Upregulation of the large conductance voltage- and Ca2+-activated K+ channels by Janus kinase 2

2014 ◽  
Vol 306 (11) ◽  
pp. C1041-C1049 ◽  
Author(s):  
Zohreh Hosseinzadeh ◽  
Ahmad Almilaji ◽  
Sabina Honisch ◽  
Tatsiana Pakladok ◽  
GuoXing Liu ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Brefeldin A ◽  
Bk Channels ◽  
Bk Channel ◽  
Janus Kinase ◽  
Protein Abundance ◽  
Janus Kinase 2 ◽  
Channel Protein ◽  
Jak2 Inhibitor ◽  
Whole Cell Patch Clamp

The iberiotoxin-sensitive large conductance voltage- and Ca2+-activated potassium (BK) channels (maxi-K+-channels) hyperpolarize the cell membrane thus supporting Ca2+ entry through Ca2+-release activated Ca2+ channels. Janus kinase-2 (JAK2) has been identified as novel regulator of ion transport. To explore whether JAK2 participates in the regulation of BK channels, cRNA encoding Ca2+-insensitive BK channels (BKM513I+Δ899–903) was injected into Xenopus oocytes with or without cRNA encoding wild-type JAK2, gain-of-function V617FJAK2, or inactive K882EJAK2. K+ conductance was determined by dual electrode voltage clamp and BK-channel protein abundance by confocal microscopy. In A204 alveolar rhabdomyosarcoma cells, iberiotoxin-sensitive K+ current was determined utilizing whole cell patch clamp. A204 cells were further transfected with JAK2 and BK-channel transcript, and protein abundance was quantified by RT-PCR and Western blotting, respectively. As a result, the K+ current in BKM513I+Δ899–903-expressing oocytes was significantly increased following coexpression of JAK2 or V617FJAK2 but not K882EJAK2. Coexpression of the BK channel with V617FJAK2 but not K882EJAK2 enhanced BK-channel protein abundance in the oocyte cell membrane. Exposure of BK-channel and V617FJAK2-expressing oocytes to the JAK2 inhibitor AG490 (40 μM) significantly decreased K+ current. Inhibition of channel insertion by brefeldin A (5 μM) decreased the K+ current to a similar extent in oocytes expressing the BK channel alone and in oocytes expressing the BK channel and V617FJAK2. The iberiotoxin (50 nM)-sensitive K+ current in rhabdomyosarcoma cells was significantly decreased by AG490 pretreatment (40 μM, 12 h). Moreover, overexpression of JAK2 in A204 cells significantly enhanced BK channel mRNA and protein abundance. In conclusion, JAK2 upregulates BK channels by increasing channel protein abundance in the cell membrane.

scholarly journals Regulation of Large Conductance Voltage-and Ca2+-Activated K+ Channels by the Janus Kinase JAK3

2015 ◽  
Vol 37 (1) ◽  
pp. 297-305 ◽  
Author(s):  
Jamshed Warsi ◽  
Yogesh Singh ◽  
Bernat Elvira ◽  
Zohreh Hosseinzadeh ◽  
Florian Lang
Keyword(s):  
Cell Proliferation ◽  
Cell Membrane ◽  
Bk Channel ◽  
Janus Kinase ◽  
Negative Regulator ◽  
K Channel ◽  
Protein Abundance ◽  
Wild Type ◽  
Protein Insertion ◽  
Channel Protein

Background/Aims: Janus kinase 3 (JAK3), a tyrosine kinase contributing to the regulation of cell proliferation and apoptosis of lymphocytes and tumour cells, has been shown to modify the expression and function of several ion channels and transport proteins. Channels involved in the regulation of cell proliferation include the large conductance voltage- and Ca2+-activated K+ channel BK. The present study explored whether JAK3 modifies BK channel protein abundance and current. Methods: cRNA encoding Ca2+-insensitive BK channel (BKM513I+Δ899-903) was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild-type JAK3, constitutively active A568VJAK3, or inactive K851AJAK3. Voltage gated K+ channel activity was measured utilizing dual electrode voltage clamp. Moreover, BK channel protein abundance was determined utilizing flow cytometry in CD19+ B lymphocyte cell membranes from mice lacking functional JAK3 (jak3-/-) and corresponding wild-type mice (jak3+/+). Results: BK activity in BKM513I+Δ899-903 expressing oocytes was slightly but significantly decreased by coexpression of wild-type JAK3 and of A568VJAK3, but not by coexpression of K851AJAK3. The BK channel protein abundance in the cell membrane was significantly higher in jak3-/- than in jak3+/+ B lymphocytes. The decline of conductance in BK and JAK3 coexpressing oocytes following inhibition of channel protein insertion by brefeldin A (5 µM) was similar in oocytes expressing BK with JAK3 and oocytes expressing BK alone, indicating that JAK3 might slow channel protein insertion into rather than accelerating channel protein retrieval from the cell membrane. Conclusion: JAK3 is a weak negative regulator of membrane BK protein abundance and activity.

scholarly journals Decrease of Store-Operated Ca2+ Entry and Increase of Na+/Ca2+ Exchange by Pharmacological JAK2 Inhibition

2016 ◽  
Vol 38 (2) ◽  
pp. 683-695 ◽  
Author(s):  
Jing Yan ◽  
Zohreh Hosseinzadeh ◽  
Bingbing Zhang ◽  
Mirjam Froeschl ◽  
Klaus Schulze-Osthoff ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Protein Abundance ◽  
Transcript Levels ◽  
Migratory Activity ◽  
Jak2 Inhibitor ◽  
Whole Cell Patch Clamp ◽  
And Migration ◽  
The Impact ◽  
Powerful Mechanism ◽  
Jak2 Inhibition

Background/Aims: Cell proliferation and migration are regulated by cytosolic Ca2+ activity ([Ca2+]i). Mechanisms modifying [Ca2+]i include store-operated Ca2+-entry (SOCE) accomplished by the pore-forming ion channel unit Orai1 and its regulator STIM1, as well as Ca2+ extrusion by Na+/Ca2+ exchanger NCX1. Kinases participating in the orchestration of cell proliferation include the Janus activated kinase JAK2. The present study explored the impact of pharmacological JAK2 inhibition on SOCE and Na+/Ca2+ exchange. Methods: MCF-7 breast carcinoma cells were studied in the absence and presence of the JAK2 inhibitors TG101348 (250 nM - 1 µM) or of AG490 (20 - 40 µM). Transcript levels were quantified with RT-PCR, protein abundance with immunoblotting, [Ca2+]i with Fura-2-fluorescence, SOCE from increase of [Ca2+]i following Ca2+ re-addition after Ca2+-store depletion with sarcoendoplasmatic Ca2+-ATPase (SERCA) inhibitor thapsigargin (1 µM), and Na+/Ca2+ exchanger activity from increase of [Ca2+]i as well as Ca2+ current in whole cell patch clamp following extracellular Na+ removal. Migratory activity was determined by a wound healing assay. Results: JAK2 inhibitor TG101348 (1 µM) decreased Orai1 and STIM1 protein abundance, increased NCX1 transcript levels, decreased Ca2+ release from intracellular stores, decreased SOCE, increased Ca2+ entry as well as Ca2+-current following extracellular Na+-removal, and decreased migration. Similar effects on Ca2+ release, SOCE, and Ca2+-entry following extracellular Na+-removal were observed following treatment with AG490. Conclusion: The present observations disclose a novel powerful mechanism regulating intracellular Ca2+ release, cellular Ca2+ entry, cellular Ca2+ extrusion and cell migration.

scholarly journals Regulation of Voltage Gated K+ Channel KCNE1/KCNQ1 by the Janus Kinase JAK3

2015 ◽  
Vol 37 (6) ◽  
pp. 2476-2485
Author(s):  
Jamshed Warsi ◽  
Abeer Abousaab ◽  
Myriam Fezai ◽  
Bernat Elvira ◽  
Florian Lang
Keyword(s):  
Cell Membrane ◽  
Brefeldin A ◽  
Janus Kinase ◽  
K Channel ◽  
Wild Type ◽  
Protein Insertion ◽  
Voltage Gated ◽  
Electrode Voltage ◽  
Jak3 Inhibitor ◽  
The Difference

Background/Aims: Janus kinase 3 (JAK3), a kinase mainly expressed in hematopoietic cells, has been shown to down-regulate the Na+/K+ ATPase and participate in the regulation of several ion channels and carriers. Channels expressed in thymus and regulating the abundance of T lymphocytes include the voltage gated K+ channel KCNE1/KCNQ1. The present study explored whether JAK3 contributes to the regulation of KCNE1/KCNQ1. Methods: cRNA encoding KCNE1/KCNQ1 was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild-type JAK3, constitutively active A568VJAK3, or inactive K851AJAK3. Voltage gated K+ channel activity was measured utilizing two electrode voltage clamp. Results: KCNE1/KCNQ1 activity was significantly increased by wild-type JAK3 and A568VJAK3, but not by K851AJAK3. The difference between oocytes expressing KCNE1/KCNQ1 alone and oocytes expressing KCNE1/KCNQ1 with A568VJAK3 was virtually abrogated by JAK3 inhibitor WHI-P154 (22 µM) but not by inhibition of transcription with actinomycin D (50 nM). Inhibition of KCNE1/KCNQ1 protein insertion into the cell membrane by brefeldin A (5 µM) resulted in a decline of the voltage gated current, which was similar in the absence and presence of A568VJAK3, suggesting that A568VJAK3 did not accelerate KCNE1/KCNQ1 protein retrieval from the cell membrane. Conclusion: JAK3 contributes to the regulation of membrane KCNE1/KCNQ1 activity, an effect sensitive to JAK3 inhibitor WHI-P154.

scholarly journals Blockade of BK channels attenuates chronic visceral hypersensitivity in an IBS-like rat model

2021 ◽  
Vol 17 ◽  
pp. 174480692110403
Author(s):  
F Fan ◽  
Y Chen ◽  
Z Chen ◽  
L Guan ◽  
Z Ye ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Nmda Receptors ◽  
Inhibitory Interneuron ◽  
Bk Channels ◽  
Bk Channel ◽  
Visceral Hypersensitivity ◽  
Potassium Ion Channels ◽  
Whole Cell Patch Clamp ◽  
Underlying Mechanisms ◽  
Irritable Bowel

Background Visceral hypersensitivity in irritable bowel syndrome (IBS) is still poorly understood, despite that chronic abdominal pain is the most common symptoms in IBS patients. To study effects of BK channels on visceral hypersensitivity in IBS rats and the underlying mechanisms, IBS rats were established by colorectal distention (CRD) in postnatal rats. The expression of large-conductance calcium and voltage-dependent potassium ion channels (BK channels) of the thoracolumbar spinal cord was examined in IBS and control rats. The effects of BK channel blockade on visceral hypersensitivity were evaluated. The interaction of BK channels and N-methyl-D-aspartate acid (NMDA) receptors was explored, and synaptic transmission at superficial dorsal horn (SDH) neurons of the thoracolumbar spinal cord was recorded by whole-cell patch clamp in IBS rats. Results The expression of the BK channels of the thoracolumbar spinal cord in IBS rats was significantly reduced. The blockade of BK channels could reduce the visceral hypersensitivity in IBS rats. There was an interaction between BK channels and NMDA receptors in the spinal cord. The frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in SDH neurons is significantly reduced in IBS rats. The blockade of BK channels depolarizes the inhibitory interneuron membrane and increases their excitability in IBS rats. Conclusions BK channels could interact with NMDA receptors in the thoracolumbar spinal cord of rats and regulate visceral hypersensitivity in IBS rats.

scholarly journals Expression of JAK3 Sensitive Na+ Coupled Glucose Carrier SGLT1 in Activated Cytotoxic T Lymphocytes

2016 ◽  
Vol 39 (3) ◽  
pp. 1209-1228 ◽  
Author(s):  
Shefalee K. Bhavsar ◽  
Yogesh Singh ◽  
Piyush Sharma ◽  
Vishal Khairnar ◽  
Zohreh Hosseinzadeh ◽  
...  
Keyword(s):  
T Cells ◽  
T Lymphocytes ◽  
Cell Membrane ◽  
Glucose Uptake ◽  
Xenopus Oocytes ◽  
Cytotoxic T Cells ◽  
Brefeldin A ◽  
Protein Abundance ◽  
Jurkat T Cells ◽  
Glucose Carrier

Background: Similar to tumor cells, activated T-lymphocytes generate ATP mainly by glycolytic degradation of glucose. Lymphocyte glucose uptake involves non-concentrative glucose carriers of the GLUT family. In contrast to GLUT isoforms, Na+-coupled glucose-carrier SGLT1 accumulates glucose against glucose gradients and is effective at low extracellular glucose concentrations. The present study explored expression and regulation of SGLT1 in activated murine splenic cytotoxic T cells (CTLs) and human Jurkat T cells. Methods: FACS analysis, immunofluorescence, confocal microscopy, chemiluminescence and Western blotting were employed to estimate SGLT1 expression, function and regulation in lymphocytes, as well as dual electrode voltage clamp in SGLT1 ± JAK3 expressing Xenopus oocytes to quantify the effect of janus kinase3 (JAK3) on SGLT1 function. Results: SGLT1 is expressed in murine CTLs and also in human Jurkat T cells. 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose uptake was significantly decreased by SGLT1-blocker phloridzin (0.2 mM) and by pharmacological inhibition of JAK3 with WHI-P131 (156 µM), WHI-P154 (11.2 µM) and JAK3 inhibitor VI (0.5 µM). Electrogenic glucose transport (Iglucose) in Xenopus oocytes expressing human SGLT1 was increased by additional expression of human wild type JAK3, active A568VJAK3 but not inactive K851AJAK3. Coexpression of JAK3 enhanced the maximal transport rate without significantly modifying affinity of the carrier. Iglucose in SGLT1+JAK3 expressing oocytes was significantly decreased by WHI-P154 (11.2 µM). JAK3 increased the SGLT1 protein abundance in the cell membrane. Inhibition of carrier insertion by brefeldin A (5 µM) in SGLT1+JAK3 expressing oocytes resulted in a decline of Iglucose, which was similar in presence and absence of JAK3. Conclusions: SGLT1 is expressed in murine cytotoxic T cells and human Jurkat T cells and significantly contributes to glucose uptake in those cells post activation. JAK3 up-regulates SGLT1 activity by increasing the carrier protein abundance in the cell membrane, an effect enforcing cellular glucose uptake into activated lymphocytes and thus contributing to the immune response.

Ca2+ dependence of flow-stimulated K secretion in the mammalian cortical collecting duct

2007 ◽  
Vol 293 (1) ◽  
pp. F227-F235 ◽  
Author(s):  
Wen Liu ◽  
Tetsuji Morimoto ◽  
Craig Woda ◽  
Thomas R. Kleyman ◽  
Lisa M. Satlin
Keyword(s):  
Apical Membrane ◽  
Collecting Duct ◽  
Brefeldin A ◽  
Bk Channels ◽  
Bk Channel ◽  
Sk Channels ◽  
Cortical Collecting Duct ◽  
Intracellular Ca ◽  
K Secretion ◽  
High Conductance

Apical low-conductance SK and high-conductance Ca2+-activated BK channels are present in distal nephron, including the cortical collecting duct (CCD). Flow-stimulated net K secretion ( JK) in the CCD is 1) blocked by iberiotoxin, an inhibitor of BK but not SK channels, and 2) associated with an increase in [Ca2+]i, leading us to conclude that BK channels mediate flow-stimulated JK. To examine the Ca2+ dependence and sources of Ca2+ contributing to flow-stimulated JK, JK and net Na absorption ( JNa) were measured at slow (∼1) and fast (∼5 nl·min−1·mm−1) flow rates in rabbit CCDs microperfused in the absence of luminal Ca2+ or after pretreatment with BAPTA-AM to chelate intracellular Ca2+, 2-aminoethoxydiphenyl borate (2-APB), to inhibit the inositol 1,4,5-trisphosphate (IP3) receptor or thapsigargin to deplete internal stores. These treatments, which do not affect flow-stimulated JNa (Morimoto et al. Am J Physiol Renal Physiol 291: F663–F669, 2006), inhibited flow-stimulated JK. Increases in [Ca2+]i stimulate exocytosis. To test whether flow induces exocytic insertion of preformed BK channels into the apical membrane, CCDs were pretreated with 10 μM colchicine (COL) to disrupt microtubule function or 5 μg/ml brefeldin-A (BFA) to inhibit delivery of channels from the intracellular pool to the plasma membrane. Both agents inhibited flow-stimulated JK but not JNa (Morimoto et al. Am J Physiol Renal Physiol 291: F663–F669, 2006), although COL but not BFA also blocked the flow-induced [Ca2+]i transient. We thus speculate that BK channel-mediated, flow-stimulated JK requires an increase in [Ca2+]i due, in part, to luminal Ca2+ entry and ER Ca2+ release, microtubule integrity, and exocytic insertion of preformed channels into the apical membrane.

scholarly journals Evidence for a role for BK channels in the regulation of ADAM17 activity

2019 ◽  
Author(s):  
Minae Yoshida ◽  
Dean Willis
Keyword(s):  
Cell Membrane ◽  
Cell Biology ◽  
Cancer Biology ◽  
Bk Channels ◽  
Bk Channel ◽  
Potassium Ions ◽  
Excitable Cells ◽  
Tnf Α ◽  
A Disintegrin And Metalloproteinase ◽  
Physical And Chemical

AbstractLarge-conductance voltage and calcium activated channels, KCa1.1, have a large single conductance (~p250) and are highly selective for potassium ions. As a result they have been termed big potassium channels (BK channels). Because of the channel’s ability to integrate multiple physical and chemical signals they have received much attention in excitable cells. In comparison they have received relatively little attention in non-excitable cells in those of the immune system. Here we report evidence that the BK channel regulates ADAM17 activity. Upon macrophage activation, BK channels translocate to the cell membrane. Genetic or pharmacological inhibition of the cell membrane BK channels resulted in elevated TNF-α release and increased metalloproteinase a disintegrin and metalloproteinase domain 17 (ADAM17) activity. Inhibitors of BK channels also increased IL-6Rα release, a second ADAM17 substrate. In comparison, a BK channel opener decreases TNF-α release. Taken together, our results demonstrate a novel mechanism by which ion channel regulates ADAM17 activity. Given the broad range of ADAM17 substrates, this finding has implications in many fields of cell biology including immunology, neurology and cancer biology.

scholarly journals MiR-339-3p aggravates rat vascular inflammation induced by AT1R autoantibodies by down-regulating BKα protein expression

2021 ◽  
Author(s):  
Huirong Liu ◽  
Yang Li ◽  
Yan Sun ◽  
Mingming Yue ◽  
Ming Gao ◽  
...  
Keyword(s):  
Protein Expression ◽  
Protein Level ◽  
Vascular Inflammation ◽  
Bk Channels ◽  
Bk Channel ◽  
The Body ◽  
Neural Precursor Cells ◽  
Α Subunit ◽  
Channel Protein ◽  
Calcium Activated Potassium Channels

The abnormality of large-conductance calcium-activated potassium channels (BK channels) is an important factor in inducing vascular inflammation. BK channel agonists can readily recover BK channel function and improve vascular inflammation. However, it is not clear how to improve BK dysfunction caused by downregulation of BK channel protein expression. This study found that angiotensin II-1 receptor autoantibodies (AT1-AA), which are widely present in the body of various types of cardiovascular diseases, can down-regulate the expression of BK channel protein and induce vascular inflammation. Further research found that the elevated neural precursor cells expressed developmentally downregulated 4-like (NEDD4L) protein level is involved in the down-regulation of BK channel α subunit (BKα) protein level by AT1-AA. Bioinformatics analysis and experiments have confirmed that miR-339-3p plays an irreplaceable role in the high expression of NEDD4L and the low expression of BKα, and aggravates the vascular inflammation induced by AT1-AA. Overall, AT1-AA increased miR-339-3p expression (targeting BKα via the miR-339-3p/NEDD4L axis or miR-339-3p alone), reduced BKα protein expression in VSMCs, and induced vascular inflammation. The results of the study indicate that miR-339-3p may become a new target for reversing vascular inflammation in AT1-AA-positive patients.

Janus Kinase-2 Inhibition Induces Durable Tolerance to Alloantigen by Dendritic Cell-Stimulated Human T Cells, Yet Preserves Immunity to Recall Antigen

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1903-1903
Author(s):  
Brian C. Betts ◽  
Omar Abdel-Wahab ◽  
Shane A Curran ◽  
Erin T St. Angelo ◽  
Priya Koppikar ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Th17 Cells ◽  
Janus Kinase ◽  
Effector Memory ◽  
Solid Organ ◽  
Janus Kinase 2 ◽  
Jak2 Inhibitor ◽  
Human T Cells ◽  
Jak2 Inhibition

Abstract Abstract 1903 Janus kinase 2 (JAK2) conveys signals from receptor binding by several inflammatory cytokines, including IL-6, IL-12, and IL-23, via phosphorylation of signal transducer and activator of transcription 3 (STAT3). These cytokines promote the development and expansion of T helper 1 (Th1) cells, which use IL-12, and Th17 cells, which use IL-6 and IL-23. Th1 and Th17 cells can in turn induce alloreactive end organ damage in GvHD. JAK2 is therefore a principle gatekeeper of alloreactivity and inflammation, and it represents an attractive target to control GvHD. We demonstrate that TG101348, a synthetic small molecule, highly selective JAK2 inhibitor, when present during initial and secondary encounters between human T cells and allogeneic monocyte-derived dendritic cells (moDCs), induces durable, profound, and specific T cell tolerance on reexposure to the same alloantigens. TG101348 ablates IL-6/JAK2-mediated phosphorylation of STAT3 in T cells (24% v 0%, P<0.05, n=5) but has no off-target effects on IL-2 or IL-15/JAK3/pSTAT5-dependent signaling, which would interfere with Tregs and other effector T cell responses. JAK2 inhibition preserves Treg numbers and thereby enhances the ratio of CD4+ Tregs to CD8+CD25+ effector T cells in favor of Tregs (Treg:Effector 1:2 vs 1:1, P<0.05, n=5). JAK2 inhibition also reduces the production of IL-6 (255 vs 157 pg/ml, P<0.05, n=5) and TNF-alpha (50 vs 15 pg/ml, P<0.05, n=4) in allogeneic MLRs, impairing the activation of central and effector memory T cells as well as the expansion of responder Th1 (24% vs 14%, P<0.05, n=4) and Th17 (2% vs 1%, P<0.05, n=4). T cells, stimulated by moDCs in the presence of TG101348, demonstrate a marked reduction in proliferative capacity upon reexposure to fresh allogeneic moDCs from the original stimulating party, even in the absence of the JAK2 inhibitor during the secondary MLR (P<0.05, n=4). Responses to stimulation de novo by influenza matrix peptide (fluMP), a pathogenic nominal antigen, remain intact (P=NS, n=4). Conversely, TG101348 also inhibits secondary MLR responses by T cells, which have already been sensitized to alloantigens in a primary MLR without the JAK2 inhibitor (P<0.05, n=4). The durable tolerance against full HLA disparities in each of these settings provides preclinical data in vitro that JAK2 represents a relevant biologic target for preventing or treating graft-versus-host disease (GvHD), or even solid organ allograft rejection, without broader immune impairment. Disclosures: No relevant conflicts of interest to declare.

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