T cell functions: phosphoinositide-3-kinase, class 2, beta polypeptide (PI3K-C2B), nucleoside diphosphate kinase B (NDPK-B), phosphohistidine phosphatase 1 (PHPT-1) and myotubularin related protein 6 (MTMR-6). We recently described that the up-regulation of KCa3.1 and NDPK-B might constitute an initiation step in CD4 Tlymphocyte proliferation in acute IBD and might be one of important mechanisms underlying the pathogenesis of IBD (Ohya et al., Am J Physiol Gastrointest Liver Physiol. 306:G873-G885). KCa3.1 K channel and its regulators may be potential therapeutic targets for inflammatory diseases such as IBD.


Introduction
In the immune cells such as lymphocytes, mast cells and macrophages, Ca 2+ -activated K + channel is characterized by an intermediate-conductance KCa3.1 channel [1] .In Tlymphocytes, the activation of KCa3.1 channels by the intracellular Ca 2+ rise during T cell receptor (TCR)-evoked Ca 2+ signaling hyperpolarizes the membrane potential, and thereby promotes Ca 2+ influx via Ca 2+ -release activated Ca 2+ (CRAC) channels composed of the complex of Orai and STIM families [2][3][4] .KCa3.1 channel plays an essential role for the positive-feedback mechanism required for the enhancement of Ca 2+ signaling in activated T-lymphocyte.KCa3.1 expression is relatively low in quiescent human naive (TN), central memory (TCM), and effector memory (TEM) T cells.Upon T cell activation, KCa3.1 channel is upregulated in TN and TCM cells, whereas voltage-gated K + channel, KV1.3 instead of KCa3.1 is up-regulated in TEM cells [5,6] .In addition, KCa3.1 currents are significant higher in helper T (Th1) cells compared with that in Th2 cells, corresponding to the larger Ca 2+ signaling in Th1 cells [7] .Similar to the case with T cells, quiescent naive and early memory B cells express low levels of KCa3.1 channels, and

RESEARCH HIGHLIGHT
effector cells derived from naive memory B cells express high levels of them [8] .KCa3.1 channel is a potential molecular target for pharmacological intervention in a variety of diseases, e.g.autoimmune diseases such as multiple sclerosis and rheumatoid arthritis, transplant rejection, sickle cell anemia, asthma, atherosclerosis, fibrosis, and traumatic brain injury [9][10][11] .
As transcriptional factors of KCa3.1, activator protein-1 (AP-1) (Fos/Jun heterodimers) and repressor element-1 silencing transcription factor (REST) have been identified [16, 17] .KCa3.1 gene expression is positively and negatively regulated by AP-1 and REST, respectively.Diseaseassociated alternative splicing of KCa3.1 has not been identified yet, however, the increase in KCa3.1 channel activity by the down-regulation of KCa3.1-ΔN may have a critical role in the pathogenesis of inflammatory diseases.
Senicapoc has been evaluated in clinical trials for sickle cell anemia and asthma, however, phase II and III studies of Senicapoc for patients of these diseases have been recently terminated [21,22] .Nevertheless, Senicapoc is recognized as a safe and well-tolerated drug because longterm treatment with Senicapoc has shown no serious toxic and adverse effects in preclinical and clinical trials.Human lung mast cells (HLMC), which are implicated in the pathology of asthma, express high levels of KCa3.1 channels, and pharmacological blockade of KCa3.1 channels attenuates HLMC proliferation and migration [23] .Senicapoc has indicated the significant efficacy for the late asthmatic response (LAR) (www.pfizer.com).In addition to allergic asthma, KCa3.1 is a potential therapeutic target for inflammatory and autoimmune diseases.Genetic disruption and/or pharmacological blockade of KCa3.1 have indicated the significant efficacy for IgE-mediated anaphylaxis and DTH (delayed type hypersensitivity) [24,25] , and also developed less severe colitis in two different IBD model mice [26] .In KCa3.1 knock-out mice, Th1 and Th2 cells show smaller Ca 2+ influx and cytokine production during TCR activation, whereas Th17 and regulatory T cells possess normal function.Moreover, agenome-wide association study has identified a certain KCa3.1 SNP (Single Nucleotide Polymorphism) (rs2306801) as a potential susceptibility factor in ileal Crohn's disease (CD) in the Australian and New Zealand populations [27] .Clinical trials to evaluate the efficacy of Senicapoc and the other KCa3.1 blockers for inflammatory diseases will be conducted in near future.

Localization of KCa3.1 channel and its interacting molecules in immunological synapse
The immunological synapse (IS) is a highly organized structure triggered by sustained TCR engagement on the surface of an antigen-presenting cell (APC), and is characterized by an extensive reorganization of signaling proteins to the T cell-APC contact zone.The IS formation plays an essential role in both the initiation and the maintenance of immune responses [34] .Similar to ion channels contributing to T-lymphocyte Ca 2+ signaling, such as CRAC and KV1.3 channels, KCa3.1 channels are recruited to the IS upon antigen stimulation, and become part of the signaling complex that facilitates T cell proliferation and cytokine production [35] .However, the distribution of KCa3.1 channels to the IS is not prevented by pharmacological blockade of KCa3.1 channel activity, and the increase in Ca 2+ influx mediated by KCa3.1 channel activation does not contribute to the initiation and the maintenance of IS formation.
Functional regulators of KCa3.1 channel, NDPK-B, PHPT-1, PI3K-C2B, and MTMR6 are recruited to the IS, and spatial arrangement in the IS of them is critical to KCa3.1 channel regulation.NDPK-B and PHPT-1, which are coupled in the C-terminus of KCa3.1 channel, form the clustering at the IS together with KCa3.1 channel [28,29] (Fig. 1).At the IS, NDPK-B interacts with the integrin cytoplasmic domain-associated protein A (ICAP1A), linking KCa3.1 to β-integrin [36] .Moreover, PI3K-C2B colocalizes with Zap70 (ζ-Chain-Associated Protein Kinase 80) and p56 lck in peripheral micro clusters, and is recruited to the IS during antigen stimulation [31] .Recruitment of NDPK-B and PI3K-C2B with KCa3.1 to the peripheral zone, p-SMAC (peripheral supramolecular activating complex) in the IS is critical for the KCa3.1 channel activation.In contrast, localization of MTMR6 and PHPT-1 to the central supramolecular activating complex (c-SMAC) segregates KCa3.1 from p-SMAC, and could provide one possible mechanism underlying the negative regulation of KCa3.1 channel activity [31] .Further comprehensive study on the molecules forming Ca 2+ signal complex sorted to the p-SMAC of the IS may enable the novel drug design for inflammatory, autoimmune and allergic diseases.

Ubiquitin-mediated KCa3.1 channel protein degradation
A balance between the translation and the degradation of ion channels is of importance to regulate its activity.Smallconductance KCa2.3 channel protein is rapidly endocytosed and recycled back to the plasma membrane.Recycling of KCa2.3 channel protein is dependent upon REM1 and Rab35 [37] .In contrast, KCa3.1 channel protein is rapidly (60-90 min after membrane trafficking) endocytosed, however, does not enter the recycling pathway.Subsequent to endocytosis, KCa3.1 channel proteinis targeted to the lysosomes for degradation in Rab7-and ESCRT (Endosomal Sorting Complex Required for Transport)dependent pathway [38] .Therefore, the inhibition of ubiquitin-activating enzymes results in reduced ubiquitylation and internalization of KCa3.1 channel proteins, and accordingly the ubiquitylase inhibitors attenuate KCa3.1 degradation.Deubiquitylase USP-8 (Ubiquitin Specific Protease 8) interacts with KCa3.1 channel protein, and siRNA-mediated USP-8 knock-out enhances the accumulation of ubquitylated KCa3.1 channel proteins [39] .Interestingly, a high-throughput screening method has been developed to identify small-molecule modulators of KCa3.1 channel endocytosis using fluorescence-tagged KCa3.1.Using this novel method, the ubiquitin-activating enzyme, E1 inhibitor, UBEI-41 has been identified as an inhibitor of KCa3.1 channel endocytosis [40] .The inhibition of deubiquitylases and the activation of ubiquitin-activating enzymes for KCa3.1 channels by pharmacological and/or genetic methods may possibly be novel strategies for drug development of inflammatory, autoimmune and allergic diseases.

Future direction
We recently showed that KCa3.1 inhibitors may be a therapeutic potential for DTH [41] and IBD [42] .In both auricular lymph node CD4 + T-lymphocytes of DTH model and mesenteric lymph node CD4 + T-lymphocytes in IBD model, an increase in KCa3.1 activity concomitant with an up-regulation of KCa3.1 was observed.Pharmacological blockade of KCa3.1 elicited the significant decrease in disease severity.It is of important that the pharmacological blockade of KCa3.1 is associated with the down-regulation of KCa3.1 in lymph node CD4 + T-lymphocytes in both Th1mediated disease models.These findings may provide novel information about radical treatments for inflammatory, autoimmune and allergic diseases.In addition, we suggest that KCa3.1 regulators such as NDPK-B may be potential therapeutic targets to decrease the risk of the disease development.