1 Division of Cellular and Molecular Biology, Toronto Western Research Institute, University Health Network, and 2 Department of Physiology, University of Toronto, Toronto, Ontario, Canada
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ABSTRACT |
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Microglial activation following central nervous system damage or disease often culminates in a respiratory burst that is necessary for antimicrobial function, but, paradoxically, can damage bystander cells. We show that several K+ channels are expressed and play a role in the respiratory burst of cultured rat microglia. Three pharmacologically separable K+ currents had properties of Kv1.3 and the Ca2+/calmodulin-gated channels, SK2, SK3, and SK4. mRNA was detected for Kv1.3, Kv1.5, SK2, and/or SK3, and SK4. Protein was detected for Kv1.3, Kv1.5, and SK3 (selective SK2 and SK4 antibodies not available). No Kv1.5-like current was detected, and confocal immunofluorescence showed the protein to be subcellular, in contrast to the robust membrane localization of Kv1.3. To determine whether any of these channels play a role in microglial activation, a respiratory burst was stimulated with phorbol 12-myristate 13-acetate and measured using a single cell, fluorescence-based dihydrorhodamine 123 assay. The respiratory burst was markedly inhibited by blockers of SK2 (apamin) and SK4 channels (clotrimazole and charybdotoxin), and to a lesser extent, by the potent Kv1.3 blocker agitoxin-2.
calcium-activated potassium channels; small-conductance K+ channels; Kv1.3; reactive oxygen intermediates; microglial activation
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INTRODUCTION |
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MICROGLIA, THE MACROPHAGE-LIKE CELLS of the central nervous system (CNS), are often the first to respond to brain injury or disease (17, 53, 54). Their activated responses include proliferation, migration to the site of damage, phagocytosis, antigen presentation, and release of cytokines, as well as morphological and immunophenotypical changes. One key function of activated microglia is in generating a NADPH-mediated respiratory burst (5, 6), a metabolic cascade that generates antimicrobial superoxide and other reactive oxygen intermediates (49). These products can damage bystander cells through lipid peroxidation, membrane disruption, cell necrosis, and DNA oxidation, and such detrimental events have been documented in spinal cord injury, multiple sclerosis, Alzheimer's disease, and stroke (2, 17, 19). Because microglia can be either helpful or harmful, developing ways of selectively regulating their functions would be valuable.
In seeking to determine whether ion channels regulate microglial activation, we have shown that proliferation is inhibited by blocking anion channels in pure cultures (46) and by 4-aminopyridine (a broad-spectrum blocker of voltage-gated K+ channels) or agitoxin-2 (a potent Kv1.3 blocker) in tissue prints made from hippocampal slices (31). Despite numerous studies describing ion currents in microglia [see review by Eder (12)], there is little known about which specific channel proteins are expressed and whether they are involved in microglial functions. There are two voltage-gated K+ current (Kv) channels expressed, based on biophysical and pharmacological features of the currents. One resembles Kv1.3 (4; for review, see Ref. 12) and the other resembles Kv1.5 (31). Expression of Kv1.3 mRNA (4, 31, 38) and protein (4, 31), as well as Kv1.5 mRNA and protein (31, 41), have been documented. Two important factors might limit the roles of these currents in microglia. The Kv1.5 current decreased during culturing of tissue prints, corresponding with a decrease in membrane localization of the protein (31), and the Kv1.3 current in cultured rat microglia was strongly inhibited by tyrosine phosphorylation and by oxygen-glucose deprivation. Two additional K+ currents, both Ca2+ activated, have been reported in cultured microglia (13, 36), but their molecular basis is unknown. There is limited evidence of a role for K+ channels in the microglial respiratory burst, with one report showing inhibition by a broad-spectrum K+ channel blocker, tetraethylammonium (TEA) (52), and our preliminary work using more selective blockers, which implicated Kv1.3 and one or more Ca2+-activated K+ channels (KCa) (27, 43).
We have now used essentially pure rat microglial cultures to study the expression and roles of K+ channels without the influence of other CNS cells, including neurons, astrocytes, and oligodendrocytes. We examined expression of specific K+ channels and found that Kv1.3, Kv1.5, SK2, and/or SK3, and SK4 were present at the mRNA level, and Kv1.3, Kv1.5 and SK3 protein were detected using available antibodies. Whole cell currents with biophysical and pharmacological properties of Kv1.3, SK2, SK3, and SK4 were detected. We then used a phorbol ester to activate the microglia respiratory burst, and, using a panel of K+ channel blockers, obtained evidence that both Kv1.3 and small-conductance KCa (SK) channels regulate the generation of a NADPH-mediated respiratory burst.
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MATERIALS AND METHODS |
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Culturing microglia.
Microglia were isolated from brain explants of 2- to 3-day-old Wistar
rats as previously described (31, 46). Briefly, neopallial
tissue was digested with agitation in MEM (GIBCO BRL, Grand Island, NY)
that contained 2.5% trypsin (Sigma, St. Louis, MO) and 100 units of
DNase I (Pharmacia, Baie d'Urfé, PQ) for 30 min at room
temperature, triturated, digested a further 30 min, and triturated
until no tissue clumps were visible. This mixture was pelleted,
resuspended in MEM, passed through a cell strainer (40-µm-diameter
holes), seeded in 75-cm2 flasks in 30 ml of endotoxin-free
culture medium that contained MEM, 5% fetal bovine serum, 5% horse
serum (both from Sigma), and 0.05 mg/ml gentamicin (GIBCO), and re-fed
on day 2. After 10-12 days without feeding, the
floating cells (>95% microglia) were plated on sterile coverslips,
allowed to adhere 1.5-2 h, and then shaken by hand for 5 min to
remove residual astrocytes. The remaining adherent cells were
98-100% microglia, as determined by their labeling with the
microglia-specific markers, isolectin B4 (54) conjugated
to Texas red (EY Labs, San Mateo, CA) or ED1 antibody
(11), or ED1 antibody (11), a Cy3-conjugated secondary
antibody (both from Sigma). Total cell counts were obtained by labeling
cell nuclei with acridine orange or propidium iodide (Molecular Probes,
Eugene, OR). The small number of contaminating cells were usually
astrocytes, as judged by their labeling with an antibody against glial
fibrillary acidic protein (Sigma). Endotoxin (lipopolysaccharide)
levels in all media and cell cultures were periodically tested
(E-Toxate detection kit; Sigma) and used only if there was 1
endotoxin U/ml.
Chemicals.
To separate the different K+ currents, well-known
K+ channel blockers were used, consisting of charybdotoxin
(Peptides International, Louisville, KY), agitoxin-2 (Alomone Labs,
Jerusalem, Israel), and apamin and clotrimazole (both from Sigma).
These compounds were stored lyophilized at 20°C, then made up in
extracellular (bath) solution that contained 0.1% BSA to reduce drug
adsorption onto the perfusion lines. For all patch-clamp recordings, we
used 500 µM NPPB [5-nitro-2-(3-phenylpropylamino)benzoic acid; RBI, Natick, MA] to fully block the anion current (46). The
phorbol ester, phorbol 12-myristate 13-acetate (PMA), and its inactive analog, 4
-phorbol 12-myristate 13-acetate (4
-PMA), were from Biomol (Plymouth Meeting, PA). Stocks of these reagents were prepared in DMSO, frozen at
20°C, and diluted in bath solution before use.
Where appropriate, control solutions contained 0.1% DMSO, the highest
concentration used in the test solutions.
Patch-clamp electrophysiology.
Whole cell recordings were made from microglia within 2 days after
preparing 98-100% pure cultures. The extracellular (bath) solution contained (in mM) 145 sodium aspartate, 5 KCl, 1 MgCl2, 1 CaCl2, and 5 HEPES adjusted with NaOH
to pH 7.4. The osmolarity (270-283 mosM) was measured with a
freezing point depression osmometer (model 3MO; Advanced Instruments,
Norwood, MA). To study KCa currents, the intracellular
(pipette) solution contained 140 potassium aspartate, 1 K41,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid, 2 K2ATP, 0.9 CaCl2, 1 MgCl2,
and 5 HEPES adjusted to pH 7.2 with KOH, 260-270 mosM, with a free
Ca2+ concentration of 1.1 µM to fully activate
small-conductance (SK) channels (26, 28). During
patch-clamp recordings, the bath chamber (150 µl; model RC-25; Warner
Instruments, Hamden, CT) was continuously perfused at 1-2 ml/min
using a gravity feed, and recordings were made at room temperature
(20-25°C). We used 8- to 12-M pipettes pulled from
borosilicate glass (WPI, Sarasota, FL) and an Axopatch 200 (Axon
Instruments, Foster City, CA) patch-clamp amplifier, which was used to
filter the currents at 2 kHz, to cancel capacitive currents by analog
subtraction and to compensate for 50-70% of the series
resistance. Voltages were applied and currents recorded using pCLAMP
software (version 6.0; Axon Instruments). Because we wanted to record
time-independent KCa currents, we did not use leak
subtraction. All voltages were corrected to account for the junction
potential between bath and pipette solutions. Data were stored on
computer and analyzed using the Origin program (version 5; Microcal,
Northampton, MA).
RT-PCR.
We used the same general procedures as previously described
(28) and cultures that were >98% pure microglia.
Briefly, total RNA was isolated using the guanidinium isothiocyanate
method, subjected to DNase I digestion (0.1 U/ml for 15 min at 37°C;
Pharmacia) to eliminate genomic contamination, and then first-strand
cDNA was synthesized using an oligo(dT)-based primer (Pharmacia). The RT-PCR reaction was conducted with 1.5 mM MgCl2, 0.5 µM
forward and reverse primers (see Table
1), and 10% of the cDNA reaction mixture, using the GeneAmp PCR 2400 system (Perkin Elmer, Mississauga, ON). After incubating the mixture at 85°C for 1 min, 1.25 units of
Taq DNA polymerase (BioBasic, Toronto, ON) was added, heated to 94°C for 5 min, and the mixture subjected to 32 cycles of a 15-s
denaturing phase at 94°C, a 20-s annealing phase (see Table 1 for
temperatures), and a 30-s extension phase at 72°C. After a final
extension for 5 min at 72°C, the samples were incubated at 4°C
until further processing. The resulting DNA products were resolved in
2% agarose gels with 0.5 mg/ml ethidium bromide and their identities
confirmed by restriction endonuclease digestion or by sequencing.
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Transfected Chinese hamster ovary cells. Chinese hamster ovary (CHO) cells, grown to ~60% confluency, were transfected (Lipofectamine, GIBCO BRL) with cDNAs for rSK1, rSK2, hSK4, or the vector pRC/CMV (Invitrogen, Carlsbad, CA). A CHO cell line stably expressing rSK3 (from Dr. G. Moss, University College, London, UK) was provided by Drs. W. J. Joiner and L. K. Kaczmarek (Yale Univ., New Haven, CT). Western blotting was performed 48 h after transient transfections.
Western blot analysis. Pure cultured microglia were lysed in ice-cold solubilization buffer containing (in mM) 25 Tris (pH 7.5), 150 NaCl, 100 NaF, 5 EDTA, 1 Na3VO4, 1 phenylmethylsulfonyl fluoride, 1% Triton X-100, 1 µg/ml leupeptin, and 2 µg/ml aprotinin. The lysate was triturated, centrifuged (15,000 g for 5 min at 4°C) to remove cellular debris, and the supernatant retrieved as described previously (4). Total protein concentration was determined with DC Protein Assay (Bio-Rad, Mississauga, ON) with BSA as the standard. Proteins were resolved on a 10% acrylamide gel by SDS-PAGE, electrotransferred to nitrocellulose, blocked in 5% nonfat milk, and incubated overnight with the appropriate primary antibody at 4°C [Kv1.3, 1:100; Kv1.5, 1:100; SK2, 1:250; SK3, 1:250 (all from Alomone)]. Blots were incubated with horseradish peroxidase-conjugated secondary antibody (Cedarlane, Hornby, ON) for 1 h at room temperature. Enhanced chemiluminescence (Amersham, Arlington Heights, IL) on XAR-2 film (Kodak, Rochester, NY) was used to visualize labeled proteins. Unless otherwise indicated, reagents for Western immunoblotting were from Sigma.
Confocal microscopy.
The subcellular location of Kv1.3 and Kv1.5 proteins was assessed by
two-color immunofluorescence and confocal microscopy as previously
described (31). Microglia were washed (3 times for 5 min
each) with PBS, then rapidly fixed and permeabilized in cold methanol
on dry ice (20 min at 20°C). Subsequent steps were performed at
room temperature unless otherwise stated. The fixed microglia were
washed (3 times for 5 min each) in PBS and then incubated for several
hours in 10% BSA as a blocking agent. To label microglial membranes, a
mouse monoclonal antibody (OX-42; 1:200 dilution) that recognizes the
CR3 complement receptor was added in a 1% BSA solution and incubated
overnight at 4°C. This membrane-associated label was compared with
fluorescent signals generated by either an anti-Kv1.3 polyclonal
antibody (1:75) or an anti-Kv1.5 polyclonal antibody (1:75) (both from
Alomone). To produce fluorescent green membranes, the microglia were
washed in 5% BSA, incubated for 3 h in a biotinylated anti-mouse
secondary antibody with FITC-conjugated streptavidin (1:100; Vector,
Carlsbad, CA), and washed again in 5% BSA. Finally, to visualize the
red fluorescent Kv channels, the fixed microglia were incubated for 1 h in a 1% BSA solution that contained Cy3-conjugated
anti-rabbit antibody (1:100; Vector); they were then washed, first with
5% BSA, then with PBS. For each negative control, the primary antibody was omitted. All stained slides were treated with Slow Fade (Molecular Probes) and stored in the dark at 4°C. To further reduce the
background staining of microglia-free regions, multiple washes in a
high concentration of 5% BSA were performed. To assess the membrane vs. subcellular distribution of each antibody, we used a scanning confocal microscope (MRC-600; Bio-Rad) equipped with an argon ion
laser, fluorescein, Cy3 filter sets, and a ×63 objective. Fluorescence
images were superimposed to show OX-42 and Kv channel staining using
the same focal plane and instrument gain. Digitized images were
prepared using the software provided with the confocal microscope and
Adobe Photoshop (version 5.0; Adobe Systems, Mountainview, CA).
Respiratory burst assay. Pure cultured microglia, plated on sterile coverslips, were transferred to serum-free medium [1% L-glutamine (GIBCO BRL) and 0.05 mg/ml gentamicin]. This was supplemented with 2% B27 medium (GIBCO BRL), which we found reduces the spontaneous respiratory burst compared with serum-containing medium. Just before each experiment, microglia were transferred to a closed perfusion chamber (Warner Instruments) that was maintained at 37°C and perfused with a standard solution that contained (in mM) 140 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 HEPES, and 3.5 D-glucose. The morphology of cells in several microscope fields was viewed with Hoffman modulation contrast optics (Austin, TX), and a representative field was recorded using a digital charge-coupled device camera (Cohu, San Diego, CA). All image acquisition and analyses were performed with Axon Imaging Workbench (version 2.1; Axon Instruments).
To measure the respiratory burst, 2 µM of the uncharged, nonfluorescent dye dihydrorhodamine 123 (DHR 123; Molecular Probes) was added to the bathing solution. This dye readily diffuses into cells, where it reacts with hydrogen peroxide in the presence of peroxidase, cytochrome C, or Fe2+ to form an oxidized green fluorescent product (rhodamine 123) (1, 42). Dye photobleaching was prevented by using a 3% neutral density filter. We initially observed a high background fluorescence in the bath solution, presumably from oxidation of extracellular DHR 123, because it was essentially eliminated by exchanging the bath solution at least five times to wash the cells. Fluorescence levels were quantified by drawing a "region-of-interest" mask around each of 4-5 cells/field and subtracting the background fluorescence of one cell-free zone. Recordings were made from the same cells every 30 s for 5 min to obtain the basal, unstimulated fluorescence level, and only cells with stable baselines were subsequently analyzed. The respiratory burst was then stimulated by switching the perfusion bath solution to one that contained 100 nM PMA, which is one of the most robust among the known stimuli for microglia (1, 6, 51), and the fluorescence signal was recorded continuously for a further 50 min. In separate experiments, either 100 nM of the inactive analog 4Statistics. Where appropriate, data are presented as means ± SE for the number of cells or experiments indicated. To test for significant differences (P < 0.05) in experiments including only two conditions, a Student's t-test was used. In pharmacological experiments where each cell was its own control, paired t-tests were used. Statistical analyses were performed using InStat (version 2.04; GraphPad Software, San Diego, CA).
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RESULTS |
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Voltage-gated K+
current.
Numerous studies of cultured microglia have described a
depolarization-activated K+ current with biophysical and
pharmacological features similar to the cloned Kv1.3 channel (4, 39, 46; see Ref. 12 for review). We recently found a
Kv1.5-like current in hippocampal microglia shortly after tissue prints
were prepared from rat brain slices (31). Thus we were
interested in determining whether these two channels are expressed in
cultured microglia. Within 2 wk of brain dissection and dissociation,
we observed a Kv1.3-like current in 78% (38/49) of microglia. That is,
the current activated in a time- and voltage-dependent manner at 40
mV or above and inactivated during long depolarizing steps (Fig.
1A). The tail currents
reversed at ~
87 mV (Fig. 1B), which is close to the
Nernst potential for K+ in these solutions (
89 mV).
Cumulative inactivation (a hallmark of Kv1.3 current) was seen as a
decrease in peak current with repetitive voltage steps; i.e., when
voltage pulses were applied at 5-s intervals from
90 to +40 mV, the
current amplitude decreased by ~50% after the second step (Fig.
1C). This Kv current was strongly inhibited by the scorpion
toxin, agitoxin-2 (Fig. 1A), which is one of the most potent
and selective Kv1.3 blockers (16), having a
Kd (dissociation constant) = 0.5 pM in T
lymphocytes (31). The microglia whole cell current was
reduced 79 ± 3% (n = 14) by 5 nM agitoxin-2, and
the residual current lacked time- or voltage-dependent activation (Fig.
1A, right), and thus was not a Kv current. After blocking Kv1.3 with agitoxin-2, there was no sign of a Kv1.5 current like that previously observed in tissue-printed microglia
(31).
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KCa.
Much less is known about KCa currents in microglia, with
one report each of big-conductance (BK) channels in human cells
(36) and charybdotoxin-sensitive channels in murine cells
(13). To study KCa currents in cultured rat
microglia, we used high Ca2+ (1.1 µM) in the pipette
solution, a concentration that fully activates both small- and
intermediate-conductance members of the SK family (26, 28,
29). For all KCa recordings, 5 nM agitoxin-2 was
added to block Kv1.3, and 500 µM NPPB was used to block anion
currents. With an approximately resting level of internal
Ca2+ (100 nM, Fig.
2A), the currents, in response
to voltage steps between 50 and +70 mV, did not display
time-dependent activation or inactivation during 500-ms-long test
pulses. The currents were very small, except at the most positive
potentials tested (<50 pA at +70 mV). With elevated Ca2+,
the currents were similar but larger: almost four times as large in
this cell. Under the physiological Na+-K+
gradient used, the current-voltage (I-V) relationship was
nearly linear between the reversal potential (~
85 mV, Fig.
2C) and +30 mV, but the total conductance decreased above
this potential, despite the 30-s-long intervals between pulses.
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Several K+ channels
are present in cultured rat microglia.
With the use of gene-specific primers (see Table 1) in RT-PCR, we
amplified cDNAs from two families of K+ channels. As
illustrated in Fig. 3A,
cultured rat microglia expressed mRNA for two Kv1 family members (Kv1.3
and Kv1.5) and for three SK channels (SK2, SK3, and SK4). The identity
of each correct-size band was verified by restriction enzyme digestion
and dideoxy sequencing (data not shown). The amplified portions of SK2
and SK3 were identical in sequence to those regions of cloned SK2 and
SK3 cDNA from rat brain (29), and SK4 was identical to
that from a human placental library and human lymphocytes (26,
28, 33). No transcripts for SK1 were detected, despite
optimizing the RT-PCR conditions by varying the number of cycles,
Mg2+ concentration, and annealing temperatures, whereas
clear bands were obtained from several positive control tissues: rat
brain, cerebellum, kidney, and heart (not shown).
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Specific K+ channel
blockers inhibit the NADPH-dependent respiratory burst.
Phorbol esters stimulate a respiratory burst in microglia (5, 6,
51, 52), thus we exposed cultured rat microglia to PMA and
monitored the respiratory burst as the intracellular green fluorescent
signal produced by rhodamine 123, after reaction of DHR 123 with
hydrogen peroxide (see MATERIALS AND METHODS). For each
trial, 3-5 cells per microscope field were chosen for analysis
using the following criteria: a stable baseline fluorescence level for
5 min after adding DHR 123 (Fig.
5A), the presence of cell
processes before treatment (Fig. 5B, i), and viability at the end of the experiment (ethidium homodimer exclusion). By 45 min
after PMA treatment, there was a 56% increase in respiratory burst
(n = 44 cells, Fig. 5A, paired Student's
t-test), compared with the baseline signal in the absence of
PMA. This is entirely consistent with reports that PMA treatment
increases the rhodamine 123 signal by 40-95% in microglia
(1, 42). Under the same experimental conditions, cells
treated with the inactive analog 4-PMA did not produce a respiratory
burst (n = 10 cells from 2 litters, P < 0.001 compared with PMA). To quantify the NADPH-mediated respiratory
burst, microglia were incubated with the inhibitor diphenylene iodonium
(5 µM) for 5 min before perfusing in 100 nM PMA. The PMA-stimulated
respiratory burst was completely abolished (23 cells from 2 litters, P < 0.0002). In addition to stimulating a
respiratory burst, over the same time course, PMA treatment changed the
morphology of 76% of the microglia examined (31/41 cells; 3 rat
litters). They retracted their processes, flattened, and became more
granular.
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DISCUSSION |
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In the present study, we assessed the expression and roles of K+ channels in the respiratory burst of essentially pure rat microglial cultures. Kv1.3, Kv1.5, SK2, SK3, and SK4 were present at the mRNA level, and Kv1.3, Kv1.5, and SK3 protein were detected. Three biophysically and pharmacologically distinguishable K+ currents were observed, with features similar to Kv1.3, SK4, and SK2 and/or SK3. All of these K+ channels appear to play a significant role in the NADPH-mediated respiratory burst generated by treating microglia with a phorbol ester.
Expression of Kv1.3 and SK channels in cultured rat microglia. Based on patch-clamp electrophysiology, we (4, 31, 46, 57) and others (for a recent review, see Ref. 12) have identified several K+ currents in microglia, including an inward rectifier, a human ether-à-go-go-related gene (HERG)-like current, two types of voltage-gated outward rectifiers (Kv1.3-like and Kv1.5-like), and both large- and intermediate-conductance Ca2+-dependent K+ currents. However, with the exception of Kv1.3 and Kv1.5, there was previously little information on which genes underlie these currents.
Microglia can express different Kv currents, which have been proposed to reflect their activation status. However, there is at present no clear consensus as to which channels correlate with which state. In most studies, cultured microglia express a Kv1.3-like current (12, 38) that is blocked by margatoxin, charybdotoxin (39, 46), or agitoxin-2 (31). This K+ current was present in rat microglia for several days after removing them from astrocyte beds, but disappeared a few days after adding colony-stimulating factor to stimulate proliferation (46), suggesting an inverse correlation with expression and no role in proliferation. However, when hippocampal tissue prints were cultured, nonproliferating microglia expressed a Kv1.5-like current that disappeared and was replaced by a Kv1.3-like current as the microglia proliferated in culture. Both currents were necessary, because K+ channel blockers inhibited proliferation, and the pharmacology of this inhibition correlated with the type of Kv current expressed. Further complications arise because some reports show induction of a Kv1.3-like current by proinflammatory stimuli such as lipopolysaccharide or interferon-A role for K+ channels in the NADPH-dependent respiratory burst. Protein kinase C activation by phorbol esters is a well-characterized stimulus of the respiratory burst in microglia and other phagocytes (6, 51, 52). In this study, when rat microglia were exposed to PMA, there was a significant increase in respiratory burst and a concomitant change in morphology, with retracted cell processes and increased granularity. As in other phagocytes, the respiratory burst was mediated by NADPH because it was abolished by the inhibitor diphenylene iodonium (8, 22). Our results are consistent with the previously reported extent and time course of the PMA-induced respiratory burst (1, 6, 42, 44) and morphological changes, which are characteristic of microglial activation (17, 53, 54). We found that the respiratory burst was inhibited by several K+ channel blockers, each used at a concentration expected to block most specific target channels: 5 nM agitoxin-2 to block Kv1.3, 1.2 nM apamin to block SK2 (with poor block of SK3), 500 nM clotrimazole to block SK4, and 50 nM charybdotoxin to block both Kv1.3 and SK4. Whenever a blocker substantially inhibited the respiratory burst, the microglial cell failed to undergo the morphological changes associated with activation. Thus blocking Kv1.3, SK4, or SK2 channels appears to have prevented microglia activation by PMA. However, "activation" is an extremely broad term, and morphological changes do not necessarily correlate with functional outcomes.
Kv1.3 blockade was less effective than blocking KCa channels, and the apamin-sensitive current was more important than its whole cell amplitude would have predicted. There are several likely explanations. First, patch-clamp analysis suggested that individual cells express different proportions of the different currents (not examined in detail). Kv1.3 current is especially variable in cultured microglia and is active over a restricted range of membrane potentials, thus it is not surprising that its contribution to the respiratory burst is less than the voltage-independent KCa channels. Second, the amplitudes of currents seen in whole cell recordings may differ from those of intact cells (used for respiratory burst assays), particularly if channel activity is maintained by cytoplasmic components that can wash out. We previously observed substantial rundown of SK2-like currents within 15-20 min after excising patches from lymphocytes (35) but not when the patches remained attached (45). Heterologously expressed SK2 channels also ran down after patch excision (24). Finally, the phorbol ester used to trigger the respiratory burst might itself have increased the apamin-sensitive current in intact cells. There is currently no simple model to explain the role of K+ channels in the NADPH-mediated respiratory burst. In principle, K+ channels can act directly by regulating membrane potential and cell volume, but they also act indirectly by affecting ion transport through other channels, exchangers, and pumps. There is scant data on microglia; however, because they are considered to be brain macrophages, some comparison with peripheral macrophages is warranted. Consistent with our results, the broad-spectrum K+ channel blocker TEA inhibited the PMA-stimulated respiratory burst in microglia after a hypoxic/reperfusion treatment (52). Because numerous K+ channel types are blocked by TEA, the specific channel could not be identified. Charybdotoxin dramatically inhibited the respiratory burst stimulated in cultured macrophages by opsonized zymosan (47). Although this supports a role for SK4 channels, a contribution of Kv1.3 could not be ruled out. Kv1.3 is present in macrophages and blocked by charybdotoxin (9). Because blocking K+ channels is expected to depolarize cells, it is worth considering effects of membrane potential on the respiratory burst. Under pathological conditions, such as ischemic injury, there is a substantial rise in extracellular K+ that can depolarize microglia. Colton and colleagues (6) found that the microglial respiratory burst was increased by depolarizing the cells with high K+. Thus it appears that K+ channel blockade differs from artificial depolarization. Some processes associated with the respiratory burst are voltage dependent, such as Ca2+ entry, and this might explain the need for K+ channel activity. A rise in intracellular Ca2+ is important for full activation of the respiratory burst (18). Moreover, protein kinase C can act synergistically with Ca2+ (10) and can also elevate Ca2+ in microglia (7, 56, and our unpublished observations). Ca2+ entry via Ca2+ channels is voltage dependent, either due to depolarization-dependent gating or to voltage determining the driving force. Ca2+ channels have been implicated in the respiratory burst (37, 50); however, most nonexcitable cells, including microglia (21), have Ca2+ channels that are activated by depletion of internal Ca2+ stores (Ca2+ release-activated Ca2+ channels) rather than depolarization. In this case, Ca2+ entry is increased by hyperpolarization (32). A simple model, then, is that K+ channel activity is required to counteract the depolarizing Ca2+ current and to maintain the driving force for Ca2+ influx. For microglia, the model would be that when Ca2+ rises, SK2 and SK4 channels open (voltage-independent gating), and the membrane potential hyperpolarizes and closes the Kv1.3 channels. If the membrane potential and Ca2+ concentration in microglia oscillate, as they do during lymphocyte activation (32), both SK and Kvl.3 channels would be expected to activate alternately and contribute to the respiratory burst. A plausible speculation is that both SK2 and SK4 channels are needed because their activities are regulated differently during the respiratory burst. For instance, reduced internal pH strongly inhibits SK4 channel activity (40). There is some evidence that voltage-gated Ca2+ channels may also be involved, because the PMA-induced respiratory burst was increased by high external K+ (6, 52), an effect potentiated by a Ca2+ channel opener and inhibited by a Ca2+ channel blocker (6). Because some depolarization is required to activate these channels, but depolarization also reduces the driving force for Ca2+ influx, the K+ channels may function to prevent too much depolarization. Nor can we rule out a direct effect of voltage on the release of Ca2+ from internal stores (34). In conclusion, our results support the view that K+ channel activity is important in the respiratory burst of microglia, and we have identified likely candidates for the channels involved in cultured rat microglia. By modulating the ability of microglia to produce reactive oxygen intermediates, these channels may be good targets for therapeutic control of microglia functions that exacerbate brain damage. ![]() |
ACKNOWLEDGEMENTS |
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We thank Brent Clark and Liane Chen for early technical support, and Frank Lee (Dept. of Pharmacology, Univ. of Toronto) for help with confocal imaging. cDNAs were provided by Drs. L. Kaczmarek and W. Joiner at Yale University (rSK1, rSK2, hSK4) and G. Moss (Univ. College, London; rSK3).
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FOOTNOTES |
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This work was supported by Medical Research Council of Canada Grant MOT-13657 (to L. C. Schlichter) and Heart and Stroke Foundation Grant T-3726 (to L. C. Schlichter).
R. Khanna was the recipient of an Ontario Graduate Scholarship and a University of Toronto Santalo Doctoral Fellowship.
Present address of R. Khanna: Dept. of Physiology, UCLA School of Medicine, 53-231 Center for Health Sciences, 10833 Le Conte Ave., Los Angeles, CA 90095-1751.
Present address of L. Roy: AstraZeneca R & D, Boston, 3 Biotech Park, 1 Innovation Dr., Worcester, MA 01605.
Address for reprint requests and other correspondence: L. C. Schlichter, Toronto Western Hospital, 399 Bathurst St., Toronto, Ontario, Canada M5T 2S8 (E-mail: schlicht{at}uhnres.utoronto.ca).
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.
Received 2 August 2000; accepted in final form 30 October 2000.
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