Biochemical and Biophysical Research Communications 352 (2007) 913–918 www.elsevier.com/locate/ybbrc
Kv1.3/Kv1.5 heteromeric channels compromise pharmacological responses in macrophages Nu´ria Villalonga a, Artur Escalada b, Rube´n Vicente a, Ester Sa´nchez-Tillo´ c, Antonio Celada c, Carles Solsona b, Antonio Felipe a,* a
b
Molecular Physiology Laboratory, Departament de Bioquı´mica i Biologia Molecular, Universitat de Barcelona, Avda. Diagonal 645, E-08028 Barcelona, Spain Departament de Patologia i Terape`utica Experimental, Universitat de Barcelona-Campus de Bellvitge, E-08907 Hospitalet de Llobregat, Spain c Institut de Recerca Biome´dica, Parc Cientı´fic de Barcelona, Universitat de Barcelona, E-08028 Barcelona, Spain Received 14 November 2006 Available online 4 December 2006
Abstract Voltage-dependent K+ (Kv) channels are involved in the immune response. Kv1.3 is highly expressed in activated macrophages and T-effector memory cells of autoimmune disease patients. Macrophages are actively involved in T-cell activation by cytokine production and antigen presentation. However, unlike T-cells, macrophages express Kv1.5, which is resistant to Kv1.3-drugs. We demonstrate that mononuclear phagocytes express different Kv1.3/Kv1.5 ratios, leading to biophysically and pharmacologically distinct channels. Therefore, Kv1.3-based treatments to alter physiological responses, such as proliferation and activation, are impaired by Kv1.5 expression. The presence of Kv1.5 in the macrophagic lineage should be taken into account when designing Kv1.3-based therapies. Ó 2006 Elsevier Inc. All rights reserved. Keywords: K+ channels; Macrophages; Drug therapy; Kv1.3; Kv1.5; Autoimmune diseases
Macrophages turn the immune response toward inflammation or tolerance. These cells, which also act as antigenpresenting cells, modify the cytokine milieu and the intensity of T-cell signaling. K+ channels are involved in the immune response [1,2] and their functional activity is important for cellular responses [3–5]. Therefore, K+ channels are considered pharmacological targets in autoimmune diseases [6–8]. Altered expression of Kv1.3 channels is associated with multiple sclerosis [8–10]. Kv1.3-based therapies are effective in experimental models [10,11] and the beneficial effects of Kv1.3 channel blockers might be due, in part, to their inhibitory action on microglia and macrophages [8]. However, either channel redundancy or different heteromeric structures may allow these cells to escape the inhibitory
effects of Kv1.3 blockers. Macrophages express Kv1.3 and Kv1.5 voltage-dependent K+ channels, and Kv1.3/ Kv1.5 hybrid channels contribute to K+ currents [12]. The aim of the present study was to determine whether various Kv heterotetrameric compositions in several mononuclear phagocyte cell lines compromised the physiological response to Kv1.3-based pharmacological treatments. Bone marrow-derived macrophages (BMDM) and Raw 264.7 macrophages express different Kv1.3/Kv1.5 ratios. The heteromeric structure of the Kv complex varies, leading to biophysically and pharmacologically distinct channels. Our results demonstrate that different channel composition changes biophysical properties and could alter the use of potential drug therapies in autoimmune diseases. Materials and methods
*
Corresponding author. Fax: +34 934021559. E-mail address:
[email protected] (A. Felipe).
0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.11.120
Animals and cell culture. Murine bone marrow-derived macrophages (BMDM) were isolated as described elsewhere [3,4]. Cells were cultured in
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DMEM containing 20% FBS and 30% supernatant of L-929 fibroblast (Lcell) conditioned media as a source of Macrophage-Colony Stimulating Factor. Raw 264.7 macrophages were cultured in RPMI culture media, containing 5% FBS supplemented with 10 U/ml penicillin and streptomycin, and 2 mM L-glutamine. In some experiments, macrophages were incubated with 100 ng/ml of lipopolysaccharide (LPS, Sigma) for 24 h. For proliferation experiments, BMDM were arrested at G0 by M-CSF deprivation in DMEM supplemented with 10% FBS for at least 18 h [3,4]. G0-arrested cells were further incubated, in the absence or presence of recombinant murine M-CSF (1200 U/ml), for the times indicated. Raw cells were also G0-arrested by incubation for 36 h in RPMI supplemented with 0.2% bovine serum albumin (BSA). Resting cells were further incubated with RPMI, in the absence or presence of 5% FBS, for the times indicated. Cells were exposed to rMargatoxin (MgTx) and DNA synthesis was measured as the incorporation of [3H]thymidine (Amersham Pharmacia Biotech) to DNA [3,4,12]. All animal handling was approved by the Ethics Committee of the University of Barcelona and was in accordance with EU regulations. RNA isolation and RT-PCR analysis. Total RNA was isolated by use of the Tripure isolation reagent (Roche) and treated with DNaseI. Readyto-Go RT-PCR Beads (Amersham Pharmacia Biotech) were used. Total RNA and Kv1.3, Kv1.5 and 18S primers were added to the beads, as described elsewhere [3,4,13,14]. For real-time RT-PCR, cDNA synthesis was performed with Transcriptor (Roche), according to the manufacturer’s instructions. PCR primers were: Kv1.5 (F: 5 0 -TCCGACGGCTGGACTCAATAA-3 0 ; R: 5 0 CAGATGGCCTTCTAGGCTGTG-3 0 ); Kv1.3 (F: 5 0 -AGTATATG GTGATCGAAGAGG-3 0 ; R: 5 0 -AGTGAATATCTTCTTGATGTT-3 0 ). LightCycler FastStart DNA MasterPLUS SYBR Green I (Roche) was used, and the reactions were performed under the following conditions: 95 °C for 5 s, 55 °C for 8 s, and 72 °C for 9 s, preceded by 10 min at 95 °C and followed by 10 min at 95 °C. Melting curves were performed to verify the specificity of the product and 18S was included as an internal reference [3,4]. Protein extracts and Western blot. Cells were washed twice in cold phosphate-buffered saline (PBS) and lysed with lysis solution (1% NP40, 10% glycerol, 50 mmol/L Hepes, pH 7.5, and 150 mmol/L NaCl) supplemented with 1 lg/ml aprotinin, 1 lg/ml leupeptin, 86 lg/ml iodoacetamide, and 1 mM phenylmethylsulfonyl fluoride as protease inhibitors. To obtain enriched membrane preparations, homogenates were centrifuged at 3000g for 10 min and the supernatant was further centrifuged at 150,000g for 90 min. The pellet was resuspended in 30 mM Hepes (pH 7.4). Membrane proteins (50 lg) were boiled in Laemmli SDS loading buffer and separated on 10% SDS–PAGE. They were transferred to nitrocellulose membranes (Immobilon-P, Millipore) and blocked in 5% dry milksupplemented 0.1% Tween 20 PBS before immunoreaction. Polyclonal antibodies against Kv1.3 (1/200, Alomone), Kv1.5 (1/500, Alomone), and inducible Nitric Oxide Synthase (1/500, Santa Cruz Biotechnology) were used. A monoclonal anti-b-actin antibody (Sigma) was used as a control. The specificity of antibodies was tested with control antigen peptides provided by the manufacturer. Electrophysiology. Whole-cell currents were measured in an EPC-9 (HEKA) amplifier, as described elsewhere [3,4]. Electrodes of 2–4 MX were manufactured in a P-97 puller (Sutter Instruments). Electrodes were filled with the following solution (in mM): 120 KCl, 1 CaCl2, 2 MgCl2, 10 Hepes, 11 EGTA, 20 D-glucose, adjusted to pH 7.3 with KOH. The extracellular solution contained (in mM): 120 NaCl, 5.4 KCl, 2 CaCl2, 1 MgCl2, 10 Hepes, 25 D-glucose, adjusted to pH 7.4 with NaOH. Macrophages were clamped to a holding potential of 60 mV. Recordings were subtracted for leak currents at 50 mV online. To calculate inactivation time constants, cells were held at 60 mV and pulse potentials of 5 s were applied. Inactivation adjustment was calculated from the peak of the current at 50 mV to steady-state inactivation. To analyze the cumulative inactivation, currents were elicited by a train of 8 depolarizing voltage steps of 200 ms to +50 mV once every 400 ms. To characterize the voltage-dependent outward K+ current pharmacologically, dose effect plots of recombinant Margatoxin (MgTx) were
done by adding the toxin to the external solution [3,4,12]. Recordings were taken at room temperature (20–23 °C).
Results and discussion Kv1.3 is a pharmacological target in human autoimmune diseases and Kv1.3 blockers ameliorate symptoms in animal models [10,11]. Activated effector memory T-cells (TEM) express large amounts of Kv1.3 [8]. T-cells also express Kv1.1, Kv1.2, and Kv1.6 [15]. Although these subunits may form heteromeric structures with Kv1.3 [16–18], any Kv1.3-based drug therapy would be more effective in TEM. Macrophages and dendritic cells are also actively involved in the immune response, since they produce activating cytokines and present antigens to T-lymphocytes. While a large body of information focuses on T-cell Kv channels, less is known about Kv in macrophages. Furthermore, there is a controversy between reports on the expression pattern and the functional roles of Kv in these cells. While some studies support the unique expression of Kv1.3 without an apparent function [19], others claim a pivotal role for Kv1.5 [20]. However, most of the literature describes Kv1.3 and Kv1.5 in the myeloid lineage [3,4,12,21–26] and an association of the two proteins have been confirmed [12]. Therefore, the present study addresses whether macrophages express structurally different Kv channels that might compromise potential Kv1.3-based therapies. BMDM and Raw cells expressed outward K+ currents (Fig. 1A). Peak amplitude (+50 mV) is 4 times higher in BMDM than in Raw cells (Fig. 1B). This result was not related to the size of the cell. Although Raw cells are about 30% smaller than BMDM (Fig. 1C), the last still had three times more K+ current density (Fig. 1D). BMDM and Raw cells express Kv1.3 and Kv1.5 mRNA (Fig. 1E), and protein (Fig. 1F). Expression and biophysical and pharmacological properties indicated that, unlike Raw cells, BMDM currents were mainly generated by Kv1.3 and the presence of Kv1.5 was much lower (Table 1). Variations due to cell origin or between species should be taken into account when extrapolating results to other models. K+ channel phenotype differences between human and mouse T-lymphocytes exist [8]. However, data on human macrophages are limited and quite disperse. While some contributions describe low potassium conductance similar to that found in murine macrophages [27,28], others found large conductance [19,29]. While human and rodent myeloid-lineage express Kv1.3 and Kv1.5 [3,4,12,22–26,30], the presence of Kv1.5 in human alveolar macrophages is questioned [19]. Further extensive work has to be performed on macrophages to evaluate significant differences. Bearing all this in mind, we calculated the channel density in both cell lines. Fig. 1G shows that Raw cells expressed fewer channels per cell, no matter which conductance was used (13 or 8 pS for Kv1.3 and Kv1.5, respectively) [31]. Although Kv1.5 is the main subunit in Raw cells (Table 1), both values were used to obtain upper and lower limits
N. Villalonga et al. / Biochemical and Biophysical Research Communications 352 (2007) 913–918
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A 100 pA
BMDM
50 ms
50 mV 25 pA
Raw 264.7
-60 mV
200 ms
C
200 150 100
***
50 0
Raw +RT
10 5
BMDM
-RT
Raw 264.7
BMDM
F
-RT
+RT
1.38 0.95 0.83
10 8 6
***
4 2 0
Raw 264.7
BMDM
Kv1.3
Kv1.5
Raw BMDM
Raw BMDM
kDa 100
Kv1.3
0.56 0.50 0.40 0.30
50 β-act
Kv1.5
0.20
*
50
***
25 0 BMDM
Raw 264.7
*
0.04
0.02
0.00
100
current inhibition (%)
75
I
0.06
2
H
100
Channels /μ membrane
Molecular weigth (kb)
**
15
0
E
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20
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BMDM
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D
25
Cell size (pF)
Peak amplitude (pA)
B
Current density (pA/pF)
50 ms
80
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60 40
Raw 264.7
20 0 -6
BMDM Raw 264.7
10
-5
10
-4
10
-3
10
-2
10
MgTx [μM]
Fig. 1. Macrophages express Kv1.3 and Kv1.5. (A) Representative traces of delayed-rectifier K+ currents. (B) Currents were elicited in BMDM and Raw 264.7 cells, as shown in (A), and peak amplitudes were calculated from at least 15 cells. (C) Cell size (pF) from the same cell population from (B). (D) Current density (pA/pF) was calculated as a function of peak amplitude and the size for each recorded cell. (E) mRNA expression of Kv1.3 and Kv1.5 in BMDM and Raw cells. PCRs were performed in the presence (+) or the absence of the retrotranscriptase reaction (). (F) Kv1.3 and Kv1.5 protein expression in BMDM and Raw macrophages. (G) Channel density was calculated from the conductance of Kv1.3 (13 pS) in BMDM and either Kv1.3 (13 pS, black) or Kv1.5 (8 pS, grey) in Raw cells. (H) The results in (G), as a function of the plasma cell membrane surface. (I) Dose-dependent inhibition curves of the K+ current by MgTx. Currents were evoked at +50 mV from a holding potential of 60 mV during 200 ms. Symbols are: s, BMDM, d, Raw macrophages. Values are means ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 vs BMDM (Student’s t test).
for channel densities. Since Kv1.3/Kv1.5 heteromers exhibit intermediate biophysical properties compared with homomeric channels [12,31], cells expressing heterotetramers should have channel densities in between. When channel density was calculated in Raw cells as a function of the cell surface area (channels/lm2 of membrane), only data obtained with Kv1.5 conductance led to similar values to BMDM, expressing higher amounts of Kv1.3 (Fig. 1H). However, Kv1.5 is resistant to MgTx [12,31] and K+ cur-
rents were inhibited (Fig. 1I). Therefore, Kv1.5 does not form homomeric channels in macrophages. The IC50 values were 50 ± 5.1 and 772 ± 40 pM for BMDM and Raw, respectively (Table 1). Raw and BMDM differ in the K+ channel composition, since pharmacological differences were clear. In addition to pharmacology, biophysics might also throw some light on possible differences in the K+ channel phenotype. Kv1.3 is well characterized by its C-type
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Table 1 Biophysical and pharmacological properties of Kv currents and expression of Kv1.3 and Kv1.5 channels in BMDM and Raw 264.7 cells BMDM
Raw 264.7
39 ± 5 7.2 ± 2 365 ± 70 46.5 ± 9.1
21 ± 3** 6.3 ± 2*** 1740 ± 50*** 19.8 ± 6.0*
50 ± 5
772 ± 40***
mRNA expression Ct(Kv1.3) Ct(Kv1.5) Ct(Kv1.5) Ct(Kv1.3) Normalized Kv1.3 (relative to Raw) Normalized Kv1.5 (relative to Raw)
25.0 ± 1.1 30.2 ± 1.1 5.16 ± 1.56 2.10 0.03
26.3 ± 1.5 26.4 ± 2.0 0.14 ± 0.53** 1 1
Protein expression (arbitrary units) Normalized Kv1.3 (relative to Raw) Normalized Kv1.5 (relative to Raw)
1.75 ± 0.16 0.29 ± 0.01
1.00 ± 0.14** 1.00 ± 0.07***
Biophysics Threshold for activation (mV)a V1/2 (mV)a Inactivation time (ms) Cumulative inactivation (%) Pharmacology (Margatoxin) IC50 (pM) b
a
Threshold for activation and half-activation voltages are taken from [12] for comparison. Time constant for inactivation and cumulative inactivation parameters were calculated, as described in Materials and methods. The IC50 for MgTx was calculated from data in Fig. 1. b mRNA levels were quantified by real-time RT-PCR in each cell line. For each primer set, a standard curve was made and the slope factor calculated. The corresponding real-time PCR efficiency (E) of one cycle in the exponential phase was calculated according to the equation: E = 10(1/slope). The normalized Kv1.3 and Kv1.5 expression was calculated by the following equation: EDCt(Raw-BMDM), where Ct means threshold cycle. * p < 0.05. ** p < 0.01. *** p < 0.001 vs BMDM (Student’s t test).
inactivation, which is absent in Kv1.5 [31,32]. Since C-type inactivation involves the cooperative interaction of all four subunits, this parameter could be used to determine heterotetramers. While K+ currents inactivated with a time constant of inactivation of 365 ± 70 ms in BMDM (Fig. 2A), in Raw cells (Fig. 2B) this was much slower
(1740 ± 50 ms). Channel inactivation is a good way of distinguishing between diverse types of K+ channels. Inactivation values of Kv1.3 in T-lymphocytes and in Xenopus oocytes range from 107 to 250 ms, respectively [33], which are faster than in macrophages. Our results further support that Kv are generated by different subunit stoichiometry and confirm that, unlike BMDM, Raw cells express Kv1.5 as the main subunit. Inactivation of certain K+ channels accumulates during repetitive depolarizing pulses because recovery during the inter-pulse interval is incomplete. This property is termed cumulative inactivation [34]. After a train of repetitive pulses, the standardized peak current amplitude rapidly diminished in BMDM (Fig. 2C). However, this was much less apparent in Raw cells (Fig. 2D). Once again, these results indicate that Kv1.5 associates with Kv1.3 forming different heteromeric structures. Table 1 summarizes the biophysical and pharmacological properties as well as the expression of Kv1.3 and Kv1.5 in BMDM and Raw cells. BMDM express two times higher Kv1.3 levels than Raw cells do. However, Raw cells express much more Kv1.5. These results corroborate the pharmacological and biophysical properties that we observed. Thus, while BMDM characteristics are closer to Kv1.3 in T-cells and heterologous systems, such as inactivation, cumulative inactivation, and IC50 for MgTx [31], in Raw macrophages these values varied as a consequence of higher expression of Kv1.5. Other biophysical parameters, such as the threshold for activation and half activation potential, further supported this [3,4]. Differential expression of Kv1.3 and Kv1.5 could modify the Kv1.3/Kv1.5 ratio leading to functional consequences. High Kv1.3/Kv1.5 ratios, as observed in BMDM (Fig. 3A), confirm more effective pharmacological effects. Thus, proliferation of BMDM, unlike Raw cells, was sensitive to MgTx (IC50 of 2 nM). In Raw cells less than 20% of proliferation was inhibited (Fig. 3B). K+ channels
Fig. 2. Biophysical properties of K+ current inactivation in BMDM and Raw cells. (A,B) C-type inactivation. Cells were held at 60 mV and pulse potentials were applied from 60 to +50 mV for 5 s. (C,D) Cumulative inactivation. Currents were elicited by a train of eight depolarizing voltage steps (200 ms) to +50 mV once every 400 ms.
N. Villalonga et al. / Biochemical and Biophysical Research Communications 352 (2007) 913–918
B 1.00 0.75 0.50
**
0.25
100
% proliferation
Kv1.3/Kv1.5 ratio in macrophages
A
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0.00
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0
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1000
Margatoxin [nM]
C
BMDM LPS MgTx
-
+
-
Raw
+ +
-
+
-
+ +
iNOS β-act Fig. 3. Functional consequences of the differential expression of Kv1.3 and Kv1.5. (A) BMDM and Raw cells express different Kv1.3/Kv1.5 ratios. Values were calculated from the expression of both proteins in four independent Western blots. **p < 0.01 vs BMDM, Student’s t test. (B) MgTx significantly inhibits proliferation in BMDM, but not in Raw cells. Proliferating macrophages were cultured in the presence of increasing concentrations of MgTx. Values are means ± SEM of four different assays, each done in triplicate. Symbols are: s, BMDM, d, Raw 264.7 macrophages. (C) Effects of MgTx on LPS-induced iNOS expression in macrophages. Cells were cultured for 24 h in the absence () or the presence (+) of LPS with or without 10 and 100 nM MgTx for BMDM and Raw cells, respectively. A representative Western blot of three independent experiments is shown.
in leukocytes are involved in proliferation and activation [3,7]. In fact, TEM express larger amounts of Kv1.3 [8,35]. To explore whether differences in the Kv1.3/Kv1.5 ratio also affect the activation of the cells, we incubated macrophages in the presence of LPS (Fig. 3C). LPS increased iNOS expression and MgTx inhibited iNOS induction in BMDM, but not in Raw cells. Our results give further evidence that low Kv1.3/Kv1.5 ratios, as observed in Raw cells, alter the pharmacological properties, leading to an impaired response to Kv1.3-based treatment. Based on our results, we hypothesized that, although both mononuclear phagocyte cell lines express Kv1.3 and Kv1.5, the stoichiometry of the complex is different. While in BMDM Kv1.3 is highly expressed, in Raw cells Kv1.5 is predominant. BMDM express about two times more Kv1.3 channels. However, Raw macrophages have up to 5 times more Kv1.5 (Table 1). A diagram of heteromeric structures in both cell lines is depicted in Supplementary Fig. 1. Our data also argue against homotetrameric channels consisting of Kv1.5, but the presence of Kv complexes generated only by Kv1.3 cannot be ruled out. In myeloid cells, the Kv1.3/Kv1.5 ratio may vary in the Kv complex, leading to pharmacologically distinct channels. Our results have physiological significance since the channels might control the cell physiology and their activity may be modulated by pharmacological treatments. K+ channels in leukocytes are pharmacological targets in autoimmune diseases [7,8,10,36] and, as we demonstrate, different channel composition changes biophysical properties and alters the use of potential drug therapies. Therefore, although further development of Kv1.3 blockers for autoimmune disease-therapy is warranted, their effectiveness in the macrophagic lineage could be compromised.
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