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 Kvβ subunits coassociate with Kvα subunits, increasing channel diversity and function. In this study we have identified Kvβ 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 Kvβ1 (Kvβ1.1, Kvβ1.2, and Kvβ1.3) and Kvβ2 (Kvβ2.1), but not Kvβ4, the alternatively spliced murine Kvβ3 variant. M-CSF-dependent proliferation induced all Kvβ isoforms. However, LPS- and TNF-α-induced activation differentially regulated these subunits. Although LPS increased Kvβ1.3, reduced Kvβ1.2, and maintained Kvβ1.1 mRNA levels constant, TNF-α up-regulated Kvβ1.1, down-regulated Kvβ1.2, and left Kvβ1.3 expression unchanged. Moreover, in contrast to TNF-α, M-CSF- and LPS- up-regulated Kvβ2.1. K+ currents from M-CSF- and LPS-stimulated BMDM exhibited faster inactivation, whereas TNF-α increased τ values. Although in M-CSF-stimulated cells the half-inactivation voltage shifted to more positive potentials, the incubation with LPS and TNF-α 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 Kvβ 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.
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 Kvβ regulatory subunits (7). Up to four different forms of these cytoplasmic proteins can form part of the heteromeric structure (α4βn). Different Kvβ genes, namely Kvβ1, Kvβ2, and Kvβ3 generate up to six different Kvβ proteins. Although Kvβ1.1, Kvβ1.2, and Kvβ1.3 are alternatively spliced forms from the Kvβ1gene, Kvβ2.1 is the only variant known to be generated from the Kvβ2 gene (7). In addition, Kvβ3 generates two alternatively spliced isoforms, Kvβ3 and Kvβ4, 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 Kvα subunit occurs via the conserved C-terminal end. Furthermore, Kvβ 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-α-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-α-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). Kvβ 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 Kvβ 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 Kvβ subunits expressed in immune system cells. Thus, F5, an IL-2-induced cDNA in Th lymphocytes, turned out to be the Kvβ2.1 subunit (14). In addition, Kvβ1.1 and Kvβ2.1 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 Kvβ 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 Kvβ subunits that may coassemble with Kv1.3 in BMDM to generate different functional Kv channel complexes. Macrophages express all isoforms of Kvβ1 (Kvβ1.1, -1.2, and -1.3) and Kvβ2 (Kvβ2.1). In contrast, Kvβ4, the murine isoform of Kvβ3 was absent. Although M-CSF-dependent proliferation increased the expression of all Kvβ subunits, it was differentially regulated by LPS- and TNF-α-induced activation. Kvβ 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
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-α (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 Kvβ 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, Kvβ1.2, and 18S) or 60°C (rest of Kvβ) 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 Kvβ 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.
|Gene .||Accession No. .||Sequencea .||Base Pairs .||Length (bp) .|
|Gene .||Accession No. .||Sequencea .||Base Pairs .||Length (bp) .|
F, forward; R, reverse.
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 [α-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 Kvβ subunits, the following Abs were donated by Dr. J. Trimmer (University of California, Davis, CA): anti-Kvβ1.1 rabbit polyclonal, anti-Kvβ1.2 rabbit polyclonal, anti-pan Kvβ rabbit polyclonal, and anti-Kvβ2.1 mouse monoclonal (11, 21, 22, 23). The anti-Kvβ1.3 rabbit polyclonal Ab was produced and characterized in the Tamkun laboratory (20). An anti-β-actin mAb (Sigma-Aldrich) was used as a loading and transfer control.
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/τ)) for activation τ, calculated from the end of the compensated capacitive transient to the peak of the current and I(t) = Aexp(−t/τ) for inactivation and deactivation τ. 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.
Recombinant murine TNF-α 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.
Delayed rectifier K+ currents were evoked in BMDM by depolarizing pulses (Fig. 1,A) 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-α further increased the current amplitude (∼600 and ∼350 pA, respectively; n = 60). Resting and M-CSF-treated BMDM showed similar voltage dependence (Fig. 1,B). 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-α were activated (10), and the steady-state activation curve of normalized conductances changed substantially (Fig. 1,B). 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-α, respectively; n = 12), k slope values were significantly different (9.6 ± 1.6 and 9.1 ± 2.2 mV for LPS and TNF-α, respectively; p < 0.001 vs M-CSF, by Student’s t test; n = 12). RT-PCR experiments (Fig. 1,C) 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. 1 D) suggested that outward conductances were principally mediated by Kv1.3, as demonstrated previously (10).
The oligomeric structure of the Kv complex is a critical determinant of electrical activity in mammalian neurons (26). Because modulation by accessory β subunits has dramatic consequences on Kv1 functional activities (7), we studied the expression of Kvβ proteins in crude membrane preparations using several specific Abs. Unfortunately, none of the anti-Kvβ1 subunit-specific Abs produced a reliable signal (data not shown). Only the polyclonal anti-pan-β subunit and the monoclonal anti-Kvβ2.1 were useful in our BMDM samples. In macrophages, the anti-pan-β subunit Ab recognizes two polypeptide species of ∼38 and ∼41 kDa, similar to those described by Trimmer and collegues (11, 22) in the brain. Kvβ2.1 protein corresponds to the lower band, and the higher band corresponds to Kvβ1 peptides sharing a common core (Fig. 2 A). HEK-293 cells were negative. Although M-CSF and LPS induced Kvβ1 and Kvβ2.1 expression in macrophages, TNF-α did not. The anti-Kvβ2.1 mAb gave similar results (data not shown).
To further determine Kvβ expression in BMDM, we designed oligonucleotides to amplify the N-terminal domain for each subunit and generated specific Kvβ cDNA probes (Table I). To control for undesirable cross-hybridizations, each Kvβ fragment was run on a 2% agarose gel and further hybridized against the rest of the Kvβ cDNAs. No signal was detected other than that expected with the appropriate specific Kvβ probe (data not shown). Fig. 2 B demonstrates that BMDM expressed several Kvβ isoforms similar to the brain. All splice variants of the Kvβ1 gene (Kvβ1.1, Kvβ1.2, and Kvβ1.3) were detected, as well as the only Kvβ2 gene product described to date, Kvβ2.1. In contrast, the murine isoform Kvβ4, identified in brain as a spliced mRNA from Kvβ3, 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 Kvβ subunits was induced (Fig. 3 A). The three splice variants of the Kvβ1 gene (Kvβ1.1, Kvβ1.2, and Kvβ1.3) 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, Kvβ2.1 increased steadily throughout the study, and the level of induction after 24 h of incubation was ∼5-fold (p < 0.001, by ANOVA).
When Kvβ subunits are coexpressed heterologously with Kvα, 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. 3 B). 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-α activate macrophages, leading to cell growth arrest (10, 27). LPS-induced activation differentially regulated Kvβ subunits in BMDM (Fig. 4,A). Under these conditions, the three splice variants of the Kvβ1 gene showed different expression patterns. Although Kvβ1.1 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 Kvβ1.1 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). Kvβ1.2 expression decreased during endotoxin treatment (p < 0.001, by ANOVA). In contrast, soon after LPS incubation, Kvβ1.3 mRNA increased (p < 0.001, by ANOVA). In addition, Kvβ2.1 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. 4,B). However, the presence of the endotoxin further decreased τ inactivation values. Fig. 4 C 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).
Several lines of evidence indicate that LPS exerts Kv regulation via TNF-α-dependent and -independent mechanisms (10, 28). Therefore, we analyzed the effects of TNF-α on the expression of Kvβ subunits (Fig. 5,A). TNF-α induced an up-regulation of Kvβ1.1 mRNA (p < 0.001, by ANOVA), Kvβ1.2 expression decreased steadily throughout the study (p < 0.001, by ANOVA), and Kvβ1.3 and Kvβ2.1 remained constant. These results indicate that in contrast to Kv1.3 and other plasma membrane proteins (10, 27, 28), LPS-mediated Kvβ subunit regulation is TNF-α independent in BMDM. Inactivation time constants of K+ currents evoked in macrophages incubated with TNF-α were higher than those in its absence, with a significant change in the pattern (Fig. 5,B). The half-inactivation voltage in TNF-α-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-α and with TNF-α 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-α and with TNF-α, respectively (Fig. 5 C).
In addition to inactivation, accessory Kvβ 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-α increased τ 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. 6,A). 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. 6,F) and normalized tail currents obtained upon repolarization from +50 to −50 mV (Fig. 6,G), indicated that K+ currents deactivated faster in LPS-activated BMDM (τ = 26 ms) than in TNF-α-stimulated cells (τ = 140 ms). Furthermore, tail current amplitudes were significantly different upon the mode of activation (Fig. 6 H).
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 Kvβ subunits from Kvβ1 and Kvβ2, but not Kvβ3. This expression is similar to that in muscle, but different from that in neural tissues, because Kvβ3 and Kvβ4 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 Kvβ subunits. However, LPS and TNF-α, 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 Kvβ1.1 and Kvβ1.2 accelerate the rate of inactivation of K+ currents arising from Kv1 channels (32, 33, 34). However, Kvβ2 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 Kvβ1 subunits (34). In this context, it is surprising that, in contrast to M-CSF-dependent proliferation, Kvβ1 gene products are clearly differentially regulated in BMDM upon the mode of activation. Thus, distinct Kvβ1 N-terminal domains could be responsible for conferring the heterogeneity of the modulatory activity that we observed.
We found that, in contrast to TNF-α, 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 (τ = 100–200 ms). Kv1.3 expressed in HEK 293 cells, in the absence of Kvβ subunits, inactivates much more slowly (τ = 1800 ms at +40 mV) (36). In contrast, in T lymphocytes, which express Kvβ 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 Kvβ 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-α (10, 27). Our results are in agreement with those described when Kv1 proteins are coexpressed with individual Kvβ 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 Kvβ2.1, is that Kvβ2.1 inhibits Kvβ1-mediated inactivation and promotes Kv surface expression (35, 41, 42). In fact, Kvβ2 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 Kvβ2.1 subunits. This structure would highlight the complex scenario in which the insertion of Kvβ2.1 could change the electrical properties of the Kv channel, leading to multiple physiological effects on the mode of activation.
αβ interactions may be additionally influenced by differential posttranslational modifications. Kvβ 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-α regulation activates different signal transduction pathways involving Ser/Thr protein kinases (45, 46, 47). In this context, the inactivation conferred on Kv1.3 by Kvβ1 could be modulated not only by α subunit phosphorylation, but also by phosphorylation of the β subunit itself, which is known to regulate β-mediated effects (7). Thus, in the Kvβ-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 Kvβ1.3 effects on Kv1.5 (48), and protein kinase A reduces Kvβ1.3-induced activation of Kv1.5 (49). This is in agreement with our data in which LPS, but not TNF-α, induced Kvβ1.3 and decreased the inactivation time constant. Furthermore, LPS induced Kvβ2.1 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 Kvβ2.1 up-regulation could trigger inhibition of the Kvβ1-induced inactivation, increasing current amplitudes. In addition, Kvβ1.2-mediated inactivation is not altered by protein kinase A activation (48), and macrophage activation by LPS and TNF-α triggered a notable decrease in Kvβ1.2 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 Kvβ1.2 (31, 34, 49).
As mentioned above, the cellular redox state represents another important level of Kv posttranslational control. It has been demonstrated that Kvβ oxidoreductase activity and the biophysics of Kvβ inactivating activity are coupled (32). LPS and TNF-α, 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-α could modulate Kvβ 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 Kvβ1.2 and Kvβ1.1 are sensitive to the redox state, Kvβ1.3 is not (30, 34, 49). Such differential sensitivity of Kvβ 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 Kvβ1.2 and Kvβ1.1 would be lost, whereas Kvβ1.3 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 Kvβ1.3 expression, TNF-α does not. In addition, it is intriguing that both the growth factor and the endotoxin exhibit similar inactivation behaviors and show an increase in Kvβ2.1 subunit expression. We suggest that different Kvβ 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 Kvβ 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 αβ 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.
The authors have no financial conflict of interest.
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.
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.
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.
Abbreviations used in this paper: Kv, voltage-dependent potassium channel; BMDM, bone marrow-derived macrophage; MgTx, rMargatoxin.