Am J Physiol Cell Physiol 308: C264–C276, 2015. First published December 4, 2014; doi:10.1152/ajpcell.00176.2014.

The inward rectifier potassium channel Kir2.1 is expressed in mouse neutrophils from bone marrow and liver Ricard Masia,1,2 Daniela S. Krause,1 and Gary Yellen2 1

Department of Pathology and Laboratory Medicine, Massachusetts General Hospital, Boston, Massachusetts; and 2Department of Neurobiology, Harvard Medical School, Boston, Massachusetts Submitted 9 June 2014; accepted in final form 26 November 2014

Masia R, Krause DS, Yellen G. The inward rectifier potassium channel Kir2.1 is expressed in mouse neutrophils from bone marrow and liver. Am J Physiol Cell Physiol 308: C264 –C276, 2015. First published December 4, 2014; doi:10.1152/ajpcell.00176.2014.—Neutrophils are phagocytic cells that play a critical role in innate immunity by destroying bacterial pathogens. Channels belonging to the inward rectifier potassium channel subfamily 2 (Kir2 channels) have been described in other phagocytes (monocytes/macrophages and eosinophils) and in hematopoietic precursors of phagocytes. Their physiological function in these cells remains unclear, but some evidence suggests a role in growth factor-dependent proliferation and development. Expression of functional Kir2 channels has not been definitively demonstrated in mammalian neutrophils. Here, we show by RT-PCR that neutrophils from mouse bone marrow and liver express mRNA for the Kir2 subunit Kir2.1 but not for other subunits (Kir2.2, Kir2.3, and Kir2.4). In electrophysiological experiments, resting (unstimulated) neutrophils from mouse bone marrow and liver exhibit a constitutively active, external K⫹-dependent, strong inwardly rectifying current that constitutes the dominant current. The reversal potential is dependent on the external K⫹ concentration in a Nernstian fashion, as expected for a K⫹-selective current. The current is not altered by changes in external or internal pH, and it is blocked by Ba2⫹, Cs⫹, and the Kir2-selective inhibitor ML133. The singlechannel conductance is in agreement with previously reported values for Kir2.1 channels. These properties are characteristic of homomeric Kir2.1 channels. Current density in short-term cultures of bone marrow neutrophils is decreased in the absence of growth factors that are important for neutrophil proliferation [granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF)]. These results demonstrate that mouse neutrophils express functional Kir2.1 channels and suggest that these channels may be important for neutrophil function, possibly in a growth factor-dependent manner. inward rectifier potassium channel; Kir2 channels; neutrophil; granulocyte colony-stimulating factor CHANNELS OF THE INWARD RECTIFIER potassium family, subtype 2 (Kir2 channels) are expressed in excitable cells and play an important role in regulating the resting membrane potential and modulating excitability (1, 23). Dysregulation of Kir2 channel function leads to disease in humans such as cardiac arrhythmias (1). A defining characteristic of Kir2 channels is strong inward rectification: they preferentially conduct K⫹ in the inward direction and exhibit only a very small outward K⫹ conductance, due to voltage-dependent block by intracellular polyamines (32). There are several Kir2 channel subunits with varying properties such as differential sensitivity to pH or channel blockers (23). Since functional Kir2 channels are tetramers, channels with different characteristics can be gen-

Address for reprint requests and other correspondence: R. Masia, Dept. of Pathology and Laboratory Medicine, Massachusetts General Hospital, 55 Fruit St. Warren 219, Boston, MA 02114 (e-mail: [email protected]). C264

erated by coassembly of multiple subunits within the same cell, as occurs in the mammalian heart (15, 38). Kir2 channels are also expressed in nonexcitable cell types such as immune system cells, but their role in these cells is not well understood. In particular, functional Kir2 channels have been demonstrated in mammalian phagocytic cells, including macrophages (17, 20, 43), macrophage-derived cell lines (13, 34, 35), and eosinophils (49). These cells are derived from a common myeloid precursor and, when activated by inflammatory stimuli, contribute to innate immunity by engulfing and destroying pathogens (33). This occurs primarily via generation of reactive oxygen species by NADPH oxidase in a process known as the respiratory burst (9). Human CD33⫺ CD34⫹ hematopoietic progenitor cells, which give rise to erythroid and myeloid progenitors, also express functional Kir2 channels, and blocking these channels inhibits growth factor-dependent proliferation and production of myeloid progeny (46). This is intriguing because growth factor-dependent phosphorylation has been shown to increase Kir2 channel activity (61, 62). Thus Kir2 channels may play a role in growth factor-dependent proliferation of phagocytic cells. Neutrophils are also phagocytic cells that are derived from myeloid precursors, and they play a key role in host defense against bacterial pathogens (33). Currents with properties typical of Kir2 channels have been demonstrated in newt neutrophils (26). In mouse neutrophils, experiments with symmetrical high-K⫹ solutions have shown inward currents compatible with Kir channels (18), but this possibility has not been examined in detail. Detection of mRNA for multiple Kir2 subunits has been reported in human neutrophils (14), but this has not been substantiated by functional studies. Thus whether functional Kir2 channels are present in mammalian neutrophils remains unclear. Understanding the physiological relevance of Kir2 channels in mammalian neutrophils may yield important insight into their function and possibly uncover strategies for modulating neutrophil activity in the clinical setting, for example, enhancing innate immunity in immunocompromised states or preventing tissue injury in idiopathic inflammatory diseases. In the present study, we demonstrate that resting (unstimulated) mouse neutrophils express Kir2.1 mRNA and exhibit currents with the characteristic properties of homomeric Kir2.1 channel currents. These currents represent the dominant conductance in neutrophils isolated from bone marrow as well as proliferating neutrophils derived from juvenile liver. Our results suggest that Kir2.1 channels may be important for neutrophil function. MATERIALS AND METHODS

Animals. Experiments were conducted on 22 C57Bl/6N mice (3 for bone marrow preparations and 19 for liver preparations; Charles River Laboratories, Wilmington, MA). Animals were held in a 12-h light-

0363-6143/15 Copyright © 2015 the American Physiological Society

http://www.ajpcell.org

KIR2.1 CHANNELS IN MOUSE NEUTROPHILS

dark cycle at room temperature (20 –25°C) and fed ad libitum. Mice were deeply anesthetized with isofluorane and euthanized by decapitation. Animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee at Harvard University. All chemicals and reagents were purchased from Sigma (St. Louis, MO) unless otherwise indicated. Bone marrow neutrophil isolation and culture. Bone marrow was collected from femurs and tibiae of adult female C57BL/6N mice (6 –10 wk old) by flushing the open bones with chilled Dulbecco’s phosphate-buffered saline supplemented with 2% FBS (StemCell Technologies, Vancouver, BC, Canada) and 1 mM EDTA (referred to as “DFE”). Neutrophils were isolated by negative-selection magneticassociated cell sorting (MACS) with the EasySep Mouse Neutrophil Enrichment Kit (StemCell Technologies), following manufacturer’s instructions. Briefly, 2 ⫻ 108 resuspended bone marrow cells (2 ml of 1 ⫻ 108 cells/ml suspension in DFE) were incubated at 4°C for 15 min with rat serum and neutrophil enrichment cocktail containing biotinylated antibodies against non-neutrophils (CD4, CD5, CD11c, CD45R/B220, CD49b, CD117, TER119, and F4/80). Cells were pelleted, resuspended in DFE, and incubated with neutrophil selection cocktail (containing bispecific anti-biotin and anti-dextran antibodies) and magnetic dextran-coated beads. Non-neutrophils are bound to beads via tetrameric antibody complex formation. Beads were removed with a magnet, and isolated neutrophils were resuspended in DFE at 5 ⫻ 106 cells/ml. Cells were immediately used for total RNA isolation or plated on glass coverslips coated with 0.01% collagen by weight in 1⫻ PBS in 12-well plates (1–5 ⫻ 105 cells/well). Plated cells were incubated at 37°C and 5% CO2 in RPMI 1640 (Invitrogen, Waltham, MA) supplemented with 10% BCS (Thermo Scientific, Waltham, MA), 24 mM NaHCO3, 2 mM HEPES, 100 U/ml penicillin plus 100 ␮g/ml streptomycin (Lonza, Basel, Switzerland), 10 ng/ml recombinant mouse stem cell factor (SCF; Invitrogen), and 10 ng/ml recombinant mouse granulocyte colony-stimulating factor (G-CSF). Where indicated, growth factors were omitted. Plated cells were used for electrophysiological experiments. Liver-derived neutrophil isolation and culture. Livers were dissected from male and female C57BL/6N mice (12–16 days old) and minced into small fragments with a razor blade in a Petri dish containing chilled Krebs’ Ringer with glucose (referred to as “KRG”) plus 0.1 mM EGTA. KRG contained the following in mM: 120 NaCl, 24 NaHCO3, 20 glucose, 5 HEPES, 4.8 KCl, 1.2 MgSO4, and 1.2 KH2PO4; pH 7.4 with 1 M NaOH; 300 mosM. Osmolarity was measured with a VAPRO 5520 vapor pressure osmometer (Wescor Biomedical Systems, Logan, UT). Liver fragments were washed with KRG and incubated at 37°C in KRG plus 1.3 mM CaCl2 and 2 mg/ml collagenase type II (Worthington, Lakewood, NJ) with gentle rocking for 10 min. Fragments were triturated by sequential pipetting in the same solution at 37°C. Collagenase digestion was stopped by addition of DMEM (Invitrogen) supplemented with 10% BCS (Thermo Scientific), 25.6 mM glucose, 17.9 mM NaHCO3, 2 mM HEPES, and 100 U/ml penicillin plus 100 ␮g/ml streptomycin (Lonza; referred to as “culture medium”) and transferred to ice. The cell suspension was filtered through a 40-␮m cell strainer (BD Falcon, San Jose, CA), and the low-speed fraction (hepatocyte-enriched fraction) was precipitated by centrifugation at 50 g for 10 min at room temperature. Cells were resuspended in culture medium and plated on glass coverslips coated with 0.01% collagen by weight in 1⫻ PBS in six-well plates (1–3 ⫻ 106 cells/well). Cells were incubated at 37°C and 5% CO2 in culture medium; medium was replaced after 4 h and again after 2 days. Cells were used for RT-PCR, electrophysiology, or live cell immunofluorescence experiments. Reverse transcription-polymerase chain reaction. For reverse transcription-polymerase chain reaction (RT-PCR) from bone marrow neutrophils, total RNA was purified from freshly isolated neutrophils (5 ⫻ 106 cells) using the RNeasy Plus Mini kit (Qiagen, Valencia, CA), following the manufacturer’s instructions. This kit removes

C265

genomic DNA with a spin column, which obviates the need for DNAse digestion. Purified RNA was quantified with a NanoDrop ND-1000 spectrophotometer (Thermo Scientific). One microgram of RNA was reverse transcribed in a 20-␮l reaction using random hexamers (Roche, Indianapolis, IN) and SuperScript III Reverse Transcriptase (RT) (Invitrogen), following the manufacturer’s instructions (5 min at 25°C, 60 min at 50°C, 15 min at 70°C), in a PTC-100 thermal cycler (MJ Research, Waltham, MA). As a positive control, total RNA was isolated from mouse hippocampus, which is known to express Kir2.1, Kir2.2, Kir2.3, and Kir2.4 (60). In RT-negative controls, RT was replaced with nuclease-free water. Two microliters of the RT reaction were used for 30 cycles of PCR [5 s at 98°C, 30 s at the annealing temperature of the primer pair (58 – 62°C), and 30 s at 72°C] in a 50-␮l reaction with Phusion HF PCR master mix with High-Fidelity buffer (New England Biolabs, Ipswich, MA) and gene-specific primers, as follows: mouse Kir2.1 (GenBank Accession No. NM_008425) forward 5=-CTACAGCATCGTCTCTTCG-3=, reverse 5=-TATCAACCAAAACACACAGC-3= (amplicon ⫽ 295 bp); mouse Kir2.2 (NM_010603.6) forward 5=-TTGCCTCCTGGTTGTTGT-3=, reverse 5=-CCTCCACGATGTGACTCTTAC-3= (amplicon ⫽ 402 bp) (22); mouse Kir2.3 (NM_008427.4) forward 5=-CCTCTTCTCAGTGGAGACC-3=, reverse 5=-TCATGTAGGGTTTGATGAGC-3= (amplicon ⫽ 311 bp) or forward 5=-CCAGTGTAACGTCTACTTCG-3=, reverse 5=-TCCACTGAGAAGAGGAAGG-3= (amplicon ⫽ 309 bp); mouse Kir2.4 (NM_145963.2) forward 5=-CCCAAGAAACGCAACGAG-3=, reverse 5=-CGATGGAAGTGGCGATAGT-3= (amplicon ⫽ 482 bp) (22). As positive control, ␤-actin expression was assessed with the QuantumRNA ␤-actin Internal Standards kit (Ambion, Waltham, MA) at a primer:competimer ratio of 2:8, which corresponds to moderately expressed transcripts (amplicon ⫽ 294 bp). For RT-PCR from liver-derived neutrophils, neutrophils were aspirated from mouse liver preparations after 3–5 days in culture by placing a glass coverslip into a plastic dish with 2 ml DFE on an inverted microscope (Zeiss, Jena, Germany). Large-diameter-tip electrodes were made from borosilicate glass capillaries (Warner Instruments, Hamden, CT) using a Flaming-Brown P-97 micropipette puller (Sutter Instrument, Novato, CA) and were coated with FCS (Invitrogen). Electrodes were loaded onto a CV 203BU headstage (Molecular Devices, Sunnyvale, CA) mounted on a MP-285 micromanipulator (Sutter Instrument), and neutrophils were aspirated singly into the electrode using gentle mouth suction under microscopic observation. Total RNA was immediately purified from aspirated liver neutrophils (⬃1 ⫻ 102 cells) using the RNeasy Plus Mini kit (Qiagen). Five microliters of purified RNA (from an eluate of 30 ␮l) were reverse transcribed and amplified by PCR in the same 25-␮l reaction using gene-specific primers (see above) and the OneStep RT-PCR kit (Qiagen), following the manufacturer’s instructions [30 min at 50°C, 15 min at 95°C, and 40 cycles of PCR amplification each consisting of 30 s at 94°C, 30 s at the annealing temperature of the primer pair (57–59°C), and 1 min at 72°C], in a PTC-100 thermal cycler (MJ Research). PCR products were resolved by 1.5% agarose gel electrophoresis, visualized with GelRed (Biotium, Hayward, CA), sized with 1 kb Plus DNA ladder (Invitrogen), and imaged in an enclosed ultraviolet hood with Quantity One 4.6.1 software (both from Bio-Rad, Hercules, CA). Electrophysiological methods. Electrophysiological recordings were performed at room temperature in a diamond-shaped solution chamber (150-␮l vol) mounted on the headstage of an inverted microscope (Zeiss) under continuous perfusion (1 ml/min flow rate). On the day of experiments, glass coverslips were cracked with a diamond-tip pen and shards were placed in the recording chamber. Bone marrow neutrophils were used 2–30 h after plating, and liverderived neutrophils were used 2–5 days after plating. No timedependent differences were observed in the electrophysiological properties of either cell type.

AJP-Cell Physiol • doi:10.1152/ajpcell.00176.2014 • www.ajpcell.org

C266

KIR2.1 CHANNELS IN MOUSE NEUTROPHILS

The bath solution (external) contained the following in mM: 135 NaCl, 10 HEPES, 10 glucose, 5 KCl, 2 CaCl2, and 1 MgCl2; pH 7.4 with 1 M NaOH (⬃4.5 mM Na⫹ added); 290 mosM. For experiments with external pH 8.2, the final pH was titrated to 8.2 using 1 M NaOH (⬃8.4 mM Na⫹ added). For experiments with external pH 6.6, HEPES was replaced by BES, and the final pH was titrated to 6.6 with 1 M NaOH (⬃2.4 mM Na⫹ added). For experiments with extracellular K⫹ concentration ([K⫹]o) ⫽ 0 and 2 mM, KCl in the bath solution was replaced equimolarly with NaCl. For experiments with [K⫹]o ⫽ 12, 27, 60, and 140 mM, NaCl was replaced equimolarly with KCl. BaCl2, CsCl, and ZnCl2 were prepared as 1-M stock solutions in water. ML133 was prepared as a 20-mM stock solution in DMSO. For perforated patch-clamp recordings, the pipette solution (internal) contained the following in mM: 130 K-gluconate, 10 NaCl, 10 HEPES, 1 MgCl2, and 0.1 EGTA; pH 7.4 with 1 M KOH (⬃3.2 mM K⫹ added); 270 mosM. Amphotericin B was prepared as a 20 mg/ml stock solution in DMSO and added to the pipette solution on the day of the experiment to achieve 200 ␮g/ml amphotericin B. For conventional whole cell recordings, 4 mM MgATP and 0.3 mM NaGTP were added to the pipette solution. For experiments with internal pH 6.6, HEPES was replaced by BES and the final pH was titrated to 6.6 with 1 M KOH (⬃0.6 mM K⫹ added). For singlechannel recordings in the cell-attached configuration, the pipette solution contained the following in mM: 140 KCl, 10 HEPES, 2 CaCl2, and 1 MgCl2; pH 7.4 with 1 M KOH (⬃3.6 mM K⫹ added); 285 mosM. Recording electrodes were made from thin-walled borosilicate glass capillaries (Warner Instruments) using a Flaming-Brown P-97 micropipette puller (Sutter Instrument), and their resistance was 2–3 M⍀ when filled with pipette solution. The perforated-patch configuration was obtained within 5 min of G⍀ seal formation. Capacitive currents were elicited by 5-mV pulses from a holding potential of 0 mV and were compensated using the “whole cell parameters” functions (whole cell capacitance and series resistance) of the amplifier. The readout of the whole cell capacitance knob was used to calculate current density. The average whole cell capacitance (Cm) was 3.1 ⫾ 0.2 pF (n ⫽ 59 cells). Macroscopic currents were acquired in the voltage-clamp mode using a CV 203BU headstage and Axopatch 200B amplifier, filtered at 1 kHz, and digitized at 5 kHz using a DigiData 1200 Series Interface board (all from Molecular Devices). Single-channel recordings were filtered at 1 kHz and digitized at 1–5 kHz. Membrane potentials were corrected for liquid junction potentials. The holding potential was ⫺45 mV. Data analysis. Offline data analysis was performed using the pClamp10.3.1.4 software suite (Molecular Devices) and Excel 2010 (Microsoft, Redmond, WA). Steady-state currents were obtained from voltage-step protocols by calculating mean current within a 10-ms interval at the end of the step. Current density (Izeroed/Cm, in pA/pF) was calculated at Vm ⫽ ⫺115 mV by subtracting current in [K⫹]o ⫽ 0 mM from current in [K⫹]o ⫽ 5 mM (control bath solution) and dividing by whole cell capacitance (Cm). Reversal potential (Erev) was determined from continuous 1-s voltage ramps from ⫺115 to ⫹25 mV by fitting binomial functions to the current in the vicinity of Erev and calculating the zero-intercept voltage with the solver function of Excel. To determine the relationship between Erev and [K⫹]o, data were fitted with a Nernst equation: Erev ⫽ 2.303

RT F

log

关 K ⫹兴 o 关 K ⫹兴 i

where Erev is the reversal potential, 2.303RT/F is the Nernstian slope (in mV/decade), [K⫹]o is the bath K⫹ concentration, and [K⫹]i is the pipette K⫹ concentration. The solver function of Excel was used to obtain the value for the Nernstian slope via least-squares fit. To determine dose-responses for Kir blockers (Ba2⫹, Cs⫹, and ML133), data were fitted with a Hill equation:

Irel ⫽ 1⫹

冉关

1 blocker兴 K1/2

冊

H

where Irel is the current relative to that in the absence of blocker, K1/2 is the half-maximal inhibitory concentration, and H is the Hill coefficient. The solver function of Excel was used to obtain values for H and K1/2 via least-squares fit. Single-channel conductance was obtained from cell-attached recordings containing up to 4 channels by manually determining singlechannel current amplitude (Isc) at pipette voltages (Vp) from ⫹75 to ⫹155 mV and fitting the data with a linear fit (where the slope is the single-channel conductance) in Excel. Data are shown as means ⫾ SE. Statistical significance was assessed by Student’s t-test, with P ⬍ 0.05 considered statistically significant. Figures were generated using Excel and PowerPoint 2010 (Microsoft). Live cell immunofluorescence. Liver preparations plated on glass coverslips and cultured for 2–5 days were incubated for 1 h at 37°C and 5% CO2 in 24-well plates containing 300 ␮l culture medium plus 1 ␮l of each antibody (1:300 dilution) and 5 ␮g/ml DAPI. Antibodies used were CD11b-PE and CD11b-FITC (BioLegend, San Diego, CA) and Ly-6G(Gr1)-FITC and Ly-6G(Gr1)-PE (eBioscience, San Diego, CA). Cells were washed twice with 1⫻ PBS and placed in a recording chamber filled with control bath solution at room temperature, mounted on an upright Olympus BX51WI microscope (Olympus, Center Valley, PA) with a ⫻40 dipping objective. The fluorescence microscopy setup is essentially as described previously (48). In brief, fluorophores were excited using a Xenon lamp and monochromator with a 12.5-nm Polychrome IV band-pass (TILL Photonics, Hillsboro, OR) using the following excitation wavelengths (in nm): 494 for FITC, 564 for PE, and 358 for DAPI. Emitted light was separated with a triple-band dichroic (69002; Chroma, Bellows Falls, VT) and collected with a CCD camera (TILL Photonics, IMAGO-QE) using a dual-view image splitter (Optical Insights, Tucson, AZ) with 529/24 and 629/53 nm band-pass filters (Semrock, Rochester, NY) for PE and FITC. For DAPI, a DCLP410 ⫹ LP400 filter was used (Semrock). Images were acquired with TILLVision v4.0.1 (TILL Photonics) at 200-ms exposure with a 50-ms live cycle and no binning. Figures were generated with Image J 1.37a and Powerpoint 2010. RESULTS

Mouse bone marrow neutrophils express mRNA for the Kir2 subunit Kir2.1. Mouse neutrophils isolated from bone marrow are functionally mature and exhibit greater in vitro proliferative capacity than peripheral blood neutrophils (3). We thus isolated mouse neutrophils from bone marrow and examined whether they express inward rectifier potassium channels of the Kir2 subfamily. End-point RT-PCR using total RNA extracted from mouse bone marrow neutrophils showed robust expression of the Kir2 subunit Kir2.1 (Fig. 1A). No expression was detected for other Kir2 subunits (Kir2.2, Kir2.3, and Kir2.4). Total RNA from mouse hippocampus was used as positive control and showed expression of all four Kir2 subunits (Fig. 1B), as expected (60). Based on these results, we hypothesized that bone marrow neutrophils express functional homomeric Kir2.1 channels. Mouse bone marrow neutrophils exhibit K⫹-dependent currents with characteristic properties of Kir2 channel currents. To examine whether mouse neutrophils exhibit functional Kir2 channels, amphotericin B perforated patch-clamp experiments were performed using short-term cultures of bone marrow neutrophils. Neutrophils were isolated using negative selection,

AJP-Cell Physiol • doi:10.1152/ajpcell.00176.2014 • www.ajpcell.org

KIR2.1 CHANNELS IN MOUSE NEUTROPHILS

C267

Fig. 1. Mouse bone marrow neutrophils express mRNA for the Kir2 subunit Kir2.1. A and B: representative end-point RT-PCR from total RNA isolated from mouse bone marrow neutrophils (A) or hippocampus (B), with gene-specific primers as indicated, with and without reverse transcriptase (RT). Amplicon sizes (in bp): ␤-actin 294, Kir2.1 295, Kir2.2 402, Kir2.3 311, and Kir2.4 482. Samples were resolved by 1.5% agarose gel electrophoresis. Each sample lane contains 10 ␮l of a 50 ␮l-volume reaction after 30 cycles of PCR. Ladder lanes contain 1.5 ␮l ladder. Exposure time is the same for both gels.

which prevents surreptitious stimulation by antibodies bound to surface receptors during isolation. The perforated patchclamp configuration was chosen because it preserves intracellular conditions, including cytoskeletal integrity, organelle function, and cell signaling pathways (8). Bone marrow neutrophils were cultured for up to 30 h in RPMI 1640 medium supplemented with growth factors. The majority of neutrophils were rounded in shape

and nonadherent (Fig. 2A), consistent with resting (unstimulated) neutrophils. This is similar to what is observed in overnight cultures of human neutrophils (11). Cells with this appearance were selected for patch-clamp experiments, and their macroscopic current properties were examined. We found that the dominant current in resting bone marrow neutrophils is a strong inwardly rectifying current that is

Fig. 2. Mouse bone marrow neutrophils exhibit K⫹-dependent currents with characteristic properties of Kir2 channel currents. A: bright-field image of mouse bone marrow neutrophils. Scale bar ⫽ 20 ␮m. B: representative current traces obtained from mouse bone marrow neutrophil in the amphotericin B perforated patch-clamp configuration (top: [K⫹]o ⫽ 5 mM; middle: [K⫹]o ⫽ 0 mM; bottom: [K⫹]o ⫽ 5 mM in the presence of 100 ␮M Ba2⫹). The voltage-step protocol is shown above the traces. C: representative current-voltage relationships (raw currents). Data are steady-state currents from traces in B, normalized to whole cell capacitance (Cm). D: representative current-voltage relationships (external K⫹-dependent currents). Data are steady-state currents from traces in B, zeroed by subtracting current in [K⫹]o ⫽ 0 mM, and normalized to whole cell capacitance (Cm). E: Kir2 current density in mouse bone marrow neutrophils cultured in the presence and absence of growth factors [10 ng/ml granulocyte colony-stimulating factor (G-CSF) and 10 ng/ml stem cell factor (SCF)] at Vm ⫽ ⫺115 mV and [K⫹]o ⫽ 5 mM. Data are means ⫾ SE for n ⫽ 6 –13 cells. *Statistically significant (P ⫽ 4.8 ⫻ 10⫺4). AJP-Cell Physiol • doi:10.1152/ajpcell.00176.2014 • www.ajpcell.org

C268

KIR2.1 CHANNELS IN MOUSE NEUTROPHILS

dependent on external K⫹ (Fig. 2, B and C). The reversal potential (Erev) of the current is ⫺82.0 ⫾ 0.6 mV (n ⫽ 9 cells), which approximates the predicted equilibrium potential for K⫹ (EK) in the near-physiological conditions we employed (predicted EK ⫽ ⫺84.3 mV for [K⫹]o ⫽ 5 mM and [K⫹]i ⫽ 133 mM). The current is constitutively active and stable for ⬎30 min of recording time and was observed in all examined cells. The rectification properties are in keeping with Kir2 channels, with strong and “steep” inward rectification (1), i.e., no outward current is detected at membrane potentials more positive than ⫺20 mV (Fig. 2D). At Vm values more negative than approximately ⫺120 mV, currents exhibit slow time-dependent “inactivation” (Fig. 2B), which likely reflects Na⫹-dependent block exhibited by Kir2 channels at very negative Vm values (28). At Vm ⫽ ⫺115 mV and [K⫹]o ⫽ 5 mM, the current density is ⫺24.0 ⫾ 3.0 pA/pF (n ⫽ 13 cells; Fig. 2E). The current is fully and reversibly blocked by 100 ␮M Ba2⫹ in the bath solution (97.8 ⫾ 0.8% block at Vm ⫽ ⫺115 mV; n ⫽ 8 cells; Fig. 2, B and D). Blocking the current with 100 ␮M Ba2⫹ leads to depolarization of Vm, as indicated by a signifi-

cant shift in Erev from ⫺81.4 ⫾ 0.7 to ⫺65.7 ⫾ 2.4 mV (n ⫽ 6 cells; P ⫽ 4.9 ⫻ 10⫺4). These electrophysiological properties are consistent with Kir2 channels. Functional Kir2 expression has been documented in human hematopoietic progenitor cells and has been proposed to play a role in the regulation of proliferation and generation of myeloid progeny in a growth factor-dependent manner (46). It is thus possible that Kir2 plays a role in the proliferation of mouse neutrophils. We tested whether the presence of growth factors (G-CSF and SCF) in the culture medium has an effect on Kir2 current density. G-CSF is the key growth factor that stimulates neutrophil proliferation and development (2). SCF is a monocytic/granulocytic growth factor that is not lineage specific but enhances the effect of other growth factors including G-CSF (53). Indeed, current density is significantly decreased when cells are cultured in the absence of growth factors (⫺10.1 ⫾ 1.6 pA/pF at Vm ⫽ ⫺115 mV and [K⫹]o ⫽ 5 mM; n ⫽ 6 cells; P ⫽ 4.8 ⫻ 10⫺4; Fig. 2E). This may indicate that regulation of Kir2 channel activity is one of the mechanisms by which growth factors affect neutrophil proliferation and function.

Fig. 3. Proliferating neutrophils in vitro can be obtained from preparations of mouse liver. A: neutrophils in cultured low-speed fraction (hepatocyte-enriched fraction) from juvenile mouse liver after 4 days in culture (left: bright field; right: DAPI fluorescence; 2 neutrophils are magnified for clarity). Note segmented nuclei. Scale bar ⫽ 20 ␮m. B: representative live cell immunofluorescence images of liver-derived neutrophils after 5 days in culture, showing positive staining for CD11b-FITC (green) and Ly-6G(Gr1)-PE (red). Scale bar ⫽ 20 ␮m. AJP-Cell Physiol • doi:10.1152/ajpcell.00176.2014 • www.ajpcell.org

KIR2.1 CHANNELS IN MOUSE NEUTROPHILS

C269

Fig. 4. Mouse liver-derived neutrophils express mRNA for Kir2.1 and exhibit K⫹-dependent currents with characteristic properties of Kir2 channel currents. A: representative end-point RT-PCR from total RNA isolated from liver-derived neutrophils, with gene-specific primers as indicated. Neutrophils were isolated by aspiration of single cells under microscopic observation from liver preparations after 3–5 days in culture. Amplicon sizes (in bp): ␤-actin 294, Kir2.1 295, Kir2.2 402, Kir2.3 309, and Kir2.4 482. Samples were resolved by 1.5% agarose gel electrophoresis. Each sample lane contains 10 ␮l of a 25 ␮l-volume reaction after 40 cycles of PCR. Ladder lane contains 1.5 ␮l ladder. B: representative current traces obtained from mouse liver-derived neutrophil in the amphotericin B perforated patch-clamp configuration (top: [K⫹]o ⫽ 5 mM; middle: [K⫹]o ⫽ 0 mM; bottom: [K⫹]o ⫽ 5 mM in the presence of 100 ␮M Ba2⫹). Currents were elicited by the same voltage-step protocol shown in Fig. 2B. C: representative current-voltage relationships. Data are steady-state currents from traces in B, zeroed by subtracting current in [K⫹]o ⫽ 0 mM, and normalized to whole cell capacitance (Cm).

Fig. 5. Kir2 currents in liver-derived neutrophils are dependent on external K⫹. A: representative current traces obtained from mouse liver-derived neutrophil in the amphotericin B perforated patch-clamp configuration at varying [K⫹]o, as indicated (mM). Currents were elicited by the same voltage-step protocol shown in Fig. 2B. B: representative current-voltage relationships at varying [K⫹]o, as indicated (mM). Data are steady-state currents from traces in A, zeroed by subtracting current in [K⫹]o ⫽ 0 mM, and normalized to whole cell capacitance (Cm). C: representative continuous current-voltage relationships showing dependence of reversal potential (Erev) on [K⫹]o, as indicated (mM). Currents were elicited by continuous 1-s voltage ramps from ⫺115 to ⫹25 mV. Traces are truncated for clarity. D: Nernst equation describing relationship between Erev and [K⫹]o. Data points indicate means ⫾ SE for n ⫽ 4 cells (error bars are not visible). Solid line is the least-squares fit to the mean data (Nernstian slope ⫽ ⫺57.4 mV/decade). AJP-Cell Physiol • doi:10.1152/ajpcell.00176.2014 • www.ajpcell.org

C270

KIR2.1 CHANNELS IN MOUSE NEUTROPHILS

Proliferating neutrophils in vitro can be obtained from preparations of mouse liver. We were interested in examining Kir2.1 channel activity in proliferating neutrophils. Although greater than that of peripheral blood neutrophils, the in vitro proliferative capacity of bone marrow neutrophils is still limited (3). In mice, the liver is the main site of hematopoiesis during fetal development, and hematopoiesis in the liver persists after birth until ⬃2 wk of age (21, 51). Thus juvenile mouse liver could potentially be a source of developing neutrophils. Indeed, we found that a preparation of proliferating neutrophils can be obtained from the liver of 12- to 16-day-old mice. We isolated liver cells by collagenase digestion and cultured the low-speed fraction, which is enriched for hepatocytes but may contain small proportions of nonparenchymal cells such as endothelial cells or resident liver macrophages (Kupffer cells) (31). Four hours after plating, the preparation consists almost entirely of large adherent epithelial cells with the typical appearance of cultured hepatocytes. After 1–2 days in culture, small clusters of rounded nonadherent cells are easily identified, and these continue to grow in number for up to 5 days in culture. Live cell staining with DAPI revealed that cells in these clusters have segmented nuclei (Fig. 3A) characteristic of mature neutrophils (59). To confirm that these cells are neutrophils, we performed live cell immunofluorescence (IF) by incubating the preparation with fluorescently labeled antibodies to neutrophil surface markers. As expected, cells are positive for the monocyte/ granulocyte marker CD11b and the mature neutrophil marker

Ly-6G (Gr1; Fig. 3B) (53, 55). Staining for Gr1 is variable in intensity, possibly reflecting some variability in the degree of maturation. In conjunction with the nuclear morphology, the IF results confirm that these cells are neutrophils. Mouse liver-derived neutrophils express mRNA for Kir2.1 and exhibit K⫹-dependent currents with characteristic properties of Kir2.1 channel currents. As with bone marrow neutrophils, end-point RT-PCR using total RNA extracted from mouse liver-derived neutrophils showed robust expression of Kir2.1 but not of the other Kir2 subunits (Kir2.2, Kir2.3, and Kir2.4; Fig. 4A). Based on this result, we hypothesized that liver-derived neutrophils express functional homomeric Kir2.1 channels and examined their electrophysiological properties in the amphotericin B perforated patch-clamp configuration. Like bone marrow neutrophils, liver-derived neutrophils express a constitutively active, external K⫹-dependent, strong inwardly rectifying current, which constitutes the dominant current under physiological conditions (Fig. 4, B and C). At Vm ⫽ ⫺115 mV and [K⫹]o ⫽ 5 mM, the current density is ⫺18.5 ⫾ 0.4 pA/pF (n ⫽ 37 cells), which is not significantly different from the current density in bone marrow neutrophils (P ⫽ 0.14). The rectification properties are also in keeping with Kir2 channels, with no outward current detected at Vm values more positive than ⫺20 mV (Fig. 4C). The current is fully and reversibly blocked by 100 ␮M Ba2⫹ in the bath solution (98.4 ⫾ 0.8% block at Vm ⫽ ⫺115 mV; n ⫽ 7 cells; Fig. 4, B and C). These electrophysiological properties are consistent with Kir2 channels.

Fig. 6. Kir2 currents in liver-derived neutrophils are blocked by Ba2⫹, Cs⫹, and ML133. A–C: representative current-voltage relationships with [K⫹]o ⫽ 60 mM (A and B) or 140 mM (C) and varying Ba2⫹ (A), Cs⫹ (B), or ML133 (C), as indicated, for liver-derived neutrophils in the amphotericin B perforated patch-clamp configuration. Data are steady-state currents elicited by 20- or 10-mV voltage steps of 0.5-s duration from a holding potential of ⫺45 mV (A and B) or 0 mV (C). D–F: Hill plots describing block by Ba2⫹ (D), Cs⫹ (E), or ML133 (F), with [K⫹]o ⫽ 60 mM and Vm ⫽ ⫺90 mV (D and E) or [K⫹]o ⫽ 140 mM and Vm ⫽ ⫺100 mV (F). Currents are normalized to current recorded in the absence of blocker and are expressed as relative current (Irel). Data points indicate means ⫾ SE for n ⫽ 6 cells (Ba2⫹), 4 cells (Cs⫹), or 3 cells (ML133). Solid lines are least-squares fit to the mean data (Ba2⫹: K1/2 ⫽ 2.66 ␮M and H ⫽ 1.25; Cs⫹: K1/2 ⫽ 156 ␮M and H ⫽ 1.07; ML133: K1/2 ⫽ 1.98 ␮M and H ⫽ 1.82). AJP-Cell Physiol • doi:10.1152/ajpcell.00176.2014 • www.ajpcell.org

KIR2.1 CHANNELS IN MOUSE NEUTROPHILS

Dependence on [K⫹]o was examined by varying the K⫹ concentration in the bath solution via equimolar replacement of NaCl with KCl. As expected for a K⫹-selective channel of the Kir2 family, with increasing [K⫹]o the magnitude of the inward current increases (Fig. 5, A and B) and the reversal potential (Erev) becomes more positive (Fig. 5, C and D). Fitting the relationship between Erev and [K⫹]o with the Nernst equation yields a Nernstian slope close to the predicted value (⫺57.4 ⫾ 0.2 mV/decade vs. the predicted ⫺59.1 mV/decade; n ⫽ 4 cells; Fig. 5D). To confirm that the molecular identity of these channels is homomeric Kir2.1, as suggested by RT-PCR results, we carried out a more detailed analysis of current properties. We examined block by known blockers of Kir2 channels (Ba2⫹ and Cs⫹) and the Kir2-selective small molecule inhibitor ML133 (Fig. 6, A–C). At [K⫹]o ⫽ 60 mM and Vm ⫽ ⫺90 mV, half-maximal inhibitory concentrations (K1/2) are 2.68 ⫾ 0.20 ␮M for Ba2⫹ (H ⫽ 1.27 ⫾ 0.08; n ⫽ 6 cells) and 156 ⫾ 11 ␮M for Cs⫹ (H ⫽ 1.08 ⫾ 0.06; n ⫽ 4 cells; Fig. 6, D and E). These results are in good agreement with previously reported values for homomeric Kir2.1 channels (30, 41). At [K⫹]o ⫽ 140 mM, Vm ⫽ ⫺100 mV, and pH 7.4, the K1/2 for ML133 is 2.14 ⫾ 0.68 (H ⫽ 2.06 ⫾ 0.15; n ⫽ 3 cells; Fig. 6F). This result is also in good agreement with the previously reported value for homomeric Kir2.1 channels (54). Single-channel conductance was determined from singlechannel recordings in the cell-attached configuration with 140 mM K⫹ in the pipette. Channels were detected as negativecurrent deflections (inward current) at negative voltages, but no channel activity was seen at positive voltages, as expected for Kir2 channels exhibiting strong inward rectification (Fig. 7A). A linear fit of the single-channel current-voltage relationship yields a single-channel conductance of 22.3 ⫾ 0.9 pS (n ⫽ 8 cells; Fig. 7B). This value is in agreement with previously reported values for homomeric Kir2.1 channels (28, 30, 40, 47). Modulation by external pH was examined by measuring current density at external pH 6.6 and 8.2. Kir2 channels containing the pH-sensitive subunits Kir2.3 and Kir2.4 exhibit significant modulation under these conditions (inhibition by pH 6.6 and stimulation by pH 8.2) (7, 25, 38, 58, 64). Neither pH 6.6 nor pH 8.2 had a significant effect on current density compared with pH 7.4 (Fig. 8, A and B). This is consistent with

C271

homomeric Kir2.1 channels, which are not modulated by external pH. Kir2 currents in mouse neutrophils are unaffected by internal pH. We were interested in examining modulation by internal pH, given that neutrophils undergo intracellular acidification (to approximately pH 6.6) during the respiratory burst that initiates phagocytosis (10, 36). In these conditions, Kir2 channels containing pH-sensitive subunits would be inhibited, while homomeric Kir2.1 channels would not be affected (42, 64). However, manipulation of intracellular pH in the perforated patch-clamp configuration is challenging. We opted to perform experiments in the conventional whole cell patchclamp configuration, where intracellular pH is determined by the pipette solution. Although this approach disrupts intracellular conditions and subjects Kir2 currents to rundown, it has been successfully applied to the study of Kir2 channels in immune system cells (13, 20, 43, 49). Measurements obtained shortly after breaking in (⬍5 min) provide a reasonable assessment of current density as a function of intracellular pH. As expected from perforated patch-clamp experiments, the Kir2 channel current comprises the dominant current in liverderived neutrophils in the whole cell configuration with internal pH 7.4 (Fig. 8C). With internal pH 6.6, there is no significant difference in Kir2 current density compared with pH 7.4 (⫺17.3 ⫾ 2.1 pA/pF vs. ⫺16.9 ⫾ 1.7 pA/pF, respectively, at Vm ⫽ ⫺115 mV and [K⫹]o ⫽ 5 mM; n ⫽ 3–9 cells; P ⫽ 0.87; Fig. 8, C and D). Neither whole cell current density is significantly different from current density in the perforated patch-clamp configuration (P ⫽ 0.55 and 0.69, respectively). This indicates that Kir2 currents are not affected by intracellular pH, as expected for homomeric Kir2.1 channels. Additionally, with internal pH 6.6 cells exhibit large, slowly activating outward currents at positive membrane voltages, and these currents are blocked by external 100 ␮M Zn2⫹ (Fig. 8C). A detailed examination of these currents is beyond the scope of this study, but their properties are in keeping with voltagegated proton channels (Hv1), which have been extensively characterized in neutrophils (11, 12). Hv1 channels are activated by intracellular acidification and play a key role in charge and pH compensation during the respiratory burst in phagocytes (9, 10). Their detection is expected and distinct under these conditions.

Fig. 7. Single channel recordings in liver-derived neutrophils reveal strong inwardly rectifying channels with single-channel conductance in agreement with reported values for Kir2.1. A: representative single-channel recording from mouse liver-derived neutrophil in the cell-attached configuration, at indicated voltages, with pipette [K⫹] ⫽ 140 mM and bath [K⫹] ⫽ 5 mM (VP ⫽ pipette voltage). Dotted lines indicate zero current. Single-channel openings are observed as downward current deflections. B: relationship between single-channel current amplitude and voltage. Data points indicate means ⫾ SE for n ⫽ 8 cells. Solid line is a linear fit to the mean data (slope ⫽ 22.7 pS). AJP-Cell Physiol • doi:10.1152/ajpcell.00176.2014 • www.ajpcell.org

C272

KIR2.1 CHANNELS IN MOUSE NEUTROPHILS

Fig. 8. Kir2 currents in liver-derived neutrophils are unaffected by external or internal pH. A: representative current traces from mouse liver-derived neutrophil in the amphotericin B perforated patch-clamp configuration, at varying external pH. Currents were elicited by the same voltage step protocol shown in Fig. 2B. B: Kir2 current density at varying external pH (from steady-state currents at Vm ⫽ ⫺115 mV and [K⫹]o ⫽ 5 mM). Data are means ⫾ SE for n ⫽ 4 –5 cells; ns: not statistically significant compared with pH 7.4 (P ⫽ 0.95 for pH 6.6 and P ⫽ 0.80 for pH 8.2). C: representative current traces from mouse liver-derived neutrophils in the conventional whole cell patch-clamp configuration, with internal pH 6.6 (left) or 7.4 (right). Cells were exposed to control bath solution (top), control plus 100 ␮M Ba2⫹ (middle), or control plus 100 ␮M Zn2⫹ (bottom). Currents are shown normalized to Cm to allow comparison between cells. The voltage-step protocol is shown below the pH 7.4 traces. Note that there is a prepulse to ⫺75 mV and voltage steps are longer in duration compared with previous protocols, to better demonstrate Hv1 currents. D: Kir2 current density at varying internal pH (from steady-state currents at Vm ⫽ ⫺115 mV and [K⫹]o ⫽ 5 mM). Data are means ⫾ SE for n ⫽ 3–9 cells; ns: not statistically significant compared with pH 7.4 (P ⫽ 0.87).

DISCUSSION

Mouse neutrophils express functional Kir2.1 channels. In the present study we demonstrate that mouse neutrophils express functional Kir2.1 channels and that these constitute the dominant conductance in resting (unstimulated/nonactivated) conditions. We conclude that these channels consist of homomeric Kir2.1 on the basis of RT-PCR results (showing detection of mRNA for Kir2.1 but no other Kir2 subunit), electrophysiological characterization of macroscopic currents (which exhibit strong and “steep” inward rectification, block by Ba2⫹ in the low-␮M range, inhibition by ML133, and lack of modulation by internal or external pH), and single-channel currents (yielding a single-channel conductance of 22.3 pS). Inhibition of these currents by the Kir2-selective inhibitor ML133 with K1/2 values of ⬃2 ␮M confirms that they are Kir2 channel currents. ML133 binds to residues on the inner face of the pore-lining M2 helix and presumably blocks the K⫹ permeation pathway at that level (54). In the same conditions we employed ([K⫹]o ⫽ 140 mM, Vm ⫽ ⫺100 mV, and pH 7.4), ML133 inhibits Kir2 channels with K1/2 values in the range of ⬃2– 4 ␮M, while other Kir subfamilies (Kir1, Kir4, Kir6, and Kir7) exhibit K1/2 values of ⬃8

␮M or higher (54). ML133 does not precisely discriminate between Kir2 isotypes, but the K1/2 value we obtained (2.1 ␮M) is close to the reported value for Kir2.1 (1.9 ␮M), which is lower than the values for other Kir2 isotypes (2.9 ␮M for Kir2.2 and 4.0 ␮M for Kir2.3) (54). Although specific blockers of Kir2 channel isotypes are not available, Kir2 subunit composition can be discerned on the basis of differential sensitivity to Kir channel blockers such as Ba2⫹. A caveat is that strength of block is dependent on [K⫹]o and Vm, such that direct comparison across studies is not always feasible. Our experimental conditions are very similar to two studies where Ba2⫹ block of multiple Kir2 subunits was examined at [K⫹]o ⫽ 60 mM and Vm ⫽ ⫺100 mV (30, 41). The K1/2 value we obtained for Kir2 channels in neutrophils (2.68 ␮M) is in good agreement with the reported value of 3.2 ␮M for Kir2.1. K1/2 values are significantly lower for Kir2.2 (0.5 ␮M) and higher for both Kir2.3 and Kir2.4 (10.3 and 235 ␮M, respectively) (30, 41). Coexpression of Kir2.1 with Kir2.2 or Kir2.3 significantly shifts the K1/2 to 0.6 or 6.3 ␮M, respectively (41). Our Ba2⫹ block data argue against a contribution of Kir2.2, Kir2.3, or Kir2.4 to the neutrophil Kir2 channel.

AJP-Cell Physiol • doi:10.1152/ajpcell.00176.2014 • www.ajpcell.org

KIR2.1 CHANNELS IN MOUSE NEUTROPHILS

The pH-sensitive Kir2 subunits (Kir2.3 and Kir2.4) are inhibited by acidification and stimulated by alkalinization of either the intracellular or extracellular milieu, whereas Kir2.1 and Kir2.2 are not (7, 25, 38, 42, 58, 64). Macroscopic Kir2 currents exhibit significant pH modulation when as few as 20% of the subunits in the population are pH sensitive (58). The lack of pH sensitivity of neutrophil Kir2 channels argues against a contribution of these subunits. Finally, single-channel recordings in the cell-attached configuration with high K⫹ in the pipette reveal strong inwardly rectifying channels with a single-channel conductance of 22.3 pS. This value is in good agreement with reported values of 22–31 pS for homomeric Kir2.1 channels (28, 30, 40, 47). Kir2.2 exhibits larger single-channel conductances (34 – 42 pS) while Kir2.3 and Kir2.4 exhibit smaller conductances (13–14 pS and 15 pS, respectively) (30, 37, 40, 47, 50). Thus the single-channel conductance data, in conjunction with blocker data, RT-PCR, and pH modulation experiments, confirm that the molecular identity of Kir2 channels in mouse neutrophils is homomeric Kir2.1. In human eosinophils, mRNA for Kir2.1 was detected by RT-PCR (49), but the expression of other Kir2 subunits was not assessed. Kir2 channels in eosinophils are inhibited by intracellular acidification (49), which may indicate a contribution from pH-sensitive subunits. By contrast, our data show that Kir2 currents in neutrophils are unaffected by intracellular acidification, as expected for homomeric Kir2.1 channels. Kir2 channels may contribute to regulation of membrane potential and Ca2⫹ entry in neutrophils. The physiological role of Kir2 channels in mammalian neutrophils remains to be elucidated, but a likely possibility is that they contribute to regulation of the resting membrane potential (Vm) by stabilizing it close to EK. This would be analogous to their role in excitable cells and has been previously proposed in phagocytes (26, 35, 49). While excitable cells express voltage-gated Ca2⫹ channels, Ca2⫹ influx into nonexcitable cells generally occurs via Ca2⫹ release-activated Ca2⫹ (CRAC) channels, also known as store-operated Ca2⫹ entry (SOCE). In cultured human macrophages, cells that express large Kir2 channel currents exhibit a more negative Vm (20); similarly, we observed a shift in Erev towards more positive values when Kir2 channels were blocked by Ba2⫹. By maintaining a relatively hyperpolarized Vm, Kir2 channels may increase the electrochemical driving force for Ca2⫹ influx into neutrophils via SOCE. This role for Kir2 channels has been demonstrated in another nonexcitable cell type, the endothelial cell, where Kir2 channels are important determinants of the resting membrane potential and the driving force for Ca2⫹ entry (39). SOCE plays an important role in neutrophil recruitment at sites of inflammation, as it contributes to the increase in intracellular Ca2⫹ needed for neutrophil adhesion and migration through the vascular endothelium (16, 44). Kir2 channels could potentially contribute to the regulation of these processes by facilitating SOCE. SOCE is also important for activation of NADPH oxidase during the respiratory burst in phagocytes (4, 63). During the burst, electron flux into the phagosome mediated by NADPH oxidase leads to marked depolarization of Vm (9). This depolarization is expected to decrease the driving force for SOCE. Since homomeric Kir2.1 channels would not be inhibited by intracellular acidification during the respiratory burst, they could help maintain the driving force for Ca2⫹ entry. However,

C273

Ba2⫹ does not significantly impair the respiratory burst in human eosinophils (49) or mouse macrophage-derived cells (34), arguing against a significant role of Kir2 channels in this process. In fact, activation of macrophages by exposure to LPS or TNF-␣ leads to downregulation of Kir2 currents (34, 52). Kir2 channels in neutrophils may thus be selectively expressed in the resting (unstimulated) state but not in the activated state. Electrophysiological experiments with activated mouse neutrophils may be informative in this regard. Modulation of Kir2 channel function by growth factors. Kir2 channel expression is modulated by growth factors in mammalian phagocytes and their precursors (46, 52), and knockdown or block of Kir2.1 inhibits generation of myeloid progeny from human hematopoietic progenitor cells (46). Our findings that growth factors increase Kir2.1 current density in mouse neutrophils and that proliferating neutrophils exhibit robust Kir2.1 channel activity suggest that Kir2 channels may play a role in growth factor-dependent proliferation and development of neutrophils. This may occur via hyperpolarization of Vm and facilitation of SOCE, as mentioned above. In human myoblasts, upregulation of Kir2.1 channels leads to hyperpolarization of Vm, increased intracellular Ca2⫹ via SOCE, and activation of transcription factors necessary for differentiation into myocytes (24, 29). Kir2-dependent hyperpolarization of Vm is important for regulation of proliferation in other cell types such as microglia (45) and cardiac myofibroblasts (5). Neutrophils can be produced in two ways, both of which are stimulated by G-CSF: development from precursor cells in the bone marrow (steady-state granulopoiesis) or expansion of the existing neutrophil population in the setting of bacterial infection (“emergency” or “stress” granulopoiesis) (2, 33). In HEK293 cells, activation of the epidermal growth factor receptor (EGFR) leads to tyrosine kinase-dependent, site-specific phosphorylation of Kir2 channels, which in turn results in increased channel activity (61, 62). Although the G-CSF receptor lacks intrinsic tyrosine kinase activity, its activation results in engagement of cytoplasmic tyrosine kinases (2). Thus a similar mechanism could underlie the increase in Kir2 current density caused by growth factors in neutrophils. Paradoxically, however, others have reported that Kir2 channel activity is decreased by EGFR-dependent tyrosine phosphorylation in COS-7 cells, Xenopus oocytes, and human myoblasts (29, 57). Thus, although there is growing evidence of direct modulation of Kir2 channel activity by growth factor-dependent tyrosine phosphorylation, the functional consequence appears to depend on cell type. The mechanistic details of such phosphorylation in neutrophils remain to be investigated. Proliferating neutrophils can be derived from juvenile mouse liver. Mammalian neutrophils exhibit limited in vitro proliferation, which hinders experimental examination of these questions. We found that culturing the hepatocyte-enriched fraction of juvenile mouse liver for 2–5 days yields proliferating neutrophils suitable for electrophysiological experiments. In mice, the liver is the primary site of blood cell production in the fetal period. Following birth, hematopoiesis shifts to the bone marrow and spleen, but extramedullary hematopoiesis persists in the liver for ⬃2 wk (21, 51). Indeed, histological sections of juvenile mouse liver (12–16 days old) show frequent clusters of developing hematopoietic cells (data not shown). Small numbers of hematopoietic precursors present in the preparation could give rise to the neutrophils we observed.

AJP-Cell Physiol • doi:10.1152/ajpcell.00176.2014 • www.ajpcell.org

C274

KIR2.1 CHANNELS IN MOUSE NEUTROPHILS

G-CSF can be secreted by many cell types, including endothelial cells and tissue macrophages (2), and this presumably sustains neutrophil proliferation in our preparation. Large numbers of neutrophils can be isolated from the liver of adult mice after stimulation with adenovirus (6), but we are unaware of expansion of mature neutrophils in the mouse liver in the absence of inflammatory stimuli. We favor that the neutrophils in our preparation are derived from precursors via steady-state granulopoiesis. The focus of this study is the electrophysiological characterization of Kir2 channels in mouse neutrophils. We designed the liver preparation with the goal of obtaining cells suitable for electrophysiology experiments, rather than rigorously quantifying proliferation, which is needed to examine whether Kir2 channels play a role in neutrophil proliferation. We are currently modifying the preparation to allow quantitation of proliferation and expand the utility of this model. Implications for expression of Kir2 channels in human neutrophils. This study constitutes the first detailed characterization of Kir2 channels in mammalian neutrophils. Currents with the properties of Kir2 channels have been demonstrated in newt neutrophils, which are considered analogous to mammalian neutrophils (26). In mouse neutrophils isolated from peripheral blood, experiments with symmetrical high-K⫹ solutions have shown inward currents compatible with Kir channels (18), but these currents were not examined in detail. Human peripheral blood neutrophils express mRNA for multiple Kir2 isoforms (Kir2.1, Kir2.2, Kir2.3, and Kir2.4) (14). However, thus far functional Kir2 channels have not been demonstrated in human neutrophils. A caveat is that many electrophysiological studies have employed experimental conditions not specifically designed to investigate Kir2 channels (for example, K⫹ was omitted from the pipette solution) (11, 12, 19), but in at least some studies, detection of Kir2 channels was sought (27). Although it is possible that Kir2 channel expression in mammalian neutrophils is species specific, differences in tissue of origin, isolation procedure, and/or experimental conditions may account for these differences. Given our interest in exploring a possible role of Kir2 channels in neutrophil proliferation, we have focused on neutrophil populations that are mature but exhibit greater proliferative capacity than peripheral blood neutrophils. In mice, mature neutrophils derived from bone marrow survive significantly longer in culture than neutrophils from peripheral blood (3). Similar differences may potentially exist in humans, and electrophysiological studies of human neutrophils have been performed on peripheral blood neutrophils (11, 12, 19, 27). It is possible that expression of Kir2 channels in mammalian neutrophils correlates with proliferative capacity and is downregulated after they are released into the peripheral circulation. Comparison of Kir2 channel expression in mouse neutrophils from peripheral blood vs. bone marrow and liver may be helpful in examining this possibility. Functional expression of Kir2 channels is well documented in primary monocytes/macrophages from both mouse (17, 43) and human (20), as well as macrophage-derived cell lines of both mouse (34, 35) and human origin (13). Given the functional similarities between macrophages and neutrophils, as well as their derivation from a common precursor, it seems plausible that they would be similar in their ion channel

expression profile. However, the human promyelocytic cell line HL-60 expresses Kir2 channels when it is differentiated into macrophages but not into granulocytes (56), possibly indicating that there are important differences in ion channel expression between the two phagocytic lineages in humans. In summary, we demonstrate here that mouse neutrophils express functional Kir2.1 channels by a combination of RT-PCR and electrophysiological characterization of macroscopic and single-channel currents. These channels constitute the dominant conductance in bone marrow neutrophils and proliferating neutrophils from juvenile liver in resting (unstimulated) conditions. Further work is needed to elucidate the physiological significance of Kir2.1 channels in mammalian neutrophils, but we speculate that they may contribute to the regulation of resting membrane potential and Ca2⫹ entry, possibly in a growth factor-dependent manner. ACKNOWLEDGMENTS We thank Mathew Tantama for assistance with cell culture and live cell IF and Juan Ramón Martínez-Francois and Andrew Lutas for assistance with electrophysiological techniques. GRANTS This study was supported by the National Cancer Institute of the National Institutes of Health under award numbers T32 CA09216 (R.M.) and K08 CA138916-02 (D.S.K.). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: R.M., D.S.K., and G.Y. conception and design of research; R.M. performed experiments; R.M. analyzed data; R.M. interpreted results of experiments; R.M. prepared figures; R.M. drafted manuscript; D.S.K. and G.Y. edited and revised manuscript; R.M., D.S.K., and G.Y. approved final version of manuscript. REFERENCES 1. Anumonwo JM, Lopatin AN. Cardiac strong inward rectifier potassium channels. J Mol Cell Cardiol 48: 45–54, 2010. 2. Basu S, Dunn A, Ward A. G-CSF: function and modes of action (Review). Int J Mol Med 10: 3–10, 2002. 3. Boxio R, Bossenmeyer-Pourie C, Steinckwich N, Dournon C, Nusse O. Mouse bone marrow contains large numbers of functionally competent neutrophils. J Leukoc Biol 75: 604 –611, 2004. 4. Brechard S, Tschirhart EJ. Regulation of superoxide production in neutrophils: role of calcium influx. J Leukoc Biol 84: 1223–1237, 2008. 5. Chilton L, Ohya S, Freed D, George E, Drobic V, Shibukawa Y, Maccannell KA, Imaizumi Y, Clark RB, Dixon IM, Giles WR. K⫹ currents regulate the resting membrane potential, proliferation, and contractile responses in ventricular fibroblasts and myofibroblasts. Am J Physiol Heart Circ Physiol 288: H2931–H2939, 2005. 6. Cotter MJ, Muruve DA. Isolation of neutrophils from mouse liver: a novel method to study effector leukocytes during inflammation. J Immunol Methods 312: 68 –78, 2006. 7. Coulter KL, Perier F, Radeke CM, Vandenberg CA. Identification and molecular localization of a pH-sensing domain for the inward rectifier potassium channel HIR. Neuron 15: 1157–1168, 1995. 8. D’Ambrosio R. Perforated patch-clamp technique. In: Patch-Clamp Analysis, edited by Walz W, Boulton A, Baker G. New York: Humana, 2002, p. 195–216. 9. DeCoursey TE. Voltage-gated proton channels find their dream job managing the respiratory burst in phagocytes. Physiology (Bethesda) 25: 27–40, 2010. 10. DeCoursey TE. Voltage-gated proton channels: molecular biology, physiology, and pathophysiology of the H(V) family. Physiol Rev 93: 599 – 652, 2013.

AJP-Cell Physiol • doi:10.1152/ajpcell.00176.2014 • www.ajpcell.org

KIR2.1 CHANNELS IN MOUSE NEUTROPHILS 11. DeCoursey TE, Cherny VV, DeCoursey AG, Xu W, Thomas LL. Interactions between NADPH oxidase-related proton and electron currents in human eosinophils. J Physiol 535: 767–781, 2001. 12. DeCoursey TE, Cherny VV, Zhou W, Thomas LL. Simultaneous activation of NADPH oxidase-related proton and electron currents in human neutrophils. Proc Natl Acad Sci USA 97: 6885–6889, 2000. 13. DeCoursey TE, Kim SY, Silver MR, Quandt FN. Ion channel expression in PMA-differentiated human THP-1 macrophages. J Membr Biol 152: 141–157, 1996. 14. Dewson G, Conley EC, Bradding P. Multigene family isoform profiling from blood cell lineages. BMC Genomics 3: 22, 2002. 15. Dhamoon AS, Pandit SV, Sarmast F, Parisian KR, Guha P, Li Y, Bagwe S, Taffet SM, Anumonwo Unique JM. Kir2.x properties determine regional and species differences in the cardiac inward rectifier K⫹ current. Circ Res 94: 1332–1339, 2004. 16. Dixit N, Simon SI. Chemokines, selectins and intracellular calcium flux: temporal and spatial cues for leukocyte arrest. Front Immunol 3: 188, 2012. 17. Essin K, Gollasch M, Rolle S, Weissgerber P, Sausbier M, Bohn E, Autenrieth IB, Ruth P, Luft FC, Nauseef WM, Kettritz R. BK channels in innate immune functions of neutrophils and macrophages. Blood 113: 1326 –1331, 2009. 18. Essin K, Salanova B, Kettritz R, Sausbier M, Luft FC, Kraus D, Bohn E, Autenrieth IB, Peschel A, Ruth P, Gollasch M. Large-conductance calcium-activated potassium channel activity is absent in human and mouse neutrophils and is not required for innate immunity. Am J Physiol Cell Physiol 293: C45–C54, 2007. 19. Femling JK, Cherny VV, Morgan D, Rada B, Davis AP, Czirjak G, Enyedi P, England SK, Moreland JG, Ligeti E, Nauseef WM, DeCoursey TE. The antibacterial activity of human neutrophils and eosinophils requires proton channels but not BK channels. J Gen Physiol 127: 659 –672, 2006. 20. Gallin EK, McKinney LC. Patch-clamp studies in human macrophages: single-channel and whole-cell characterization of two K⫹ conductances. J Membr Biol 103: 55–66, 1988. 21. Grossi CE, Velardi A, Cooper MD. Postnatal liver hemopoiesis in mice: generation of pre-B cells, granulocytes, and erythrocytes in discrete colonies. J Immunol 135: 2303–2311, 1985. 22. Han Y, Chen JD, Liu ZM, Zhou Y, Xia JH, Du XL, Jin MW. Functional ion channels in mouse cardiac c-kit⫹ cells. Am J Physiol Cell Physiol 298: C1109 –C1117, 2010. 23. Hibino H, Inanobe A, Furutani K, Murakami S, Findlay I, Kurachi Y. Inwardly rectifying potassium channels: their structure, function, and physiological roles. Physiol Rev 90: 291–366, 2010. 24. Hinard V, Belin D, Konig S, Bader CR, Bernheim L. Initiation of human myoblast differentiation via dephosphorylation of Kir2.1 K⫹ channels at tyrosine 242. Development 135: 859 –867, 2008. 25. Hughes BA, Kumar G, Yuan Y, Swaminathan A, Yan D, Sharma A, Plumley L, Yang-Feng TL, Swaroop A. Cloning and functional expression of human retinal Kir2.4, a pH-sensitive inwardly rectifying K⫹ channel. Am J Physiol Cell Physiol 279: C771–C784, 2000. 26. Kawa K. Electrophysiological properties of three types of granulocytes in circulating blood of the newt. J Physiol 415: 211–231, 1989. 27. Krause KH, Welsh MJ. Voltage-dependent and Ca2(⫹)-activated ion channels in human neutrophils. J Clin Invest 85: 491–498, 1990. 28. Kubo Y, Baldwin TJ, Jan YN, Jan LY. Primary structure and functional expression of a mouse inward rectifier potassium channel. Nature 362: 127–133, 1993. 29. Leroy MC, Perroud J, Darbellay B, Bernheim L, Konig S. Epidermal growth factor receptor down-regulation triggers human myoblast differentiation. PLoS One 8: e71770, 2013. 30. Liu GX, Derst C, Schlichthorl G, Heinen S, Seebohm G, Bruggemann A, Kummer W, Veh RW, Daut J, Preisig-Muller R. Comparison of cloned Kir2 channels with native inward rectifier K⫹ channels from guinea-pig cardiomyocytes. J Physiol 532: 115–126, 2001. 31. Liu W, Hou Y, Chen H, Wei H, Lin W, Li J, Zhang M, He F, Jiang Y. Sample preparation method for isolation of single-cell types from mouse liver for proteomic studies. Proteomics 11: 3556 –3564, 2011. 32. Lopatin AN, Makhina EN, Nichols CG. Potassium channel block by cytoplasmic polyamines as the mechanism of intrinsic rectification. Nature 372: 366 –369, 1994. 33. Mayadas TN, Cullere X, Lowell CA. The multifaceted functions of neutrophils. Annu Rev Pathol 9: 181–218, 2014.

C275

34. McKinney LC, Gallin EK. Effect of adherence, cell morphology, and lipopolysaccharide on potassium conductance and passive membrane properties of murine macrophage J774.1 cells. J Membr Biol 116: 47–56, 1990. 35. McKinney LC, Gallin EK. Inwardly rectifying whole-cell and singlechannel K currents in the murine macrophage cell line J774.1. J Membr Biol 103: 41–53, 1988. 36. Morgan D, Capasso M, Musset B, Cherny VV, Rios E, Dyer MJ, DeCoursey TE. Voltage-gated proton channels maintain pH in human neutrophils during phagocytosis. Proc Natl Acad Sci USA 106: 18022–18027, 2009. 37. Morishige K, Takahashi N, Jahangir A, Yamada M, Koyama H, Zanelli JS, Kurachi Y. Molecular cloning and functional expression of a novel brain-specific inward rectifier potassium channel. FEBS Lett 346: 251–256, 1994. 38. Munoz V, Vaidyanathan R, Tolkacheva EG, Dhamoon AS, Taffet SM, Anumonwo JM. Kir2.3 isoform confers pH sensitivity to heteromeric Kir2.1/Kir2.3 channels in HEK293 cells. Heart Rhythm 4: 487–496, 2007. 39. Nilius B, Droogmans G. Ion channels and their functional role in vascular endothelium. Physiol Rev 81: 1415–1459, 2001. 40. Panama BK, McLerie M, Lopatin AN. Functional consequences of Kir2.1/ Kir2.2 subunit heteromerization. Pflügers Arch 460: 839–849, 2010. 41. Preisig-Muller R, Schlichthorl G, Goerge T, Heinen S, Bruggemann A, Rajan S, Derst C, Veh RW, Daut J. Heteromerization of Kir2.x potassium channels contributes to the phenotype of Andersen’s syndrome. Proc Natl Acad Sci USA 99: 7774 –7779, 2002. 42. Qu Z, Zhu G, Yang Z, Cui N, Li Y, Chanchevalap S, Sulaiman S, Haynie H, Jiang C. Identification of a critical motif responsible for gating of Kir2.3 channel by intracellular protons. J Biol Chem 274: 13783– 13789, 1999. 43. Randriamampita C, Trautmann A. Ionic channels in murine macrophages. J Cell Biol 105: 761–769, 1987. 44. Schaff UY, Dixit N, Procyk E, Yamayoshi I, Tse T, Simon SI. Orai1 regulates intracellular calcium, arrest, and shape polarization during neutrophil recruitment in shear flow. Blood 115: 657–666, 2010. 45. Schlichter LC, Sakellaropoulos G, Ballyk B, Pennefather PS, Phipps DJ. Properties of K⫹ and Cl⫺ channels and their involvement in proliferation of rat microglial cells. Glia 17: 225–236, 1996. 46. Shirihai O, Merchav S, Attali B, Dagan D. K⫹ channel antisense oligodeoxynucleotides inhibit cytokine-induced expansion of human hemopoietic progenitors. Pflügers Arch 431: 632–638, 1996. 47. Takahashi N, Morishige K, Jahangir A, Yamada M, Findlay I, Koyama H, Kurachi Y. Molecular cloning and functional expression of cDNA encoding a second class of inward rectifier potassium channels in the mouse brain. J Biol Chem 269: 23274 –23279, 1994. 48. Tantama M, Martinez-Francois JR, Mongeon R, Yellen G. Imaging energy status in live cells with a fluorescent biosensor of the intracellular ATP-to-ADP ratio. Nat Commun 4: 2550, 2013. 49. Tare M, Prestwich SA, Gordienko DV, Parveen S, Carver JE, Robinson C, Bolton TB. Inwardly rectifying whole cell potassium current in human blood eosinophils. J Physiol 506: 303–318, 1998. 50. Topert C, Doring F, Wischmeyer E, Karschin C, Brockhaus J, Ballanyi K, Derst C, Karschin A. Kir2.4: a novel K⫹ inward rectifier channel associated with motoneurons of cranial nerve nuclei. J Neurosci 18: 4096 –4105, 1998. 51. Velardi A, Cooper MD. An immunofluorescence analysis of the ontogeny of myeloid, T, and B lineage cells in mouse hemopoietic tissues. J Immunol 133: 672–677, 1984. 52. Vicente R, Escalada A, Coma M, Fuster G, Sanchez-Tillo E, LopezIglesias C, Soler C, Solsona C, Celada A, Felipe A. Differential voltage-dependent K⫹ channel responses during proliferation and activation in macrophages. J Biol Chem 278: 46307–46320, 2003. 53. Wang GG, Calvo KR, Pasillas MP, Sykes DB, Hacker H, Kamps MP. Quantitative production of macrophages or neutrophils ex vivo using conditional Hoxb8. Nat Methods 3: 287–293, 2006. 54. Wang HR, Wu M, Yu H, Long S, Stevens A, Engers DW, Sackin H, Daniels JS, Dawson ES, Hopkins CR, Lindsley CW, Li M, McManus OB. Selective inhibition of the K(ir)2 family of inward rectifier potassium channels by a small molecule probe: the discovery, SAR, and pharmacological characterization of ML133. ACS Chem Biol 6: 845–856, 2011. 55. Weber-Steffens D, Hunold K, Kurschner J, Martinez SG, Elumalai P, Schmidt D, Trevani A, Runza VL, Mannel DN. Immature mouse granulocytic myeloid cells are characterized by production of ficolin-B. Mol Immunol 56: 488 –496, 2013.

AJP-Cell Physiol • doi:10.1152/ajpcell.00176.2014 • www.ajpcell.org

C276

KIR2.1 CHANNELS IN MOUSE NEUTROPHILS

56. Wieland SJ, Chou RH, Gong QH. Macrophage-colony-stimulating factor (CSF-1) modulates a differentiation-specific inward-rectifying potassium current in human leukemic (HL-60) cells. J Cell Physiol 142: 643–651, 1990. 57. Wischmeyer E, Doring F, Karschin A. Acute suppression of inwardly rectifying Kir2.1 channels by direct tyrosine kinase phosphorylation. J Biol Chem 273: 34063–34068, 1998. 58. Yan DH, Nishimura K, Yoshida K, Nakahira K, Ehara T, Igarashi K, Ishihara K. Different intracellular polyamine concentrations underlie the difference in the inward rectifier K(⫹) currents in atria and ventricles of the guinea-pig heart. J Physiol 563: 713–724, 2005. 59. Yang M, Busche G, Ganser A, Li Z. Morphology and quantitative composition of hematopoietic cells in murine bone marrow and spleen of healthy subjects. Ann Hematol 92: 587–594, 2013. 60. Young CC, Stegen M, Bernard R, Muller M, Bischofberger J, Veh RW, Haas CA, Wolfart J. Upregulation of inward rectifier K⫹ (Kir2)

61.

62.

63. 64.

channels in dentate gyrus granule cells in temporal lobe epilepsy. J Physiol 587: 4213–4233, 2009. Zhang DY, Wu W, Deng XL, Lau CP, Li GR. Genistein and tyrphostin AG556 inhibit inwardly-rectifying Kir2.1 channels expressed in HEK 293 cells via protein tyrosine kinase inhibition. Biochim Biophys Acta 1808: 1993–1999, 2011. Zhang DY, Zhang YH, Sun HY, Lau CP, Li GR. Epidermal growth factor receptor tyrosine kinase regulates the human inward rectifier potassium K(IR)2.3 channel, stably expressed in HEK 293 cells. Br J Pharmacol 164: 1469 –1478, 2011. Zhang H, Clemens RA, Liu F, Hu Y, Baba Y, Theodore P, Kurosaki T, Lowell CA. STIM1 calcium sensor is required for activation of the phagocyte oxidase during inflammation and host defense. Blood 123: 2238–2249, 2014. Zhu G, Chanchevalap S, Cui N, Jiang C. Effects of intra- and extracellular acidifications on single channel Kir2.3 currents. J Physiol 516: 699 –710, 1999.

AJP-Cell Physiol • doi:10.1152/ajpcell.00176.2014 • www.ajpcell.org

Copyright of American Journal of Physiology: Cell Physiology is the property of American Physiological Society and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.