Genistein Inhibits the Activity of Kv1.3 Potassium Channels in Human T Lymphocytes

In the present study, the whole-cell patch-clamp technique was applied to follow the inhibitory effect of genistein — a tyrosine kinase inhibitor and a natural anticancer agent—on the activity of voltage-gated potassium channels Kv1.3 expressed in human T lymphocytes (TL). Obtained data provide evidence that genistein application in the concentration range of 1–80 μM reversibly decreased the whole-cell potassium currents in TL in a concentration-dependent manner to about 0.23 of the control value. The half-blocking concentration range of genistein was from 10 to 40 μM. The current inhibition was correlated in time with a significant decrease of the current activation rate. The steady-state activation of the currents was unchanged upon application of genistein, as was the inactivation rate. The inhibitory effect of genistein on the current amplitude and activation kinetics was voltage-independent. The current inhibition was not changed significantly in the presence of 1 mM of sodium orthovanadate, a tyrosine phosphatase inhibitor. Application of daidzein, an inactive genistein analogue, did not affect significantly either the current amplitudes or the activation kinetics. Possible mechanisms of the observed phenomena and their significance for genistein-induced inhibition of cancer cell proliferation are discussed.     

Reduced Kv1.3 Potassium Channel Expression in Human Prostate Cancer

The presence of Kv1.3 voltage-gated potassium channels in rat and human prostate epithelial cells has been previously reported. We examined, by immunohistochemistry, Kv1.3 levels in 10 normal human prostate, 18 benign prostatic hyperplasia (BPH) and 147 primary human prostate cancer (Pca) specimens. We found high epithelial expression of Kv1.3 in all normal prostate, 16 BPH and 77 (52%) Pca specimens. Compared to normal, Kv1.3 levels were reduced in 1 (6%) BPH specimen and in 70 (48%) Pca specimens. We found a significant inverse correlation between Kv1.3 levels and tumor grade (r = −0.25, P = 0.003) as well as tumor stage (r = −0.27, P = 0.001). Study of an additional 30 primary Pca specimens showed that 15 (50%) had reduced Kv1.3 immunostaining compared to matched normal prostate tissue. Our data suggest that in Pca reduced Kv1.3 expression occurs frequently and may be associated with a poor outcome.     

Modulation of the Kv1.3 Potassium Channel by Receptor Tyrosine Kinases

The voltage-dependent potassium channel, Kv1.3, is modulated by the epidermal growth factor receptor (EGFr) and the insulin receptor tyrosine kinases. When the EGFr and Kv1.3 are coexpressed in HEK 293 cells, acute treatment of the cells with EGF during a patch recording can suppress the Kv1.3 current within tens of minutes. This effect appears to be due to tyrosine phosphorylation of the channel, as it is blocked by treatment with the tyrosine kinase inhibitor erbstatin, or by mutation of the tyrosine at channel amino acid position 479 to phenylalanine. Previous work has shown that there is a large increase in the tyrosine phosphorylation of Kv1.3 when it is coexpressed with the EGFr. Pretreatment of EGFr and Kv1.3 cotransfected cells with EGF before patch recording also results in a decrease in peak Kv1.3 current. Furthermore, pretreatment of cotransfected cells with an antibody to the EGFr ligand binding domain (α-EGFr), which blocks receptor dimerization and tyrosine kinase activation, blocks the EGFr-mediated suppression of Kv1.3 current. Insulin treatment during patch recording also causes an inhibition of Kv1.3 current after tens of minutes, while pretreatment for 18 h produces almost total suppression of current. In addition to depressing peak Kv1.3 current, EGF treatment produces a speeding of C-type inactivation, while pretreatment with the α-EGFr slows C-type inactivation. In contrast, insulin does not influence C-type inactivation kinetics. Mutational analysis indicates that the EGF-induced modulation of the inactivation rate occurs by a mechanism different from that of the EGF-induced decrease in peak current. Thus, receptor tyrosine kinases differentially modulate the current magnitude and kinetics of a voltage-dependent potassium channel.     

Inhibition of the Activity of Human Lymphocyte Kv1.3 Potassium Channels by Resveratrol

The whole-cell patch-clamp technique was applied to study the modulatory effect of resveratrol on voltage-gated potassium channel Kv1.3 expressed in human lymphocytes. Results demonstrate that application of resveratrol in the concentration range 1–200 μM inhibited the channel activity in a concentration-dependent manner to about 18% of the control value. The half-blocking concentration of resveratrol was 40.9 μM, whereas the Hill coefficient was 1.05. The inhibition was time-dependent and slowly reversible. The inhibitory effect of resveratrol was correlated in time with a significant slowing of the current activation, whereas the inactivation rate remained unaffected upon application of resveratrol. The inhibition of Kv1.3 channels was voltage-independent. The steady-state activation of the currents remained unchanged upon resveratrol application. The magnitude of the inhibitory effect of resveratrol was not altered when resveratrol was coapplied with genistein. The possible mechanism of the inhibitory effect and its significance for biological activity of resveratrol are discussed.     

Voltage Gated Potassium Channel Kv1.3 Is Upregulated on Activated Astrocytes in Experimental Autoimmune Encephalomyelitis

Kv1.3 is a voltage gated potassium channel that has been implicated in pathophysiology of multiple sclerosis (MS). In the present study we investigated temporal and cellular expression pattern of this channel in the lumbar part of spinal cords of animals with experimental autoimmune encephalomyelitis (EAE), animal model of MS. EAE was actively induced in female Dark Agouti rats. Expression of Kv1.3 was analyzed at different time points of disease progression, at the onset, peak and end of EAE. We here show that Kv1.3 increased by several folds at the peak of EAE at both gene and protein level. Double immunofluorescence analyses demonstrated localization of Kv1.3 on activated microglia, macrophages, and reactive astrocytes around inflammatory lesions. In vitro experiments showed that pharmacological block of Kv1.3 in activated astrocytes suppresses the expression of proinflammatory mediators, suggesting a role of this channel in inflammation. Our results support the hypothesis that Kv1.3 may be a therapeutic target of interest for MS and add astrocytes to the list of cells whose activation would be suppressed by inhibiting Kv1.3 in inflammatory conditions.     

Kv1.3 钾离子通道在卵巢癌细胞株中的表达及活性

目的:研究Kv1.3钾离子通道在SKOV3卵巢癌细胞中的表达及其在细胞增殖和细胞周期中的作用。方法:应用RT-PCR和免疫细胞化学鉴别Kv1.3钾离子通道在SKOV3卵巢癌细胞中的表达。应用MTT和流式细胞技术观察KV1.3钾离子通道对SKOV3卵巢癌细胞增殖及细胞周期的影响。结果:4-氨基吡啶是Kv1.3钾离子通道特异性阻滞剂。不同浓度的4-氨基吡啶可以明显抑制SKOV3细胞的增殖,并且细胞周期也受到影响。G0/G1细胞比例增加,S期和G2/M期细胞比例下降。结论:Kv1.3钾离子通道在SKOV3卵巢癌细胞中表达,并且在细胞增殖及细胞周期变换中扮演着重要的角色。     

Influence of Specific Immunotherapy on the Activity of Human T Lymphocyte Kv1.3 Voltage-Gated Potassium Channels in Insect Venom Allergic Patients

Kv1.3 channels play an important role in T lymphocytes function. CD4⁺ and CD4⁺CD25⁺ T cells are two broad categories of T cells that are critically involved in the immunoresponse to allergens and that are also a major target for allergen immunotherapy. The aim of the study was to evaluate the effects of venom immunotherapy (VIT) on the activity of Kv1.3. channels on noncultured subsets: CD4⁺ and CD4⁺CD25⁺ T cells of insect venom allergic patients. Eleven patients with allergic reactions to bee or wasp venoms participated in the study. The patients were provided VIT according to the ultrarush protocol. CD4⁺ and CD4⁺CD25⁺ T cells were isolated from peripheral blood mononuclear cells of VIT-treated patients by an immunomagnetic method. We used the whole-cell patch clamp technique to investigate the whole potassium chord conductance (gK) of Kv1.3. channels in CD4⁺ and CD4⁺CD25⁺ T cells of venom-sensitive patients before and during the course of VIT. The conductance of Kv1.3. channels on CD4⁺CD25⁺ T cells decreased during the course of VIT. On day 0 it was 0.054 ± 0.07 [nS], and on day 70 it was 0.008 ± 0.09 [nS] (P = 0.03). The observed decrease of the gK of the Kv1.3 channels in the subpopulation of activated T cells may contribute to T cell tolerance and functional unresponsiveness of these cells to allergen in the early stages of VIT.     

Whole-Cell Recording of Calcium Release-Activated Calcium (CRAC) Currents in Human T Lymphocytes

In T lymphocytes, depletion of Ca²⁺ from the intracellular Ca²⁺ store leads to activation of plasmalemmal Ca²⁺ channels, called Calcium Release-Activated Calcium (CRAC) channels. CRAC channels play important role in regulation of T cell proliferation and gene expression. Abnormal CRAC channel function in T cells has been linked to severe combined immunodeficiency and autoimmune diseases ^{1, 2} . Studying CRAC channel function in human T cells may uncover new molecular mechanisms regulating normal immune responses and unravel the causes of related human diseases. Electrophysiological recordings of membrane currents provide the most accurate assessment of functional channel properties and their regulation. Electrophysiological assessment of CRAC channel currents in Jurkat T cells, a human leukemia T cell line, was first performed more than 20 years ago ³, however, CRAC current measurements in normal human T cells remains a challenging task. The difficulties in recording CRAC channel currents in normal T cells are compounded by the fact that blood-derived T lymphocytes are much smaller in size than Jurkat T cells and, therefore, the endogenous whole-cell CRAC currents are very low in amplitude. Here, we give a step-by-step procedure that we routinely use to record the Ca²⁺ or Na⁺ currents via CRAC channels in resting human T cells isolated from the peripheral blood of healthy volunteers. The method described here was adopted from the procedures used for recording the CRAC currents in Jurkat T cells and activated human T cells ^4-8.Download video file.^{(94M, mov)}     

Regulation of the Kv2.1 Potassium Channel by MinK and MiRP1

Kv2.1 is a voltage-gated potassium (Kv) channel α-subunit expressed in mammalian heart and brain. MinK-related peptides (MiRPs), encoded by KCNE genes, are single–transmembrane domain ancillary subunits that form complexes with Kv channel α-subunits to modify their function. Mutations in human MinK (KCNE1) and MiRP1 (KCNE2) are associated with inherited and acquired forms of long QT syndrome (LQTS). Here, coimmunoprecipitations from rat heart tissue suggested that both MinK and MiRP1 form native cardiac complexes with Kv2.1. In whole-cell voltage-clamp studies of subunits expressed in CHO cells, rat MinK and MiRP1 reduced Kv2.1 current density three- and twofold, respectively; slowed Kv2.1 activation (at +60 mV) two- and threefold, respectively; and slowed Kv2.1 deactivation less than twofold. Human MinK slowed Kv2.1 activation 25%, while human MiRP1 slowed Kv2.1 activation and deactivation twofold. Inherited mutations in human MinK and MiRP1, previously associated with LQTS, were also evaluated. D76N–MinK and S74L–MinK reduced Kv2.1 current density (threefold and 40%, respectively) and slowed deactivation (60% and 80%, respectively). Compared to wild-type human MiRP1–Kv2.1 complexes, channels formed with M54T– or I57T–MiRP1 showed greatly slowed activation (tenfold and fivefold, respectively). The data broaden the potential roles of MinK and MiRP1 in cardiac physiology and support the possibility that inherited mutations in either subunit could contribute to cardiac arrhythmia by multiple mechanisms. Electronic supplementary material  The online version of this article (doi: ) contains supplementary material, which is available to authorized users. Z. A. McCrossan and T. K. Roepke have contributed equally to this work.     

The Sensitivity of the Human Kv1.3 (hKv1.3) Lymphocyte K+ Channel to Regulation by PKA and PKC is Partially Lost in HEK 293 Host Cells

cDNA encoding the full-length hKv1.3 lymphocyte channel and a C-terminal truncated (Δ459-523) form that lacks the putative PKA Ser⁴⁶⁸ phosphorylation site were stably transfected in human embryonic kidney (HEK) 293 cells. Immunostaining of the transfected cells revealed a distribution at the plasma membrane that was uniform in the case of the full-length channel whereas clustering was observed in the case of the truncated channel. Some staining within the cell cytoplasm was found in both instances, suggesting an active process of biosynthesis. Analyses of the K⁺ current by the patch-clamp technique in the whole cell configuration showed that depolarizing steps to 40 mV from a holding potential (HP) of −80 mV elicited an outward current of 2 to 10 nA. The current threshold was positive to −40 mV and the current amplitude increased in a voltage-dependent manner. The parameters of activation were −5.7 and −9.9 mV (slope factor) and −35 mV (half activation, V _0.5) in the case of the full-length and truncated channels, respectively. The characteristics of the inactivation were 14.2 and 24.6 mV (slope factor) and −17.3 and −39.0 mV (V _0.5) for the full-length and truncated channels, respectively. The activation time constant of the full-length channel for potentials ranging from −30 to 40 mV decreased from 18 to 12 msec whereas the inactivation time constant decreased from 6600 msec at −30 mV to 1800 msec at 40 mV. The unit current amplitude measured in cells bathing in 140 mm KCl was 1.3 ± 0.1 pA at 40 mV, the unit conductance, 34.5 pS and the zero current voltage, 0 mV. Both forms of the channels were inhibited by TEA, 4-AP, Ni²⁺ and charybdotoxin. In contrast to the native (Jurkat) lymphocyte Kv1.3 channel that is fully inhibited by PKA and PKC, the addition of TPA resulted in 34.6 ± 7.3% and 38.7 ± 9.4% inhibition of the full-length and the truncated channels, respectively. 8-BrcAMP induced a 39.4 ± 5.4% inhibition of the full-length channel but had no effect (8.6 ± 8.3%) on the truncated channel. Cell dialysis with alkaline phosphatase had no effects, suggesting that the decreased sensitivity of the transfected channels to PKA and PKC was not due to an already phosphorylated channel. Patch extract experiments suggested that the hKv1.3 channel was partially sensitive to PKA and PKC. Cotransfecting the Kvβ1.2 subunit resulted in a decrease in the value of the time constant of inactivation of the full-length channel but did not modify its sensitivity to PKA and PKC. The cotransfected Kvβ2 subunit had no effects. Our results indicate that the hKv1.3 lymphocyte channel retains its electrophysiological characteristics when transfected in the Kvβ-negative HEK 293 cell line but its sensitivity to modulation by PKA and PKC is significantly reduced. Received: 18 June 1997/Revised: 7 October 1997     

Stimulation of Kv1.3 potassium channels by death receptors during apoptosis in Jurkat T lymphocytes

The loss of intracellular potassium is a pivotal step in the induction of apoptosis but the mechanisms underlying this response are poorly understood. Here we report caspase-dependent stimulation of potassium channels by the Fas receptor in a human Jurkat T cell line. Receptor activation with Fas ligand for 30 min increased the amplitude of voltage-activated potassium currents 2-fold on average. This produces a sustained outward current, approximately 10 pA, at physiological membrane potentials during Fas ligand-induced apoptosis. Both basal and Fas ligand-induced currents were blocked completely by toxins that selectively inhibit Kv1.3 potassium channels. Kv1.3 stimulation required the expression of Fas-associated death domain protein and activation of caspase 8, but did not require activation of caspase 3 or protein synthesis. Furthermore, Kv1.3 stimulation by Fas ligand was prevented by chronic stimulation of protein kinase C with 20 nm phorbol 12-myristate 13-acetate during Fas ligand treatment, which also blocks apoptosis. Thus, Fas ligand increases Kv1.3 channel activity through the same canonical apoptotic signaling cascade that is required for potassium efflux, cell shrinkage, and apoptosis.     

A Store-Operated Nonselective Cation Channel in Human Lymphocytes

1. Agonist interaction with phospholipase C-linked receptors at the plasma membrane can elicit both Ca²⁺ and Na⁺ influxes in lymphocytes. While Ca²⁺ influx is mediated by Ca²⁺ release-activated Ca²⁺ (CRAC) channels, the pathway responsible for Na⁺ influx is largely unknown.2. We show that thapsigargin, ionomycin, ADP-ribose and IP₃ activated a nonselective cation channel in lymphocytes that had a slightly outwardly rectifying I–V relationship, and a single channel conductance of 23.1 pS. We termed this channel a Ca²⁺ release-activated nonselective cation (CRANC) channel.3. On activation in cell-attached configuration, switching to an inside-out configuration abolished CRANC channel activity.4. Transfection of Jurkat T cells with antisense oligonucleotides for LTRPC2 reduced capacitative Ca²⁺ entry.5. These results suggest that CRANC channels are responsible for the Na⁺ influx as well as a portion of the Ca²⁺ influx in lymphocytes induced by store depletion, that sustained activation of CRANC channels requires some property of the environment of a cell depleted of its Ca²⁺ stores; and that LTRPC2 protein is a likely component of the CRANC channel.     

Assembly and suppression of endogenous Kv1.3 channels in human T cells

The predominant K+ channel in human T lymphocytes is Kv1.3, which inactivates by a C-type mechanism. To study assembly of these tetrameric channels in Jurkat, a human T-lymphocyte cell line, we have characterized the formation of heterotetrameric channels between endogenous wild-type (WT) Kv1.3 subunits and heterologously expressed mutant (A413V) Kv1.3 subunits. We use a kinetic analysis of C-type inactivation of currents produced by homotetrameric channels and heterotetrameric channels to determine the distribution of channels with different subunit stoichiometries. The distributions are well- described by either a binomial distribution or a binomial distribution plus a fraction of WT homotetramers, indicating that subunit assembly is a random process and that tetramers expressed in the plasma membrane do not dissociate and reassemble. Additionally, endogenous Kv1.3 current is suppressed by a heterologously expressed truncated Kv1.3 that contains the amino terminus and the first two transmembrane segments. The time course for suppression, which is maximal at 48 h after transfection, overlaps with the time interval for heterotetramer formation between heterologously expressed A413V and endogenous WT channels. Our findings suggest that diversity of K+ channel subtypes in a cell is regulated not by spatial segregation of monomeric pools, but rather by the degree of temporal overlap and the kinetics of subunit expression.     

Fine-tuning of Voltage Sensitivity of the Kv1.2 Potassium Channel by Interhelix Loop Dynamics

Many proteins function by changing conformation in response to ligand binding or changes in other factors in their environment. Any change in the sequence of a protein, for example during evolution, which alters the relative free energies of the different functional conformations changes the conditions under which the protein will function. Voltage-gated ion channels are membrane proteins that open and close an ion-selective pore in response to changes in transmembrane voltage. The charged S4 transmembrane helix transduces changes in transmembrane voltage into a change in protein internal energy by interacting with the rest of the channel protein through a combination of non-covalent interactions between adjacent helices and covalent interactions along the peptide backbone. However, the structural basis for the wide variation in the V₅₀ value between different voltage-gated potassium channels is not well defined. To test the role of the loop linking the S3 helix and the S4 helix in voltage sensitivity, we have constructed a set of mutants of the rat K_v1.2 channel that vary solely in the length and composition of the extracellular loop that connects S4 to S3. We evaluated the effect of these different loop substitutions on the voltage sensitivity of the channel and compared these experimental results with molecular dynamics simulations of the loop structures. Here, we show that this loop has a significant role in setting the precise V₅₀ of activation in K_v1 family channels.     

Control of Voltage-gated Potassium Channel Kv2.2 Expression by Pyruvate-Isocitrate Cycling Regulates Glucose-stimulated Insulin Secretion

Recent studies have shown that the pyruvate-isocitrate cycling pathway, involving the mitochondrial citrate/isocitrate carrier and the cytosolic NADP-dependent isocitrate dehydrogenase (ICDc), is involved in control of glucose-stimulated insulin secretion (GSIS). Here we demonstrate that pyruvate-isocitrate cycling regulates expression of the voltage-gated potassium channel family member Kv2.2 in islet β-cells. siRNA-mediated suppression of ICDc, citrate/isocitrate carrier, or Kv2.2 expression impaired GSIS, and the effect of ICDc knockdown was rescued by re-expression of Kv2.2. Moreover, chronic exposure of β-cells to elevated fatty acids, which impairs GSIS, resulted in decreased expression of Kv2.2. Surprisingly, knockdown of ICDc or Kv2.2 increased rather than decreased outward K⁺ current in the 832/13 β-cell line. Immunoprecipitation studies demonstrated interaction of Kv2.1 and Kv2.2, and co-overexpression of the two channels reduced outward K⁺ current compared with overexpression of Kv2.1 alone. Also, siRNA-mediated knockdown of ICDc enhanced the suppressive effect of the Kv2.1-selective inhibitor stromatoxin1 on K⁺ currents. Our data support a model in which a key function of the pyruvate-isocitrate cycle is to maintain levels of Kv2.2 expression sufficient to allow it to serve as a negative regulator of Kv channel activity.     

Mitochondrial Regulation of Store-operated Calcium Signaling in T Lymphocytes

Mitochondria act as potent buffers of intracellular Ca²⁺ in many cells, but a more active role in modulating the generation of Ca²⁺ signals is not well established. We have investigated the ability of mitochondria to modulate store-operated or “capacitative” Ca²⁺ entry in Jurkat leukemic T cells and human T lymphocytes using fluorescence imaging techniques. Depletion of the ER Ca²⁺ store with thapsigargin (TG) activates Ca²⁺ release-activated Ca²⁺ (CRAC) channels in T cells, and the ensuing influx of Ca²⁺ loads a TG- insensitive intracellular store that by several criteria appears to be mitochondria. Loading of this store is prevented by carbonyl cyanide m-chlorophenylhydrazone or by antimycin A1 + oligomycin, agents that are known to inhibit mitochondrial Ca²⁺ import by dissipating the mitochondrial membrane potential. Conversely, intracellular Na⁺ depletion, which inhibits Na⁺-dependent Ca²⁺ export from mitochondria, enhances store loading. In addition, we find that rhod-2 labels mitochondria in T cells, and it reports changes in Ca²⁺ levels that are consistent with its localization in the TG-insensitive store. Ca²⁺ uptake by the mitochondrial store is sensitive (threshold is <400 nM cytosolic Ca²⁺), rapid (detectable within 8 s), and does not readily saturate. The rate of mitochondrial Ca²⁺ uptake is sensitive to extracellular [Ca²⁺], indicating that mitochondria sense Ca²⁺ gradients near CRAC channels. Remarkably, mitochondrial uncouplers or Na⁺ depletion prevent the ability of T cells to maintain a high rate of capacitative Ca²⁺ entry over prolonged periods of >10 min. Under these conditions, the rate of Ca²⁺ influx in single cells undergoes abrupt transitions from a high influx to a low influx state. These results demonstrate that mitochondria not only buffer the Ca²⁺ that enters T cells via store-operated Ca²⁺ channels, but also play an active role in modulating the rate of capacitative Ca²⁺ entry.