Activity of Potassium Channels in CD8+ T Lymphocytes: Diagnostic and Prognostic Biomarker in Ovarian Cancer?

CD8+ T cells play a role in the suppression of tumor growth and immunotherapy. Ion channels control the Ca2+-dependent function of CD8+ lymphocytes such as cytokine/granzyme production and tumor killing. Kv1.3 and KCa3.1 K+ channels stabilize the negative membrane potential of T cells to maintain Ca2+ influx through CRAC channels. We assessed the expression of Kv1.3, KCa3.1 and CRAC in CD8+ cells from ovarian cancer (OC) patients (n = 7). We found that the expression level of Kv1.3 was higher in patients with malignant tumors than in control or benign tumor groups while the KCa3.1 activity was lower in the malignant tumor group as compared to the others. We demonstrated that the Ca2+ response in malignant tumor patients is higher compared to control groups. We propose that altered Kv1.3 and KCa3.1 expression in CD8+ cells in OC could be a reporter and may serve as a biomarker in diagnostics and that increased Ca2+ response through CRAC may contribute to the impaired CD8+ function.

A Fluorescent Peptide Toxin for Selective Visualization of the Voltage-Gated Potassium Channel KV1.3.

Upregulation of the voltage-gated potassium channel KV1.3 is implicated in a range of autoimmune and neuroinflammatory diseases, including rheumatoid arthritis, psoriasis, multiple sclerosis, and type I diabetes. Understanding the expression, localization, and trafficking of KV1.3 in normal and disease states is key to developing targeted immunomodulatory therapies. HsTX1[R14A], an analogue of a 34-residue peptide toxin from the scorpion Heterometrus spinifer, binds KV1.3 with high affinity (IC50 of 45 pM) and selectivity (2000-fold for KV1.3 over KV1.1). We have synthesized a fluorescent analogue of HsTX1[R14A] by N-terminal conjugation of a Cy5 tag. Electrophysiology assays show that Cy5-HsTX1[R14A] retains activity against KV1.3 (IC50 ∼ 0.9 nM) and selectivity over a range of other potassium channels (KV1.2, KV1.4, KV1.5, KV1.6, KCa1.1 and KCa3.1), as well as selectivity against heteromeric channels assembled from KV1.3/KV1.5 tandem dimers. Live imaging of CHO cells expressing green fluorescent protein-tagged KV1.3 shows co-localization of Cy5-HsTX1[R14A] and KV1.3 fluorescence signals at the cell membrane. Moreover, flow cytometry demonstrated that Cy5-HsTX1[R14A] can detect KV1.3-expressing CHO cells. Stimulation of mouse microglia by lipopolysaccharide, which enhances membrane expression of KV1.3, was associated with increased staining by Cy5-HsTX1[R14A], demonstrating that it can be used to identify KV1.3 in disease-relevant models of inflammation. Furthermore, the biodistribution of Cy5-HsTX1[R14A] could be monitored using ex vivo fluorescence imaging of organs in mice dosed subcutaneously with the peptide. These results illustrate the utility of Cy5-HsTX1[R14A] as a tool for visualizing KV1.3, with broad applicability in fundamental investigations of KV1.3 biology, and the validation of novel disease indications where KV1.3 inhibition may be of therapeutic value.

Role of C-Terminal Domain and Membrane Potential in the Mobility of Kv1.3 Channels in Immune Synapse Forming T Cells.

Voltage-gated Kv1.3 potassium channels are essential for maintaining negative membrane potential during T-cell activation. They interact with membrane-associated guanylate kinases (MAGUK-s) via their C-terminus and with TCR/CD3, leading to enrichment at the immunological synapse (IS). Molecular interactions and mobility may impact each other and the function of these proteins. We aimed to identify molecular determinants of Kv1.3 mobility, applying fluorescence correlation spectroscopy on human Jurkat T-cells expressing WT, C-terminally truncated (ΔC), and non-conducting mutants of mGFP-Kv1.3. ΔC cannot interact with MAGUK-s and is not enriched at the IS, whereas cells expressing the non-conducting mutant are depolarized. Here, we found that in standalone cells, mobility of ΔC increased relative to the WT, likely due to abrogation of interactions, whereas mobility of the non-conducting mutant decreased, similar to our previous observations on other membrane proteins in depolarized cells. At the IS formed with Raji B-cells, mobility of WT and non-conducting channels, unlike ΔC, was lower than outside the IS. The Kv1.3 variants possessing an intact C-terminus had lower mobility in standalone cells than in IS-engaged cells. This may be related to the observed segregation of F-actin into a ring-like structure at the periphery of the IS, leaving much of the cell almost void of F-actin. Upon depolarizing treatment, mobility of WT and ΔC channels decreased both in standalone and IS-engaged cells, contrary to non-conducting channels, which themselves caused depolarization. Our results support that Kv1.3 is enriched at the IS via its C-terminal region regardless of conductivity, and that depolarization decreases channel mobility.

Immune Synapse Residency of Orai1 Alters Ca2+ Response of T Cells.

CRAC, which plays important role in Ca2+-dependent T-lymphocyte activation, is composed of the ER-resident STIM1 and the plasma membrane Orai1 pore-forming subunit. Both accumulate at the immunological synapse (IS) between a T cell and an antigen-presenting cell (APC). We hypothesized that adapter/interacting proteins regulate Orai1 residence in the IS. We could show that mGFP-tagged Orai1-Full channels expressed in Jurkat cells had a biphasic IS-accumulation kinetics peaked at 15 min. To understand the background of Orai1 IS-redistribution we knocked down STIM1 and SAP97 (adaptor protein with a short IS-residency (15 min) and ability to bind Orai1 N-terminus): the mGFP-Orai1-Full channels kept on accumulating in the IS up to the 60th minute in the STIM1- and SAP97-lacking Jurkat cells. Deletion of Orai1 N terminus (mGFP-Orai1-Δ72) resulted in the same time course as described for STIM1/SAP97 knock-down cells. Ca2+-imaging of IS-engaged T-cells revealed that of Orai1 residency modifies the Ca2+-response: cells expressing mGFP-Orai1-Δ72 construct or mGFP-Orai1-Full in SAP-97 knock-down cells showed higher number of Ca2+-oscillation up to the 90th minute after IS formation. Overall, these data suggest that SAP97 may contribute to the short-lived IS-residency of Orai1 and binding of STIM1 to Orai1 N-terminus is necessary for SAP97-Orai1 interaction.

Periodic Membrane Potential and Ca2+ Oscillations in T Cells Forming an Immune Synapse.

The immunological synapse (IS) is a specialized contact area formed between a T cell and an antigen presenting cell (APC). Besides molecules directly involved in antigen recognition such as the TCR/CD3 complex, ion channels important in the membrane potential and intracellular free Ca2+ concentration control of T cells are also recruited into the IS. These are the voltage-gated Kv1.3 and Ca2+-activated KCa3.1 K+ channels and the calcium release-activated Ca2+ channel (CRAC). However, the consequence of this recruitment on membrane potential and Ca2+ level control is not known. Here we demonstrate that the membrane potential (MP) of murine T cells conjugated with APCs in an IS shows characteristic oscillations. We found that depolarization of the membrane by current injection or by increased extracellular K+ concentration produced membrane potential oscillations (MPO) significantly more frequently in conjugated T cells than in lone T cells. Furthermore, oscillation of the free intracellular Ca2+ concentration could also be observed more frequently in cells forming an IS than in lone cells. We suggest that in the IS the special arrangement of channels and the constrained space between the interacting cells creates a favorable environment for these oscillations, which may enhance the signaling process leading to T cell activation.

Sterol Regulation of Voltage-Gated K+ Channels.

Cholesterol is an essential lipid building block of the cellular plasma membrane. In addition to its structural role, it regulates the fluidity and raft structure of the membrane and influences the course of numerous membrane-linked signaling pathways and the function of transmembrane proteins, including ion channels. This is supported by a vast body of scientific data, which demonstrates the modulation of ion channels with a great variety of ion selectivity, gating, and tissue distribution by changes in membrane cholesterol. Here, we review what is currently known about the modulation of voltage-gated K+ (Kv) channels by changes in membrane cholesterol content, considering raft association of the channels, the roles of cholesterol recognition sites, and those of adaptor proteins in cholesterol-Kv channel interactions. We specifically focus on Kv1.3, the dominant K+ channel of human T cells. Effects of cholesterol depletion and enrichment and 7-dehydrocholesterol enrichment on Kv1.3 gating are discussed in the context of the immunological synapse and the comparison of the in vitro effects of sterol modifications on Kv1.3 function with ex vivo effects on cells from hypercholesterolemic and Smith-Lemli-Opitz patients.

7DHC-induced changes of Kv1.3 operation contributes to modified T cell function in Smith-Lemli-Opitz syndrome.

In vitro manipulation of membrane sterol level affects the regulation of ion channels and consequently certain cellular functions; however, a comprehensive study that confirms the pathophysiological significance of these results is missing. The malfunction of 7-dehydrocholesterol (7DHC) reductase in Smith-Lemli-Opitz syndrome (SLOS) leads to the elevation of the 7-dehydrocholesterol level in the plasma membrane. T lymphocytes were isolated from SLOS patients to assess the effect of the in vivo altered membrane sterol composition on the operation of the voltage-gated Kv1.3 channel and the ion channel-dependent mitogenic responses. We found that the kinetic and equilibrium parameters of Kv1.3 activation changed in SLOS cells. Identical changes in Kv1.3 operation were observed when control/healthy T cells were loaded with 7DHC. Removal of the putative sterol binding sites on Kv1.3 resulted in a phenotype that was not influenced by the elevation in membrane sterol level. Functional assays exhibited impaired activation and proliferation rate of T cells probably partially due to the modified Kv1.3 operation. We concluded that the altered membrane sterol composition hindered the operation of Kv1.3 as well as the ion channel-controlled T cell functions.

Nanovesicle-targeted Kv1.3 knockdown in memory T cells suppresses CD40L expression and memory phenotype.

Ca(2+) signaling controls activation and effector functions of T lymphocytes. Ca(2+) levels also regulate NFAT activation and CD40 ligand (CD40L) expression in T cells. CD40L in activated memory T cells binds to its cognate receptor, CD40, on other cell types resulting in the production of antibodies and pro-inflammatory mediators. The CD40L/CD40 interaction is implicated in the pathogenesis of autoimmune disorders and CD40L is widely recognized as a therapeutic target. Ca(2+) signaling in T cells is regulated by Kv1.3 channels. We have developed lipid nanoparticles that deliver Kv1.3 siRNAs (Kv1.3-NPs) selectively to CD45RO(+) memory T cells and reduce the activation-induced Ca(2+) influx. Herein we report that Kv1.3-NPs reduced NFAT activation and CD40L expression exclusively in CD45RO(+) T cells. Furthermore, Kv1.3-NPs suppressed cytokine release and induced a phenotype switch of T cells from predominantly memory to naïve. These findings indicate that Kv1.3-NPs operate as targeted immune suppressive agents with promising therapeutic potentials.

Selective inhibition of KCa3.1 channels mediates adenosine regulation of the motility of human T cells.

Adenosine, a purine nucleoside, is present at high concentrations in tumors, where it contributes to the failure of immune cells to eliminate cancer cells. The mechanisms responsible for the immunosuppressive properties of adenosine are not fully understood. We tested the hypothesis that adenosine's immunosuppressive functions in human T lymphocytes are in part mediated via modulation of ion channels. The activity of T lymphocytes relies on ion channels. KCa3.1 and Kv1.3 channels control cytokine release and, together with TRPM7, regulate T cell motility. Adenosine selectively inhibited KCa3.1, but not Kv1.3 and TRPM7, in activated human T cells. This effect of adenosine was mainly mediated by A2A receptors, as KCa3.1 inhibition was reversed by SCH58261 (selective A2A receptor antagonist), but not by MRS1754 (A2B receptor antagonist), and it was mimicked by the A2A receptor agonist CGS21680. Furthermore, it was mediated by the cAMP/protein kinase A isoform (PKAI) signaling pathway, as adenylyl-cyclase and PKAI inhibition prevented adenosine effect on KCa3.1. The functional implication of the effect of adenosine on KCa3.1 was determined by measuring T cell motility on ICAM-1 surfaces. Adenosine and CGS21680 inhibited T cell migration. Comparable effects were obtained by KCa3.1 blockade with TRAM-34. Furthermore, the effect of adenosine on cell migration was abolished by pre-exposure to TRAM-34. Additionally, adenosine suppresses IL-2 secretion via KCa3.1 inhibition. Our data indicate that adenosine inhibits KCa3.1 in human T cells via A2A receptor and PKAI, thereby resulting in decreased T cell motility and cytokine release. This mechanism is likely to contribute to decreased immune surveillance in solid tumors.

The role of PSD-95 in the rearrangement of Kv1.3 channels to the immunological synapse.

Establishment of the immunological synapse (IS) between T lymphocytes and antigen-presenting cells is a key step in the adaptive immune response. Several proteins accumulate in the IS, such as the Kv1.3 potassium channel; however, the mechanism of this translocation is unknown. PSD-95 and SAP97 are adaptor proteins that regulate the polarized cell surface expression and localization of Kv1 channels in neurons. We investigated whether these proteins affect the redistribution of Kv1.3 into the IS in non-excitable human T cells. We show here that PSD-95 and SAP97 are expressed in Jurkat and interact with the C terminus of Kv1.3. Disruption of the interaction between PSD-95 or SAP97 and Kv1.3 in Jurkat was realized by the expression of a C-terminal truncated Kv1.3, which lacks the binding domain for these proteins, or by the knockdown of the expression of PSD-95 or SAP97 using specific shRNA. Expression of the truncated Kv1.3 or knockdown of PSD-95, but not the knockdown of SAP97, inhibited the recruitment of Kv1.3 into the IS; the fraction of cells showing polarized Kv1.3 expression upon engagement in an IS was significantly lower than in control cells expressing the full-length Kv1.3, and the rearrangement of Kv1.3 did not show time dependence. In contrast, Jurkat cells expressing the full-length channel showed marked time dependence in the recruitment into the IS peaking at 1 min after the conjugation of the cells. These results demonstrate that PSD-95 participates in the targeting of Kv1.3 into the IS, implying its important role in human T-cell activation.

Analysis of the K+ current in human CD4+ T lymphocytes in hypercholesterolemic state.

Atherosclerosis involves immune mechanisms: T lymphocytes are found in atherosclerotic plaques, suggesting their activation during atherogenesis. The predominant voltage-gated potassium channel of T cells, Kv1.3 is a key regulator of the Ca(2+)-dependent activation pathway. In the present experiments we studied the proliferation capacity and functional changes of Kv1.3 channels in T cells from healthy and hypercholestaeremic patients. By means of CFSE-assay (carboxyfluorescein succinimidyl ester) we showed that spontaneous activation rate of lymphocytes in hypercholesterolemia was elevated and the antiCD3/antiCD28 co-stimulation was less effective as compared to the healthy group. Using whole-cell patch-clamping we obtained that the activation and deactivation kinetics of Kv1.3 channels were faster in hypercholesterolemic state but no change in other parameters of Kv1.3 were found (inactivation kinetics, steady-state activation, expression level). We suppose that incorporation of oxLDL species via its raft-rupturing effect can modify proliferative rate of T cells as well as the gating of Kv1.3 channels.

Membrane microdomain organization, calcium signal, and NFAT activation as an important axis in polarized Th cell function.

T helper lymphocytes become polarized upon antigen and cytokine stimuli received after their maturation in the thymus. Since the balance of Th1 and Th2 responses is critical in healthy and pathological immune responses, understanding the molecular base of T cell polarization still remained an important question. Using our Th0/Th1/Th2 hybridoma model system, we performed a comparative study on polarized Th1 and Th2 cells in terms of their membrane raft expression/composition, their TCR mediated activation signaling, and sensitivity to activation-induced cell death (AICD) using flow and image cytometric methods. We show here that the TCR stimulation induced more intense and sustained Ca(2+) -response in Th1 cells compared to Th2 ones correlates well with a shorter nuclear residence time of the Ca(2+) -dependent NFAT transcription factor in Th2 cells. In addition, NFAT translocation directly depended on lipid raft integrity/membrane cholesterol level. Expression pattern of raftophilic accessory proteins (CD4, CD59, and CD48) and lipids (GM1, cholesterol) were also different in the Th1 and Th2 hybridomas, similarly to differentiated spleen Th cells. The activation-induced, remarkably clustered and polarized membrane distribution of TCR/CD3 complex in Th1, but not in Th2 cells, together with an increased raft localization of Kv1.3 ion channels regulating the Ca(2+) -response, are consistent with the above properties of NFAT. Finally, the polarized Th cells, especially Th1, were more sensitive to AICD than their unpolarized Th0 precursor. These results suggest that the membrane microdomain organization-Ca(2+) -signaling-NFAT activation axis is an important determinant of polarized Th cell effector function and fate.

Voltage-gated sodium channel Nav1.7 maintains the membrane potential and regulates the activation and chemokine-induced migration of a monocyte-derived dendritic cell subset.

Expression of CD1a protein defines a human dendritic cell (DC) subset with unique functional activities. We aimed to study the expression of the Nav1.7 sodium channel and the functional consequences of its activity in CD1a(-) and CD1a(+) DC. Single-cell electrophysiology (patch-clamp) and quantitative PCR experiments performed on sorted CD1a(-) and CD1a(+) immature DC (IDC) showed that the frequency of cells expressing Na(+) current, current density, and the relative expression of the SCN9A gene encoding Nav1.7 were significantly higher in CD1a(+) cells than in their CD1a(-) counterparts. The activity of Nav1.7 results in a depolarized resting membrane potential (-8.7 ± 1.5 mV) in CD1a(+) IDC as compared with CD1a(-) cells lacking Nav1.7 (-47 ± 6.2 mV). Stimulation of DC by inflammatory signals or by increased intracellular Ca(2+) levels resulted in reduced Nav1.7 expression. Silencing of the SCN9A gene shifted the membrane potential to a hyperpolarizing direction in CD1a(+) IDC, resulting in decreased cell migration, whereas pharmacological inhibition of Nav1.7 by tetrodotoxin sensitized the cells for activation signals. Fine-tuning of IDC functions by a voltage-gated sodium channel emerges as a new regulatory mechanism modulating the migration and cytokine responses of these DC subsets.

Biotechnological Applications of Mushrooms under the Water-Energy-Food Nexus: Crucial Aspects and Prospects from Farm to Pharmacy.

Mushrooms have always been an important source of food, with high nutritional value and medicinal attributes. With the use of biotechnological applications, mushrooms have gained further attention as a source of healthy food and bioenergy. This review presents different biotechnological applications and explores how these can support global food, energy, and water security. It highlights mushroom's relevance to meet the sustainable development goals of the UN. This review also discusses mushroom farming and its requirements. The biotechnology review includes sections on how to use mushrooms in producing nanoparticles, bioenergy, and bioactive compounds, as well as how to use mushrooms in bioremediation. The different applications are discussed under the water, energy, and food (WEF) nexus. As far as we know, this is the first report on mushroom biotechnology and its relationships to the WEF nexus. Finally, the review valorizes mushroom biotechnology and suggests different possibilities for mushroom farming integration.

Translatability of in vitro Inhibition Potency to in vivo P-Glycoprotein Mediated Drug Interaction Risk.

P-glycoprotein (P-gp) may limit oral drug absorption of substrate drugs due to intestinal efflux. Therefore, regulatory agencies require investigation of new chemical entities as possible inhibitors of P-gp in vitro. Unfortunately, inter-laboratory and inter-assay variability have hindered the translatability of in vitro P-gp inhibition data to predict clinical drug interaction risk. The current study was designed to evaluate the impact of potential IC50 discrepancies between two commonly utilized assays, i.e., bi-directional Madin-Darby Canine Kidney-MDR1 cell-based and MDR1 membrane vesicle-based assays. When comparing vesicle- to cell-based IC50 values (n = 28 inhibitors), non-P-gp substrates presented good correlation between assay formats, whereas IC50s of P-gp substrates were similar or lower in the vesicle assays. The IC50s obtained with a cell line expressing relatively low P-gp aligned more closely to those obtained from the vesicle assay, but passive permeability of the inhibitors did not appear to influence the correlation of IC50s, suggesting that efflux activity reduces intracellular inhibitor concentrations. IC50s obtained between two independent laboratories using the same assay type showed good correlation. Using the G-value (i.e., ratio of estimated gut concentration-to-inhibition potency) >10 cutoff recommended by regulatory agencies resulted in minimal differences in predictive performance, suggesting this cutoff is appropriate for either assay format.

Developmental switch of the expression of ion channels in human dendritic cells.

Modulation of the expression and activity of plasma membrane ion channels is one of the mechanisms by which immune cells can regulate their intracellular Ca(2+) signaling pathways required for proliferation and/or differentiation. Voltage-gated K+ channels, inwardly rectifying K+ channels, and Ca(2+)-activated K+ channels have been described to play a major role in controlling the membrane potential in lymphocytes and professional APCs, such as monocytes, macrophages, and dendritic cells (DCs). Our study aimed at the characterization and identification of ion channels expressed in the course of human DC differentiation from monocytes. We report in this study for the first time that immature monocyte-derived DCs express voltage-gated Na+ channels in their plasma membrane. The analysis of the biophysical and pharmacological properties of the current and PCR-based cloning revealed the presence of Nav1.7 channels in immature DCs. Transition from the immature to a mature differentiation state, however, was accompanied by the down-regulation of Nav1.7 expression concomitant with the up-regulation of voltage-gated Kv1.3 K+ channel expression. The presence of Kv1.3 channels seems to be common for immune cells; hence, selective Kv1.3 blockers may emerge as candidates for inhibiting various functions of mature DCs that involve their migratory, cytokine-secreting, and T cell-activating potential.

Immunomagnetic separation is a suitable method for electrophysiology and ion channel pharmacology studies on T cells.

Ion channels play pivotal role in the physiological and pathological function of immune cells. As immune cells represent a functionally diverse population, subtype-specific functional studies, such as single-cell electrophysiology require proper subset identification and separation. Magnetic-activated cell sorting (MACS) techniques provide an alternative to fluorescence-activated cell sorting (FACS), however, the potential impact of MACS-related beads on the biophysical and pharmacological properties of the ion channels were not studied yet. We studied the aforementioned properties of the voltage-gated Kv1.3 K+ channel in activated CD4+ T-cells as well as the membrane capacitance using whole-cell patch-clamp following immunomagnetic positive separation, using the REAlease® kit. This kit allows three experimental configurations: bead-bound configuration, bead-free configuration following the removal of magnetic beads, and the label-free configuration following removal of CD4 recognizing antibody fragments. As controls, we used FACS separation as well as immunomagnetic negative selection. The membrane capacitance and of the biophysical parameters of Kv1.3 gating, voltage-dependence of steady-state activation and inactivation kinetics of the current were not affected by the presence of MACS-related compounds on the cell surface. We found subtle differences in the activation kinetics of the Kv1.3 current that could not be explained by the presence of MACS-related compounds. Neither the equilibrium block of Kv1.3 by TEA+ or charybdotoxin (ChTx) nor the kinetics of ChTx block are affected by the presence of the magnetics beads on the cell surface. Based on our results MACS is a suitable method to separate cells for studying ion channels in non-excitable cells, such as T-lymphocytes.

Diagnostic Performance of Procalcitonin for the Early Identification of Sepsis in Patients with Elevated qSOFA Score at Emergency Admission.

Infectious biomarkers such as procalcitonin (PCT) can help overcome the lack of sensitivity of the quick Sequential Organ Failure Assessment (qSOFA) score for early identification of sepsis in emergency departments (EDs) and thus might be beneficial as point-of-care biomarkers in EDs. Our primary aim was to investigate the diagnostic performance of PCT for the early identification of septic patients and patients likely to develop sepsis within 96 h of admission to an ED among a prospectively selected patient population with elevated qSOFA score. In a large multi-centre prospective cohort study, we included all adult patients (n = 742) with a qSOFA score of at least 1 who presented to the ED. PCT levels were measured upon admission. Of the study population 27.3% (n = 202) were diagnosed with sepsis within the first 96 h. The area under the curve for PCT for the identification of septic patients in EDs was 0.86 (95% confidence interval (CI): 0.83-0.89). The resultant sensitivity for PCT at a cut-off of 0.5 µg/L was 63.4% (95% CI: 56.3-70.0). Furthermore, specificity was 89.2% (95% CI: 86.3-91.7), the positive predictive value was 68.8% (95% CI: 62.9-74.2), and the negative predictive value was 86.7% (95% CI: 84.4-88.7). The early measurement of PCT in a patient population with elevated qSOFA score served as an effective tool for the early identification of sepsis in ED patients.

KCNE4-dependent functional consequences of Kv1.3-related leukocyte physiology.

The voltage-dependent potassium channel Kv1.3 plays essential roles in the immune system, participating in leukocyte activation, proliferation and apoptosis. The regulatory subunit KCNE4 acts as an ancillary peptide of Kv1.3, modulates K+ currents and controls channel abundance at the cell surface. KCNE4-dependent regulation of the oligomeric complex fine-tunes the physiological role of Kv1.3. Thus, KCNE4 is crucial for Ca2+-dependent Kv1.3-related leukocyte functions. To better understand the role of KCNE4 in the regulation of the immune system, we manipulated its expression in various leukocyte cell lines. Jurkat T lymphocytes exhibit low KCNE4 levels, whereas CY15 dendritic cells, a model of professional antigen-presenting cells, robustly express KCNE4. When the cellular KCNE4 abundance was increased in T cells, the interaction between KCNE4 and Kv1.3 affected important T cell physiological features, such as channel rearrangement in the immunological synapse, cell growth, apoptosis and activation, as indicated by decreased IL-2 production. Conversely, ablation of KCNE4 in dendritic cells augmented proliferation. Furthermore, the LPS-dependent activation of CY15 cells, which induced Kv1.3 but not KCNE4, increased the Kv1.3-KCNE4 ratio and increased the expression of free Kv1.3 without KCNE4 interaction. Our results demonstrate that KCNE4 is a pivotal regulator of the Kv1.3 channelosome, which fine-tunes immune system physiology by modulating Kv1.3-associated leukocyte functions.

Comparative biocompatibility and antimicrobial studies of sorbic acid derivates.

Nowadays, the sorbates are the third largest group of antimicrobial preservatives in food and pharmaceutical industries, following the parabens and benzoates whose safety is questioned by recent publications. A disadvantage of sorbates is their pH dependence, as their antimicrobial effect is greatly reduced in alkaline environment. The main, widely used sorbate derivatives are sorbic acid and potassium sorbate, no sorbic acid esters are involved in current industrial application. We aimed to test whether the esters of sorbic acid are capable to extend the antimicrobial spectrum of the original molecule while maintaining its advantageous biocompatibility profile. A comparative biocompatibility study of different derivatives (sorbic acid, potassium sorbate, isopropyl sorbate and ethyl sorbate) was carried out. In vitro cell viability assays of MTT (2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide), Neutral Red (3-amino-7-dimethylamino-2-methylphenazine hydrochloride) and flow cytometry with propidium iodide and annexin were performed on Caco-2 cells. In case of in vivo toxicity study, G. mellonella larvae were injected with different concentrations of the test compounds. Time-kill tests were executed on reference strains of C. albicans, E. coli, and S. aureus. According to the MTT-assay, the IC50 values were the following: ethyl sorbate, sorbic acid <0.045% w/w, isopropyl sorbate 0.32% w/w, potassium sorbate >0.75% w/w, while Neutral Red values were >0.75% w/w for the esters and potassium sorbate and 0.66% w/w for sorbic acid. Flow cytometry results indicated the higher cell damage in case of isopropyl sorbate. However, the cytotoxic results of isopropyl sorbate, in vivo toxicity study on G. mellonella larvae did not show significant mortality. It was found, that the antimicrobial properties of isopropyl sorbate were outstanding compared to sorbic acid and potassium sorbate. These results indicate, that the use of sorbate esters can be advantageous, hence, further toxicity studies are needed to prove their safety.