Clofazimine Inhibits Human Kv1.3 Potassium Channel by
Perturbing Calcium Oscillation in T Lymphocytes
Yunzhao R. Ren1,2, Fan Pan1, Suhel Parvez3, Andrea Fleig3, Curtis R. Chong1, Jing Xu1, Yongjun Dang1,
Jin Zhang1, Hongsi Jiang4, Reinhold Penner3, Jun O. Liu1,2,5*
1Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America, 2Program in
Biochemistry, Cellular and Molecular Biology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America, 3Center for Biomedical
Research at The Queen’s Medical Center and John A. Burns School of Medicine at the University of Hawaii, Honolulu, Hawaii, United States of America, 4Department of
Medicine, Feinberg School of Medicine, Northwestern University, Evanston, Illinois, United States of America, 5Department of Oncology, The Johns Hopkins University
School of Medicine, Baltimore, Maryland, United States of America
The Kv1.3 potassium channel plays an essential role in effector memory T cells and has been implicated in several important
autoimmune diseases including multiple sclerosis, psoriasis and type 1 diabetes. A number of potent small molecule
inhibitors of Kv1.3 channel have been reported, some of which were found to be effective in various animal models of
autoimmune diseases. We report herein the identification of clofazimine, a known anti-mycobacterial drug, as a novel
inhibitor of human Kv1.3. Clofazimine was initially identified as an inhibitor of intracellular T cell receptor-mediated
signaling leading to the transcriptional activation of human interleukin-2 gene in T cells from a screen of the Johns Hopkins
Drug Library. A systematic mechanistic deconvolution revealed that clofazimine selectively blocked the Kv1.3 channel
activity, perturbing the oscillation frequency of the calcium-release activated calcium channel, which in turn led to the
inhibition of the calcineurin-NFAT signaling pathway. These effects of clofazimine provide the first line of experimental
evidence in support of a causal relationship between Kv1.3 and calcium oscillation in human T cells. Furthermore,
clofazimine was found to be effective in blocking human T cell-mediated skin graft rejection in an animal model in vivo.
Together, these results suggest that clofazimine is a promising immunomodulatory drug candidate for treating a variety of
Citation: Ren YR, Pan F, Parvez S, Fleig A, Chong CR, et al. (2008) Clofazimine Inhibits Human Kv1.3 Potassium Channel by Perturbing Calcium Oscillation in T
Lymphocytes. PLoS ONE 3(12): e4009. doi:10.1371/journal.pone.0004009
Editor: Jose Alberola-Ila, Oklahoma Medical Research Foundation, United States of America
Received May 4, 2008; Accepted November 8, 2008; Published December 23, 2008
Copyright: ? 2008 Ren et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by the Department of Pharmacology, Johns Hopkins School of Medicine, the Keck Foundation, the Flight Attendant
Medical Research Institute Fund, the Fund for Medical Discovery from Johns Hopkins and the Johns Hopkins Malaria Research Institute (for initial funding of the
library). The project was also supported in part by Grant Number UL1 RR 025005 from the National Center for Research Resources (NCRR), a component of the
National Institutes of Health (NIH) and NIH Roadmap for Medical Research. RP was supported by NIH RO1 GM080555 and AF by NIH PO1 GM078195.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Immunosuppressive agents constitute a major class of drugs for
the treatment of undesirable or abnormal activation of T
lymphocytes and the immune system associated with organ
transplantation and autoimmune diseases. Among the most widely
used immunosuppressive drugs in the clinic are cyclosporin A
(CsA) and FK506, natural products of microbial origin that work
through inhibition of intracellular calcium signaling cascade
downstream of the T cell receptor (TCR). By recruiting abundant
cytosolic immunophilin receptors, each of these immunosuppres-
sive drugs induces the formation of a ternary complex with the
calcium, calmodulin-dependent protein phosphatase calcineurin,
thereby blocking access to the active site of calcineurin by its
substrate, nuclear factor of activated T cells (NFAT), preventing
the dephosphorylation and subsequent nuclear translocation of
NFAT [1–4]. Despite its widespread use among organ transplan-
tation patients , CsA and FK506 exhibit significant side effects,
particularly nephrotoxicity, which prevents their use for the
treatment of a wider range of autoimmune diseases. Since the
nephrotoxicity of CsA and FK506 was found to share the same
molecular basis as their immunosuppressive effect , attention
has been turned to other signal transducers downstream of TCR
as potential therapeutic targets in recent years.
The Kv1.3 potassium channel [7,8] has emerged as one of the
most promising targets for developing novel immunosuppressants.
Although no clear T cell phenotype was observed in Kv1.3
knockout mice , several lines of evidence exist in support of a
critical role of Kv1.3 channel in the activation and function of
human T cells. In particular, Kv1.3 has been shown to play a
unique role in effector memory T cell activation  and in the
pathogenesis of a myriad of important autoimmune diseases.
Notably, Kv1.3 has been shown to be highly expressed in auto-
reactive effector memory T cells from MS patients [11,12]. As a
result, extensive efforts have been made to discover and develop
small molecule inhibitors of Kv1.3 as novel immunosuppressants
and immunomodulators. A large number of structurally distinct
WIN17317-3 [14,15], correolide , verapamil , and 5-
phenylalkoxypsoralens (Psora) , among others. Several of the
inhibitors have been shown to be effective in animal models of
autoimmunity [19–21]. However, none has reached the clinic due
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to lack of potency, specificity, bioavailability or easy access due to
structural complexity [22,23].
The difficulty in finding a clinically useful Kv1.3 inhibitor is not
unexpected, as development of a new drug is a tedious and costly
process to begin with. To accelerate drug development process, we
recently assembled a library of mostly FDA-approved drugs as well
as drugs approved abroad and drug candidates that have reached in
PhaseIIclinical trials,known asthe JohnsHopkins Drug Library.By
systematically screening the library in cell-based assays, we have
identified and validated several drugs with novel and previously
unknown anti-angiogenic and anti-malarial activities [24–27].
Herein, we disclose the identification of clofazimine as a promising
hit from another cell-based screen for novel inhibitors of the
intracellular TCR signaling pathway leading to the transcriptional
activation of IL-2. Through a systematic examination of different
steps in intracellular TCR signaling, it was revealed that clofazimine
blocked calcium signaling in T cells by directly interfering with the
function of Kv1.3 channel. Importantly, it was demonstrated that
clofazimine was effective in preventing human T cell-mediated skin
graft rejection in a reconstituted mouse model of skin transplanta-
tion. As clofazimine has been used as an antibiotic in humans since
early 1960s, it has great potential as a novel treatment for many
human autoimmune diseases. The distinct structure of clofazimine
also offers a novel scaffold for the development of future generations
of immunosuppressive and immunomodulatory agents.
The signaling pathway emanating from TCR and leading to the
transcriptional activation of the IL-2 promoter is dependent on the
second messenger calcium and the calcium and calmodulin-
dependent protein phosphatase calcineurin that has been shown to
be a common target for both cyclosporine A and FK506 [2–4].
We thus engineered a reporter T cell line by stably integrating a
luciferase reporter gene under the control of the minimal human
IL-2 proximal promoter into the genome of Jurkat T cells. Upon
stimulation with PMA and ionomycin, a 20-fold increase in
luciferase activity was observed (data not shown). Using the
reporter T cell line, we screened the Johns Hopkins Drug Library
at a final concentration of 10 mM for each drug using the IL-2
reporter assay in 96-well format [24–26]. The known immuno-
suppressive drugs CsA and FK506 were both positive hits,
validating the screen. One of the most potent novel hits was
identified as clofazimine (Fig. 1), a known anti-mycobacterial drug
that has been used for the treatment of leprosy [28,29].
The ability of clofazimine to inhibit TCR-mediated IL-2
production was confirmed using transiently transfected IL-2
luciferase reporter gene in Jurkat T cells and a separately prepared
clofazimine stock solution. Clofazimine inhibited PMA/ionomycin-
stimulated IL-2 luciferase reporter gene activation with an IC50of
22 nM (Fig. 1A). It also inhibited the activation of endogenous IL-2
promoter in response to PMA and thapsigargin with an IC50of
1.1 mM (Fig. 1B). Importantly, clofazimine inhibited human mixed
lymphocyte reaction with an IC50of 0.9 mM (Fig. 1B), similar to its
effect on endogenous IL-2 production in Jurkat T cells.
The transcription activation of the IL-2 promoter is dependent
on three key transcription factors, NF-AT, NF-kB and AP-1. We
thus determined whether clofazimine affected the activation of
each of those transcription factors using their respective luciferase
reporters. As shown in Fig. 1C, while the NFAT luciferase reporter
was as sensitive to clofazimine as the IL-2 promoter-driven
luciferase reporter gene, the NF-kB luciferase reporter is about 40-
fold less sensitive to clofazimine. In contrast, the AP-1 luciferase
reporter gene activity was enhanced, rather than inhibited, by
higher concentrations of clofazimine (Fig. 1D). A similar
stimulation of the AP-1 reporter was also observed at higher
concentrations of CsA (Fig. S1A), suggesting that clofazimine may
affect the same signaling pathway as CsA.
The selective inhibitory effects of clofazimine on both NFAT
and NF-kB over AP-1 suggested that it is likely to affect the
activation of their common upstream regulator, calcineurin
[1,3,4]. To assess this possibility, we determined whether
clofazimine, like CsA and FK506, affected the dephosphorylation
of endogenous NFAT in response to ionomycin treatment. Similar
to CsA, clofazimine inhibited ionomycin-induced dephosphoryla-
tion of NFATc2 in a dose-dependent manner (Fig. 1E). In
addition, clofazimine also blocked the ionomycin-induced nuclear
translocation of NFAT in Jurkat T cells (Fig. S1B–C). Together,
these results indicated that clofazimine inhibited the activation of
calcineurin in vivo. We next examined the effects of clofazimine on
the activity of calcineurin in vitro. Clofazimine had no effect on the
enzymatic activity of calcineurin with either para-nitrophenylpho-
sphate or immunoprecipitated endogenous NFATc2 as a substrate
(Fig. S2A–B). Nor did it affect the binding of GST-NFATc2 to
recombinant calcineurin (Fig. S2C). Interestingly, when the
association between the N-terminal fragment of NFAT and the
constitutively active form of calcineurin (CnDC) was examined in a
mammalian two-hybrid assay, clofazimine inhibited the calcium-
dependent NFAT-calcineurin interaction in a dose-dependent
manner (Fig. S2D). Thus, clofazimine appeared to act at a step
upstream of calcineurin activation in vivo, raising the possibility
that it affected either the release of intracellular calcium or calcium
influx through the plasma membrane calcium channels.
We employed live cell imaging to determine the effect of
clofazimine on changes in intracellular calcium concentrations in
response to thapsigargin treatment. Using the calcium indicator dye
Fura-2AM, we were able to observe entry of calcium into Jurkat T
cells upon treatment of cells with thapsigargin followed by addition
of 2 mM Ca2+into the extracellular medium (Fig. 2A). Pretreatment
of Jurkat T cells with known CRAC channel inhibitors econazole or
gadolinium abrogated calcium entry as expected (Fig. 2B). We
noticed that Jurkat T cells exhibited heterogeneity in their response
to clofazimine with varying degrees of inhibition at a given
concentration of clofazimine. For example, when cells were
preincubated with clofazimine for 5 min, the calcium entry of only
about a quarter of cells are inhibited to near completion by the drug
while that in remaining cells was blocked to a lesser degree (Fig. 2B).
When the preincubation timewasincreased to upto 2 h,therewas a
time-dependent increase in the proportion of cells that became
sensitive to clofazimine (Fig. S3A–B). The inhibitory effect of
clofazimine on the extracellular calcium influx suggested that it
might affect the CRAC channel. We thus examined the effects of
clofazimine in reconstituted CRAC channels using ectopically
expressed CRACM1 (Orai1), CRACM2 or CRACM3 subunits
co-expressed with STIM1 in HEK 293T cells. But we observed no
effects of clofazimine on the reconstituted CRAC current (Fig. S3C),
ruling out the possibility that clofazimine directly interacts and
interferes with the known components of the CRAC channel.
Given that clofazimine inhibited calcineurin activation in T cells,
we next determined whether clofazimine affected the oscillation
frequency Ca2+entry in Jurkat T cells, which has been shown to be
critical and selective for sustained activation of calcineurin and
NFAT to drive cytokine gene expression . Indeed, addition of
clofazimine significantly disrupted the oscillation patterns of the
store-operated Ca2+entry induced by a low concentration of
thapsigargin . It both decreased the amplitude and increased the
period of the calcium oscillation (Fig. 2C). In contrast to the partial
response of calcium influx to clofazimine detected by Fura-2AM
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(Fig. 2A–B), over 80% of cells exhibited elongation of the oscillation
period upon treatment with clofazimine, indicating that this effect is
statistically significant (Fig. 2D).
The pronounced effects of clofazimine on the oscillation
patterns of calcium entry, together with the lack of effect of
clofazimine on reconstituted CRAC current, raised the possibility
that it may affect other channels, particularly potassium channels,
which are known to regulate the driving force for Ca2+through
open CRAC channels. We thus determined the effects of
clofazimine on the activity of various known channels expressed
in activated T cells. As shown in Fig. 3A and B, clofazimine had a
dramatic effect on Kv1.3 current in a time- and dose-dependent
manner. It inhibited the Kv1.3 potassium current with an IC50of
300 nM and a Hill coefficient of 0.75 (Fig. 3C), consistent with its
potency for the inhibition of both endogenous IL-2 production in
Jurkat T cells and the human mixed lymphocyte reaction (Fig. 1B).
In addition to Jurkat T cells, we also determined the effect of
clofazimine on Kv1.3 activity in primary human T cells and a
similar inhibitory effect was observed as in Jurkat T cells (Fig. 3D).
In contrast to Kv1.3, the activity of Ca2+-activated potassium
(TRPM4) , remained unaffected by clofazimine at up to
10 mM concentration when cells were perfused with intracellular
solutions in which Ca2+was buffered to 1 mM (data not shown).
To further investigate the specificity of clofazimine, we
conducted a series of experiments testing the drug against several
Figure 1. Clofazimine inhibits IL-2 production and NFAT activation in Jurkat T cells. (A) Clofazimine (structure shown in inset) inhibits IL-2
proximal promoter driven-luciferase stimulated by PMA/ionomycin in Jurkat T cells (n=6). The IC50s for reporter assay is 21.864.4 nM. (B) Clofazimine
inhibits IL-2 production in PMA/thapsigargin-stimulated Jurkat T cells (IC50=1.1060.26 mM) and human mixed lymphocyte reaction (MLR)
(IC50=0.9060.17 mM) (n=6 each). (C) Clofazimine inhibits NFAT pathway in Jurkat T cells. The IC50s of clofazimine in the IL-2, NFAT, NF-kB luciferase
reporter assays are 56.0614.8 nM, 113630 nM and 2.4361.25 mM, respectively (n=6 each). Both reporters were stimulated with PMA/ionomycin. (D)
Clofazimine significantly enhances AP-1 luciferase reporter at high concentrations (.1 mM, n=6). The reporter was also activated by PMA/ionomycin.
(E) Clofazimine inhibits dephosphorylation of endogenous NFATc2 in response to ionomycin treatment. NFATc2 and a-tubulin were detected by
Western blot using specific antibodies.
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heterologously expressed potassium channels, including mouse
Kv1.3 . As shown in Fig 3E and 3F, clofazimine strongly
suppressed mouse Kv1.3 stably expressed in L929 cells with an
IC50of 470 nM and a Hill coefficient of 0.5 (Fig. 3G). All other Kv
channel species tested (Mouse Kv1.1, rat Kv1.2, human Kv1.5
and mouse Kv3.1  proved considerably less sensitive to
clofazimine, blocking less than 50% of current at a 10 mM
concentration (Fig. S4). This indicates that their IC50values for
clofazimine are above 10 mM. Interestingly, there seems to be
some voltage dependence to the effect on Kv channels other than
Kv1.3, since the clofazimine block is smaller at 0 mV compared to
+80 mV (Fig. S5). Since most cells do not depolarize beyond
0 mV, at least not for appreciable amounts of time, this represents
the more physiologically relevant parameter in regards to the
inhibitory effect of clofazimine. Taken together, these data suggest
that clofazimine is highly selective for the Kv1.3 channel.
Kv1.3 is known to exhibit a polarized cell surface expression
pattern, which can be visualized using polyclonal antibodies in
conjunction with Cy5-labeled secondary antibodies  (Fig. 4A).
We took advantage of the intrinsic fluorescence of clofazimine,
which can be detected using the same filter as for FITC and
compared the distribution patterns of clofazimine and Kv1.3.
Indeed, clofazimine displayed the same polarized subcellular
localization pattern as that of Kv1.3 (Fig. 4A), suggesting that
clofazimine is likely to be associated with Kv1.3 in vivo.
Next, we assessed the direct interaction between clofazimine
and purified recombinant Kv1.3 in vitro. His-Kv1.3 was expressed
in 293T cells and purified to near homogeneity (Fig. 4B) as
described previously . The interaction between purified Kv1.3
protein and clofazimine was assessed by taking advantage of the
difference in their mobility in native polyacrylamide gels. Free
clofazimine migrated quite slowly in the gel (Fig. 4C, top panel).
Addition of BSA did not affect the gel mobility of clofazimine.
Upon mixing with recombinant Kv1.3, however, clofazimine co-
migrated with Kv1.3, as judged by the overlap of the Kv1.3
protein band revealed by Western blot and the colored clofazimine
band (Fig. 4C, bottom panel). The shift in gel mobility of
clofazimine by recombinant Kv1.3 strongly suggests that clofaz-
imine forms a complex with recombinant Kv1.3 by directly
binding to the protein.
To further assess the physiological relevance of Kv1.3 as a
molecular target for clofazimine, we first determined the effect of
ectopic overexpression of Kv1.3 on the sensitivity of the IL-2
reporter gene to clofazimine. As shown in Fig. 5A, overexpression of
Kv1.3 led to a gain in resistance of the IL-2 reporter to clofazimine
in a dose-dependent manner. At the highest concentration of Kv1.3
expression plasmid used (2 mg), there was a 35-fold increase in the
IC50value ofclofazimine.Next,wedownregulatedtheexpression of
endogenous Kv1.3 using lentivirus-mediated RNA interference. Of
a total of nine RNAi constructs tested, the most effective construct,
shKv1.3-4, partially downregulated the protein level of Kv1.3by ca.
70 % (Fig. 5B, C). A comparison of the dose-response curves of
Jurkat cells transduced with shKv1.3-4 lentiviruses and those
transduced with viruses carrying a control shRNA against EGFP
revealed that knockdown of Kv1.3 increased the sensitivity of the
IL-2 luciferase reporter to clofazimine with a nearly 5-fold decrease
in the IC50values (Fig. 5D). In contrast, knockdown of Kv1.3 had
no effect onthesensitivityof the IL-2 luciferase reporter toCsA(Fig.
S6). The changes in the sensitivity of the IL-2 reporter gene to
clofazimine upon overexpression or knockdown of Kv1.3 provide
strong support for the notion that Kv1.3 is a specific molecular
target of clofazimine.
Kv1.3 has been implicated in T cell activation and has served as
a molecular target for developing novel immunosuppressive agents
[22,23]. Given that clofazimine is already used in the clinic, albeit
for a completely different indication, we wondered whether it is
efficacious in animal models of organ transplantation. Initial
experiments using mouse skin or heart transplant models revealed
Figure 2. Clofazimine interferes with calcium influx in Jurkat T
cells. (A) Calcium influx was inhibited by clofazimine in a heteroge-
neous fashion. Clofazimine was added 5 minutes before stimulation
with 1 mM TG. Representative images were taken 30 minutes after
2 mM calcium was added. The color gradient represents fura-2 350 nm/
380 nm excitation ratio. (B) Effects of clofazimine on store-depletion
induced calcium influx. Typically the cells can be divided into 2 groups,
the responsive (red) and none-responsive (pink) populations. Each
curve represents average signal of 20 cells from the same field in (A)
(The results were reproduced 10 times under the same condition). (C)
Effect of clofazimine on calcium oscillation in Jurkat T cells (represen-
tative of 56 cells). The oscillation was stimulated by 10 nM TG. (D)
Clofazimine elongated oscillation period in more than 80% of Jurkat T
cells (Results represented 3 experiments, at least 80 cells were counted
for each experiment).
Clofazimine Inhibits Kv1.3
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Figure3. Clofazimine inhibits Kv1.3 channel. (A) Averaged time course of Kv1.3currents measured in Jurkat T cells in the absence and the presence
of clofazimine(CLF) ormargatoxin (MGX). VariousconcentrationsofCLF (n=5 each) or 10 nMMGX (open squares,n=5)wereappliedas indicatedbythe
black bar. Currents were elicited by applying a ramp protocol from 2100 mV to +100 mV over a span of 50 ms and acquired every 2 s. Holding potential
was 280 mV. Current amplitudes were extracted at +80 mV and plotted versus the time of the experiment. Currents were normalized to cell size and
plotted as pA/pF. Error bars indicate S.E.M. (B) Current-voltage relationship of Kv1.3 currents taken from example cells and extracted before (30 s) or after
application(120 s)of either 10 mM CLF (thick trace) or 10 nMMGX (thin trace). (C) Dose-response curve of the inhibitory effect of CLF (closed circles, n=5
each). Datawere normalized tothe current measured before applicationat30 s as I/I30s. Theinhibitionwas measured at the end of applicationat120 s. A
dose-response fit to the data resulted in an IC50of 300 nM and a Hill coefficient of 0.75. The maximum inhibitory effect of CLF was compared to the
inhibitoryeffectof10 nMMGX(open circle,n=5),asaturatingconcentrationtoassureKv1.3inhibition.Thedashedlineindicatesthemaximuminhibitory
effect of MGX. (D) Clofazimine inhibits Kv1.3 in primary human T cells. Average development of Kv1.3 currents measured in human T cells (protocol
approval number RIRC QMC RA-2004-048) in control cells (closed circles, n=5) and with superfusion of external standard solution supplemented with
1 mM clofazimine (open circles, n=5) as indicated by the black bar. Error bars indicate S.E.M. Data were normalized to the time of application, averaged
and plotted versus time of the experiment. Holding potential was 280 mV. Voltage ramp protocol and solutions are described in methods. (E) Current
behavior of heterologous mouse Kv1.3 expressed in L929 cells plotted over time in response to increasing concentrations of clofazimine, applied through
a wide-mouth glass pipette at the time indicated by the black bar (control, closed circles, no application, n=7; 10 mM, closed triangles, n=4; 1 mM, open
square, n=6; 100 nM, open circles, n=5). Currents were measured by application of a ramp protocol from 2100 mV to +100 mV over 500 ms and given
at 5 s intervals. Current amplitudes were assessed at +80 mV, normalized to the current amplitude at 40 s, averaged and plotted versus time. Error bars
indicateS.E.M.(F)I/V curveofarepresentativecellexpressingmouseKv1.3withcontrolI/V(black) extractedat40 s afterwhole-cellestablishmentandthe
I/V for 10 mM clofazimine extracted at the end of application (red, 160 s). (G) Concentration-response behavior of mouse Kv1.3 expressed in L929 cells to
increasing concentrations of clofazimine (n=4–7). A fit to the data gave an IC50of 470 nM with a Hill coefficient of 0.5.
Clofazimine Inhibits Kv1.3
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no beneficial effects of clofazimine in those models. These negative
results are not surprising given that Kv1.3 plays distinct roles in
humans and rodents, as it has been shown that Kv1.3 is
dispensable in mice due to the up-regulation of other chloride
channels . Consistent with this notion, we also failed to observe
a dose-dependent inhibition of IL-2 production in primary mouse
T cells (Fig. S7A) and murine mixed lymphocyte reaction (Fig.
S7B). Moreover, similar results were obtained for mixed
lymphocyte reaction using cells derived from rats, making it
difficult to evaluate the in vivo effects of clofazimine using well-
established animal models. To overcome this problem, we turned
to a model of reconstituted human T cell-mediated human skin
rejection in immunodeficient mice . We thus transplanted
human foreskin into Pfb-Rag22/2 mice that lack T, B and NK
cells. Upon healing of the skin graft for about 7 days, a total of 100
million human peripheral blood lymphocytes from an unrelated
donor were adoptively transferred into the same animals. The
animals were administered orally either olive oil (control) or
clofazimine at 50 mg/kg/day for a total of 10 days (Fig. 6A). For
the control group, the transplanted foreskin was rejected with a
median survival time of 11 days (Fig. 6B). For the group treated
with clofazimine, the skin survived even beyond the cessation of
the drug treatment with a mean survival time of 35 days (Fig. 6B),
which is comparable to the efficacy for FK506 treatment (data not
shown). It is noteworthy that in a parallel experiment using murine
skin and total murine T cells, clofazimine had no effect on the
survival of murine skin transplant (Fig. 6B). Together, these results
demonstrated that clofazimine is uniquely effective in inhibiting
human T cell-mediated graft rejection with no significant effect on
murine T cells.
The intracellular TCR-mediated signal transduction pathway
leading to IL-2 transcription is essential for the activation of
quiescent T cells and as such has served as a reliable model system
to discover and evaluate new immunosuppressive agents. In
addition to the discovery of FK506 using this model system ,
other immunosuppressive agents have been discovered from
different chemical libraries [39,40]. By screening a library of
known drugs (HDL), we identified clofazimine as a novel inhibitor
of this signaling pathway. Further mechanistic deconvolution by
systematically examining the known steps in this signaling pathway
led to the identification of Kv1.3 as a physiologically relevant
target for clofazimine. The selective inhibition of Kv1.3 by
clofazimine accounts for the perturbation of calcium oscillation
patterns by the drug and the distinct effects of clofazimine on the
intrinsic enzymatic activity of calcineurin in vitro and the
calcineurin-mediated NFAT dephosphorylation in vivo.
Several lines of evidence were obtained that support Kv1.3 as a
major molecular target for clofazimine. In addition to the
inhibition of channel activity of ectopically expressed Kv1.3, it
was found that clofazimine demonstrated remarkable selectivity
for Kv1.3 over other related Kv channels including Kv1.1, Kv1.2,
Kv1.5 and Kv3.1 (Fig. S4, S5). More importantly, we not only
observed colocalization of clofazimine with Kv1.3 in live cells, but
also denmonstrated that clofazimine could directly associate with
purified recombinant Kv1.3 protein in native polyacrylamide gel
as judged by the co-migration of the otherwise less mobile
clofazimine and faster migrating Kv1.3 protein (Fig. 4C). To
further assess the physiological relevance of Kv1.3 as a target of
clofazimine, we also determined the activities of a well-known
Kv1.3 inhibitor, Psora-4, in several assays (Fig. S8). Gratifyingly,
Psora-4 displayed an activity profile quite similar to clofazimine,
including heterologous inhibition of calcium influx in Jurkat T
cells (Fig. S8A–B vs. Fig. 2A), selective inhibition of IL-2 and
NFAT luciferase reporter genes over the NF-kB reporter (Fig. S8
C vs. Fig. 1C), and stimulation of the AP-1 luciferase reporter gene
at higher concentrations (Fig. S8 D vs. Fig. 1D). It is worth noting
that unlike Psora-4, clofazimine does not significantly cross inhibit
Kv1.5, which should make clofazimine less toxic. Although all
existing experimental evidence is consistent with Kv1.3 as a
molecular target for clofazimine, we cannot rule out other
molecular targets for clofazimine, since it has not been possible
to take a unbiased approach, such as photoaffinity labeling of
whole cell lysates , to detect clofazimine binding proteins.
Although we observed excellent correlation between inhibition
of Kv1.3 (Fig. 3) and that of IL-2 reporter gene (Fig. 1) as well as
that of thapsigargin-induced calcium oscillation in Jurkat T cells
(Fig. 2C–D), one discrepancy remains—the calcium influx in only
a subpopulation of Jurkat T cells, as measured by the fluorescent
Figure 4. Interaction between clofazimine and Kv1.3 in vivo and
in vtro. (A) Clofazimine colocalizes with Kv1.3 on the plasma membrane
of Jurkat T cells. Clofazimine was visualized in FITC channel while Kv1.3
was stained by Cy5 secondary antibody. (B) SDS-PAGE analysis of His-
Kv1.3 purified from 293 T cells upon staining with Coomassie blue.
Recombinant Kv1.3 protein was found in the 200 and 300 mM
imidazole elution fractions. (C) Gel shift of clofazimine by Kv1.3 on
native polyacrylamide gel. 10 mM Clofazimine was incubated with
buffer alone (lane 1), 2 mM BSA (lane 2) and 2 mM Kv1.3 (lane 3) for
30 minutes before the samples were resolved on a native polyacryl-
amide gel. Proteins and clofazimine were transferred to nitrocellulose
membrane. The presence of clofazimine was apparent by its intense
organge/yellow color (member picture) and Kv1.3 was detected by
Western blot analysis. The membrane image was taken in the same field
after HRP detection was completed.
Clofazimine Inhibits Kv1.3
PLoS ONE | www.plosone.org6 December 2008 | Volume 3 | Issue 12 | e4009
dye Fura-2AM, is completely inhibited (Fig. 2A–B). In addition to
Kv1.3, T cells express calcium-activated potassium channel that is
responsible for setting up the membrane potential to drive calcium
influx and that, together with TRPM4 and Kv1.3, is responsible
for calcium oscillation in T cells . In Jurkat T cells, this
calcium-activated potassium channel is SKCa2 . The varying
degrees of sensitivity of individual Jurkat T cells in a given
population to clofazimine could be attributed to the different levels
of expression of SKCa2 in them.
There are two related but distinct parameters for measuring
calcium influx, one being the amount of calcium ions entering T
cells via the CRAC channel, which is measured by the Fura-2AM
dye (Fig. 2A–B) and the other being calcium oscillation frequency
(Fig. 2C–D). It has been shown that both a sufficient amplitude of
calcium current and an appropriate oscillation frequency are
essential for activating downstream signaling events . In Jurkat
T cells, three ion channels other than CRAC have been shown to
modulate calcium influx and oscillations, namely TRPM4, Kv1.3
and small-conductance calcium-activated potassium channel, or
hSKCa2 . We did observe a strong correlation between the
effect of clofazimine on calcium oscillation frequency (Fig. 2C–D)
and Kv1.3 inhibition (Fig. 3). Together with previous reports on
the important role of calcium oscillation frequency in signaling
and cytokine gene expression in T cells, these results offer a
coherent and consistent mechanism of action by clofazimine via
inhibition of Kv1.3. However, at the same concentration of
clofazime that inhibited calcium oscillation frequency in over 80%
cells, only 25–50% are affected significantly in the amount of
calcium ions getting into cells (Fig. 2A–B). Thus, even among
those ‘‘non-responsive’’ cells as judged by the change in net
cytosolic calcium concentrations, clofazimine inhibited the calci-
um oscillation in them (Fig. 2. A–B vs. C–D). The same
discrepancy was observed with Psora-4, a well-established Kv1.3
inhibitor (Fig. S8A–B and data not shown). On the other hand, it
has been reported that several selective and potent pharmacolog-
ical inhibitors of KCa channels, but not KV channels, reduce Ca2+
entry in Jurkat and in mitogen-activated human T cells .
Together, these results strongly suggest that the two potassium
channels involved in regulating CRAC current have distinct
functions. While it is unclear whether KCa channels regulate both
the amount of Ca2+entry and oscillation frequency, it is apparent
that Kv1.3 is mainly responsible for enabling oscillation frequency
with less influence on the amount of Ca2+entry (Fig. 2). To the
best of our knowledge, this is the first time that a causal
relationship between Kv1.3 and calcium oscillation frequency
has been directly demonstrated experimentally.
As a new member of a growing family of inhibitors for Kv1.3,
clofazimine is unique in its chemical structure. The tricyclic
phenazine core of clofazimine has not been seen in any other
known inhibitors of Kv1.3, thus providing a new pharmacophore
for structure/activity relationship studies and the development of
new generations of more potent inhibitors. On the other hand,
clofazimine does share some similarity with known Kv1.3
inhibitors in certain structural features. For example, the
polycyclic core is reminiscent of the tricyclic core of Psora-4 and
bicyclic cores of WIN173173. The chlorophenyl substituents on
clofazimine can also be found in WIN173173. Whether these gross
structural similarities can be translated into similar binding modes
of these Kv1.3 inhibitors remains to be determined.
Clofazimine has been used as an anti-leprosy drug since the
early 1960s. In addition to leprosy, clofazimine has also been
tested and found to be efficacious in several autoimmune diseases
including lupus erythematosus [43,44], Crohn’s disease ,
ulcerative colitis , and pustular psoriasis . In particular, it
Figure 5. (A) Over-expression of Kv1.3 in Jurkat T cells resulted in resistance of the IL-2 luciferase reporter to CLF. The IC50s for different curves (Kv1.3
overexpression from low to high) are 41.668.4 nM, 41.0614.5 nM, 3866214 nM, 1.3760.52 mM and 1.4660.63 mM (n=6). (B) Kv1.3 knockdown
resulted in an increase in sensitivity of the IL-2 luciferase reporter to CLF. The IC50s for control EGFP-siRNA lentiviral transduced Jurkat T cells (control)
was 49.9613.0 nM, while that for shKv1.3-4 transduced cells was 10.562.4 nM (n=6). (C) Lentivirus-mediated knockdown of Kv1.3 assessed by
Western blot. (D) Quantitation of Kv1.3 knockdown efficiency. After normalization against GAPDH, lenti-Kv1.3-4 decreased Kv1.3 protein level by 70%
in comparison to the control (Lenti-EGFP).
Clofazimine Inhibits Kv1.3
PLoS ONE | www.plosone.org7 December 2008 | Volume 3 | Issue 12 | e4009
was shown to be effective in human graft-versus-host disease .
To date, however, the molecular mechanism of its immunomod-
ulatory activity as well as its anti-mycobacterial activity has
remained unknown. The surprising connection between clofazi-
mine and the Kv1.3 potassium channel made in this manuscript
not only unraveled a fundamental molecular mechanism of action
of clofazimine in the human immune system, but also suggested
potentially new expanded therapeutic applications of clofazimine.
It is fascinating that clofazimine has been used in a recent trial as
part of a combined antibiotic regimen in Crohn’s disease to test
the hypothesis that Mycobacterium avium subspecies paratuber-
culosis is a possible cause of the disease . Although a
suboptimal dose of clofazimine (50 mg/day) was administered
along with other drugs, the outcome of the clinical trial may have
to be reinterpreted in light of the inhibitory effect of clofazimine on
effector memory T cells uncovered in the present study . In
addition to T cell-mediated graft rejection, Kv1.3 has been shown
to play a uniquely important role in the proliferation and survival
of effector memory T cells which have been implicated in the
pathogenesis of a number of autoimmune diseases. Thus,
clofazimine is likely to be effective on a number of autoimmune
disorders through the same molecular mechanism.
Although clofazimine does have a number of side effects
including GI and skin discoloration [51–53], it is well tolerated
and relatively safe . In particular, it lacks the nephrotoxicity
and neurotoxicity associated with both CsA and FK506 . As
such, clofazimine has great potential in the treatment of
autoimmune diseases such as multiple sclerosis, type 1 diabetes
and psoriasis as well as organ transplantation. The fact that
clofazimine, unlike other known inhibitors of Kv1.3, has been used
in humans for decades may allow for its accelerated clinical
evaluation and eventual introduction as new treatments for
different autoimmune and other pertinent diseases.
Materials and Methods
Cell culture and IL2-Luciferase reporter cell line
Jurkat E6.1 (ATCC TIB152) T cells were maintained in RPMI
medium 1640 (Invitrogen) supplemented with 10% FBS, 2 mM-L-
glutamine, penicillin (50 mg/ml) and streptomycin (50 mg/ml).
HFF-1 (ATCC SCRC1041) fibroblast was maintained in low
glucose DMEM (Invitrogen) supplemented with 10% FBS, 2 mM-
L-glutamine, penicillin (50 mg/ml) and streptomycin (50 mg/ml).
Jurkat E6.1 cells were transfected with linearized pIL2-Luc-Neo
and linearized pMEP4 by electroporation, followed by selection
for resistance to 400 mg/ml hygromycin. The stably transfected
IL-2 reporter cell line, Jurkat/IL-2Luc, was maintained in
hygromycin, which was omitted from medium for the screening
and other assays.
The Hopkins Drug Library (HDL)
An early version of the library has been described .
Clofazimine was purchased from Sigma.
Jurkat/IL-2Luc cells were seeded in 96 well plates (Nunc
136102) at 26105(180 mL) per well. Cells were incubated with
10 mM (final concentration) drugs from the library for 1 h before
they were stimulated with 1 mM ionomycin and 40 nM phorbol
myristate acetate (PMA) for an additional 16 h. Cells were pelleted
by centrifugation. Upon removal of the culture medium, lysis/
assay buffer was added into each well. The luciferase activity was
determined as per manufacture’s instructions.
Calcein incorporation assay
HFF-1 (human forebrain fibroblast) cells (26103in 190 ml) were
seeded into 96-well plates and were incubated with drugs from the
library for 4 days. Cells were washed with PBS twice before they
were treated with 1 mM (final concentration) Calcein-AM
(invitrogen C1430) for 4 h. Plates were directly counted by a
fluorescent plate reader.
Detection of IL-2 secreted from T cells
Jurkat E6.1 T cells (16105in 180 ml) were seeded into 96-well
plates. Cells in each well were treated with different concentrations
of clofazimine for 1 h before 1 mM ionomycin and 40 nM PMA
were added. The incubation was continued for 2 days. The plates
were centrifuged at 1,2006g for 5 min, and the supernatant from
each well was collected followed by ELISA detection of IL-2. The
primary antibody, biotinylated secondary antibody and HRP
conjugated avidin were purchase from BD/Pharmingen.
Dephosphorylation of endogenous NFATc2
Jurkat T cells were pretreated with indicated compounds for 1 h
before 3 mM (final concentration) ionomycin was added. Cells
were harvested after 30 min and lysed in a lysis buffer [40 mM
Tris (pH 7.8), 1% NP-40, 10 mM EDTA, 60 mM Na3P4O7and
common protease inhibitors] by sonication. NFATc2 was resolved
by 8% SDS-PAGE followed by Western blot analysis using anti-
NFAT antibodies (Santa-cruz Sc-7296, 1:100 dilution).
Figure 6. Clofazimine inhibits human T cell-mediated skin graft
rejection in immunodeficient mice. (A) Representative skin grafts
at different days post-transplantation. Human foreskin was transplanted
onto Pfp/Rag22/2 mice. 1.06108human peripheral blood lympho-
cytes (PBL) were adoptively transferred into each animal at Day 7 post-
transplantation. Administration of clofazimine or carrier control (olive
oil) was also initiated at Day 7. * Days after skin transplantation/cell
transfer. (B) Effect of clofazimine on the mean survival time of
transplanted human (n=5) and mouse (n=4) skin grafts. The mouse
skin transplantation was performed using Balb/c mice as skin donors,
B6 Rag12/2 mice as recipients and PBL from B6 for adoptive transfer.
Clofazimine Inhibits Kv1.3
PLoS ONE | www.plosone.org8 December 2008 | Volume 3 | Issue 12 | e4009
Calcium imaging assay
Jurkat cells were attached to L-lysine-coated glass dishes
(MatTek). Cells were loaded with 1 mM fura-2-AM (Invitrogen
F-1201) for 45 min before they were washed sequentially with
growth medium and Ca2+-free HBSS (Hank’s buffer with 2 g/L
glucose and 20 mM HEPES, pH 7.4). Fluorescence excitation
wavelengths were set at 350 and 380 nm, respectively, on a Zeiss
Axiovert 200M microscope while emission wavelength was set at
510 nm. Changes in intracellular calcium concentrations were
determined by fluorescence intensity ratio (F350/F380).
Measurement of calcium oscillation period
Calcium oscillation was stimulated by 10 nM TG and 5 mM
clofazimine was added 30 min after initiation of oscillation by TG.
The average oscillation periods before and after drug addition
were determined by total time divided by peak numbers. Changes
in average oscillation period larger than 10 seconds were
considered significant while those smaller than 10 seconds were
ignored for statistical analysis.
Patch pipettes, pulled from glass capillaries (inner diameter
1.5 mm, Kimble products) with a horizontal puller (Sutter
instruments, Modell P-97), were fire-polished, and had resistances
between 2 and 4 MOhm. Patch-clamp experiments were performed
in the tight-seal whole-cell configuration at room temperature (22–
24uC). High-resolution current recordings were acquired by a
computer-based patch-clamp amplifier system (EPC-9, HEKA).
Immediately following establishment of the whole-cell configuration,
every two seconds voltage ramps of 50 ms duration spanning the
voltage range of 2100 to +100 mV for Jurkat T cells and 2150 to
+150 mV for HEK293 were delivered from a holding potential (Vh)
of 270 mV for Jurkats and 0 mV for HEK293 cells over a period of
300–600 s. Heterologously expressed potassium currents (Kv1.1,
Kv1.2, Kv1.3, Kv1.5 and Kv3.1) were acquired using a voltage
ramp from 2100 mV to +100 mV over 500 ms and at 2 s (Kv3.1 &
Kv1.2) or 5 s intervals from a Vhof 280 mV. All voltages were
corrected for a liquid junction potential of 10 mV. Currents were
filtered at 2.9 kHz and digitized at 100 ms intervals. Capacitive
currents and series resistance were determined and corrected before
each voltage ramp using the automatic capacitance compensation of
the EPC-9. Kv1.3 currents were normalized to the point of
application of 5 mM clofazimine. Kv1.3 currents in Jurkat T cells
were measured at +80 mV. Heterologously expressed Kv1.1, Kv1.2,
Kv1.3, Kv1.5 and Kv3.1 were assessed at +80 mV except in Fig. S5,
where currentswereadditionallymeasured at the morephysiological
voltage of 0 mV. CRAC currents in HEK293 cells were assessed at
280 mV. Standard external solution contained (in mM): 140 NaCl,
1 CaCl2, 2 MgCl2, 2.8 KCl, 10 HEPES-NaOH, with pH at 7.2 and
osmolarity 300–320 mosm. The solution contained 10 mM CaCl2
in the case of CRAC measurements. Internal standard solution
contained (in mM): 120 K-glutamate, 1 MgCl2, 8 NaCl, 10HEPES-
KOH, 10 K-BAPTA, with pH 7.2 and osmolarity 290–310 mosm.
The internal solution was supplemented with 20 mM IP3for CRAC
measurements. Substance application was performed on individual
cells using a wide-mouth glass pipette connected to a pneumatic
Jurkat T cells treated with clofazimine were centrifuged onto L-
lysine-coated coverslips. Cells were fixed with 4% p-formaldehyde
for 15 min. The cells were washed in PBS, permeabilized by 220uC
methanol and blocked with 10% donkey serum in PBS. The fixed
cells were then incubated with Kv1.3 primary antibody (sc-17239 at
1:100 dilution) for 1 h. Cells were washed in PBS (365 min) and
incubated with donkey anti-goat Cy5 antibodies for an additional
1 h. Cells were then washed three times in PBS, mounted, and
photographed. Mounting was performed using Vectashield mount-
ing medium (Vector Laboratories) and images were captured using
either a Zeiss LSM510 confocal microscope. Merged images were
compiled using LSM5 Image Examiner or Adobe Photoshop CS.
shRNA Lentivirus Production
The targeting sequence of sh Kv1.3-4 is 59-GCCACCTT-
CTCGCGAAACAT-39. Recombinant lentiviruses were generated
using a three-plasmid system described previously . Virus was
harvested and concentrated (1:50) 2 days after transfection. Jurkat
T cells were transduced by concentrated virus at a ratio of 56106
cells/150 ml virus. Cells were cultured for two days before they
were harvested for Western blot analysis and other experiments.
Clofazimine gel-shift assay
The lower resolving gel was cast with 6% acrylamide, 375 mM
Tris (pH 8.5), 0.1% APS and 0.04% TEMED. The upper stacking
gel was cast with 5% acrylamide, 125 mM Tris (pH 6.8), 0.1% APS
and 0.1% TEMED. Clofazimine was incubated with or without
indicated protein in a buffer (20 mM Tris (pH 7.4), 200 mM KCl,
0.1% phosphatidylcholine, 1 mM iodoacetamide and 2% CHAPS)
for 30 minutes before aliquots of 56loading buffer (50% glycerol,
1M Tris (pH 8.5)) was added. The running buffer was made from
14.4 g/l glycine and pH was adjusted by Tris to 8.5. Electrophoresis
was carried out overnight at 4uC with stable current of 11 mA. The
proteins and clofazimine were transferring onto a nitrocellulose
membrane in regular transfer buffer with 0.1% SDS. Once on the
membrane, clofazimine can be visualized directly as a light yellow
band. The membrane was then subject to staining with anti-Kv1.3
antibodies to locate the protein band.
Western blot analysis of Kv1.3
Jurkat T cells were washed once in PBS and lysed by RIPA
buffer with 5 mM PMSF, 10 mg/ml each of pepstatin, leupeptin
and aprotinin. The protein concentrations were determined by
Bradford assay before the lysate was boiled with loading buffer and
resolved by SDS-PAGE. Proteins were transferred onto a
nitrocellulose membrane under 100 volts for 1 h. The membrane
was blotted with anti-kv1.3 antibody (sc-17241, Santa Cruz) with a
dilution of 1:200 in 5% milk followed by wash and incubation with
HRP-conjugated donkey anti-goat IgG (sc-2020). The membrane
was developed using an ECL kit (Pierce 34078).
Human mixed lymphocyte reaction (MLR)
The human MLR was established by coculturing normal
human PBMN responder lymphocytes (0.56106) with an equal
number of miomycin C-treated stimulator cells in RPMI1640
complete medium. The cells were incubated with varying doses of
clofazimine at 37uC in a humidified atmosphere of 5% CO2for 4
days. Then 1 mCi of [3H]-thymidine was added into culture and
incubation was continued for 6 h. The cells were harvested and
[3H]-thymidine incorporated into cells was measured in a liquid
Mouse model of human skin transplantation
The experimental procedure is similar to that described
given p.o. at 50 mg/kg/day in a total volume of 0.2 ml per
Clofazimine Inhibits Kv1.3
PLoS ONE | www.plosone.org9 December 2008 | Volume 3 | Issue 12 | e4009
For additional experimental procedures, see Materials and
and the nuclear translocation of NFAT in response to ionomycin
treatment. (A) Dose-dependent enhancement of the AP-1 luciferase
reporter gene by CsA (n=6). (B) Clofazimine inhibits EGFP-
NFATc3 nuclear translocation in Jurkat T cells stimulated by 1 mM
ionomycin. Images were taken 30 minutes after addition of
ionomycin. (C) Dose-dependent inhibition of ionomycin-stimulated
NFAT nuclear translocation by clofazimine (n=3).
Found at: doi:10.1371/journal.pone.0004009.s001 (0.18 MB TIF)
Effects of clofazimine on AP-1 luciferase reporter gene
calcineurin in vitro. (A) Clofazimine does not inhibit the protein
phosphatase activity of calcineurin in vitro. 20 mM p-nitrophe-
nylphosphate was incubated with purified recombinant calcineurin
A/B and calmodulin in the presence of 1 mM calcium or 5 mM
EGTA at 30uC. The progress of the reaction was followed by
absorbance at 410 nm every 0.5 second. Representative curves of
three different experiments. (B) Clofazimine does not inhibit
NFATc2 dephosphorylation by calcineurin in vitro. NFATc2 was
immuno-precipitated from Jurkat lysate and incubated with
recombinant calcineurin A/B and calmodulin for 30 min at room
temperature in the presence of 1 mM Ca2+ or 5 mM EGTA. The
reaction mixtures were subjected to SDS-PAGE, followed by
Western blot using anti-NFAT antibodies. (C) Clofazimine does
not interfere with calcineurin-NFATc2 interaction. GST-NFATc2
(1–415) was purified by glutathione-sepharose beads and incubat-
ed with Jurkat cell lysate. The pull-down products were resolved
by SDS-PAGE and detected by a-calcineurin antibody. (D)
Clofazimine does not affect binding calcineurinA (1–400,
H160N) and NFATc2 (1–415) in Jurkat T cells in a mammalian
two-hybrid assay. But it inhibits the calcium-dependent enhance-
ment of the calcineurin-NFATc2 interaction. (n=6)
Found at: doi:10.1371/journal.pone.0004009.s002 (0.35 MB TIF)
Clofazimine does not affect the enzymatic activity of
Jurkat T cells without affecting reconstituted ICRAC in HEK293
cells. (A, B) Time-dependent increase in the population of cells
that are sensitive to clofazimine. Jurkat T cells were incubated with
clofazimine for varied lengths of time before 1 mM TG was added.
Images were taken 30 min after 2 mM calcium was added. (C)
Average CRAC current densities at 280 mV induced by IP3
(20 mM) in stable STIM1 expressing HEK293 cells transiently
overexpressing CRACM1, CRACM2 and CRACM3. Holding
potential was at 0 mV. The bar indicates the time for clofazimine
Found at: doi:10.1371/journal.pone.0004009.s003 (0.56 MB TIF)
Effect of 10 mM clofazimine on heterologous Kv1.1,
Kv1.2 Kv1.5 and Kv3.1. (A) Average time course of mouse Kv1.1
currents stably expressed in L929 cells. Control cells (closed circles,
n=5, no application) and cells superfused with 10 mM clofazimine
added to the standard extracellular solution (open circles, n=6) as
indicated by the black bar. Voltage protocol, solutions and analysis
as outlined in Fig. 3D. (B) Current-voltage relationship (I/V) of a
representative cell expressing mouse Kv1.1 with control I/V (black)
extracted at 40 s after whole-cell establishment and the I/V for
clofazimine extracted at the end of application (red, 160 s). (C)
Average time course of heterologous rat Kv1.2 expressed in B82
cells. Control cells (closed circles, n=5, no application) and cells
superfused with 10 mM clofazimine (open circles, n=5, black bar
indicates application time) are shown. Acquisition and analysis as in
Clofazimine alters calcium oscillation patterns in
(A). (D) I/V of a representative cellexpressing rat Kv1.2 with control
I/V (black) extracted at 40 s after whole-cell establishment and the
I/V for clofazimine extracted at the end of application (red, 160 s).
(E) Average time course of heterologous human Kv1.5 expressed in
MEL cells. Control cells (closed circles, n=5, no application) and
cells superfused with 10 mM clofazimine (open circles, n=5, black
in (A). (F) I/V of a representative cell expressing human Kv1.5 with
control I/V (black) extracted at 40 s after whole-cell establishment
and the I/V for clofazimine extracted at the end of application (red,
160 s). (G) Average time course of heterologous mouse Kv3.1
expressed in L929 cells. Control cells (closed circles, n=5, no
application) and cells superfused with 10 mM clofazimine (open
circles, n=5, black bar indicates application time) are shown.
Acquisition and analysis as in (A). (H) I/V of a representative cell
expressing mouse Kv3.1 with control I/V (black) extracted at 40 s
after whole-cell establishment and the I/V for clofazimine extracted
at the end of application (red, 160 s).
Found at: doi:10.1371/journal.pone.0004009.s004 (0.41 MB TIF)
Inhibition of Kv channels by 10 mM clofazimine
assessed at 0 mV (red bars) or +80 mV (black bars). Same cells as
in Fig. 3D and Fig. S4 were used. Note the increased inhibitory
effect at 0 mV for all Kv channels displayed except Kv1.3.
Found at: doi:10.1371/journal.pone.0004009.s005 (0.19 MB TIF)
IL-2 luciferase reporter to CsA. The IC50 for control EGFP-siRNA
lentiviral transduced Jurkat T cells was 2.560.6 nM, and that for
Kv1.3 lentiviral 4 transduced T cells was 2.660.7 nM (n=6).
Found at: doi:10.1371/journal.pone.0004009.s006 (0.00 MB TIF)
Knockdown of Kv1.3 did not affectthe sensitivity of the
production and mixed lymphocyte reaction in murine T cells. (A)
Dose response of IL-2 production from anti-CD3/anti-CD28-
stimulated mouse primary T cells to different concentrations of
clofazimine (n=3). (B) Biphasic effects of clofazimine on mouse
mixed lymphocyte reaction (n=3).
Found at: doi:10.1371/journal.pone.0004009.s007 (0.00 MB TIF)
Effects of clofazimine on mouse TCR-mediated IL-2
of different reporter genes in Jurkat T cells. (A) Calcium influx in
Jurkat T cells was inhibited by 10 mM psora-4 in a heterogeneous
fashion. Psora-4 was added 5 minutes before stimulation with 1 mM
TG. Representative images were taken 30 minutes after 2 mM
calcium wasadded.Thecolor gradient represents fura-2 at 350 nm/
380 nm excitation ratio. (B) Quantitation of 350 nm/380 nm ratios
for calcium imaging results shown in (A). Jurkat T cells can be
divided into psora-4 responsive (R) and psora-4 non-responsive (NR)
groups. (C) Psora-4 inhibits NFAT pathway in Jurkat T cells. The
IC50s of psora-4 for IL-2, NFAT reporters are 9.361.7 mM and
29.668.2 mM, respectively. And the IC50 of clofazimine for NF-kB
luciferase reporter assay is over 1 mM (n=6 each). All the reporters
were stimulated with PMA/ionomycin. (D) Psora-4 significantly
enhances AP-1 luciferase reporter at high concentrations (.50 mM,
n=6), similar to clofazimine.
Found at: doi:10.1371/journal.pone.0004009.s008 (0.77 MB TIF)
Effects of Psora-4 on calcium influx and the activation
Materials and Methods S1
Found at: doi:10.1371/journal.pone.0004009.s009 (0.10 MB
Supporting Materials and Methods
We thank Drs. Murali Prakriya, Shaoyu Ge, and Hongjun Song for helpful
discussions and Dr. George Chandy for helpful comments during the
revision of this manuscript.
Clofazimine Inhibits Kv1.3
PLoS ONE | www.plosone.org10 December 2008 | Volume 3 | Issue 12 | e4009
Author Contributions Download full-text
Conceived and designed the experiments: YRR FP JL. Performed the
experiments: YRR FP SP AF JX YD HJ RP. Analyzed the data: YRR FP
AF JZ HJ RP JL. Contributed reagents/materials/analysis tools: CRC JZ.
Wrote the paper: YRR AF HJ RP JL.
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Clofazimine Inhibits Kv1.3
PLoS ONE | www.plosone.org11 December 2008 | Volume 3 | Issue 12 | e4009