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Am J Physiol Cell Physiol 293: C45-C54, 2007. First published February 28, 2007; doi:10.1152/ajpcell.00450.2006
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Large-conductance calcium-activated potassium channel activity is absent in human and mouse neutrophils and is not required for innate immunity

Kirill Essin,1,2 Birgit Salanova,2 Ralph Kettritz,2 Matthias Sausbier,4 Friedrich C. Luft,2 Dirk Kraus,3 Erwin Bohn,3 Ingo B. Autenrieth,3 Andreas Peschel,3 Peter Ruth,4 and Maik Gollasch1,2

1Department of Nephrology and Medical Intensive Care, 2Franz Volhard Clinic and Max Delbrück Center for Molecular Medicine, HELIOS Klinikum Berlin, Charité-University Medicine Berlin, Humboldt University of Berlin, Berlin; and 3Institute of Medical Microbiology and Hygiene and 4Institute of Pharmacy, Department of Pharmacology and Toxicology, University of Tübingen, Tübingen, Germany

Submitted 23 August 2006 ; accepted in final form 22 February 2007


   ABSTRACT
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 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Large-conductance Ca2+-activated K+ (BK) channels are reported to be essential for NADPH oxidase-dependent microbial killing and innate immunity in leukocytes. Using human peripheral blood and mouse bone marrow neutrophils, pharmacological targeting, and BK channel gene-deficient (BK–/–) mice, we stimulated NADPH oxidase activity with 12-O-tetradecanoylphorbol-13-acetate (PMA) and performed patch-clamp recordings on isolated neutrophils. Although PMA stimulated NADPH oxidase activity as assessed by O2– and H2O2 production, our patch-clamp experiments failed to show PMA-activated BK channel currents in neutrophils. In our studies, PMA induced slowly activating currents, which were insensitive to the BK channel inhibitor iberiotoxin. Instead, the currents were blocked by Zn2+, which indicates activation of proton channel currents. BK channels are gated by elevated intracellular Ca2+ and membrane depolarization. We did not observe BK channel currents, even during extreme depolarization to +140 mV and after elevation of intracellular Ca2+ by N-formyl-L-methionyl-L-leucyl-phenylalanine. As a control, we examined BK channel currents in cerebral and tibial artery smooth muscle cells, which showed characteristic BK channel current pharmacology. Iberiotoxin did not block killing of Staphylococcus aureus or Candida albicans. Moreover, we addressed the role of BK channels in a systemic S. aureus and Yersinia enterocolitica mouse infection model. After 3 and 5 days of infection, we found no differences in the number of bacteria in spleen and kidney between BK–/– and BK+/+ mice. In conclusion, our experiments failed to identify functional BK channels in neutrophils. We therefore conclude that BK channels are not essential for innate immunity.

killing assay; reactive oxygen species; BK-deficient mice; mice infection

NEUTROPHILS ARE THE FIRST-LINE cell defense of the innate immune system. Neutrophils kill microorganisms by ingesting them into phagocytic vacuoles and bombarding them with reactive oxygen species (ROS; e.g., O2– and H2O2) and enzymatic contents of cellular granules (23). ROS generation is mediated through the activation of NADPH oxidase in the plasma membrane. NADPH oxidase transfers electrons out of the cell into the phagocytic vacuole to reduce O2 to O2–. The resulting negative charge movement (44) should be compensated. Two currents have been suggested to compensate electron flux into the vacuole: an H+ current through voltage-gated proton channels (16, 25, 34) and a K+ current (38). K+ flux activates neutrophil granule proteases, which are necessary to resist staphylococcal and candidal infections (38, 45). Ahluwalia et al. (1) recently proposed that the K+ flux is caused by opening of big-conductance K+ (BK) channels in neutrophils.

BK channels, also known as slo and maxi-K+ channels, are broadly distributed among different cell types (for recent review see Refs. 21 and 32). BK channels are activated by membrane depolarization and by an increase in cytosolic Ca2+ and can be suppressed by potent blockers: iberiotoxin (19), charybdotoxin (22), and tetraethylammonium (TEA) (48). The BK channel consists of four pore-forming {alpha}-subunits and tissue-specific modulatory beta-subunits. BK channel {alpha}-subunit-knockout (BK–/–) mice suffer from cerebral ataxia and Purkinje cell dysfunction (42), elevated blood pressure (41), progressive hearing loss (40), and erectile dysfunction (49). Immune disorders in these mice have not been reported.

Ahluwalia et al. (1) found that the phorbol ester phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC)-dependent NADPH oxidase activator (8), stimulated a rise in intracellular free Ca2+ concentration ([Ca2+]i) and BK channel activity in isolated neutrophils. PMA-activated currents were blocked by iberiotoxin, but not by Zn2+ (1), an effective inhibitor of proton current (13). In the study of Ahluwalia et al., iberiotoxin did not affect ROS production but completely inhibited the killing of Staphylococcus aureus, Serratia marcescens, and Candida albicans by human neutrophils. The authors concluded that the BK channel is essential for innate immunity.

K+ channels in neutrophils are not as well studied as those in neuronal or muscle cells (26) and even in other leukocytes such as T and B cells (2, 20). The most comprehensive study of K+ channels in neutrophils was published in 1990 by Krause and Welsh (29). They did not find BK channels in human neutrophils but found two separate K+ currents: a voltage-dependent current and a Ca2+-activated current. Neither current was sensitive to charybdotoxin. Subsequently, Ca2+-activated intermediate-conductance K+ channels were identified in cultured HL-60-derived neutrophils (47). ATP-sensitive K+ channels, which may play a role in neutrophil migration and chemotaxis in the inflammatory response, may also be present in neutrophils (10). BK channels had been detected in macrophages but had not been described in neutrophils (15, 18).

Given the proposed importance of BK channels for innate immunity (1), we used BK channel gene-deleted (BK–/–) mice to study the role of the BK channel in the immune system. We failed to find that PMA stimulated BK channel activity in mouse and human neutrophils. Iberiotoxin, a specific blocker of the BK channel, did not decrease ROS production and microorganism killing in human neutrophils. Survival of bacteria was not increased in mice after genetic depletion of the BK channel. Thus our results are in agreement with the data obtained by Femling et al. (17), who also could not confirm an essential role of BK channels in neutrophil function.


   METHODS
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 METHODS
RESULTS
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Mice. All the experiments were performed with 2-to 3-mo-old male and female wild-type (BK+/+) and BK–/– mice. BK–/– mice were generated as described elsewhere (42). BK+/+ and BK–/– mice were of the hybrid SV129/C57BL6 background. Litter- or age-matched animals were randomly assigned to the experimental procedures. All animal experimental protocols were approved by the local animal care committees (Regierungspräsidium, Tübingen and LaGetSi, Berlin, Germany).

Purification of cells. Heparinized venous blood was obtained from healthy volunteers after written informed consent was obtained. Human neutrophils were isolated from blood as previously described (28, 43). Smooth muscle cells were enzymatically isolated from cerebral arteries from mice as previously described (41). Tibial smooth muscle cells were isolated using the same protocol. However, incubation with papain was prolonged to 45 min and isolation with collagenases to 10 min. Mouse neutrophils were isolated from bone marrow by a Ficoll-Histopaque gradient. Mice were killed, the femur and the tibia from both hindlegs were removed and freed of soft tissue attachments, and the extreme distal tip of each extremity was removed. Ca2+- and Mg2+-free Hanks’ balanced salt solution (HBSS; GIBCO) was forced through the bone with a syringe. The resulting cell suspension was passed over a 70-µm sterile nylon filter (BD Falcon) for removal of cell aggregates and other debris. The filtrate was layered over a double Ficoll-Histopaque gradient (1:1 Histopaque 1119 and 1083, Sigma) and centrifuged at 700 g for 30 min. The intermediate layer was collected and washed three times with HBSS. Purity of isolated bone marrow neutrophils was 70% as assessed by staining with Hemacolor (Merck). Neutrophils for further experiments were selected on the basis of their morphology.

Mice infection. Freshly thawed, plasmid-harboring Yersinia enterocolitica strain WA-314 serotype O:8 organisms suspended in 0.1 ml of sterile PBS, pH 7.4, were used for intravenous infection as described previously (5, 7). To determine the actual number of bacteria administered, we plated serial dilutions of the inoculum on Mueller-Hinton agar and counted colony-forming units (CFUs) after 36 h of incubation at 26°C. BK+/+ and BK–/– mice were killed by carbon dioxide asphyxiation at 5 days after infection with 5 x 103 bacteria. The spleens were aseptically removed, and a single-cell suspension was prepared using 5 ml of PBS containing 0.1% BSA. Duplicates of 0.1 ml of serial dilutions of these preparations were plated on Mueller-Hinton agar. The limit of detectable CFUs was 25 (log10 = 1.4). A total of nine mice were infected per group.

BK+/+ and BK–/– mice were also infected intravenously with 107 S. aureus SA113 and killed 3 days later. The number of bacteria in the spleen was determined by serial dilution and plating on Luria broth plates.

Electrophysiology. Patch-clamp studies were performed on freshly isolated neutrophils and smooth muscle cells. Membrane currents were recorded with an Axopatch 200B amplifier (Axon Instruments). Data were acquired and analyzed with a CED1401 interface and CED Patch and Voltage Clamp Software (version 6.08, Cambridge Electronic Design). Cells were voltage clamped at –30, –40, or 0 mV and pulsed for 300 ms from –100 to +140 or +100 mV in 20-mV increments every 2 s. Currents were measured at the end of the pulse at +140 or +100 mV. In some experiments, currents were recorded from a holding potential of –40 mV during linear voltage ramps at 0.5 V/s from –100 to +100 mV applied every 10 s. In experiments designed to measure proton currents, cells were voltage clamped at –60 mV and pulsed for 8 s in 20-mV increments every 20 s.

If not otherwise indicated, the experiments were performed in the perforated-patch configuration. A stock solution of 100 mg/ml amphotericin B in DMSO was prepared and diluted in the pipette solution to give a final concentration of 200 µg/ml. Stable access was obtained after 10–20 min. Cell capacitance was 3.1 ± 0.2 (n = 36, range 1.7–5 pF) for human neutrophils and 2.0 ± 0.1 (n = 54, range 1.5–2.5 pF) for mouse neutrophils. We did not routinely correct for series resistance. Agents were applied to the bath with a gravity-driven perfusion system. Several extracellular and pipette solutions were used. In first set of experiments, we used solutions identical to a high-Na+ extracellular solution containing (in mM) 140 NaCl, 2.5 KCl, 0.5 MgCl2, 1.2 CaCl2, 10 HEPES, and 5 glucose (with pH adjusted to 7.4 with NaOH) and a high-K+ pipette solution containing (in mM) 140 KCl, 10 NaCl, 2 MgCl2, 0.7 CaCl2, 1 EGTA, and 10 HEPES (with pH adjusted to pH 7.3 with KOH) to record currents. In another set of experiments, we used a high-Na+ extracellular solution containing (in mM) 134 NaCl, 6 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES, and 10 glucose (with pH adjusted to 7.4 with NaOH) and a high-K+ pipette solution containing (in mM) 110 K+-aspartate, 30 KCl, 10 NaCl, 1 MgCl2, 10 HEPES, and 0.05 EGTA (with pH adjusted to 7.2 with KOH), which is routinely used in our laboratory to record BK channel currents (37, 41).

We performed some experiments using symmetrical high-K+ aspartate solutions. The bath solution contained (in mM) 110 K-aspartate, 30 KCl, 10 NaCl, 1 MgCl2, 2 CaCl2, 10 HEPES, and 20 glucose (with pH adjusted to 7.2 with KOH), and the pipette solution contained (in mM) 110 K+-aspartate, 30 KCl, 10 NaCl, 1 MgCl2, 10 HEPES, and 0.05 EGTA (with pH adjusted to 7.2 with KOH). In another set of experiments, the symmetrical high-K+ aspartate solutions were supplemented with NH4 (14) to facilitate the measurements of proton currents. The extracellular solution contained (in mM) 80 K+-aspartate, 25 (NH4)2SO4, 2 MgCl2, 2 CaCl2, 10 HEPES, and 1 EGTA (with pH adjusted to 7.0 with KOH), and the pipette solution contained (in mM) 80 K+-aspartate, 25 (NH4)2SO4, 2 MgCl2, 5 HEPES, and 1 EGTA (with pH adjusted to 7.0 with KOH).

Values are means ± SE. Statistical analysis was performed by one-way analysis of variance and paired t-test.

Confocal imaging. For Ca2+ measurements, cells were loaded with 5 µM fluo 4-AM (Molecular Probes) and 0.01% pluronic acid (Calbiochem) for 30 min at 5°C in HEPES-PSS (in mM: 134 NaCl, 6 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES, and 10 glucose, with pH adjusted to 7.4 with NaOH). After they were loaded, the cells were washed with HEPES-PSS three times for removal of extracellular fluo 4-AM and plated at low density on 22-mm glass coverslips, which were mounted on the stage of an inverted Nikon microscope equipped with an UltraVIEW spinning-disk confocal system (Perkin Elmer). Fluo 4-AM was excited at the 488-nm line of an argon laser, and the fluorescence was measured at >510-nm emission. Ca2+ signals were monitored in individual cells before and after drug application. Drugs were added to the chamber as concentrated stock solutions to reach the desired final concentration. Two-dimensional fluorescence images were recorded at a rate of 2 frames/s and analyzed with the temporal mode of the UltraVIEW software (Perkin Elmer).

Measurement of O2– generation by ferricytochrome c reduction. O2– was measured using the assay of SOD-inhibitable reduction of ferricytochrome c, as described elsewhere (36). Neutrophils (0.75 x 106) were preincubated with 100 nM iberiotoxin (Sigma) or 10 µM diphenylene iodonium for 15 min at 37°C and then activated with 0.025 or 1 µg/ml PMA or buffer control. Experiments were done in duplicate. Samples were incubated in 96-well plates at 37°C for up to 60 min, and the absorption of samples with and without 300 U/ml SOD was scanned repetitively at 550 nm using a Microplate Autoreader (Molecular Devices, Munich, Germany). The final ferricytochrome c concentration was 250 µM, and the final cell concentration was 3.75 x 106/ml.

Measurement of cellular oxidant stress by dihydrorhodamine oxidation. The generation of reactive oxygen radicals was additionally assessed using dihydrorhodamine-1,2,3 (DHR), as described previously (28). Briefly, prewarmed neutrophils [1 x 107/ml HBSS with Ca2+ and Mg2+ (HBSS++, Biochrom)] were loaded with 1 µM DHR for 10 min at 37°C and then incubated with 100 nM iberiotoxin for 15 min at 37°C. Cells were activated with 0.025 or 1 µg/ml PMA or buffer control at 37°C. After 45 min, the reactions were stopped by addition of ice-cold 1% BSA-PBS. Samples were analyzed using a FACScan (Becton Dickinson, Heidelberg, Germany). Data were collected from 10,000 cells per sample. The shift of green fluorescence in the FL-1 mode was determined, and the mean fluorescence intensity (representing the amount of generated rhodamine 123) is reported.

Killing of C. albicans by human neutrophils. Killing of C. albicans was assessed as previously described (12, 35). C. albicans were selected from single colonies grown on Sabouraud-agar plates, inoculated into Sabouraud broth, and grown overnight at 30°C. The microorganisms were washed twice in HBSS++-HSA and adjusted to a density of 5 x 107 cells/ml. Pooled human serum (Sigma) was added to a final concentration of 10%, and microorganisms were opsonized for 10 min at 37°C. Neutrophils were isolated from peripheral blood as described above and resuspended in HBSS++ containing 0.05% human serum albumin (HSA) and 10% pooled human serum. Neutrophils were preincubated for 15 min with 100 nM iberiotoxin at 37°C. Opsonized microorganisms were added at a microorganism-to-neutrophil ratio of 2:1. A sample without neutrophils served as a control. The samples were shaken for 90 min at 37°C, and incubation was stopped by addition of 2 ml of ice-cold distilled water to disrupt the neutrophils. Aliquots (25 µl) were spread on Sabouraud agar plates, and colonies were counted after 24 h of incubation at 30°C. The percent killing was calculated as follows: [CFU sample (microorganisms) – CFU sample (neutrophils + microorganisms)]/CFU sample (microorganism) x 100.

Killing of S. aureus by human neutrophils. Neutrophils were isolated from peripheral blood of healthy volunteers as described previously (43) and resuspended in HBSS containing 0.05% HSA. To prepare bacteria, basic medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl, 0.1% glucose, and 0.1% K2HPO4) was inoculated with 1:100 dilution of an overnight culture and shaken at 37°C until midlogarithmic phase. The bacteria were washed twice in 10 mM potassium phosphate (KPi) buffer (pH 7.0) containing 0.01% HSA and adjusted to 5 x 107 bacteria/ml. Bacterial and neutrophil suspensions were mixed to final concentrations of 5 x 106/ml and 2.5 x 106/ml, respectively. Bacteria were opsonized by addition of pooled human serum (Sigma) to a final concentration of 10%. Samples (500 µl) with 100 nM iberiotoxin and without iberiotoxin were shaken at 37°C. Incubation was stopped by dilution of aliquots in ice-cold, distilled water. The neutrophils were disrupted by vigorous vortexing. Appropriate sample volumes were plated on basic medium agar plates, and colonies were counted after 24 h of incubation at 37°C.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
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Membrane currents in human and mouse neutrophils are not inhibited by iberiotoxin. Iberiotoxin is a potent and specific blocker of the BK channel (19). We did not observe iberiotoxin-sensitive currents in human blood and mouse bone marrow neutrophils (Fig. 1A), even after stimulation with PMA. We used the amphotericin B perforated-patch mode of the patch-clamp technique to measure currents in isolated neutrophils. We used the same solutions and protocols routinely used for BK channel current measurements (37, 41) and the solutions and protocols used by Ahluwalia et al. (1) but failed to find iberiotoxin-sensitive currents in neutrophils. Representative currents measured in cells clamped at –30 mV and depolarized for 300 ms from –100 to +140 mV in 20-mV increments in the absence and presence of 1 µg/ml PMA, in the presence of 1 µg/ml PMA + 100 nM iberiotoxin, and in the presence of 1 µg/ml PMA + 3 mM ZnCl2 are shown in Fig. 1A. Depolarization-evoked current amplitudes increased on stimulation with PMA and were not inhibited by iberiotoxin (Fig. 1B) but were blocked by Zn2+, which abolishes proton currents (17). The BK channel can be also blocked by 1 mM TEA (30, 48). TEA, at 1 mM, had no effect on depolarization-evoked currents in human neutrophils (n = 6 cells; data not shown).


Figure 1
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  		Fig. 1. Big-conductance Ca2+-activated K+ (BK) channel activity is absent in human and mouse neutrophils. A: representative currents in human and mouse BK+/+ and BK–/– neutrophils recorded in the absence (Ctr) and presence of 1 µg/ml phorbol 12-myristate 13-acetate (PMA, 5–7 min after application), in the presence of 1 µg/ml PMA + 100 nM iberiotoxin (Ibtx, 10–15 min after application), and in the presence of 1 µg/ml PMA + 3 mM ZnCl2 (Zn, 1–3 min after application). Iberiotoxin did not inhibit PMA-induced currents, indicating that BK channels are not present. Effects of PMA and Zn2+ were significant, since only small currents were induced by "sham bath change" within 25 min. Amphotericin-perforated cells were voltage clamped at a holding potential of –30 mV and pulsed for 300 ms from –100 to +140 mV in 20-mV increments every 2 s. High-Na+ external solution and high-K+ internal solution were used (1). B: mean current amplitudes recorded in human and BK+/+ and BK–/– mouse neutrophils in response to step depolarization to +140 mV in the absence and presence of 1 µg/ml PMA, in the presence of 1 µg/ml PMA + 100 nM iberiotoxin, and in the presence of 1 µg/ml PMA + 3 mM ZnCl2. Numbers of cells are indicated above bars. *P < 0.05. ns, Not significant.

 
Next, using symmetrical high-K+ aspartate solutions, we attempted to find BK channel currents. Representative currents measured in mouse neutrophils clamped at 0 mV and depolarized for 300 ms from –100 to +100 mV in 20-mV increments in the absence and presence of 1 µg/ml PMA (5–6 min) and in the presence of 1 µg/ml PMA + 100 nM iberiotoxin (10–12 min) are shown in Fig. 2A. Depolarization-evoked current amplitudes tended to increase on stimulation with PMA and were not inhibited by iberiotoxin (Fig. 2B). External application of 1 mM Ba2+ blocked the amplitude of the inward component of the current significantly but did not affect the outward component (n = 3 cells each for BK+/+ and BK–/– mice; data not shown), indicating the presence of inwardly rectifying K+ currents previously reported in newt blood neutrophils (27).


Figure 2
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  		Fig. 2. Currents in symmetrical high-K+ aspartate solutions were not sensitive to iberiotoxin. A: representative currents in BK+/+ and BK–/– mouse neutrophils recorded in the absence and presence of 1 µg/ml PMA (5–7 min after stimulation) and in the presence of 1 µg/ml PMA + 100 nM iberiotoxin (10–15 min after application). Amphotericin-perforated cells were voltage clamped at a holding potential of 0 mV and pulsed for 300 ms from –100 to +100 mV in 20-mV increments every 2 s. Symmetrical high-K+ aspartate solutions were used in bath and pipette. B: mean current amplitudes recorded in BK+/+ and BK–/– mouse neutrophils in response to step depolarization to +100 mV in the absence and presence of 1 µg/ml PMA and in the presence of 1 µg/ml PMA + 100 nM iberiotoxin. Numbers of cells are indicated above bars.

 
The effects of PMA, a well-known PKC activator (39), on BK channel activity are not clear: inhibition (46) and activation have been reported (6). Ahluwalia et al. (1) found that PMA increased [Ca2+]i in human neutrophils and, therefore, activated BK channels that are Ca2+ sensitive. We also found that PMA induced a Ca2+ increase in human neutrophils (Fig. 3A). Changes in [Ca2+]i were measured in fluo 4-AM-loaded neutrophils using Nipkow disk confocal microscopy. The time course of changes in fluo-4 fluorescence intensity evoked by 1 µM PMA averaged from five cells in a representative experiment is shown in Fig. 3A. The relative fluorescence (F/F0) increase in the presence of PMA was <20% but was, nonetheless, statistically significant. To achieve a more significant increase in [Ca2+]i, we used N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLP). [Ca2+]i in neutrophils was increased much more effectively by 1 µM fMLP than by PMA at the same concentration. Despite a significant increase in [Ca2+]i, fMLP failed to induce appropriate BK channel activity in human neutrophils (Fig. 3B).


Figure 3
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  		Fig. 3. N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLP) elevates cytosolic Ca2+ concentration ([Ca2+]i) but fails to stimulate BK channels in human neutrophils. A: [Ca2+]i changes induced by 1 µM PMA and 1 µM fMLP measured using Nipkow spinning-disk confocal microscopy in human neutrophils loaded with the Ca2+ indicator dye fluo 4-AM. Time course of mean fluorescence intensity changes was averaged for 5 cells each. Increases in global [Ca2+]i in human neutrophils induced by 1 µM PMA and 1 µM fMLP are expressed as percentage of fluorescence intensity changes: (F – F0) * 100/F0, where F is the average intensity over the whole area of the cell in the presence of substance and F0 is fluorescence before substance application. Values are means ± SE of ≥100 cells for each substance. B: representative currents recorded in symmetrical high-K+ aspartate solutions in a human neutrophil in the absence of fMLP, in the presence of 1 µM fMLP (5–7 min after stimulation), and in the presence of 1 µM fMLP + 100 nM iberiotoxin (10–15 min after application). Amphotericin-perforated cell was voltage clamped at a holding potential of 0 mV and pulsed for 300 ms from –100 to +100 mV in 20-mV increments. Mean current amplitudes were recorded in human neutrophils in response to step depolarization to +100 mV in the absence and presence of 1 µM fMLP and in the presence of 1 µM fMLP + 100 nM iberiotoxin. Numbers of cells in each experimental condition are indicated above bars. *P < 0.05.

 
As a control, we measured BK currents in smooth muscle cells isolated from mouse tibial and cerebral arteries (Fig. 4). In contrast to the neutrophils, in arterial smooth muscle cells, depolarization-evoked currents were effectively blocked by 100 nM iberiotoxin. BK channel activity in BK+/+ cerebral smooth muscle cells was abolished by iberiotoxin (Fig. 4A). In contrast, BK channel currents were absent in BK–/– cells. BK channel currents in BK+/+ tibial smooth muscle cells were effectively blocked by iberiotoxin (Fig. 4B). In tibial and cerebral smooth muscle cells, iberiotoxin also blocked spontaneous transient outward currents, which represent coordinated openings of a cluster of BK channels caused by Ca2+ sparks (37, 41).


Figure 4
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  		Fig. 4. BK channel activity in arterial smooth muscle cells. A: BK channel activity in a smooth muscle cell isolated from a cerebral artery of a BK+/+ mouse (top trace) was blocked by 100 nM iberiotoxin (10 min after application, middle trace). BK channel activity was absent in a smooth muscle cell isolated from a cerebral artery of a BK–/– mouse (bottom trace). Amphotericin-perforated cells were voltage clamped at a holding potential of +20 mV. High-Na+ external and high-K+ internal pipette solutions were used (41). Data were filtered at 1 kHz and sampled at 2 kHz. Arrow indicates coordinated openings of several BK channels, which are known as spontaneous transient outward current (STOC) (37). Similar results were obtained in 3 other BK+/+ and 3 other BK–/– cerebral smooth muscle cells (see Ref. 41 for our previous recordings). B: 100 nM iberiotoxin blocked STOCs and whole cell K+ current in a smooth muscle cell isolated from the tibial artery of a BK+/+ mouse. Amphotericin-perforated cell was voltage clamped at –40 mV, and linear voltage ramps at 0.5 V/s from –100 to +100 mV were applied every 10 s. High-Na+ external and high-K+ internal pipette solutions were used (41). The 6 superimposed records are shown in the absence and presence of 100 nM iberiotoxin (10 min after application). Similar results were obtained in 3 other cells.

 
As another control, proton-like currents were recorded in human and mouse neutrophils. Symmetrical high-K+ aspartate solutions were supplemented with NH4 (14). Representative current traces are shown in Fig. 5. Amphotericin-perforated cells were voltage clamped at a holding potential of –60 mV and pulsed for 8 s from –60 to +60 mV in 20-mV increments every 20 s. PMA-stimulated currents were not sensitive to iberiotoxin. However, the currents were almost completely blocked by Zn2+, which is consistent with previously reported results (17).


Figure 5
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  		Fig. 5. Proton-like currents in human and mouse neutrophils were not sensitive to iberiotoxin but were blocked by Zn2+. Currents in human and BK+/+ and BK–/– mouse neutrophils were recorded in the absence and presence of 1 µg/ml PMA (5–7 min after stimulation), in the presence of 1 µg/ml PMA + 100 nM iberiotoxin (10–15 min after application), and in the presence of 1 µg/ml PMA + 3 mM ZnCl2 (1–3 min after application). Amphotericin-perforated cells were voltage clamped at a holding potential of –60 mV and pulsed for 8 s from –60 to +60 mV in 20-mV increments every 20 s. Recordings were performed in symmetrical high-K+ aspartate solutions supplemented with NH4 (14). PMA-stimulated currents were not sensitive to iberiotoxin. However, they were blocked by Zn2+. Similar results were obtained in 4 other human and 3 other BK+/+ and 3 other BK–/– mouse neutrophils.

 
Iberiotoxin does not inhibit killing of human neutrophils. Ahluwalia et al. (1) reported that iberiotoxin is able to inhibit neutrophil killing. We tested the effect of iberiotoxin on the ability of human neutrophils to kill S. aureus and C. albicans (Fig. 6, A and B). Survival of S. aureus at the end of 15, 30, and 60 min of incubation with neutrophils at 37°C compared with the initial number of bacteria is shown in Fig. 6A. The ratio of bacteria to neutrophils was taken as 2:1. Almost all bacteria were killed at 60 min. Bacterial survival was not significantly changed by 100 nM iberiotoxin at 15, 30, or 60 min. The ability of neutrophils to kill C. albicans at the end of 90 min of incubation with neutrophils at 37°C is shown in Fig. 6B. Opsonized microorganisms were added at a microorganism-to-neutrophil ratio of 2:1. The ~40% of C. albicans killed in control was not decreased by 100 nM iberiotoxin. Therefore, our data indicate that iberiotoxin does not inhibit killing of S. aureus and C. albicans by neutrophils, in contrast to the data presented by Ahluwalia et al. (1) but in agreement with other reports (11, 17).


Figure 6
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  		Fig. 6. BK channel inhibition does not influence killing activity of neutrophils and reactive oxygen species production. A: effect of BK channel inhibition on killing of opsonized Staphylococcus aureus by human neutrophils. Number of viable S. aureus colony-forming units (CFUs) after incubation with human neutrophils is expressed as percentage of initial count. Samples were incubated in the absence and presence of 100 nM iberiotoxin. Values are means ± SE of 2 independent experiments run in duplicate. BK channel inhibitor iberiotoxin had no effect on killing of S. aureus by human neutrophils. B: effect of BK channel inhibition on the killing of C. albicans by human neutrophils. BK channel inhibitor iberiotoxin had no effect on killing of C. albicans by human neutrophils. C: BK channel knockout does not reduce resistance to Yersinia infection in mice. Number of bacteria were counted in spleens isolated 5 days after BK+/+ and BK–/– mice were infected with Yersinia. Values are means ± SE for 9 mice. BK channel is not essential for Yersinia killing. D: BK channel knockout does not reduce resistance to S. aureus infection in mice. Number of bacteria were counted in spleens and kidneys isolated 3 days after BK+/+ and BK–/– mice were infected with S. aureus. Values are means ± SE from 10 mice. BK channel is not essential for S. aureus killing. E: effect of BK channel inhibition on O2– generation. Data are from samples incubated for 45 min. Values are means ± SE (n = 7). BK channel inhibitor iberiotoxin had no effect on respiratory burst activity of PMA-treated neutrophils. F: effect of BK channel inhibition on oxidant generation of PMA-treated neutrophils. Oxidants were assessed using dihydrorhodamine (DHR) oxidation assay. Values are means ± SE (n = 6). Independent assay to estimate respiratory burst activity indicates that BK channel inhibitor iberiotoxin had no effect on respiratory burst of PMA-stimulated neutrophils. MFI, mean fluorescence units (i.e., amount of rhodamine-1,2,3 generated).

 
Iberiotoxin does not inhibit ROS production in human neutrophils. Effective killing of S. aureus and C. albicans requires NADPH oxidase activity and generation of ROS (4, 24). We tested the effect of iberiotoxin on generation of ROS in human neutrophils with two independent assays. O2– was measured using the assay of SOD-inhibitable reduction of ferricytochrome c (Fig. 6E). At 25 ng/ml–1 µg/ml, PMA, a known NADPH oxidase activator (8), stimulated O2– production. Iberiotoxin (100 nM) did not block O2– production in neutrophils in the absence or presence of PMA. Similar results were obtained with the DHR oxidation assay. The generation of reactive oxidants was stimulated by 25 ng/ml and 1 µg/ml PMA and was not inhibited by 100 nM iberiotoxin (Fig. 6F). In contrast to iberiotoxin, diphenylene iodonium, a classical inhibitor of NADPH-dependent ROS production (9), strongly reduced the oxidant generation in PMA-stimulated cells (n = 6; data not shown). Therefore, our data show that ROS production was not inhibited by iberiotoxin. The data are in agreement with the absence of an inhibitory effect of iberiotoxin on killing activity of human neutrophils and confirm data obtained in previous studies (1, 17).

BK channel knockout does not reduce resistance to S. aureus and Yersinia infection in mice. If BK channels are essential for innate immunity, a reasonable expectation would be that BK–/– mice are less resistant to infections than BK+/+ mice. We performed experiments with S. aureus- and Yersinia-infected mice to explore this possibility (Fig. 6C). BK+/+ and BK–/– mice were intravenously infected with S. aureus or Y. enterocolitica. Three days after infection with S. aureus and 5 days after infection with Yersinia, the mice were killed, and the number of viable bacteria recovered from spleen and kidney was determined. The number of viable Yersinia and S. aureus was not increased by the absence of BK channels in mice (Fig. 6, C and D). Thus the data do not support the idea that BK channels are essential for innate immunity and, thus, for protection against bacterial infections.


   DISCUSSION
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 ABSTRACT
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 DISCUSSION
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 REFERENCES
 
We did not find that PMA stimulated BK channel activity in human or mouse neutrophils. For a positive control, we recorded iberiotoxin-sensitive BK channel currents in arterial smooth muscle cells, where their existence is well established (for recent review see Ref. 31). Electrophysiological measurements were done using the protocol and solutions routinely used in our laboratory (37, 41) and the solutions and protocol used by Ahluwalia et al. (1). They found that PMA significantly increases intracellular Ca2+ in neutrophils and activates Ca2+-dependent K+ currents blocked by iberiotoxin. In contrast to iberiotoxin, PMA is not a classical pharmacological tool to study BK channels. The action of PMA is variable, ranging from channel activation (6) to inhibition (46), depending on the cell type. The reported effects were not associated with a rise in [Ca2+]i but, rather, with PKC-dependent protein phosphorylation. For example, in pulmonary arterial smooth muscle cells, PMA stimulated PKC and, thereby, activated BK channels via cGMP-dependent protein kinase (6), which directly phosphorylates the pore-forming channel {alpha}-subunits (3). Also, the increase in [Ca2+]i in neutrophils is not the commonly observed effect of PMA (33). We detected the PMA-induced increase in neutrophil [Ca2+]i; however, the response we observed was much more modest than that reported by Ahluwalia et al. On the other hand, a similar concentration of fMLP increased [Ca2+]i significantly but failed to stimulate BK channel activity.

Although BK channel activity was not detected in neutrophils in our study and in the report by Femling et al. (17), these cells successfully kill S. aureus and C. albicans (17) (Fig. 6, A and B). Iberiotoxin, a specific blocker of the BK channel, did not decrease the ability of neutrophils to eliminate microorganisms. The killing assays were performed in two independent laboratories (Berlin and Tübingen) using different experimental methods and conditions. Although Femling et al. (17) also obtained their results in two separate laboratories, four independent sources report no essential role for BK channels in the neutrophil killing function. Our experiments with BK–/– mice also do not support the idea that BK channels are essential for innate immunity. BK–/– mice were not less resistant to S. aureus and Yersinia infection than their BK+/+ littermates. The notion that neutrophils function via BK channel activity should be revised.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
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This work was supported by the Deutsche Forschungsgemeinschaft.


   ACKNOWLEDGMENTS
 
We thank Dr. S. Herroeder and A. Nuernberg for demonstrating the method of neutrophil extraction from mouse bone marrow, Yoland Anistan for help with cell isolation, and Juliane Krejci for expert technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Gollasch, Charité Campus Virchow Klinikum, Humboldt Univ., Augustenburger Platz 1, 13353 Berlin, Germany (e-mail: maik.gollasch{at}charite.de)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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Electrophysiology of the phagocyte respiratory burst. Focus on "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, July 1, 2007; 293(1): C30 - C32.
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