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The Journal of Immunology, 2005, 174: 4736-4744.
Copyright © 2005 by The American Association of Immunologists

Pattern of Kv{beta} Subunit Expression in Macrophages Depends upon Proliferation and the Mode of Activation1

Rubén Vicente2,*, Artur Escalada2,§, Concepció Soler{dagger}, Maribel Grande*, Antonio Celada{ddagger}, Michael M. Tamkun, Carles Solsona§ and Antonio Felipe3,*

* Molecular Physiology Laboratory, Departament de Bioquímica i Biologia Molecular and {dagger} Departament de Fisiologia, Universitat de Barcelona, and {ddagger} Macrophage Biology Group, Biomedical Research Institute of Barcelona, Barcelona, Spain; § Cellular and Molecular Neurobiology Laboratory, Departament de Biologia Cellular i Anatomia Patològica, Universitat de Barcelona-Campus de Bellvitge, Hospitalet de Llobregat, Spain; and Departments of Physiology and Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Voltage-dependent potassium channels (Kv) in leukocytes are involved in the immune response. In bone marrow-derived macrophages (BMDM), proliferation and activation induce delayed rectifier K+ currents, generated by Kv1.3, via transcriptional, translational, and posttranslational controls. Furthermore, modulatory Kv{beta} subunits coassociate with Kv{alpha} subunits, increasing channel diversity and function. In this study we have identified Kv{beta} subunits in mouse BMDM, studied their regulation during proliferation and activation, and analyzed K+ current parameters influenced by these proteins. BMDM express all isoforms of Kv{beta}1 (Kv{beta}1.1, Kv{beta}1.2, and Kv{beta}1.3) and Kv{beta}2 (Kv{beta}2.1), but not Kv{beta}4, the alternatively spliced murine Kv{beta}3 variant. M-CSF-dependent proliferation induced all Kv{beta} isoforms. However, LPS- and TNF-{alpha}-induced activation differentially regulated these subunits. Although LPS increased Kv{beta}1.3, reduced Kv{beta}1.2, and maintained Kv{beta}1.1 mRNA levels constant, TNF-{alpha} up-regulated Kv{beta}1.1, down-regulated Kv{beta}1.2, and left Kv{beta}1.3 expression unchanged. Moreover, in contrast to TNF-{alpha}, M-CSF- and LPS- up-regulated Kv{beta}2.1. K+ currents from M-CSF- and LPS-stimulated BMDM exhibited faster inactivation, whereas TNF-{alpha} increased {tau} values. Although in M-CSF-stimulated cells the half-inactivation voltage shifted to more positive potentials, the incubation with LPS and TNF-{alpha} resulted in a hyperpolarizing displacement similar to that in resting BMDM. Furthermore, activation time constants of K+ currents and the kinetics of the tail currents were different depending upon the mode of activation. Our results indicate that differential Kv{beta} expression modifies the electrical properties of Kv in BMDM, dependent upon proliferation and the mode of activation. This could determine physiologically appropriate surface channel complexes, allowing for greater flexibility in the precise regulation of the immune response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The activation and proliferation of cells in the immune system are modulated by membrane transduction of extracellular signals. Some interactions occur via the regulation of transmembrane ion fluxes, and several studies suggest that some signaling occurs through ion movements in macrophages. Changes in the membrane potential are among the earliest events after stimulation of macrophages, and ion channels underlie the Ca2+ signal involved in the leukocyte activation (1). Potassium channels indirectly determine the driving force for Ca2+ entry (2). The resting membrane potential in leukocytes is about –50 to –60 mV, and these channels serve to hyperpolarize the membrane even further to –80 mV (2). This hyperpolarization accentuates Ca2+ influx to promote Ca2+-dependent signal transduction pathways that depend upon the activity of ion channels (1, 2, 3, 4).

Voltage-dependent potassium channels (Kv) 4 play a crucial role in excitable cells by determining resting membrane potential and controlling action potentials (5). In addition, they are involved in the activation and proliferation of leukocytes (1, 2, 3, 4). The mammalian Shaker family (Kv1) contains at least eight different genes (Kv1.1 to Kv1.8) that can form functional homo- and heterotetrameric complexes (6). Thus, Kv1 channels can assemble promiscuously, yielding a wide variety of biophysically and pharmacologically distinct channels (5). In addition, assigning specific K+ channel clones to native currents is often difficult, because this complexity is enhanced by the presence of Kv{beta} regulatory subunits (7). Up to four different forms of these cytoplasmic proteins can form part of the heteromeric structure ({alpha}4{beta}n). Different Kv{beta} genes, namely Kv{beta}1, Kv{beta}2, and Kv{beta}3 generate up to six different Kv{beta} proteins. Although Kv{beta}1.1, Kv{beta}1.2, and Kv{beta}1.3 are alternatively spliced forms from the Kv{beta}1gene, Kv{beta}2.1 is the only variant known to be generated from the Kv{beta}2 gene (7). In addition, Kv{beta}3 generates two alternatively spliced isoforms, Kv{beta}3 and Kv{beta}4, expressed in rat and mouse brain, respectively (8, 9). All subunits are ~300 aa in length and share a common conserved core (>85% amino acid identity), with the highest degree of variability in the N termini. Their association with the Kv{alpha} subunit occurs via the conserved C-terminal end. Furthermore, Kv{beta} regulatory subunits generally assemble with channels from the Kv1 family (7).

We previously described the expression of Kv1.3 and Kv1.5 in bone marrow-derived macrophages (BMDM) (10). Macrophage K+ channels are tightly regulated during M-CSF-dependent proliferation, and LPS- or TNF-{alpha}-induced activation and their functional activity are important for cellular responses. Proliferation and activation induce outward K+ currents under transcriptional and translational control. Several lines of evidence led to the conclusion that LPS-induced activation regulates Kv via TNF-{alpha}-dependent and -independent mechanisms (10). In addition, our results indicate that posttranslational events are involved in the differential Kv regulation in response to different stimuli (10). Kv{beta} subunits confer rapid inactivation, alter current amplitude and gating, and promote Kv cell surface expression. In fact, both Kv1.3 and Kv1.5 are able to assemble with Kv{beta} subunits to form functional Kv channels, increasing the variety of electrical responses (11, 12, 13). Nevertheless, only a few studies have been undertaken to identify Kv{beta} subunits expressed in immune system cells. Thus, F5, an IL-2-induced cDNA in Th lymphocytes, turned out to be the Kv{beta}2.1 subunit (14). In addition, Kv{beta}1.1 and Kv{beta}2.1 subunits are up-regulated during mitogen-stimulated activation of mouse T cells (15). Furthermore, the heterologous expression of Kv1.3 and Kv1.5 together with Kv{beta} subunits in Xenopus oocytes and mammalian cells, respectively, dramatically modifies the rate of inactivation and the amplitude of the K+ current (12, 13).

In this study we describe, for the first time, the expression of Kv{beta} subunits that may coassemble with Kv1.3 in BMDM to generate different functional Kv channel complexes. Macrophages express all isoforms of Kv{beta}1 (Kv{beta}1.1, -1.2, and -1.3) and Kv{beta}2 (Kv{beta}2.1). In contrast, Kv{beta}4, the murine isoform of Kv{beta}3 was absent. Although M-CSF-dependent proliferation increased the expression of all Kv{beta} subunits, it was differentially regulated by LPS- and TNF-{alpha}-induced activation. Kv{beta} subunits triggered a finely tuned modulation of macrophage electrical activity dependent upon proliferation and mode of activation. These data suggest that by changing the heteromeric Kv channel structure, macrophages could physiologically set the membrane potential to achieve precise immune responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Isolation of BMDM and cell culture

BMDM from 6- to 10-wk-old BALB/c mice (Charles River Laboratories) were isolated and cultured as described previously (16). Briefly, animals were killed by cervical dislocation, and both femurs were dissected. The ends of the bones were removed, and the marrow tissue was flushed out by irrigation with DMEM. Once dispersed by passing through a 25-gauge needle, the cells were cultured in plastic dishes (150 mm) in DMEM containing 20% FBS and 30% L cell supernatant of L-929 fibroblast (L cell)-conditioned medium as a source of M-CSF and kept at 37°C in a humidified 5% CO2 atmosphere. Macrophages were obtained as a homogeneous population of adherent cells after 7 days of culture. For experiments, they were cultured with the same tissue culture differentiation medium (DMEM, 20% FBS, and 30% L cell medium) or arrested at G0 by M-CSF deprivation in DMEM supplemented with 10% FBS for at least 18 h (basal). Resting (G0-arrested) cells were further incubated in the absence or the presence of recombinant murine M-CSF (1200 U/ml), with or without LPS (100 ng/ml) or TNF-{alpha} (100 ng/ml), for the indicated times. All animal handling was approved by the ethics committee of University of Barcelona in accordance with European Union regulations.

HEK-293 cells that lack expression of Kv{beta} subunits were used as negative controls and cultured as described previously (17).

RNA isolation and RT-PCR analysis

Total RNA from mouse macrophages, brain, and liver was isolated using the Tripure reagent (Roche), according to the manufacturer’s instructions. Samples were also treated with the DNA-free kit from Ambion to remove DNA.

Ready-to-Go RT-PCR Beads (Amersham Biosciences) were used in a one-step RT-PCR as described previously (18). Total RNA and selected primers (1 µM) were added to the beads. The reverse transcription reaction was initiated by incubating the mixture at 42°C for 30 min. Once first-strand cDNA had been synthesized, the conditions were set for additional PCR: 92°C for 30 s, either 55°C (Kv1.3, Kv1.5, Kv{beta}1.2, and 18S) or 60°C (rest of Kv{beta}) for 1 min, and 72°C for 2 min. These settings were applied for 40 cycles. After every 10 cycles, 10 µl of the total reaction were collected in a separate tube for electrophoresis and further analysis. A range of RNA dilutions from each independent sample was performed to obtain an exponential phase of amplicon production (data not shown), as described previously (19). The same independent RNA aliquot was used to analyze mRNA expression and the respective amount of 18S rRNA. In all cases negative controls were performed in the absence of the reverse transcription reaction. Because Kv{beta} isoforms have a highly homologous core, we designed specific primers to the N terminus for each subunit. Primer sequences, accession numbers, and cDNA lengths are shown in Table I.


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  		Table I. Genes, accession numbers, and sequences of oligonucleotide primers, base pairs, and cDNA length

 
Once the exponential phase of amplicon production had been determined the specificity of each product was confirmed in test RT-PCRs using the appropriate cDNA probe in a Southern blot analysis. PCR-generated cDNA probes from mouse brain were subcloned using the pSTBlue-1 acceptor vector kit (Novagen), and the sequences were confirmed using the Big Dye Terminator Cycle Sequencing kit and an ABI 377 sequencer (Applied Biosystems). EcoRI-digested [{alpha}-32P]CTP random primer-labeled cDNAs were used as probes, as described previously (19). At least three different filters were prepared from independent samples, and representative blots are shown. Results were analyzed with Phoretix software (Nonlinear Dynamics).

Preparation of crude membrane fractions from BMDM and Western blot analysis

Crude membrane preparations were obtained as described previously (20). Macrophages and HEK-393 cells were washed twice in cold PBS and lysed on ice with lysis solution containing 0.32 M sucrose, 5 mM Na2HPO4, and the following protease inhibitors: 0.31 mg/ml benzamidine, 0.62 mg/ml N-ethylmaleimide, 1 mg/ml bacitracin (Sigma-Aldrich), 1 µg/ml pepstatin, 1 µg/ml leupeptin, and 0.07 µg/ml Pefablock (Roche). Brain samples were also homogenized in the same lysis solution in a glass homogenizer. Lysates were centrifuged at 4°C at 3000 rpm for 10 min to remove large debris and nuclei. The supernatant was collected and further centrifuged for 1 h at 4°C at 15,000 rpm. The resulting membrane pellet was resuspended in ice-cold PBS and stored at –80°C. The sample protein concentration was determined by Bio-Rad protein assay. Crude membrane proteins (25 µg) were boiled at 95°C in Laemmli SDS-loading buffer and separated by 10% SDS-PAGE. They were transferred to nitrocellulose membranes (Immobilon-P; Millipore) and blocked in 5% dry milk-supplemented 0.2% Tween 20 PBS before immunoreaction. To monitor Kv{beta} subunits, the following Abs were donated by Dr. J. Trimmer (University of California, Davis, CA): anti-Kv{beta}1.1 rabbit polyclonal, anti-Kv{beta}1.2 rabbit polyclonal, anti-pan Kv{beta} rabbit polyclonal, and anti-Kv{beta}2.1 mouse monoclonal (11, 21, 22, 23). The anti-Kv{beta}1.3 rabbit polyclonal Ab was produced and characterized in the Tamkun laboratory (20). An anti-{beta}-actin mAb (Sigma-Aldrich) was used as a loading and transfer control.

Electrophysiological recordings

Whole-cell currents were measured using the patch-clamp technique. An EPC-9 (HEKA) with the appropriate software was used for data recording and analysis. Currents were filtered at 2.9 kHz. Patch electrodes of 2–4 MOhm were produced from borosilicate glass (outer diameter, 1.2 mm; inner diameter, 0.94 mm; Clark Electromedical Instruments) with a P-97 puller (Sutter Instruments). Electrodes were filled with the following solution: 120 mM KCl, 1 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, 11 mM EGTA, and 20 mM D-glucose, adjusted to pH 7.3 with KOH. The extracellular solution contained the following: 120 mM NaCl, 5.4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, and 25 mM D-glucose, adjusted to pH 7.4 with NaOH. After establishing a whole-cell configuration, macrophages were clamped to a holding potential of –60 mV, with seal resistances of at least 2.5 GOhm. All recordings were routinely subtracted for leak currents online. Only cells with a series resistance compensation of 80–90% were selected for analysis. Uncompensated series resistances were 4–8 MOhm, as currents evoked were <1 nA; voltage errors from uncompensated series resistance were <2 mV.

To evoke voltage-dependent currents, cells were stimulated with specific square pulses ranging from –60 to +50 mV in 10-mV steps. Protocols are detailed in the figures.

To pharmacologically characterize the voltage-dependent outward K+ current, rMargatoxin (rMgTx) and ShK-Dap22 were added to the external solution (24, 25). Toxins were reconstituted at 10 µM in Tris buffer (0.1% BSA, 100 mM NaCl, and 10 mM Tris, pH 7.5). All recordings were performed at room temperature (20–23°C).

Analysis and statistics

According to the solutions used, the calculated equilibrium potential for potassium was –79 mV (EK) using the Nernst equation. The normalized G/Gmax vs the voltage curve was fitted using Boltzmann’s equation: G/Gmax = 1/(1 + exp((V1/2 – V/k)), where V1/2 is the voltage at which the current is half-activated, and k is the slope factor of the activation curve.

Steady-state inactivation plots were fitted to a Botlzmann equation as follows: I/Imax = 1/(1 – exp((V1/2 – V/k)).

Activation, inactivation, and deactivation time constants were adjusted to single exponential functions: I(t) = A(1 – exp(–t/{tau})) for activation {tau}, calculated from the end of the compensated capacitive transient to the peak of the current and I(t) = Aexp(–t/{tau}) for inactivation and deactivation {tau}. Inactivation adjustment was calculated from the peak of the current to the steady-state inactivation, and deactivation was fitted from the peak of the tail current to steady state; traces were fitted with SigmaPlot (SPSS).

Values are expressed as the mean ± SEM. The significance of the differences was established either by Student’s t test or one-way ANOVA (PRISM 3.0; GraphPad) for either two-group or two-factor comparisons, respectively. Where indicated, a Tukey post hoc test was performed. A value of p < 0.05 was considered significant.

Reagents

Recombinant murine TNF-{alpha} was obtained from PeproTech EC; recombinant murine M-CSF was purchased from R&D Systems; LPS was purchased from Sigma-Aldrich; rMgTx was obtained from Alomone Laboratories; ShK-Dap22 was purchased from Bachem; other reagents were of analytical grade.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Delayed rectifier K+ currents were evoked in BMDM by depolarizing pulses (Fig. 1A) as described previously (10). Resting macrophages (n = 40) exhibited an outward conductance with ~40 pA peak current amplitude. M-CSF enhanced K+ currents ~2-fold (~90 pA; n = 120). Both LPS and TNF-{alpha} further increased the current amplitude (~600 and ~350 pA, respectively; n = 60). Resting and M-CSF-treated BMDM showed similar voltage dependence (Fig. 1B). Channels opened at depolarizing potentials (–40 to –30 mV) with V1/2 values of –12.2 ± 1.3 and –12.4 ± 2.1 mV and k slopes of 20.5 ± 2.5 and 22.0 ± 3.0 mV for resting and M-CSF-treated macrophages, respectively (n = 30). Macrophages incubated with LPS and TNF-{alpha} were activated (10), and the steady-state activation curve of normalized conductances changed substantially (Fig. 1B). Although channels were open at the same depolarizing pulse potentials with V1/2 values similar to those observed with M-CSF (–14.3 ± 3.4 and –15.1 ± 3.1 mV for LPS and TNF-{alpha}, respectively; n = 12), k slope values were significantly different (9.6 ± 1.6 and 9.1 ± 2.2 mV for LPS and TNF-{alpha}, respectively; p < 0.001 vs M-CSF, by Student’s t test; n = 12). RT-PCR experiments (Fig. 1C) showed that Kv1.3 and Kv1.5 mRNAs are present in brain and BMDM but not in liver. Moreover, the MgTx- and ShK-Dap22-mediated inhibition of K+ currents evoked in response to different stimuli (Fig. 1D) suggested that outward conductances were principally mediated by Kv1.3, as demonstrated previously (10).



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  		FIGURE 1. M-CSF, LPS, and TNF-{alpha} increase delayed rectifier K+ currents generated by Kv1.3 in macrophages. A, Representative traces of delayed rectifier K+ currents. Resting cells were incubated for 24 h in the absence (basal) or the presence of M-CSF, with or without LPS or TNF-{alpha}. Outward traces were elicited by a depolarizing step as indicated. B, Steady-state activation curves. Cells were held at –60 mV, and pulse potentials were applied from –60 to +50 mV in 10-mV steps of 200 ms. Conductances were normalized to the peak current density at +50 mV. {circ}, basal; •, M-CSF; {square}, LPS; {blacksquare}, TNF-{alpha}. C, Macrophages express Kv1.3 and Kv1.5. One microgram of total RNA from mouse brain, macrophages, and liver was used in RT-PCRs as described in Materials and Methods. D, Inhibition of K+ currents by MgTx and ShK-Dap22. Currents were elicited in BMDM as described in A. The percentage of peak current was calculated by comparing the current at a given concentration of toxin vs that obtained in its absence. Concentrations of 50 and 500 pM toxin were selected to block the peak current either partially (~50%) or totally (~100%), respectively. {blacksquare}, MgTx; {square}, ShK-Dap22.

 
The oligomeric structure of the Kv complex is a critical determinant of electrical activity in mammalian neurons (26). Because modulation by accessory {beta} subunits has dramatic consequences on Kv1 functional activities (7), we studied the expression of Kv{beta} proteins in crude membrane preparations using several specific Abs. Unfortunately, none of the anti-Kv{beta}1 subunit-specific Abs produced a reliable signal (data not shown). Only the polyclonal anti-pan-{beta} subunit and the monoclonal anti-Kv{beta}2.1 were useful in our BMDM samples. In macrophages, the anti-pan-{beta} subunit Ab recognizes two polypeptide species of ~38 and ~41 kDa, similar to those described by Trimmer and collegues (11, 22) in the brain. Kv{beta}2.1 protein corresponds to the lower band, and the higher band corresponds to Kv{beta}1 peptides sharing a common core (Fig. 2A). HEK-293 cells were negative. Although M-CSF and LPS induced Kv{beta}1 and Kv{beta}2.1 expression in macrophages, TNF-{alpha} did not. The anti-Kv{beta}2.1 mAb gave similar results (data not shown).



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  		FIGURE 2. Macrophages express Kv{beta} subunits. A, Protein expression of Kv{beta} subunits in response to different stimuli in BMDM. Crude membrane samples were obtained from mouse brain, HEK-293 cells, and BMDM as described in Materials and Methods. Brain and HEK samples were used as positive and negative controls, respectively. Expression of {beta}-actin was used as a loading control. Representative Western blot analyses are shown in the upper panels. Lower panels, The densitometric analysis of the data. Values expressed as arbitrary units are the mean ± SEM of three independent experiments. B, Kv{beta} subunit mRNA expression in brain, macrophages, and liver from mouse. One-step RT-PCR was performed on 1 µg of total RNA, and amplicons were electrophoresed and analyzed as described in Materials and Methods. Representative filters are shown.

 
To further determine Kv{beta} expression in BMDM, we designed oligonucleotides to amplify the N-terminal domain for each subunit and generated specific Kv{beta} cDNA probes (Table I). To control for undesirable cross-hybridizations, each Kv{beta} fragment was run on a 2% agarose gel and further hybridized against the rest of the Kv{beta} cDNAs. No signal was detected other than that expected with the appropriate specific Kv{beta} probe (data not shown). Fig. 2B demonstrates that BMDM expressed several Kv{beta} isoforms similar to the brain. All splice variants of the Kv{beta}1 gene (Kv{beta}1.1, Kv{beta}1.2, and Kv{beta}1.3) were detected, as well as the only Kv{beta}2 gene product described to date, Kv{beta}2.1. In contrast, the murine isoform Kv{beta}4, identified in brain as a spliced mRNA from Kv{beta}3, was absent in BMDM. Liver was used as a negative control.

The addition of M-CSF to resting macrophages triggers cell growth (10, 16), and under these conditions the expression of Kv{beta} subunits was induced (Fig. 3A). The three splice variants of the Kv{beta}1 gene (Kv{beta}1.1, Kv{beta}1.2, and Kv{beta}1.3) followed the same pattern, with similar values. Thus, the expression peaked after 6 h (~3-fold induction), and remained high throughout treatment (p < 0.001, by ANOVA). In contrast, Kv{beta}2.1 increased steadily throughout the study, and the level of induction after 24 h of incubation was ~5-fold (p < 0.001, by ANOVA).



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  		FIGURE 3. M-CSF induces Kv{beta} subunit mRNA expression and modulates K+ current inactivation kinetics in macrophages. A, M-CSF induces Kv{beta} subunit mRNA in macrophages. One-step RT-PCR was performed and analyzed as described in Materials and Methods. Samples were collected at different times after the addition of M-CSF. Total RNA concentration and PCR cycles were as follows: Kv{beta}1.1 and Kv{beta}1.2, 1 µg and 40 cycles; Kv{beta}1.3, 0.1 µg and 30 cycles; Kv{beta}2.1, 0.5 µg and 20 cycles; and 18S, 0.1 µg and 10 cycles. These conditions represent the exponential phase of amplicon production. Representative filters are shown. Values expressed as arbitrary units (AU) are the mean ± SEM of four independent experiments. Significant differences were found with Kv{beta}1.1, Kv{beta}1.2, Kv{beta}1.3, and Kv{beta}2.1 (p < 0.001, by ANOVA). B, Plot of inactivation time constant vs test potential. Cells were incubated for 24 h in the presence (•) or the absence ({circ}) of M-CSF. Macrophages were held at –60 mV, and pulse potentials were applied from –60 to +50 mV in 10-mV steps of 4 s. The inset illustrates current traces at +50 mV for comparison. C, Steady-state inactivation levels of K+ currents in the presence (•) or the absence ({circ}) of M-CSF. Cells were treated as described in B. Pulse protocols and current traces for both situations are depicted on the right. Currents were measured by varying the holding potential from –50 to 40 mV over 4 s, then applying a 200-ms test pulse to 50 mV. To ensure complete recovery from inactivation, cells were repolarized for 45 s at –60 mV. Values are the mean ± SEM of at least four independent cells.

 
When Kv{beta} subunits are coexpressed heterologously with Kv{alpha}, proteins modulate the inactivation properties of Kv (7). The K+ current inactivation time constants from BMDM incubated with M-CSF were slightly lower than those in its absence, but showed similar voltage dependence (Fig. 3B). The normalized steady-state inactivation indicated that incubation with the growth factor resulted in a 10-mV positive displacement in the V1/2 (–19.6 ± 0.6 and –10.0 ± 0.8 mV for without M-CSF and with M-CSF, respectively; p < 0.001, by Student’s t test) without changes in the k slope (–6.0 ± 0.7 and –6.9 ± 1.0 mV for without M-CSF and with M-CSF, respectively).

LPS and TNF-{alpha} activate macrophages, leading to cell growth arrest (10, 27). LPS-induced activation differentially regulated Kv{beta} subunits in BMDM (Fig. 4A). Under these conditions, the three splice variants of the Kv{beta}1 gene showed different expression patterns. Although Kv{beta}1.1 mRNA remained almost constant throughout the study (not significant by ANOVA), both an additional Tukey post hoc test and Student’s t test indicated that Kv{beta}1.1 levels were statistically higher at 6 h of incubation (1.0 ± 0.1 vs 1.7 ± 0.2 for 0 and 6 h, respectively; p < 0.05; n = 4). Kv{beta}1.2 expression decreased during endotoxin treatment (p < 0.001, by ANOVA). In contrast, soon after LPS incubation, Kv{beta}1.3 mRNA increased (p < 0.001, by ANOVA). In addition, Kv{beta}2.1 mRNA increased steadily throughout the study (p < 0.001, by ANOVA). Inactivation of K+ currents in macrophages incubated with or without LPS showed similar voltage dependence (Fig. 4B). However, the presence of the endotoxin further decreased {tau} inactivation values. Fig. 4C shows that in LPS-treated cells, the half-inactivation voltage shifted in the negative direction by 10 mV, similar to that obtained with nonproliferating BMDM (–10.0 ± 1 and –22.0 ± 1 mV for without LPS and with LPS, respectively; p < 0.01, by Student’s t test) without changes in k slope (–6.9 ± 1.0 and –6.6 ± 0.9 mV for without LPS and with LPS, respectively).



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  		FIGURE 4. Differential regulation of Kv{beta} isoforms and functional analysis of K+ current inactivation in LPS-treated macrophages. A, BMDM incubated with M-CSF were treated with LPS, and RNA samples were collected at different times before RT-PCR analysis, performed as described in Materials and Methods. Total RNA concentration and PCR cycles were as follows: Kv{beta}1.1 and Kv{beta}1.2, 1 µg and 40 cycles; Kv{beta}1.3, 0.1 µg and 30 cycles; Kv{beta}2.1, 0.5 µg and 20 cycles; and 18S, 0.1 µg and 10 cycles. These conditions represent the exponential phase of amplicon production. Representative filters are shown. Arbitrary unit (AU) values are the mean ± SEM of four independent experiments. Significant differences were found with Kv{beta}1.2, Kv{beta}1.3, and Kv{beta}2.1 (p < 0.001, by ANOVA) and Kv{beta}1.1 at 6 h vs 0 h (p < 0.05, by Student’s t test and ANOVA plus Tukey post hoc test). B, Plot of inactivation time constant vs test potential. Macrophages were incubated with M-CSF for 24 h in the presence (•) or the absence ({circ}) of LPS. Cells were held at –60 mV, and pulse potentials were applied from –60 to +50 mV in 10-mV steps of 4 s. The inset illustrates current traces at +50 mV for comparison. C, Steady-state inactivation levels of K+ currents in the presence (•) or the absence ({circ}) of LPS. Cells were treated as described in B. Pulse protocols and current traces for both situations are depicted on the right. Currents were measured by varying the holding potential from –50 to 40 mV over 4 s, then applying a 200-ms test pulse to 50 mV. To ensure complete recovery from inactivation, cells were repolarized for 45 s at –60 mV. Values are the mean ± SEM of at least four independent cells.

 
Several lines of evidence indicate that LPS exerts Kv regulation via TNF-{alpha}-dependent and -independent mechanisms (10, 28). Therefore, we analyzed the effects of TNF-{alpha} on the expression of Kv{beta} subunits (Fig. 5A). TNF-{alpha} induced an up-regulation of Kv{beta}1.1 mRNA (p < 0.001, by ANOVA), Kv{beta}1.2 expression decreased steadily throughout the study (p < 0.001, by ANOVA), and Kv{beta}1.3 and Kv{beta}2.1 remained constant. These results indicate that in contrast to Kv1.3 and other plasma membrane proteins (10, 27, 28), LPS-mediated Kv{beta} subunit regulation is TNF-{alpha} independent in BMDM. Inactivation time constants of K+ currents evoked in macrophages incubated with TNF-{alpha} were higher than those in its absence, with a significant change in the pattern (Fig. 5B). The half-inactivation voltage in TNF-{alpha}-stimulated cells shifted to negative potentials, similar to those obtained with nonproliferating macrophages in the absence of M-CSF (see above). Thus, V1/2 values were –10.0 ± 1 and –22.2 ± 1 mV for without TNF-{alpha} and with TNF-{alpha} respectively (p < 0.001, by Student’s t test), and k values were –6.9 ± 1.0 and –7.0 ± 1.0 mV for without TNF-{alpha} and with TNF-{alpha}, respectively (Fig. 5C).



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  		FIGURE 5. TNF-{alpha} differentially regulates Kv{beta} subunit mRNAs and alters the biophysical properties of K+ current inactivation. A, BMDM incubated with M-CSF were treated with TNF-{alpha}, and RNA samples were collected at different times before RT-PCR analysis, performed as described in Materials and Methods. Total RNA concentration and PCR cycles were as follows: Kv{beta}1.1 and Kv{beta}1.2, 1 µg and 40 cycles; Kv{beta}1.3, 0.1 µg and 30 cycles; Kv{beta}2.1, 0.5 µg and 20 cycles; and 18S, 0.1 µg and 10 cycles. These conditions represent the exponential phase of amplicon production. Representative filters are shown. Values are the mean ± SEM of four independent experiments. Significant differences were found with Kv{beta}1.1 and Kv{beta}1.2 (p < 0.001, by ANOVA). B, Plot of inactivation time constant vs test potential. Macrophages were incubated with M-CSF for 24 h in the presence (•) or the absence ({circ}) of TNF-{alpha}. Cells were held at –60 mV, and pulse potentials were applied from –60 to +50 mV in 10-mV steps of 4 s. The inset illustrates current traces at +50 mV for comparison. C, Steady-state inactivation levels of K+ currents in the presence (•) or the absence ({circ}) of TNF-{alpha}. Cells were treated as described in B. Pulse protocols and current traces for both situations are depicted on the right. Currents were measured by varying the holding potential from –50 to 40 mV over 4 s, then applying a 200-ms test pulse to 50 mV. To ensure complete recovery from inactivation, cells were repolarized for 45 s at –60 mV. Values are the mean ± SEM of at least four independent cells.

 
In addition to inactivation, accessory Kv{beta} subunits modulate K+ current kinetics by altering activation and deactivation time constants (7). Activation time constants showed voltage dependence (Fig. 6, A–E). However, although LPS exhibited a voltage dependence similar to that obtained with resting and M-CSF-treated macrophages, TNF-{alpha} increased {tau} values at negative potentials (–20 to 0 mV; p < 0.05 vs M-CSF, by ANOVA Tukey post hoc test and Student’s t test; n = 4; Fig. 6A). Analysis of the deactivation time constant was performed as a function of the presence of a tail current. Tail currents were only apparent when cells were treated with activating agents, but not in resting or proliferating macrophages (Fig. 6, F–H). Deactivation time constants (Fig. 6F) and normalized tail currents obtained upon repolarization from +50 to –50 mV (Fig. 6G), indicated that K+ currents deactivated faster in LPS-activated BMDM ({tau} = 26 ms) than in TNF-{alpha}-stimulated cells ({tau} = 140 ms). Furthermore, tail current amplitudes were significantly different upon the mode of activation (Fig. 6H).



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  		FIGURE 6. Functional analysis of activation and deactivation kinetics in BMDM in response to different stimuli. Macrophages were cultured in the absence of M-CSF for 18 h, then G0-arrested cells were further incubated for 24 h in the absence (basal) or the presence of M-CSF, with or without LPS and TNF-{alpha}. A, Voltage dependence of the activation time constant in BMDM. Currents were evoked as described. B–E, Representative families of currents at –20 and 50 mV. Traces have been normalized for detail. F, Plot of deactivation time constant vs repolarizing pulses in BMDM incubated for 24 h with M-CSF in the presence of LPS (•) and TNF-{alpha} ({circ}). Cells were held at –60 mV over 5 s, and a 50-mV pulse of 200 ms was applied, then stepped from –60 to –30 mV over 1 s. G, Representative normalized tail currents obtained upon repolarization from +50 to –50 mV in the presence of LPS and TNF-{alpha}. Note that the deactivation is slower in the presence of TNF-{alpha} ({tau} = 140 ms) compared with LPS ({tau} = 26 ms). H, Tail current amplitude vs repolarizing pulse in BMDM treated with LPS (•) and TNF-{alpha} ({circ}). Pulse protocols are described in F. Values are the mean ± SEM of at least four independent cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Proliferation and activation regulate Kv expression in macrophages via transcriptional, translational, and posttranslational controls (10). The outward K+ current is mainly generated by Kv1.3. However, similar to brain macrophages, a role for Kv1.5 should not be discounted in BMDM. In fact, we observed that K+ current kinetics in macrophages are significantly different from those described in T cells and heterologous expression systems. Activation and inactivation of Kv1.5 exhibit more depolarized potentials than Kv1.3 currents (29). Heteromeric formation of K+ channels has been appreciated as a mechanism to increase channel functional diversity, and hybrid channels show a mixture of characteristics of homomeric subunits. Because Kv1.3 and Kv1.5 assemble in macrophages (R. Vicente and A. Felipe, unpublished observations), Kv1.3/Kv1.5 heterotetramers may explain these discrepancies.

We report, for the first time, that macrophages express Kv{beta} subunits from Kv{beta}1 and Kv{beta}2, but not Kv{beta}3. This expression is similar to that in muscle, but different from that in neural tissues, because Kv{beta}3 and Kv{beta}4 have only been described in brain (8, 9, 19). This represents another mechanism for regulating K+ current diversity in BMDM. M-CSF-dependent proliferation does not modify steady-state activation kinetics, but increases current amplitude concomitant with an up-regulation of all Kv{beta} subunits. However, LPS and TNF-{alpha}, which further increased current amplitude, reduced the k slope factor. This could be in agreement with changes in gating (30, 31). The expression level, gating, and conductance properties of expressed channels are profoundly influenced by the presence of auxiliary subunits (7, 17, 30, 31). Coexpression of Kv{beta}1.1 and Kv{beta}1.2 accelerate the rate of inactivation of K+ currents arising from Kv1 channels (32, 33, 34). However, Kv{beta}2 exhibits little modulation of fast inactivation and mostly facilitates surface expression (7, 35). The degree of channel inactivation correlates with the charge density of the N terminus of Kv{beta}1 subunits (34). In this context, it is surprising that, in contrast to M-CSF-dependent proliferation, Kv{beta}1 gene products are clearly differentially regulated in BMDM upon the mode of activation. Thus, distinct Kv{beta}1 N-terminal domains could be responsible for conferring the heterogeneity of the modulatory activity that we observed.

We found that, in contrast to TNF-{alpha}, K+ current inactivation in M-CSF- and LPS-stimulated cells showed similar voltage dependence. However, although resting and M-CSF-treated macrophages exhibit inactivation time constants of between 250 and 400 ms at positive potentials, Kv1.3 currents in LPS-stimulated cells clearly inactivate much faster ({tau} = 100–200 ms). Kv1.3 expressed in HEK 293 cells, in the absence of Kv{beta} subunits, inactivates much more slowly ({tau} = 1800 ms at +40 mV) (36). In contrast, in T lymphocytes, which express Kv{beta} proteins (15), Kv1.3 inactivates with a time constant of 150–200 ms at +40mV (37), similar to that in LPS-stimulated BMDM. However, this could be as a result of the model because human T cells were activated with PHA A to increase K+ channel expression (37, 38). Thus, Kv1.3 in BMDM and T cells inactivates faster than in HEK cells, suggesting a role for Kv{beta} subunits in the leukocyte membrane excitability. In addition, Kv currents in M-CSF-stimulated cells exhibited a shift in V1/2 inactivation to more positive potentials, but upon activation, values returned to more negative potentials, which are characteristic of K+ currents in nonproliferating cells.

Macrophage activation also led to important modifications in K+ current activation kinetics and tail currents. These parameters indicated enormous differences between proliferation and the mode of activation and are in agreement with the cell growth arrest triggered by LPS and TNF-{alpha} (10, 27). Our results are in agreement with those described when Kv1 proteins are coexpressed with individual Kv{beta} subunits in heterologous systems (32, 33, 34, 35, 39, 40). A possible interpretation, taking into account the fact that M-CSF and LPS both induce Kv{beta}2.1, is that Kv{beta}2.1 inhibits Kv{beta}1-mediated inactivation and promotes Kv surface expression (35, 41, 42). In fact, Kv{beta}2 subunits self-associate, presumably forming a tetramer (43). This suggests that in the presence of M-CSF and LPS, the heteromeric structure of the channel may contain mainly Kv{beta}2.1 subunits. This structure would highlight the complex scenario in which the insertion of Kv{beta}2.1 could change the electrical properties of the Kv channel, leading to multiple physiological effects on the mode of activation.

{alpha}{beta} interactions may be additionally influenced by differential posttranslational modifications. Kv{beta} subunits are oxidoreductases that are under oxidative stress and protein kinase regulation (7, 30, 44). In contrast to that by M-CSF, LPS and TNF-{alpha} regulation activates different signal transduction pathways involving Ser/Thr protein kinases (45, 46, 47). In this context, the inactivation conferred on Kv1.3 by Kv{beta}1 could be modulated not only by {alpha} subunit phosphorylation, but also by phosphorylation of the {beta} subunit itself, which is known to regulate {beta}-mediated effects (7). Thus, in the Kv{beta}-negative HEK-293 cell line, Kv1.3 sensitivity to protein kinase C and protein kinase A is significantly reduced, in contrast to observations in Jurkat T cells (36). Moreover, protein kinase C is required for Kv{beta}1.3 effects on Kv1.5 (48), and protein kinase A reduces Kv{beta}1.3-induced activation of Kv1.5 (49). This is in agreement with our data in which LPS, but not TNF-{alpha}, induced Kv{beta}1.3 and decreased the inactivation time constant. Furthermore, LPS induced Kv{beta}2.1 in BMDM, and in fibroblasts the presence of this regulatory subunit resulted in a hyperpolarizing displacement of the normalized inactivation curve similar to that we observed in macrophages (17). Moreover, high levels of phosphorylation and Kv{beta}2.1 up-regulation could trigger inhibition of the Kv{beta}1-induced inactivation, increasing current amplitudes. In addition, Kv{beta}1.2-mediated inactivation is not altered by protein kinase A activation (48), and macrophage activation by LPS and TNF-{alpha} triggered a notable decrease in Kv{beta}1.2 levels. In fact, this could be related to the decrease in peak current amplitudes of Kv1 subunits in whole-cell and macropatch recordings in the presence of Kv{beta}1.2 (31, 34, 49).

As mentioned above, the cellular redox state represents another important level of Kv posttranslational control. It has been demonstrated that Kv{beta} oxidoreductase activity and the biophysics of Kv{beta} inactivating activity are coupled (32). LPS and TNF-{alpha}, but not M-CSF, trigger NO production in BMDM through the inducible NO synthase (10). However, the endotoxin is the most powerful NO-stimulating agent (50). High intracellular concentrations of NO would increase the oxidative stress of BMDM, thereby contributing to the pathogenesis of septic shock (51). Thus, an increase in oxidative stress generated by LPS and, to a lesser extent, TNF-{alpha} could modulate Kv{beta} activity, leading to a precise Kv functional role. In this context, treatment of macrophages with the antioxidant N-acetylcysteine improves the immune response (52). Furthermore, although Kv{beta}1.2 and Kv{beta}1.1 are sensitive to the redox state, Kv{beta}1.3 is not (30, 34, 49). Such differential sensitivity of Kv{beta} subunits to the redox state may be important in some pathophysiological conditions, such as ischemia and endotoxic shock. With an abnormal increase in cellular oxygen radicals, the effects of Kv{beta}1.2 and Kv{beta}1.1 would be lost, whereas Kv{beta}1.3 could still modulate Kv1 currents (34). These results suggest important changes in the functional activity of the Kv complex, because although M-CSF and LPS induce Kv{beta}1.3 expression, TNF-{alpha} does not. In addition, it is intriguing that both the growth factor and the endotoxin exhibit similar inactivation behaviors and show an increase in Kv{beta}2.1 subunit expression. We suggest that different Kv{beta} subunits extend the range of Kv1.3 modulation and may provide a variable mechanism for adjusting K+ currents in response to alterations in cellular conditions.

In summary, Kv channels are important in BMDM, where they contribute to diverse processes, such as proliferation and activation. We have described, for the first time, the characterization of Kv{beta} expressed in BMDM and studied their regulation. Stoichiometry may ultimately serve as a key determinant in shaping the repertoire of the Kv1 channels present in the plasma membrane of leukocytes. These data will be critical for further determination of the molecular composition of individual Kv currents and the physiological relevance of these {alpha}{beta} interactions. Investigation of the mechanisms involved in the regulation of potassium ion conduction is, therefore, essential for the understanding of potassium channel functions in the immune response to infection and inflammation.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Acknowledgments
 
We thank L. Martín and J. Bertrán for their help with macrophage cultures. The editorial assistance of Robin Rycroft (University of Barcelona, Language Advisory Service) is also acknowledged.


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

1 This work was supported by the Universitat de Barcelona and Ministerio de Ciencia y Tecnología, Spain (to A.F., C.F., A.C., and Co.S.), the Fundació August Pi i Sunyer and Generalitat de Catalunya (to C.F.), the National Institutes of Health (to M.M.T.), and Fondo de Investigaciones Sanitarias (to Co.S.). R.V. holds a fellowship from the Universitat de Barcelona. A.E. was supported by a fellowship from the Fundació Marató TV3. Back

2 R.V. and A.E. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Antonio Felipe, Molecular Physiology Laboratory, Departament de Bioquímica i Biologia Molecular, Universitat de Barcelona, Avinguda. Diagonal 645, E-08028 Barcelona, Spain. E-mail address: afelipe{at}ub.edu Back

4 Abbreviations used in this paper: Kv, voltage-dependent potassium channel; BMDM, bone marrow-derived macrophage; MgTx, rMargatoxin. Back

Received for publication September 30, 2004. Accepted for publication February 3, 2005.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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