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

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.

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

Cellular Physiology and Biochemistry ◽

10.1159/000438600 ◽

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.

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

AJP Cell Physiology ◽

10.1152/ajpcell.00209.2013 ◽

2014 ◽

Vol 306 (11) ◽

pp. C1041-C1049 ◽

Cited By ~ 4

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.

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SPAK and OSR1 Sensitive Cell Membrane Protein Abundance and Activity of KCNQ1/E1 K+ Channels

Cellular Physiology and Biochemistry ◽

10.1159/000438563 ◽

2015 ◽

Vol 37 (5) ◽

pp. 2032-2042 ◽

Cited By ~ 1

Author(s):

Bernat Elvira ◽

Jamshed Warsi ◽

Myriam Fezai ◽

Carlos Munoz ◽

Florian Lang

Keyword(s):

Cell Membrane ◽

Cell Volume ◽

Epithelial Transport ◽

Cell Volume Regulation ◽

K Channel ◽

Protein Abundance ◽

Wild Type ◽

E1 Protein ◽

Cell Membrane Protein ◽

Constitutively Active

Background/Aims: KCNQ1/E1 channels are expressed in diverse tissues and serve a variety of functions including endolymph secretion in the inner ear, cardiac repolarization, epithelial transport and cell volume regulation. Kinases involved in regulation of epithelial transport and cell volume include SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1), which are under control of WNK (with-no-K[Lys]) kinases. The present study explored whether KCNQ1/E1 channels are regulated by SPAK and/or OSR1. Methods: cRNA encoding KCNQ1/E1 was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild-type SPAK, constitutively active T233ESPAK, WNK insensitive T233ASPAK, catalytically inactive D212ASPAK, wild-type OSR1, constitutively active T185EOSR1, WNK insensitive T185AOSR1 and catalytically inactive D164AOSR1. Voltage gated K+ channel activity was quantified utilizing dual electrode voltage clamp and KCNQ1/E1 channel protein abundance in the cell membrane utilizing chemiluminescence of KCNQ1/E1 containing an extracellular Flag tag epitope (KCNQ1-Flag/E1). Results: KCNQ1/E1 activity and KCNQ1-Flag/E1 protein abundance were significantly enhanced by wild-type SPAK and T233ESPAK, but not by T233ASPAK and D212ASPAK. Similarly, KCNQ1/E1 activity and KCNQ1-Flag/E1 protein abundance were significantly increased by wild-type OSR1 and T185EOSR1, but not by T185AOSR1 and D164AOSR1. Conclusions: SPAK and OSR1 participate in the regulation of KCNQ1/E1 protein abundance and activity.

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OSR1 and SPAK Sensitivity of Large-Conductance Ca2+ Activated K+ Channel

Cellular Physiology and Biochemistry ◽

10.1159/000443105 ◽

2016 ◽

Vol 38 (4) ◽

pp. 1652-1662 ◽

Cited By ~ 1

Author(s):

Bernat Elvira ◽

Yogesh Singh ◽

Jamshed Warsi ◽

Carlos Munoz ◽

Florian Lang

Keyword(s):

Cell Volume ◽

Volume Regulation ◽

Cell Volume Regulation ◽

Bk Channels ◽

Bk Channel ◽

K Channel ◽

Xenopus Laevis Oocytes ◽

Channel Activity ◽

Wild Type ◽

Constitutively Active

Background/Aims: The oxidative stress-responsive kinase 1 (OSR1) and the serine/threonine kinases SPAK (SPS1-related proline/alanine-rich kinase) are under the control of WNK (with-no-K [Lys]) kinases. OSR1 and SPAK participate in diverse functions including cell volume regulation and neuronal excitability. Cell volume and neuronal excitation are further modified by the large conductance Ca2+-activated K+ channels (maxi K+ channel or BK channels). An influence of OSR1 and/or SPAK on BK channel activity has, however, never been shown. The present study thus explored whether OSR1 and/or SPAK modify the activity of BK channels. Methods: cRNA encoding the Ca2+ insensitive BK channel mutant BKM513I+Δ899-903 was injected into Xenopus laevis oocytes without or with additional injection of cRNA encoding wild-type OSR1 or wild-type SPAK, constitutively active T185EOSR1, catalytically inactive D164AOSR1, constitutively active T233ESPAK or catalytically inactive D212ASPAK. K+ channel activity was measured utilizing dual electrode voltage clamp. Results: BK channel activity in BKM513I+Δ899-903 expressing oocytes was significantly decreased by co-expression of OSR1 or SPAK. The effect of wild-type OSR1/SPAK was mimicked by T185EOSR1 and T233ESPAK, but not by D164AOSR1 or D212ASPAK. Conclusions: OSR1 and SPAK suppress BK channels, an effect possibly contributing to cell volume regulation and neuroexcitability.

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JNK1 Induces Notch1 Expression to Regulate Genes Governing Photoreceptor Production

Cells ◽

10.3390/cells8090970 ◽

2019 ◽

Vol 8 (9) ◽

pp. 970

Author(s):

Pan ◽

Hu ◽

Wang ◽

Zhou ◽

Zhang ◽

...

Keyword(s):

Cell Proliferation ◽

Transcriptional Factor ◽

Negative Regulator ◽

Potential Candidate ◽

Wild Type ◽

Cell Proliferation And Differentiation ◽

Proliferation And Differentiation ◽

Factor Expression ◽

Ophthalmic Diseases ◽

Essential Function

c-Jun N-terminal kinases (JNKs) regulate cell proliferation and differentiation via phosphorylating such transcription factors as c-Jun. The function of JNKs in retinogenesis remains to be elucidated. Here, we report that knocking out Jnk1, but not Jnk2, increased the number of photoreceptors, thus enhancing the electroretinogram (ERG) responses. Intriguingly, Notch1, a well-established negative regulator of photoreceptor genesis, was significantly attenuated in Jnk1 knockout (KO) mice compared to wild-type mice. Mechanistically, light specifically activated JNK1 to phosphorylate c-Jun, which in turn induced Notch1 transcription. The identified JNK1–c-Jun–Notch1 axis strongly inhibited photoreceptor-related transcriptional factor expression and ultimately impaired photoreceptor opsin expression. Our study uncovered an essential function of JNK1 in retinogenesis, revealing JNK1 as a potential candidate for targeting ophthalmic diseases.

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The Homodimeric Hematopoietic Cytokine Receptor C-Mpl Requires Cross Phosphorylation in Order to Signal Cell Proliferation

Blood ◽

10.1182/blood.v112.11.3734.3734 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3734-3734

Author(s):

Justin Persico ◽

Imawati Budjahardo ◽

Kenneth Kaushansky

Keyword(s):

Cell Proliferation ◽

Cell Line ◽

Cell Lines ◽

Western Blotting ◽

Control Cell ◽

Janus Kinase ◽

Cytokine Receptor ◽

Parental Cell Line ◽

Wild Type ◽

Receptor Dimerization

Abstract Objectives: The three primary regulators of hematopoiesis, erythropoietin, granulocyte colony-stimulating factor and thrombopoietin, bind to homodimeric members of the cytokine receptor superfamily and utilize Janus Kinase (JAK) 2 to initiate signaling. Recently, mutations in JAK2, particularly JAK2V617F, were found to contribute to the pathogenesis of the myeloproliferative disorders. In vitro studies have determined that only homodimeric cytokine receptors can support JAK2-mediated cytokine hypersensitivity. As part of a strategy to identify novel approaches to inhibit mutant JAK2 function we tested whether the homodimeric receptors initiate signaling by JAK2 mediated receptor trans-phosphorylation, and whether JAK2V617F escapes this requirement. Methods: We introduced the engineered receptor Myr/FKBPF36V/c-Mplcyto into hematopoietic cell lines containing either wild-type JAK2 or JAK2V617F, a receptor designed to adopt either a monomeric or dimeric state depending on the absence or presence, respectively, of the chemical dimerizer AP20187. To evaluate the effects of receptor dimerization on the growth of wild-type and V617F mutant JAK2 cell lines we measured reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). If either of the cell lines were able to support cell proliferation in absence of receptor dimerization it would indicate that signaling is initiated by ipsilateral receptor phosphorylation, as opposed to the trans-phosphorylation employed by subfamilies of heterodimeric receptors, such as that for IL-2. Results: The Myr/FKBPF36V/c-Mplcyto receptor construct was subcloned into a retroviral vector, transduced into Baf3, Baf3/JAK2 and Baf3/JAK2V617F cells and spontaneous, IL-3- and AP20187-induced cell proliferation was assessed. Equal expression of the receptor construct in each cell line was confirmed by western blotting. Both Baf3/JAK2- and Baf3/JAK2V617F-derived cell lines transduced with wild type c-Mpl served as controls, and quantitative western blotting was used to verify that equal levels of the two receptor constructs were introduced into the cell lines. Growth factor dependence was confirmed in the control cell lines with both thrombopoietin and IL-3 and was confirmed with IL-3 in the experimental cell lines. There was an increased sensitivity to growth factors in the control cell line containing the JAK2 V617F mutant, consistent with a myeloproliferative phenotype. When Myr/FKBPF36V/Mplcyto was introduced into either Baf3/JAK2 or Baf3/JAK2V617F cells, the cells remained dependent on either IL-3 or AP20187, although maximal rates of cell growth were significantly greater in the Baf3/JAK2V617F/Myr/FKBPF36V/Mplcyto cells than in Baf3/JAK2/Myr/FKBPF36V/Mplcyto cells. The maximal rate of growth of Baf3/JAK2V617F/Myr/FKBPF36V/Mplcyto cells also significantly exceeded that of the Baf3 parental cell line. Furthermore, we found that in the absence of chemically induced dimerization neither Myr/FKBPF36V/c-Mplcyto/JAK2 nor Myr/FKBPF36V/c-Mplcyto JAK2V617F cells proliferated. Conclusions: These results argue that JAK2 induces signaling by trans-phosphorylation of the cytoplasmic domains of c-Mpl and that the kinase hyperactivity displayed by JAK2V617F cannot overcome this requirement. Therefore it may be possible to alter or inhibit trans-phosphorylation and attenuate JAK2V617F-mediated myeloproliferation.

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Stimulation of N-Terminal Truncated Isoform of Androgen Receptor Stabilizes Human Ether-á-go-go-Related Gene-Encoded Potassium Channel Protein via Activation of Extracellular Signal Regulated Kinase 1/2

Endocrinology ◽

10.1210/en.2007-1802 ◽

2008 ◽

Vol 149 (10) ◽

pp. 5061-5069 ◽

Cited By ~ 17

Author(s):

Zhi-Yuan Wu ◽

Kun Chen ◽

Bernard Haendler ◽

Thomas V. McDonald ◽

Jin-Song Bian

Keyword(s):

Androgen Receptor ◽

Long Qt Syndrome ◽

Hek293 Cells ◽

K Channel ◽

Protein Abundance ◽

Related Gene ◽

Long Qt ◽

Channel Protein ◽

Qt Syndrome ◽

Stimulation Of

Proarrhythmic drugs induce long QT syndrome more frequently in women than men. The present study was designed to determine whether androgens regulate the function and expression of the human ether-á-go-go-related gene (HERG) encoded K+ channel, which is largely responsible for determining the QT interval. In a concentration-dependent manner (10−9 to 10−6m for 24 h), 5α-dihydrotestosterone (5α-DHT) increased HERG protein abundance in HEK293 cells stably expressing HERG in the presence of coexpressed cardiac androgen receptor (AR) variant [N-terminal truncated isoform of AR (AR45)]. The elevation of HERG protein was seen in endoplasmic reticulum, Golgi, and plasma membrane without clear preferential colocalization. Coexpression of the more common form of the AR did not confer 5α-DHT augmentation of HERG protein. Proteasome inhibitors, N-acetyl-L-leucyl-L-leucyl-L-norleucinal and MG132 prevented the 5α-DHT- dependent enhancement of HERG, as did the lysosome inhibitor, bafilomycin A1. Consistently, the cycloheximide-based protein chase study showed that 5α-DHT prolonged HERG protein half-life. 5α-DHT/AR45 signaling induced phosphorylation of ERK1/2. Blockade of ERK1/2 with PD98059 and U0126 prevented the effect of androgen on HERG protein abundance. Functional studies showed that 5α-DHT treatment for 24 h increased HERG K+ current density in Chinese hamster ovary cells cotransfected with cDNAs of AR45 and HERG channels. Moreover, 5α-DHT also increased ether-á-go-go-related gene-encoded K+ channel protein abundance in isolated rabbit cardiac myocytes. In conclusion, these data provide evidence that stimulation of AR45 receptors by androgens up-regulates HERG K+ channel abundance and activity mainly through stabilizing HERG protein in an ERK1/2 dependent mechanism, and suggest a mechanism to explain the sex difference in the long QT syndrome.

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Up-Regulation of the Large-Conductance Ca2+-Activated K+ Channel by Glycogen Synthase Kinase GSK3β

Cellular Physiology and Biochemistry ◽

10.1159/000447810 ◽

2016 ◽

Vol 39 (3) ◽

pp. 1031-1039 ◽

Cited By ~ 2

Author(s):

Myriam Fezai ◽

Musaab Ahmed ◽

Zohreh Hosseinzadeh ◽

Florian Lang

Keyword(s):

Cell Volume ◽

Glycogen Synthase ◽

Negative Control ◽

Bk Channels ◽

Bk Channel ◽

Glycogen Synthase Kinase ◽

K Channel ◽

Xenopus Laevis Oocytes ◽

Channel Activity ◽

Wild Type

Background/Aims: The pleotropic functions of the large conductance Ca2+-activated K+ channels (maxi K+ channel or BK channels) include regulation of neuronal excitation and cell volume. Kinases participating in those functions include the glycogen synthase kinase GSK3 ß which is under negative control of protein kinase B (PKB/Akt). GSK3ß is inhibited by the antidepressant Lithium. The present study thus explored whether GSK3ß modifies the activity of BK channels. Methods: cRNA encoding the Ca2+ insensitive BK channel mutant BKM513I+Δ899-903 was injected into Xenopus laevis oocytes without or with additional injection of cRNA encoding wild-type GSK3ß, inactive K85RGSK3ß, or wild-type GSK3ß with wild-type PKB. K+ channel activity was measured utilizing dual electrode voltage clamp. Results: BK channel activity in BKM513I+Δ899-903 expressing oocytes was significantly increased by co-expression of GSK3ß, but not by co-expression of K85RGSK3ß. The effect of wild type GSK3ß was abrogated by additional co-expression of wild-type PKB and by 24 hours incubation with Lithium (1 mM). Disruption of channel insertion into the cell membrane by brefeldin A (5 µM) was followed by a decline of the current to a similar extent in oocytes expressing BK and GSK3ß and in oocytes expressing BK alone. Conclusion: GSK3ß may up-regulate BK channels, an effect disrupted by Lithium or additional expression of PKB and possibly participating in the regulation of cell volume and excitability.

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A Role for the S0 Transmembrane Segment in Voltage-dependent Gating of BK Channels

The Journal of General Physiology ◽

10.1085/jgp.200609662 ◽

2007 ◽

Vol 129 (3) ◽

pp. 209-220 ◽

Cited By ~ 33

Author(s):

Olga M. Koval ◽

Yun Fan ◽

Brad S. Rothberg

Keyword(s):

Channel Gating ◽

Large Change ◽

Bk Channels ◽

Bk Channel ◽

Voltage Sensor ◽

K Channel ◽

Wild Type ◽

Transmembrane Segment ◽

Amino Terminal ◽

Transmembrane Segments

BK (Maxi-K) channel activity is allosterically regulated by a Ca2+ sensor, formed primarily by the channel's large cytoplasmic carboxyl tail segment, and a voltage sensor, formed by its transmembrane helices. As with other voltage-gated K channels, voltage sensing in the BK channel is accomplished through interactions of the S1–S4 transmembrane segments with the electric field. However, the BK channel is unique in that it contains an additional amino-terminal transmembrane segment, S0, which is important in the functional interaction between BK channel α and β subunits. In this study, we used perturbation mutagenesis to analyze the role of S0 in channel gating. Single residues in the S0 region of the BK channel were substituted with tryptophan to give a large change in side chain volume; native tryptophans in S0 were substituted with alanine. The effects of the mutations on voltage- and Ca2+-dependent gating were quantified using patch-clamp electrophysiology. Three of the S0 mutants (F25W, L26W, and S29W) showed especially large shifts in their conductance–voltage (G-V) relations along the voltage axis compared to wild type. The G-V shifts for these mutants persisted at nominally 0 Ca2+, suggesting that these effects cannot arise simply from altered Ca2+ sensitivity. The basal open probabilities for these mutants at hyperpolarized voltages (where voltage sensor activation is minimal) were similar to wild type, suggesting that these mutations may primarily perturb voltage sensor function. Further analysis using the dual allosteric model for BK channel gating showed that the major effects of the F25W, L26W, and S29W mutations could be accounted for primarily by decreasing the equilibrium constant for voltage sensor movement. We conclude that S0 may make functional contact with other transmembrane regions of the BK channel to modulate the equilibrium between resting and active states of the channel's voltage sensor.

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