hif-1a–mediated upregulation of task-2 k channels augments ca

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of February 14, 2018. This information is current as Mouse B Cells under Hypoxia Signaling in 2+ Channels Augments Ca + K Mediated Upregulation of TASK-2 - α HIF-1 Joon Kim Hyun Nam, Woo Kyung Kim, Yin Hua Zhang and Sung Joo Kim, Jin Young Kim, Yang Sook Chun, Jong Wan Park, Dong Hoon Shin, Haiyue Lin, Haifeng Zheng, Kyung Su ol.1301829 http://www.jimmunol.org/content/early/2014/10/10/jimmun published online 10 October 2014 J Immunol average * 4 weeks from acceptance to publication Speedy Publication! Every submission reviewed by practicing scientists No Triage! from submission to initial decision Rapid Reviews! 30 days* ? The JI Why Subscription http://jimmunol.org/subscription is online at: The Journal of Immunology Information about subscribing to Permissions http://www.aai.org/About/Publications/JI/copyright.html Submit copyright permission requests at: Email Alerts http://jimmunol.org/alerts Receive free email-alerts when new articles cite this article. Sign up at: Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved. Copyright © 2014 by The American Association of 1451 Rockville Pike, Suite 650, Rockville, MD 20852 The American Association of Immunologists, Inc., is published twice each month by The Journal of Immunology by guest on February 14, 2018 http://www.jimmunol.org/ Downloaded from by guest on February 14, 2018 http://www.jimmunol.org/ Downloaded from

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Page 1: HIF-1a–Mediated Upregulation of TASK-2 K Channels Augments Ca

of February 14, 2018.This information is current as

Mouse B Cells under Hypoxia Signaling in2+ Channels Augments Ca+K

Mediated Upregulation of TASK-2−αHIF-1

Joon KimHyun Nam, Woo Kyung Kim, Yin Hua Zhang and Sung

JooKim, Jin Young Kim, Yang Sook Chun, Jong Wan Park, Dong Hoon Shin, Haiyue Lin, Haifeng Zheng, Kyung Su

ol.1301829http://www.jimmunol.org/content/early/2014/10/10/jimmun

published online 10 October 2014J Immunol 

        average*  

4 weeks from acceptance to publicationSpeedy Publication! •    

Every submission reviewed by practicing scientistsNo Triage! •    

from submission to initial decisionRapid Reviews! 30 days* •    

?The JIWhy

Subscriptionhttp://jimmunol.org/subscription

is online at: The Journal of ImmunologyInformation about subscribing to

Permissionshttp://www.aai.org/About/Publications/JI/copyright.htmlSubmit copyright permission requests at:

Email Alertshttp://jimmunol.org/alertsReceive free email-alerts when new articles cite this article. Sign up at:

Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2014 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

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Page 2: HIF-1a–Mediated Upregulation of TASK-2 K Channels Augments Ca

The Journal of Immunology

HIF-1a–Mediated Upregulation of TASK-2 K+ ChannelsAugments Ca2+ Signaling in Mouse B Cells under Hypoxia

Dong Hoon Shin,*,† Haiyue Lin,* Haifeng Zheng,* Kyung Su Kim,* Jin Young Kim,*

Yang Sook Chun,*,‡ Jong Wan Park,‡,x Joo Hyun Nam,{ Woo Kyung Kim,{

Yin Hua Zhang,*,‡ and Sung Joon Kim*,‡

The general consensus is that immune cells are exposed to physiological hypoxia in vivo (PhyO2, 2–5% PO2). However, functional

studies of B cells in hypoxic conditions are sparse. Recently, we reported the expression in mouse B cells of TASK-2, a member of

pH-sensitive two-pore domain K+ channels with background activity. In this study, we investigated the response of K+ channels to

sustained PhyO2 (sustained hypoxia [SH], 3% PO2 for 24 h) in WEHI-231 mouse B cells. SH induced voltage-independent

background K+ conductance (SH-Kbg) and hyperpolarized the membrane potential. The pH sensitivity and the single-channel

conductance of SH-Kbg were consistent with those of TASK-2. Immunoblotting assay results showed that SH significantly

increased plasma membrane expressions of TASK-2. Conversely, SH failed to induce any current following small interfering

(si)TASK-2 transfection. Similar hypoxic upregulation of TASK-2 was also observed in splenic primary B cells. Mechanistically,

upregulation of TASK-2 by SH was prevented by si hypoxia-inducible factor-1a (HIF-1a) transfection or by YC-1, a pharmaco-

logical HIF-1a inhibitor. In addition, TASK-2 current was increased in WEHI-231 cells overexpressed with O2-resistant HIF-1a.

Importantly, [Ca2+]c increment in response to BCR stimulation was significantly higher in SH-exposed B cells, which was

abolished by high K+-induced depolarization or by siTASK-2 transfection. The data demonstrate that TASK-2 is upregulated

under hypoxia via HIF-1a–dependent manner in B cells. This is functionally important in maintaining the negative membrane

potential and providing electrical driving force to control Ca2+ influx. The Journal of Immunology, 2014, 193: 000–000.

Ion channels are widely accepted to play critical roles inregulating proliferation, apoptosis, or cytokine production inimmune cells (1, 2). B cells are located in lymph nodes and

spleen, where oxygen pressure is low (2–5%, depending on thedistance to the perfusion vasculature) (3, 4). Such a hypoxic mi-croenvironment in vivo (PhyO2) can be graded from hypoxic toanoxic sites such as poorly perfused cancer and atheroscleroticregions (5). Despite this physiological hypoxia is well acknowl-edged, most biological experiments and cell culture processes areperformed at atmospheric PO2 (AtmO2, 20–21%). Whether hyp-oxia (especially sustained hypoxia [SH]) affects the membraneconductance in B cells and the types of ion channels those areresponsible for the effects are unknown.Hypoxic stimulation regulates a variety of immune cell responses.

In T cells, exposure to acute hypoxia (AH; 15 min, 1% O2) inhibits

voltage-gated K+ channel (Kv1.3) current. In addition, decrease ofKV1.3 expression was observed in T cells undergoing SH (24 h,

1% O2) condition. Inhibition of KV channels induces membrane

depolarization and suppresses the activation response of T cells

(6, 7). Hypoxia regulates various functions of immune cells

such as T cells via hypoxia-inducible factor-1a (HIF-1a) (8). In

addition, dendritic cells show impaired migration to regional

lymph node under hypoxia (9).Role of ion channels in lymphocytes is directly associated with

Ca2+ signaling mechanisms, among which the Ca2+ influx path-

ways, such as calcium-release activated Ca2+ channels, have been

rigorously investigated. To effectively support the Ca2+ influx;

however, hyperpolarized membrane potential is required. Therefore,

the role of K+ channels has been also emphasized in lymphocytes in

the context of Ca2+ signaling (1, 10). Previous studies have shown

that Kv1.3 and Ca2+-activated K+ channel (SK4) play key roles

in lymphocytes (11, 12). Our recent studies suggested that TREK-2

and TASK-2, which are K2P channel proteins, may exert additional

functions in B cells (13, 14). Because of their background activity,

K2P channels are believed to set the negative resting membrane

potential (15, 16). Therefore, it is possible that TASK-2 and TREK-2

channels are important in facilitating Ca2+ influx and increasing

the intracellular Ca2+ level ([Ca2+]c). However, involvement of

K2P channels in hypoxia regulation of intracellular Ca2+ in B cells

remains unidentified.In the current study, we investigated whether SH affects mem-

brane conductance in WEHI-231 mouse B cells and primary splenicB cells. In addition, we identified the ion channel responsible forthe change inmembrane conductance and themechanismsmediatingthe effects of SH. Changes in intracellular Ca2+ were also detectedin B cell lines after SH. We provide direct evidence to show thatin B cells, sustained hypoxia upregulates TASK-2 via HIF-1a–mediated signaling pathway.

*Department of Physiology, Seoul National University College of Medicine, Seoul110-799, Republic of Korea; †Division of Natural Medical Sciences, College of HealthScience, Chosun University, Gwangju 501-759, Republic of Korea; ‡Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul110-799, Republic of Korea; xDepartment of Pharmacology, Seoul National Univer-sity College of Medicine, Seoul 110-799, Republic of Korea; and {ChannelopathyResearch Center, Dongguk University College of Medicine, Goyang 410-773, Re-public of Korea

Received for publication July 12, 2013. Accepted for publication September 10,2014.

This work was supported by the Basic Science Research Program through the Na-tional Research Foundation of Korea funded by the Ministry of Education, Science,and Technology (Grants NRF 2011-0017370 and NRF 2012-0000809).

Address correspondence and reprint requests to Dr. Sung Joon Kim, Department ofPhysiology, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu,Seoul, 110-799, Republic of Korea. E-mail address: [email protected]

Abbreviations used in this article: AA, arachidonic acid; AH, acute hypoxia; c-a, cell-attached; HIF-1a, hypoxia-inducible factor-1a; mTASK-2, mouse TASK-2; sc,scrambled; SH, sustained hypoxia; si, small interfering.

Copyright� 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301829

Published October 10, 2014, doi:10.4049/jimmunol.1301829 by guest on February 14, 2018

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Materials and MethodsCell culture and mouse B cell isolation

WEHI-231 cells (American Type Culture Collection, Manassas, VA) weregrown in DMEM (Invitrogen Life Technologies, Grand Island, NY) sup-plemented with 10% FBS(v/v) (Invitrogen, Carlsbad, CA), 50 mM 2-ME(Sigma-Aldrich, St. Louis, MO), and 1% penicillin/streptomycin (Invi-trogen) at 37˚C in 20% O2/10% CO2 (control condition) and 3% O2/10%CO2 (SH condition). The pH was set to 7.4 when equilibrated with 48 mMNaHCO3 in DMEM.

The total population of mouse primary B cells was prepared from thespleens of 6 wk-old C57BL/6 mice. Mice were sacrificed using 100% CO2,and the spleens were removed immediately. The spleens were dissociatedinto single-cell suspensions, and B cells were isolated using Spin-SepB cell enrichment kits (Stem Cell Technologies, Vancouver, BC, Canada).Isolated B cells were kept in AIM-V medium (Invitrogen) supplementedwith 10% FBS (v/v) at 37˚C in the control or SH condition.

Plasmids and gene transfection

mKCNK5 small interfering RNA (siRNA), a mixture of four siRNAsagainst mKCNK5 (mouse TASK-2 gene), was purchased from Dharmacon(Lafayette, LA) and mouse HIF-1a siRNA was purchased from SantaCruz Biotechnology (Santa Cruz, CA). The Pro-Ser-Thr-rich oxygen-de-pendent degradation domain determines the protein stability of HIF-1a,and O2-stable form of HIF-1a cDNA (sh-HIF-1a) was generated by de-leting 3 degradation motifs (aa 397–405, 513–553, and 554–595) (17). Plasmidsand siRNAs were transiently transfected using a nucleofector and corre-sponding kit (AMAXA Biosystems, Cologne, Germany). Briefly, WEHI-231cells were resuspended in nucleofector solution and 100 ml cells (23 106–5 3 106 /ml) were mixed with 1 mg pmax GFP (for electrophysiology) or byyellow fluorescent protein (for fura-2 fluorimetry) vector and siRNA (200nM), transferred to a cuvette, and nucleofected with AMAXA nucleofector.The cells transfected with scrambled (sc)RNA (Invitrogen) were used asnegative controls. Forty-eight hours after transfection, TASK-2 and HIF-1aknockdown by siRNA was verified using a patch clamp and RT-PCR.

Electrophysiology

Cells were transferred into a bath mounted on the stage of a model IX-70inverted microscope (Olympus, Osaka, Japan). The bath (∼0.15 ml) wassuperfused at 5 ml/min, and voltage clamp experiments were performed atroom temperature (22–25˚C). Patch pipettes with a free-tip resistance of2.5–3.5 MV were connected to the head stage of an Axopatch-1D patch-clamp amplifier (Molecular Devices, Sunnyvale, CA). pCLAMP softwareversion 9.2 and a Digidata-1322A apparatus (Molecular Devices) wereused to acquire data and apply command pulses. Throughout the whole-cell clamp experiments, 3 mM MgATP was included in the pipette solutionto minimize the influence of the activity of TREK-2 (18).

Single-channel activities were recorded at 20 kHz in cell-attached (c-a)configurations using patch pipettes with free-tip resistance 6–7 MV.Voltage and current data were low-pass filtered at 1 kHz. Current traceswere stored and analyzed using Clampfit version 9.2 and Origin version 7.0software (OriginLab, Northampton, MA). Single-channel recordings wereanalyzed to obtain amplitude histograms and total channel activities (NPo)where N and Po are the observed level of channel opening and the openprobability, respectively.

[Ca2+]i measurement

WEHI-231 cells or splenic B cells were loaded with fura-2 acetoxymethylester (5 mM, 30 min, room temperature) and washed twice with fresh NTbath solution (Table I). The Fura-2 loaded cells were transferred intoa microscope stage bath (∼100 ml) mounted on the stage of an invertedmicroscope (IX 70; Olympus) and perfused with NT solution at 5 ml/min.Fluorescence was monitored using a Polychrome IV monochromator (TILLPhotonics, Martinsried, Germany), a Cascade 650 charge-coupled devicecamera (Roper Scientific, Sarasota, FL) and Metafluor software (UniversalImaging, Downingtown, PA) at excitation wavelengths of 340 and 380 nm,and the ratio of emission light at 510 nm was measured (R340/380). Atthe end of each experiment, Ca2+-free solution with 5 mM EGTA wereapplied to produce a minimum fluorescence ratio (Rmin). Then, 2 mMionomycin and 10 mM CaCl2 were applied to confirm a maximum ratio offluorescence (Rmax). In each cell, R340/380 were normalized against theRmax and Rmin [(R340/380 2 Rmin)/Rmax].

Western blot

WEHI-231 cells or splenic B cells were incubated under SH conditions (3%PO2). For control experiments, mouse TASK-2 transfected HEK293T cells

were also prepared. Abs against TASK-2 (APC-037; Alomone Labs,Jerusalem, Israel) and anti-tubulin (2146S; Cell Signaling Technology,Beverly, MA) were obtained as outlined.

WEHI-231 cells were biotinylated using membrane impermeable Sulfo-NHE-SS-Biotin (Pierce, Rockford, IL) in PBS was added to the cell sus-pension and gently shaken for 1 h at 4˚C. After quenching free biotin by theaddition of 50 mM Tris-Cl (pH 7.4), WEHI-231 cells were lysed in lysisbuffer (0.5 M EDTA, 25 mM Tris-Cl [pH 7.4], 150 mM NaCl, and 1%Triton X-100) and centrifuged at 13,000 3 g for 10 min. Protein wasquantified at this step, and the same amounts of samples (500 mg proteinfrom control and SH cells) were used for the next procedure for total andmembrane fractions analyses, respectively. Supernatants were incubatedwith solution containing NeutrAvidin agarose resin (Pierce) for 1 h at roomtemperature. Beads were washed twice with 0.1% TBST. Avidin bindingproteins were eluted with elution buffer (62.5 mM Tris-Cl [pH 6.8], 1%SDS, 10% glycerol, and 50 mM DTT), and immunoblotting was performedusing a conventional procedure. To obtain total proteins, cells were har-vested and suspended in homogenization buffer (0.5 M EDTA, 25 mMTris-Cl [pH 7.4], 150 mM NaCl, 1% Triton X-100, 1 mM NaVO4, and1 mM b-glycerophosphate) containing a complete protease inhibitormixture (Roche Applied Science, Mannheim, Germany) and lysed usinga 22-gauge needle. Cell debris was removed by centrifugation, and thecleared lysates were mixed with recovered in SDS sample buffer and wereseparated using 4–12% precast polyacrylamide gels. The separated pro-teins were transferred to nitrocellulose membranes, which were blocked byincubating for 1 h in a solution containing 5% nonfat dry milk in 20 mMTris-HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20. Membranes werethen incubated with the appropriate primary and secondary Abs, andprotein bands were detected using ECL. Membranes were then stripped for30 min in stripping buffer (60 mM Tris-HCl, [pH 6.8], 100 mM 2-ME, and2% SDS) and reprobed with anti-tubulin.

RT-PCR

Total RNA was isolated from WEHI-231 cells using TRizol (Invitrogen).Mouse HIF-1a mRNA and 18s rRNA were analyzed by RT-PCR. Onemicrogram of RNA was reverse transcribed at 48˚C for 20 min, and thecDNA produced was amplified over 30 PCR cycles (57˚C for 1 min, 72˚Cfor 2 min, and 95˚C for 45 s). PCR products (5 ml) were electrophoresedon a 2% agarose gel at 100 V in a 13 Tris-acetate-EDTA buffer and vi-sualized using ethidium bromide. The nucleotide sequences of the primersused are summarized in Table II.

Statistical analysis

Data are expressed as mean6 SEM. Student t test and ANOVAwas used totest for significance at the level of p , 0.05.

ResultsSH induces Kbg current in WEHI-231 cells

The names and compositions of the experimental solutions used inthe electrophysiological experiments are listed in Table I. In whole-cell configuration, cells were held at 260 mV, and ramp-like de-polarization pulses (from 2100 to 100 mV) were applied to obtainI/V curves. Basal whole-cell currents in control WEHI-231 cellsfrom atmospheric O2, showed outwardly rectifying K+ current (Kv)that was activated from depolarizing voltages (more than 220 mV;Fig. 1A, Con). In contrast, sustained hypoxia (SH, 3% PO2, 24 h)induced prominent outward K+ current with voltage-independentbackground activity (Kbg; Fig. 1A, SH). In the WEHI-231 cellsexposed to SH (SH cells), the reversal potential of I/V curve wasclose to 280 mV, which was close to the K+ equilibrium potential,indicating that the background type K+ channel (Kbg) is the mainion channel that would set the hyperpolarized membrane potential(Table II). In SH cells held at 0 mV, a large inward current atnegative voltages was immediately revealed on replacing extracel-lular Na+ with equimolar K+, consistent with the increased Kbg

activity (Fig. 1B, SH). In contrast, the control cells showed defi-nitely smaller background conductance (Fig. 1B, Con).Then, we compared the Kbg activity in WEHI-231 cells at

different time of exposure to the hypoxia culture (3% PO2). Theamplitude of outward Kbg current in NT solution was measuredat 220 mV where KV current is negligible. The increase of Kbg

2 HYPOXIC UPREGULATION OF K+ CHANNELS VIA HIF-1 IN B CELLS

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current density (pA/pF) during hypoxia showed a biphasic patternin its time course, with an initial transient increase at ∼4 h anda further increase from 16 to 24 h and a slight decline at 48 h(Fig. 1C). The peak Kbg current density was observed at 24 h.We also compared the KV currents between control and SH

cells. To analyze the KV current component underlying the total I/Vcurve in SH cells, cells were held at depolarized holding voltage(20 mV) to induce maximum inactivation of KV channels. Then,reverse (hyperpolarizing from 100 to 2100 mV) ramp pulse wasapplied, and the obtained I/V curve was subtracted from theinitial I/V curve obtained by forward (depolarizing from 2100 to100 mV, held at 260 mV) ramp pulsein the whole-cell configu-ration. Collectively, no significant difference was observed be-tween SH and control cell (Fig. 1D).Since the above patch clamp study of SH cells was performed

under ambient PO2, we also tested the effect of AH by superfusingwith 100% N2-bubbled NT solution. We confirmed that the PO2 ofperfusate in the bath was lowered to 1–2% on changing to the N2-saturated solution (data not shown). AH immediately decreasedthe amplitude of KV current of control WEHI-231 cells (n = 6;Fig. 1E). In contrast, however, the Kbg currents measured in SHcells was slightly increased by AH (n = 9; Fig. 1F). Paired com-parison of AH-induced changes in current amplitudes more clearlydemonstrated the differential responses of control and SH cellsto AH (Fig. 1G). In each cell, normalized current amplitudesat 220 mV (for comparison of mainly Kbg activity) and 20 mV(for comparison of both Kbg and Kv activities) showed that the Kbg

at negative voltage was increased by AH in SH cells.

Molecular identity of SH-induced Kbg

Mouse B cells express TREK-2 and TASK-2, two members ofthe K2P (KCNK) family (13, 14). Because TREK-2 channelsare effectively activated by arachidonic acid (AA), amplitudes of10 mM AA-activated K+ current at negative membrane voltage inthe symmetrical KCl conditions were compared between controland SH cells. The AA-activated TREK-2 current was obtained bydigital subtraction of initial I/V curve (control) or 10-s AA-treatedI/V curve (SH) from the steady-state (120 s) I/V curve. A largeincrease in the slope of I/V curve was similarly observed in bothcontrol and SH cells (Fig. 2A, 2B).Single-channel activities of AA-activated TREK-2 were also

compared in c-a recording. With symmetrical KCl solutions,10 mM AA-activated Kbg channels with relatively large amplitude(∼17 pA at 260 mV) in both control and SH cells (Fig. 2B). Theslope conductance of AA-activated channel was close to 290 pS atsymmetrical [K+] (145 mM) condition, which was consistent withthe TREK-2 channels reported in B lympchytes (13). The calcu-

lated maximum activity (NPo) of TREK-2 with 10 mMAAwas notdifferent between control and SH cells (Fig. 2C, 2D).We next investigated the single channel properties of SH-

induced Kbg channels in the c-a recording with symmetrical KClsolutions without AA. In SH cells, inward K+ channel current with5.5 pA amplitudes at 260 mV were frequently observed (Fig. 2E,bottom panel). The slope conductance of SH-induced Kbg channelwas 82 pS (Fig. 2G), which is consistent with the known unitaryconductance of TASK-2 channel in B cells (19). In control cells,however, the inward K+ channel current was rarely observed(Fig. 2E, upper panel). In total, the TASK-2–like channel activity(NPo) was markedly higher in SH cells (n = 6; Fig. 2F).The hallmark property of TASK-2 is the extracellular pH (pHe)

sensitivity (15). Although the changes of pHe had an insignificanteffect on Kv current in control cells (Fig. 3A), the Kbg current inSH cells was significantly augmented and inhibited at pHe 8.4 and6.4, respectively (Fig. 3B). The pHe-sensitive outward Kbg currentwas rarely observed in the control cells. The summarized currentamplitudes at 220 mV are shown in the bar graph depicted inFig. 3C (n = 10).An immunoblot assay using the mouse TASK-2 (mTASK-2)–

specific Ab confirmed the expression of TASK-2 in WEHI-231cells. Protein samples were also obtained from mTASK-2 over-expressed in HEK-293T cells as a positive control (T2 in Fig. 4A).TASK-2 total protein expression significantly increased in SHcells (Fig. 4A, upper panel). The normalized total TASK-2 ex-pression (TASK-2/b-tubulin) was significantly increased in theSH cells (TASK-2/ b-tubulin [SH/Con], n = 4; Fig. 4B, left panel).In addition, a surface biotinylation assay demonstrated that themembrane expression of TASK-2 was definitely increased inSH cells (Fig. 4A, lower panel). Because the same amount ofbiotinylated proteins were obtained from control and SH cells,membrane fraction signal from SH cells was normalized againstcontrol cells (Fig. 4B, TASK-2 [SH/Con], right panel, n = 4).The molecular identity of hypoxia upregulated K+ channel as

TASK-2 was further proven by TASK-2–specific siRNA (siTASK-2) transfection. scRNA or siTASK-2 was cotransfected with GFPvector in WEHI-231 cells. Twenty four hours after the transfec-tion, the two groups of cells were further incubated under hypoxia(24 h, 3% PO2) conditions. We then performed whole-cell patchclamp in the GFP-positive cells under fluorescence microscopyexamination. A representative I/V curve of scRNA-transfectedSH cells consistently showed Kbg current in addition to voltage-dependent activation of Kv current, whereas siTASK-2–trans-fected SH cells showed KV current with little Kbg current(Fig. 5A). To rule out the influence from KV current, we initiallyused margatoxin, a selective blocker of Kv1.3 that is known to be

Table I. Experimental solutions and their compositions

Solute (mM)

Bath Solution Pipette Solution

NT NT (pH) High K+ (w-c, c-a) High K+ (w-c) High K+ (c-a)

NaCl 145 145 — 5 —KCl 3.6 3.6 140 140 140CaCl2 1.3 1.3 1 — 1MgCl2 1 1 1 0.5 1Glucose 5 5 5 — 5HEPES1 10 5 5 10 10MES — 5 5 — —EGTA — — — 10 —MgATP — — — 3 —pH 7.4 6.4 ∼ 8.4 6.4 ∼ 8.4 7.2 7.4

All the units are in mM except pH. NaOH or KOH were used to adjust pH values in bath and pipette solutions. w-c,whole-cell patch clamp.

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expressed in T and B lymphocytes (1, 7, 10, 11). However, the KV

current in WEHI-231 was insensitive to margatoxin, and RT-PCRanalysis showed no expression of Kv1.3 mRNA in WEHI-231

(data not shown). Therefore, to exclude KV current in WEHI-231, the membrane voltage was held at 20 mV, which inducedalmost full inactivation of KV channels. With depolarized holding

FIGURE 1. Sustained hypoxia induces background

K+ current in B cells. (A) Comparison of membrane

currents of WEHI-231 between control and SH (3%

O2, 24 h) cells. In the whole-cell clamp conditions, I/V

curves were obtained by ramp pulses (from 2100 to

100 mV, holding voltage 260 mV). Note the increase

of background type K+ (Kbg) current in SH cells. (B)

I/V curves obtained by ramp pulses (from 2100 to 100

mV, holding voltage 0 mV) under symmetric K+ con-

ditions. The induction of Kbg conductance was also

confirmed as inward current in the SH-cells (n = 6). (C)

Time-dependent induction of Kbg current by hypoxic

culture conditions. Mean amplitudes of outward current

at 220 mV are plotted according to the duration

(hours) of hypoxia. Numbers of tested cells are indi-

cated above each point (mean 6 SEM) (**p , 0.01;

*p , 0.05). (D) Comparison KV currents between

control and SH cells. KV current component was ob-

tained by subtracting non-inactivating current from the

initial current obtained by depolarizing ramp pulse

(from 2100 to 100 mV, holding voltage 260 mV), and

averaged I/V curves are displayed (n = 11). The non-

inactivating current was obtained from the steady-state

I/V curve by reverse ramp pulse (from 100 to 2100

mV) from depolarized holding voltage (20 mV). (E)

Inhibitory effect of AH on KV currents in control cells

(n = 6). I/V curves were obtained by forward ramp

pulses (from 2100 to 60 mV, holding voltage 260

mV). (F) Augmentation of Kbg current by AH in SH

cells. Throughout the figures, I/V curves obtained in

each group were averaged and plotted with vertical

bars indicating SEM (mean 6 SEM, n = 9). (G) The

current amplitudes under AH normalized to the re-

spective control (%) at 220 and 20 mV are shown in

the bar graph (mean 6 SEM) (*p , 0.05).

Table II. Nucleotide sequences of the primers used for RT-PCR

Protein (Gene Bank No.) Primer Size (bp) Sequence (59 to 39)

Mouse GAPDH Forward 275 CCCACTAACATCAAATGGGGReverse CCTTCCACAATGCCAAAGTT

Mouse HIF-1a Forward 350 GTCGGACAGCCTCACCAGACAGReverse TCTGCATGCTAAATCGGAGGGT

Mouse 18s rRNA Forward 186 CGGCTACCACATCCAAGGAAReverse GCTGGAATTACCGCGGCT

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voltage, a reverse ramp pulse from 100 to 2100 mV was appliedto obtain an I/V curve of noninactivating Kbg current that wasalmost absent at negative membrane voltages (Fig. 5B). Thus, tomeasure the whole-cell Kbg current, the reverse-ramp protocolwas used to conduct patch clamp study. In control cells, neitherscRNA nor siTASK-2–transfected cells showed significant Kbg

current at negative voltage ranges (n = 9; Fig. 5C). In contrast,the scRNA-transfected SH cells consistently demonstrated in-creased Kbg currents, which was not observed in the siTASK-2–transfected SH cells (n = 12; Fig. 5D).

HIF-1–dependent translational regulation of TASK-2

Many adaptive cellular responses to hypoxia are mediated by HIF-1, a transcription factor activated by hypoxia. HIF-1 functions asa heterodimer, consisting of HIF-1a and HIF-1b (20, 21). Theknockdown of HIF-1a expression by transfection of mouse HIF-1a–specific siRNA markedly decreased HIF-1a mRNA in WEHI-231 cells (Fig. 6A). In the whole-cell clamp experiment, reverseramp pulse from 100 to 2100 mV (holding voltage, 20 mV) was

applied to obtain an I/V curve of Kbg. SH-induced TASK-2 currentwas inhibited by siHIF-1a but not by scRNA (n = 13; Fig. 6B). Inaddition, cotreatment with 10 mM YC-1, a HIF-1a inhibitor (22),prevented the hypoxic upregulation of TASK-2 current in SHcells (n = 10; Fig. 6C). Vice versa, overexpression with O2-stableHIF-1a (sh-HIF-1a) alone induced TASK-2 in WEHI-231 cellsunder control normoxic culture conditions (n = 10; Fig. 6D).

Physiological implication of TASK-2 upregulation

The reversal voltages of I/V curves in SH cells indicate that TASK-2 activity would hyperpolarize B lymphocytes. The membranepotential measured under zero-current clamp mode showed sig-nificantly hyperpolarized values in SH cells (2816 3.6 mV, n = 8)than control (230 6 3.8 mV, n = 9). The upregulation of TASK-2and subsequent membrane hyperpolarization may augment theelectrical driving force for Ca2+ influx. To test this inference, Ca2+

signals activated by BCR stimulation were compared. Bath ap-plication of a-IgM raised [Ca2+]c, and the change of [Ca2+]c(DR340/380) was higher in SH cells than control (n = 10; Fig. 7A,

FIGURE 2. Induction of TASK-2 like K+ channels in

WEHI-231 cells undergoing SH. (A) Activation of

TREK-2 K+ current by AA (10 mM) in WEHI-231

cells. In the whole-cell clamp conditions, I/V curves

were obtained by ramp-like pulses (from 2100 to

100 mV, holding voltage 0 mV). To reveal the voltage-

independent K+ current activity, I/V curves of K+

conductance were obtained under symmetrical KCl

conditions. (B) AA-induced current was obtained from

AA-activated currents after 120 s passed minus currents

obtained after initial I/V curve control or SH cells

(mean 6 SEM, n = 10). (C) In cell-attached configu-

ration under symmetric KCl conditions, application of

10 mM AA-activated inward K+ current with relatively

large single-channel conductance (17 pA at 260 mV),

which was consistent with TREK-2. (D) The activity of

AA-induced TREK-2 (NPo) was not different between

control and SH cells (mean 6 SEM, n = 6). (E) TASK-

2–like K+ channel current with 5.5 pA single-channel

current (at 260 mV) was rarely observed in control

cells (upper) whereas frequently observed in SH-cells

(lower). (F) The averaged values of TASK-2–like

channel activity (NPo) were definitely higher in SH

cells (mean 6 SEM, n = 6) (**p , 0.01). (G) Slope

conductance of SH-induced Kbg channel obtained by

c-a patch clamp recording under symmetrical KCl con-

ditions. A linear fitting at negative voltage produced

82 pS conductance (n = 4).

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7C). The augmented Ca2+ signal by SH was not observed whenthe membrane potential was abolished by high K+-induced de-polarization (n = 22; Fig. 7C), which further proved the role ofenhanced electrical driving force for Ca2+ influx. Finally, siTASK-2–transfected SH cells showed attenuated Ca2+ signal when com-pared with scRNA transfected SH cells (n = 17; Fig. 7B, 7C).The functional expression of TASK-2 in mouse splenic B cell

and its up-regulation by BCR-ligation was reported previously(14). Therefore, we tested whether the hypoxic upregulation ofTASK-2 is also observed in fresh isolated B cells. The isolatedsplenic B cells were divided into two groups and incubated in thecontrol or SH condition. Whole-cell current was measured be-tween 20 and 28 h after the isolation. Because of the small size ofprimary B cells (∼0.8 pF of whole-cell membrane capacitance),outward K+ current in the whole-cell configuration was verysmall (10–30 pA at 0 mV), which limited the discrimination ofpHe-sensitivity without increasing the gain of input signal. Nev-ertheless, we could sometimes record TASK-like pHe-sensitiveoutward current in splenic B cells underwent 24h SH while notin control primary B cells (Fig. 8A).To further identify the functional expression of TASK-2 in the

primary B cells, single channel activities with pHe sensitivity was

investigated in outside-out mode (i.e., recording with 50 electricalgain under the whole-cell configuration) (Fig. 8B–D). The pHe-sensitive outward currents with unitary amplitudes of ∼1.8 pA at0 mV were recorded more frequently in SH than control B cells(Fig. 8B, 8C). The TASK-2 like channels showed increase anddecrease of activities (NPo) by alkaline (pH 8.4) and acidic (pH 6.4)pHe, respectively (Fig. 8B, C). The slope conductance was 87 pSin symmetrical [K+], consistent with the property of TASK-2(Fig. 8D). In SH cells, the TASK-2–like channel activity at pH 8.4tends to be higher than the one at pH 7.4 (p value, 0.051; Fig. 8E). Incontrol cells, we rarely observed the pHe-sensitive TASK-2–likechannels; the channel activity (NPo) at pH 8.4 is higher in SH thancontrol (p value, 0.018; Fig. 8E). Protein expression of TASK-2 andits upregulation by SH was also confirmed in two of four trials ofimmunoblot assay (Fig. 8F).The physiological role of TASK-2 upregulation in SH primary

B cell was also tested in the Ca2+ signal activated by BCR ligation. Infura-2–loaded primary B cells, application of anti-IgM slowly raised[Ca2+]c, and the steady-state change of [Ca

2+]c (DR340/380) was higherin SH primary B cells than control (Fig. 8G). The difference in Ca2+

signal was abolished when [K+]e was raised to 145 mM, collapsingthe electrical driving force by K+ channel activity (Fig. 8H).

DiscussionFirst, this study demonstrates the specific modification of ionchannel activities by hypoxia in B cells. So far, studies relevant tohypoxic modulation of ion channels in the immune system havebeen limited to T cells where both AH and SH inhibited Kv1.3channels (6, 7). The inhibition of Kv current by AH was alsoobserved in this study using B cells (Fig. 1E) while not decreasedin SH cells (Fig. 1D).The novel finding is that SH upregulates TASK-2, a member of

the pH-sensitive K2P (KCNK) channel family. Although a geneticanalysis of the promoter region for human TASK-2 identified HIF-1binding regions, it was not confirmed whether SH actually upre-gulates TASK-2 expression (23). Here we directly demonstrate thehypoxic upregulation of intrinsic TASK-2 channels in the mam-malian cells. It has to be also noted that the upregulated TASK-2current in SH cells was maintained or further increased by AH(Fig. 1F, 1G). Therefore, the hyperpolarizing effect of the up-

FIGURE 3. pHe dependence of SH-induced Kbg

current. In the whole-cell clamp conditions, I/V curves

were obtained by forward ramp pulses (from 2100 to

100 mV, holding voltage 260 mV). Representative

current traces of control (A) and SH cells (B) at dif-

ferential pHe (6.4, 7.4, and 8.2). (B) The Kbg current

of SH cell was increased and decreased by pH 8.2

and 6.4, respectively. (C) Summary of current ampli-

tudes at 220 mV for each pHe (mean 6 SEM, n = 10)

(**p , 0.01).

FIGURE 4. SH increased the protein expression of TASK-2 in B cells.

(A) Immunoblot assay for mTASK-2 in WEHI-231 cells. Total and

membrane fraction of mTASK-2 protein expression were increased in SH-

cells (T2: mTASK-2 overexpressed in HEK-293T, positive control). (B)

Summary of the immunoblot density ratios (total: [TASK-2/b-tubulin]/

control; membrane fraction: TASK-2/control) normalized to the initial

level (mean 6 SEM, n = 4) (**p , 0.01; *p , 0.05).

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regulated TASK-2 would be valid in the hypoxic/anoxic envi-ronments in vivo.Among the two types of K2P channels expressed in mouse

B cells (13, 14), total activity of TREK-2 was not altered by SH(Fig. 2D). Although both TASK-2 and TREK-2 channels belongto the so-called background-type K+ channels, TREK-2 chan-nels have large ranges of activation by various physicochemicalconditions (e.g., intracellular acidification and AA) (13, 16).Ischemic/hypoxic conditions activate phospholipase A2 that areresponsible for AA production (24), which might potentiate theTREK-2 activity in vivo hypoxia. However, another confoundingfactor for TREK-2 is their putative O2 sensitivity. Previous stud-ies claimed that hypoxia directly inhibits TREK-1 (25) that hashigh homology with TREK-2. In this respect, the contribution ofTREK-2 to the ion channel–mediated immunomodulation underhypoxic/ischemic conditions in vivo requires further investigation.Hypoxic regulation of ion channels can be categorized into an

acute response and chronic, sustained up- or downregulation. Theacute inhibitions of K+ channel have been described in variouschemoreceptor cells where subsequent membrane depolarizationactivates voltage-gated L-type Ca2+ channels. In carotid bodyglomus cells, inhibitions of Kv, ether-a-go-go (hERG), Ca2+ ac-tivated K+ (MaxiK) and TASK-1/3 K+ channels were reported(26–29). In pulmonary artery smooth muscle cells, the hypoxicinhibitions of KV and TASK-1-like K+ channels have been alsowell described, and suggested as the key mechanisms for hypoxicpulmonary vasoconstriction and hypoxic arterial remodeling (30,31). In this respect, the upregulation by SH and the facilitation byAH are relatively unique properties of TASK-2 in B lymphocytes.

Recently, TASK-2 was suggested to play a role in the O2 sen-sitivity of central respiratory center. TASK-22/2 mice showed dis-turbed chemosensory function to PCO2 changes and compromisedadaptation to chronic hypoxia (32). In this study, the electrical ac-tivity of respiratory center region in the brainstem was suppressedby hypoxia, which was not observed in the TASK-22/2 mice. Theauthors suggested that the putative reactive oxygen species gener-ation during the hypoxia would have activated TASK-2 channels.Interestingly, our study also demonstrated an increase of TASK-2current by AH in the B cells (Fig. 1F, 1G). The precise cellularmechanisms of TASK-2 facilitation by AH in B cells require rig-orous investigation in future.In T cells, SH induce functional downregulation of KV1.3 by

inhibiting forward vesicular trafficking process (33). In contrast,the increased current density of TASK-2 in the SH-B cells wasassociated with the increased amount of protein expression(Figs. 4, 8). Such changes could result from transcription, trans-lation, or increased stability of channel protein. Furthermore, therelatively higher increase of membrane TASK-2 by SH suggestedthat membrane trafficking of TASK-2 was also augmented by SHin B cells (Fig. 4).In this study, both pharmacololgical (YC-1) and genetic (siHIF-

1a) inhibitions of HIF-1a indicated that the hypoxic upregulationof TASK-2 is mediated by transcriptional regulation from HIF-1a(Figs. 6B, 6C). In addition, the increase of background K+ currentunder control conditions by sh-HIF-1a overexpression stronglysuggest that HIF-1-depenent transcriptional regulation is sufficientfor the induction of TASK-2 in B cells (Fig. 6D). Consistently,potential HIF-1 binding region has been identified in the promoter

FIGURE 5. Inhibition of SH-induced Kbg current by siTASK-2 transfection. (A) Representative I/V curves obtained by depolarizing ramp pulse from

260 mV holding voltage in scRNA- and siTASK-2–transfected SH-cells. (B) Representative I/V curves showing the inactivation of KV current by

depolarized holding voltage. The voltage-dependent outward current component decreased by half on 1 s after changing the holding voltage from260 to 20

mV (②-1) and reached a steady-state inactivated at 15 s (②-2). (C) In control cells, neither scRNA- nor siTASK-2–transfected cells showed significant Kbg

current especially at 220 mV (mean 6 SEM, n = 9). (D) In contrast, the scRNA transfected SH cells consistently demonstrated increased Kbg currents,

which was not observed in the siTASK-2–transfected SH cells. Summarized results are shown for scRNA- and siTASK-2–transfected WEHI-231 cells

(mean 6 SEM, n = 12).

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regions of the TASK-2 gene (23). HIF-1 mediated cellular re-sponses cover a wide variety of adaptive and pathological changesduring hypoxia (34). Abnormal peritoneal B-1 like lymphocytes arefrequently observed in HIF-1a KO mice, and such phenomenon arepostulated to be associated with autoimmunity (35). The HIF-1-mediated upregulation of TASK-2 in B cells might also playa role in the systemic adaptation in terms of humoral immuneresponses and B cell differentiation.The present study demonstrates that hypoxia is strongly asso-

ciated with TASK-2 up-regulation and membrane hyperpolariza-

tion. In fact, WEHI-231 cells exposed to SH showed augmented[Ca2+]c elevation by BCR-ligation (Fig. 7 and Fig. 8). siTASK-2transfection largely suppressed the steady-state [Ca2+]c elevationin comparison with scRNA transfected WEHI-231 cells (Fig. 7).Since the chemical depolarization (high K+ conditions) abolishedthe difference between control and SH cells, it was suggested thatthe hyperpolarization inferred from HIF-1 mediated TASK-2 up-regulation was responsible for the augmented Ca2+ signal, whichmight implicate positive influence on B cell responses.(Fig. 7 andFig. 8).

FIGURE 6. HIF-1a mediates SH activation of

TASK-2 in B cells. (A) RT-PCR analysis for HIF-1a in

scRNA- and siHIF-1a–transfected SH cells, showing

abolished HIF-1a mRNA by siHIF-1a. (B) Comparison

of TASK-2 current between scRNA- and siHIF-1a–

transfected SH-cells. I/V curves were obtained by

repolarizing ramp pulses (holding voltage, 20 mV) and

averaged (mean 6 SEM). TASK-2 current was largely

abolished in the siHIF-1a–transfected SH cells (n = 13).

(C) Inhibitory effect of YC-1, a HIF-1 inhibitor, on

TASK-2 induced by SH. Summary of I/V curves ob-

tained by repolarizing ramp pulses (n = 9). (D) Up-

regulation of TASK-2 current by overexpression of

O2-resistant sh-HIF-1a in WEHI-231 cells under con-

trol condition (n = 10).

FIGURE 7. Augmented Ca2+ sig-

nal by BCR stimulation in SH cells.

(A) Normalized fura-2 fluorescence

ratio of single WEHI-231 cell, an

indicator of [Ca2+]c, was increased

by BCR stimulation with anti-IgM

Ab. Averaged results from 10 cells

for each group, control and SH cells,

displayed higher amplitudes of [Ca2+]cchanges (mean 6 SEM,). (B) [Ca2+]cmeasurements in SH cells transfected

with either scRNA or siTASK-2 also

showed suppressed Ca2+ signal in

siTASK-2–transfected cells (n = 17).

(C) Peak changes in [Ca2+]c (DR340/380)

were summarized for each group

(control versus SH) in NT and high

KCl solutions, respectively. It is

notable that the DR340/380 was at-

tenuated in KCl solution with no

difference between control and SH

cells (**p , 0.01).

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In T cells, however, it has been reported that HIF-1 dependentregulation reduced Ca2+ signals via upregulating the Ca2+ pumpproteins in ER membrane (SERCA2), which has an impact onthymic selection (36). The hypoxia/HIF-1 induced change ofSERCA activity has not been investigated in B cells yet. Inter-estingly, according to literature, the stimulated primary T cellsproliferate better at AtmO2 (37, 38), whereas B cells showedhigher proliferation and Ab production at PhysO2 (39). In thisrespect, the SH-induced changes of K+ conductance appear to beopposite between T and B cells. A series of studies consistentlydemonstrated that hypoxia produces both acute and long-terminhibition of Kv1.3 channels in T cells. The suppression ofKv1.3 is suggested to be associated with the hypoxic inhibitionof TCR-mediated proliferation (6). The unique hypoxic upreg-ulation of TASK-2 and the augmented Ca2+ signals by hyper-polarization might contribute to the positive regulation of B cellresponses. Both increase of TASK-2 current and protein ex-pression by SH were confirmed in the primary splenic B cells

(Fig. 8A–E). Also, the Ca2+ signal induced by BCR ligation wasaugmented in SH, which became indistinguishable by 145 KCldepolarization (Fig. 8F, 8G). Further study using TASK-2–defi-cient mice would be helpful to show a role of SH-induced up-regulation of TASK-2 in B cell development and/or functionin vivo. TASK-2 has been proposed to play a role in apoptoticvolume decrease in kidney cells, in volume regulation of glialcells and T lymphocytes, and in negative membrane potential ofchondrocytes (40).In summary, the expression of TASK-2 in WEHI-231 is markedly

upregulated by hypoxia in a HIF-1a–dependent manner. The aug-mented Ca2+ influx in the B cells underwent SH could arise fromthe enhanced electrical driving force (i.e., hyperpolarizing effect ofTASK-2). More rigorous studies about the immunological impli-cation of TASK-2 in systemic levels need to be conducted.

DisclosuresThe authors have no financial conflicts of interest.

FIGURE 8. Higher activity of TASK-2 channels

and augmented Ca2+ signal by BCR-ligation in

primary splenic B cells undergoing SH. (A) In

whole-cell configuration, mouse splenic B cells

were held at 260 mV, and ramp-like pulses (from

2100 to 60 mV) were applied to obtain brief I-V

curves. I-V curve of a control cell (left panel)

shows a KV predominant case where alkaline pHe

(8.4) had negligible effect. I-V curve of a SH

splenic B cell (right panel) demonstrates alkaline

pHe-activated background K+ current. (B) Repre-

sentative traces of pHe-sensitive K+ channels in the

o-o configuration of a mouse splenic B cell from

SH condition. The membrane voltage was held at

0 mV to selectively record K+ channel activity in

the ionic composition indicated in the figure. (C)

Slope conductance of the pHe-sensitive K+ channel

in splenic B cells underwent SH conditions. Linear

fitting of the unitary currents at negative voltage

range showed ∼87 pS (n = 3). (D) The summary of

pHe-dependent activity (NPo) of the TASK-2–like

channels. In each patch, NPo at pHe 7.4 and 8.4 was

measured during 40 s, respectively. In control cells,

pHe-sensitive TASK-22like channels were rarely

observed (2 of 11 cells cases showed .50% in-

crease by pHe 8.4). In SH cells, however, alkaline

pHe-activated channels were more frequently ob-

served (6 of 14 cells showed .50% increase by

pHe 8.4). Also the activities (NPo) of K+ channels

were generally higher than the control group at pHe

8.4 (p , 0.05). (E) Immunoblot assay for

mTASK-2 in splenic B cells. Total and membrane

fraction of mTASK-2 protein expression were in-

creased in the splenic B cells from SH incubation

(T2: mTASK-2 overexpressed in HEK-293T, pos-

itive control). (F) Augmented Ca2+ signal by BCR

stimulation (anti-IgM Ab) in splenic B cells from

SH incubation. Fura-2 fluorescence ratio was nor-

malized to Rmax ([R 2 Rmin]/Rmax). Averaged

results from 30 cells for each group displayed

higher changes in SH than control (mean 6 SEM).

(G) The steady-state increase in [Ca2+]c were

summarized for each group (control versus SH) in

NT and 145 mM KCl solutions, respectively

(**p , 0.01). The DR340/380 was attenuated in KCl

solution with no difference between control and SH

cells.

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