Kv1.3 Channels Can Modulate Cell Proliferation During
Phenotypic Switch by an Ion-Flux Independent Mechanism
Pilar Cidad,* Laura Jiménez-Pérez,* Daniel García-Arribas, Eduardo Miguel-Velado, Sendoa Tajada,
Christian Ruiz-McDavitt, José R. López-López,† M. Teresa Pérez-García†
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Objective—Phenotypic modulation of vascular smooth muscle cells has been associated with a decreased expression of all
voltage-dependent potassium channel (Kv)1 channel encoding genes but Kcna3 (which encodes Kv1.3 channels). In fact,
upregulation of Kv1.3 currents seems to be important to modulate proliferation of mice femoral vascular smooth muscle
cells in culture. This study was designed to explore if these changes in Kv1 expression pattern constituted a landmark of
phenotypic modulation across vascular beds and to investigate the mechanisms involved in the proproliferative function
of Kv1.3 channels.
Methods and Results—Changes in Kv1.3 and Kv1.5 channel expression were reproduced in mesenteric and aortic vascular
smooth muscle cells, and their correlate with protein expression was electrophysiologicaly confirmed using selective
blockers. Heterologous expression of Kv1.3 and Kv1.5 channels in HEK cells has opposite effects on the proliferation
rate. The proproliferative effect of Kv1.3 channels was reproduced by “poreless” mutants but disappeared when voltagedependence of gating was suppressed.
Conclusion—These findings suggest that the signaling cascade linking Kv1.3 functional expression to cell proliferation is
activated by the voltage-dependent conformational change of the channels without needing ion conduction. Additionally,
the conserved upregulation of Kv1.3 on phenotypic modulation in several vascular beds makes this channel a good target
to control unwanted vascular remodeling. (Arterioscler Thromb Vasc Biol. 2012;32:1299-1307.)
Key Words: gene expression  ion channels  vascular muscle  cell proliferation  phenotypic modulation
V
changes associated with the phenotypic switch in 2 proliferative models of murine femoral arteries.6 We observed a
general decrease of ion channels expression on proliferative
switch, except for the voltage-dependent potassium channel
(Kv) Kv1.3 and the Kv2 auxiliary subunit. Kv1.3 increase
parallels an enormous decrease of Kv1.5, the dominant Kv1
channel in the contractile state. Moreover, Kv1.3 functional
expression associates with proliferative phenotype because
channel blocking induces a significant inhibition of cell proliferation. Interestingly, the relevant role of Kv1.3 channels in
VSMC proliferation has recently also been shown in a human
model of neointimal hyperplasia.7
Kv channels constitute a large and ubiquitous family of
membrane proteins present in both excitable and nonexcitable
cells. In nonexcitable cells, their function as feedback regulators of resting VM has been proposed to participate in many
cellular functions ranging from secretion to cell migration,
proliferation, and apoptotic death. Kv channel genes can give
rise to an even larger number of functional Kv currents, via
heteromultimerization, association with accessory subunits,
ascular smooth muscle cells (VSMC) are essential components of the vessel wall that contribute to control blood
flow through changes in their contractile activity. This function requires the expression of a set of proteins conducive for
structural support and contraction. However, this contractile
phenotype changes during several vascular disorders toward a
proliferative phenotype, where the new set of expressed proteins provides the acquisition of a proliferative and migratory
state. Because this phenotypic switch significantly contributes
to the pathogenesis of vascular diseases, including atherosclerosis or hypertension, the study of its molecular determinants
has a special significance.
One set of proteins particularly relevant in this context
comprises ion channels required for controlling basic parameters, such as cell volume or membrane potential (VM). Their
contribution to VSMC function is well characterized in the
contractile phenotype but is just beginning to be appreciated
in the proliferative state.1–5 The diversity of channels described
as modulators of proliferation prompted our laboratory to
carry out a global portrait of ion channel gene expression
Received on: November 23, 2011; final version accepted on: February 21, 2012.
From the Departamento de Bioquímica y Biología Molecular y Fisiología e Instituto de Biología y Genética Molecular (P.C.,L.J.-P., D.G.-A., E.M.-V.,
S.T., C.R.-M., J.R.L.-L., M.T.P.-G.), Universidad de Valladolid y CSIC, Valladolid, Spain.
*P.C. and L.J.-P. contributed equally to this article.
†J.R.L.L. and M.T.P.G. are equal senior authors.
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.111.242727/-/
DC1.
Correspondence to M. Teresa Pérez-García, Departamento de Bioquímica y Biología Molecular y Fisiología, Universidad de Valladolid, Edificio IBGM,
c/ Sanz y Forés s/n, 47003 Valladolid, Spain. E-mail tperez@ibgm.uva.es
© 2012 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.orgDOI: 10.1161/ATVBAHA.111.242727
1299
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alternative splicing, and posttranslational modifications.8 Their
contribution to these “nonexcitable” functions, particularly
migration and proliferation, has been well documented for
several Kv channels in different cellular systems, including
Kv10.1, Kv11.1, and Kv1.3.9,10 Association between Kv1.3
expression and cell proliferation has been postulated in many
cell types, including several cancer cell lines,9,10 endothelial
cells,11 microglia,12 T lymphocytes,13 macrophages,14 and oligodendrocyte progenitors,15 but the underlying mechanisms linking Kv1.3 upregulation to proliferation remain controversial. In
T lymphocytes, genetic or pharmacological Kv1.3 blockade has
an antiproliferative effect due to membrane depolarization and
consequent reduced calcium entry.13 However, this may not be
the case in VSMC, because even though Kv1.3 inhibition leads
to depolarization, proliferation was not affected by changes in
VM that were not mediated by Kv1.3.6 This intriguing observation suggests that the proproliferative effect of Kv1.3 upregulation may not be related to its role as a K+ channel.
In this work, we sought to explore in more detail the functional meaning of the Kv1.5 to Kv1.3 channel switch associated with VSMC proliferation. First, we explored if this
channel switch is a conserved feature linked to proliferation
in different vascular beds. Next, we investigated if the association between Kv1.3 expression and increased cell proliferation could be reproduced in a heterologous system, which
could be exploited for studying the mechanisms involved in
this new function of Kv1.3 channels. We found that the Kv1.3
to Kv1.5 ratio is a landmark of the VSMC phenotype in all
the preparations studied. Functional expression of Kv1.3 in
HEK cells increased proliferation rate, and selective Kv1.3
blockers reverted this effect. Furthermore, the use of channel
mutants with altered permeation and/or gating suggests that
Kv1.3 could be a moonlighting protein that regulates intracellular signaling pathways leading to cell proliferation by a
mechanism that is independent of potassium flux.
Materials and Methods
VSMC Isolation and Culture
BPN mice (Jackson Laboratories) were maintained with inbred crossing in the animal facilities of the University of Valladolid. Mice were
killed by decapitation after isofluorane anesthesia, using protocols
approved by the ethical committee of our University and in accordance with the European Community guiding principles. Femoral,
aorta, or mesenteric arteries were dissected and cleaned of connective and endothelial tissues, and stored for RNA extraction or used
directly to obtain fresh dispersed VSMC (contractile VSMC) or cultured VSMC (proliferative VSMC) as previously described.3,6
Plasmids Construction and Transfection
Kv1.3 plasmids were obtained from 8860686 IMAGE mouse cDNA
clone (Source Bioscience). Mutants were generated by site-directed
mutagenesis using QuikChange (Stratagene). HEK293 cells were
transient transfected using Lipofectamine 2000 (Invitrogen).
RNA Expression Profile
mRNA levels were determined by qPCR with Taqman probes in a
Rotor-Gene 3000 instrument (Corbett Research). Data were analyzed
with the threshold cycle relative quantification method (ΔΔCt).6 Each
sample derived from ≈500 ng of mRNA from contractile or proliferative VSMC.
Protein Expression and Function
Selective antibodies were used for protein detection by Western blot
and immunocytochemistry.6 For functional studies, the whole-cell
and perforated-patch configuration of patch-clamp technique were
used in VSMC or transfected HEK cells, as previously described.6
Proliferation Assays
Proliferation was studied with Click-iT EdU incorporation assays
(Molecular Probes) and counting cells with a hemocytometer.
For an expanded Methods section, please see the online-only Data
Supplement.
Results
When characterizing the ion channels expression profile associated with the phenotypic switch of mice femoral VSMC, we
found an upregulation of Kv1.3 channels, whose functional
expression could be related to proliferation.6 However, looking at the expression levels of the Kv1 subfamily members
(Figure 1A), it is noticeable that contractile VSMC have a
predominant expression of Kv1.5 channels (whose functional contribution has been characterized16–18); whereas
Kv1.5 expression is almost absent in cultured VSMC, where
Kv1.3 expression predominates. Data from 3 different vascular beds (Figure 1B) show that the Kv1.5 to Kv1.3 switch
on proliferation is conserved in VSMC from conduit and
resistance arteries, suggesting that the ratio Kv1.3:Kv1.5 is
a representative parameter of the VSMC phenotype. We also
confirmed that the relative functional expression of channel
proteins parallels the mRNA expression levels of Kv1.3 and
Kv1.5. Figure 1C shows representative experiments obtained
from femoral artery VSMC, either freshly dissociated or cultured, when testing the effect of selective blockers of Kv1.5
channels (diphenyl phosphine oxide-1) or Kv1.3 channels
(5-(4-phenoxybutoxy) psoralen [PAP-1]). Identical results
were obtained from mesenteric VSMC (Figure 1D). Because
the Kv1.5 to Kv1.3 switch is present in all the studied VSMC
types, we hypothesized that it could be a relevant event needed
to facilitate proliferation.
VSMC in primary culture are difficult to obtain in large
amounts and hard to transfect efficiently as to study effects in
cell populations. For these reasons, we explored if our hypothesis held true in a system more amenable to modify Kv channels
expression in order to investigate the molecular mechanisms
involved in their effects on cell proliferation. With this idea, we
studied the effect of Kv1.3 and Kv1.5 overexpression on the
proliferation rate of HEK cells, using 2 alternative methods:
cell count and determination of the percentage of cells entering S-phase of the cell cycle using Click-iT EdU. Figure 2A
represents the pooled data obtained from several experiments in
which cell count was performed in HEK cells up to 4 days after
transfection with vectors expressing Kv1.3 or Kv1.5. Because no
differences in proliferation were detected among untransfected,
mock transfected, and cells transfected with the empty vector,
we have used this latter condition as control. Kv1.3 overexpression significantly increased proliferation rate up to 60 hours after
transfection, with a loss of this effect at longer times probably
due to plasmid dilution with cell divisions. On the contrary,
the expression of Kv1.5 had an antiproliferative effect at all
times explored. No evidence of increased apoptosis was
Cidad et al Mechanisms of Kv1.3 Channel-Induced Proliferation 1301
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Figure 1. A, Relative mRNA abundance of the voltage-dependent potassium channel (Kv)1 family genes in contractile and proliferating vascular smooth muscle cells (VSMC) from mice femoral arteries. Expression levels were normalized with respect to RP18S and
expressed as 2–∆Ct, where ∆Ct=Ctchannel-Ct18s. Each bar is the mean of 4 to 6 determinations obtained in 2 to 3 duplicate assays. All
through the article, *P<0.05; ** P<0.01, ***P<0.001. B, The relative abundance of Kv1.3 and Kv1.5 in 3 different vascular beds in both
conditions is illustrated by the pie charts. The bars plot shows the Kv1.3:Kv1.5 ratio expressed as log (2–∆∆Ct), where ∆∆Ct was ∆CtKv1.3–
∆CtKv1.5. A value of 0 indicates a Kv1.3:Kv1.5 ratio of 1, a value of –2 denotes Kv1.5 expression levels 100 times higher than Kv1.3, and
a value of +2 Kv1.3 expression levels 100 times higher than Kv1.5. Each data point was obtained from at least 4 independent determinations. C, Current amplitude at the end of depolarizing pulses to +40 mV from a holding potential of –80 mV, applied every 10 s, from a
freshly dissociated (contractile) and a cultured (proliferative) femoral VSMC. Diphenyl phosphine oxide-1 (DPO) or 5-(4-phenoxybutoxy)
psoralen (PAP-1) were present in the bath solution during the indicated times. The insets show sample current traces in each condition.
D, Averaged data showing the fraction of the total Kv current represented by Kv1.5 currents (DPO sensitive) and Kv1.3 currents (PAP-1
sensitive) in contractile vs proliferative VSMC from femoral and mesenteric arteries. Mean±SEM, 4 to 7 cells/condition.
observed with TUNEL assays, in spite of previous reports
suggesting a proapoptotic role of Kv1.5 channels in some
VSMC preparations19,20 (Figure II in the online-only Data
Supplement).
When transfection of HEK cells was carried out with
decreased amounts of Kv1.3 plasmid (to tritate down the current density) we found a good correlation between the rate of
proliferation and the amplitude of the Kv currents recorded in
the transfected cells (Figure 2B, open symbols), that saturated
when higher amounts of plasmid (and higher currents) were
obtained. In femoral VSMC, phenotypic switch associates with
the increased expression of both Kv1.3 and Kv2. Because
Kv2 has a chaperone effect increasing the functional expression of Kv1.3 channels,21 we have explored if Kv2 coexpression could affect HEK proliferation. Using different amounts
of Kv1.3 plasmid alone or in combination with excess Kv2
plasmid we have confirmed the chaperone effect of Kv2
in electrophysiological studies (Figure 2B, inset). However,
Kv2 coexpression (Figure 2B, filled symbols) did not elicit a
proliferative effect different from the maximal effect obtained
with Kv1.3 alone, suggesting that Kv2 effect on proliferation simply relates to its chaperon effect. Accordingly, Kv2
coexpression was able to significantly increase proliferation
at low concentrations of Kv1.3 plasmid (Figure 2B, bars plot),
but no additional effect was observed at saturating expression
levels of Kv1.3 plasmid.
The specificity of the effect of Kv1.3 on proliferation rate
was confirmed by Kv1.3 pharmacological blockade. Figure
2C plots the summary data of the proliferation rate of control
or Kv1.3-transfected cells using Click-iT EdU assay. Seruminduced proliferation of HEK cells was significantly augmented in Kv1.3 transfected cells and could be abolished in
the presence of MgTx (10 nmol/L) or PAP-1 (100 nmol/L). At
these concentrations (within the range of complete blockade
of Kv1.3 currents) the drugs had no effect on the proliferation rate of control cells. Similar results were obtained by cell
counting (Figure 2D).
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Figure 2. A, Proliferation time course of HEK cells explored in untransfected, mock transfected or cells transfected with plasmids
expressing voltage-dependent potassium channel (Kv)1.3 (dsRED2ires-Kv1.3) or Kv1.5 (Kv1.5-EGFP). Data are normalized for the proliferation of dsRED2ires-transfected HEK cells (empty vector). Each point is the mean±SEM of 5 to 8 experiments, with 4 to 8 different
data points each. B, HEK cells were transfected with increasing amounts (from 1–250 ng/35 mm dish) of Kv1.3 alone (open symbols) or
together with Kv2 (filled symbols), and both proliferation rate (determined by cell counting at 24 hours after transfection) and current
density of the transfected cells (obtained from the current amplitude elicited in depolarizing pulses to +40mV) were monitored. Each current density data point is the average of 3 to 6 cells/culture. The line shows the fit of the data to a hyperbolic function (Pmax 170.6%, P0.5
0.57 nA/pF). The inset shows the current density obtained in HEK cells transfected with different amounts of Kv1.3 without (empty dots)
or with (filled dots) Kv2 subunit. Both groups of data were fitted to linear functions. Each point is mean±SEM of 6 to 12 determinations.
The proliferation rate of HEK cells transfected with low (5 ng) and high (250 ng) amounts of Kv1.3 alone or in combination with excess of
Kv2 are represented in the bars plot. C, Proliferation rate of HEK cell transfected with dsRED2ires vector (control) or dsRED2ires-Kv1.3
was determined by measuring the fraction of cells incorporating EdU reagent after 24 h incubation in serum-free media (SF) or with 5%
FSB (5%S), alone or in the presence of MgTx or 5-(4-phenoxybutoxy) psoralen (PAP-1) (mean±SEM, n=4–10). Statistical significance was
determined against control cells incubated with 5% FBS. D, The effect of treatment with 10 nmol/L MgTx on the proliferation rate was
also explored by cell counting at two different times after transfection (mean±SEM, n=4).
Voltage-dependent ion channels sense changes in membrane potential (VM) and catalyze ion fluxes that modulate
those changes. Theoretically, the channel proproliferative
effect could require the voltage-sensing, the VM modulation,
or both. To distinguish between these options, we designed
some Kv1.3 mutant channels in which 1 or the 2 functions
were lost. We created 2 pore mutants: Kv1.3AYA (G399A/
G401A), a dominant-negative pore mutant, and Kv1.3WF
(W389F), a nonconducting point mutant with intact gating
properties. Finally, Kv1.3WF3x (R320N/L321A/R326I), was
devised as a nonconducting and voltage-insensitive channel,
as the triple mutation in S4 region shifts channel activation
to potentials below –170 mV, so that the channel remains
inactivated along the physiological range of voltages.22 All
the constructs were created as GFP fusion proteins to allow
their localization. Confocal microscopy using an extracellular Kv1.3 antibody in nonpermeabilized cells showed a clear
membrane localization of all proteins but Kv1.3AYA (Figure
3A). No differences in protein expression were observed for
all Kv1.3 constructs, and the percentage of membrane expression was similar for Kv1.3, WF, and WF3X (Figure 3B, C).
Moreover, immunoblot using anti-Kv1.3 revealed a band of the
expected molecular size and an extra band of slightly higher
molecular weight in all but Kv1.3AYA (Figure 3B). This
extra band reflects the N-glycosilation of the channel located
at the plasma membrane, as it disappeared on treatment
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Figure 3. A, Confocal images obtained in HEK cells transfected with vectors expressing GFP fusion proteins of voltage-dependent
potassium channel (Kv)1.3 or the different Kv1.3 mutants. (Mutations are depicted in the table). The panels show GFP fluorescence
(green), labeling of nonpermeabilized cells with an extracellular anti-Kv1.3 channel (red), the nuclear staining with Hoechst (blue) and
the bright field image. B, Immunoblot of cell lysates from these cultures with anti-Kv1.3 antibody shows a band (upper arrow) of the
expected molecular weight (in kDa, for the Kv1.3.GFP fusion protein) and a band of higher molecular weight in all cases but in the
Kv1.3AYA expressing cells. Loading control (-actin) of the same membrane is also shown. C, White bars show average data of Kv1.3
protein expression normalized to -actin protein from several inmunoblots as the one shown in B. The gray bars represent averaged fraction of extracellular anti-Kv1.3 labeling obtained from confocal images of nonpermeabilized cells as in panel A (see also Figure I in the
online-only Data Supplement).
of the samples with N-glycosidase (Figure III in the onlineonly Data Supplement).
Functional characterization of the channels was carried out
with the patch-clamp technique (Figure 4). Although large
voltage-dependent outward currents were recorded in Kv1.3
channels-expressing cells, only the gating currents could be
recorded at the onset and the end of the pulse when cells
expressed Kv1.3WF mutant. Gating currents were absent
in the Kv1.3WF3X mutant, as there are not conformational
changes along the range of voltages explored (Figure 4A).
Gating currents similar to the Kv1.3WF mutant were disclosed
in Kv1.3-transfected cells when K+ fluxes were almost completely blocked by replacing intra- and extracellular K+ with
N-methyl–glucamide (Figure 4B). Additional confirmation of
the behavior of the channels was obtained in current-clamp
experiments, as only Kv1.3-transfected cells showed a significant hyperpolarization that was sensitive to Kv1.3 selective
blockers (Figure 4C and 4D).
The effect of the Kv1.3 mutants on proliferation was
assessed by cell counting and by Click-iT EdU assays with
comparable results (Figure 5). Only Kv1.3WF was able to
induce proliferation, being the magnitude and the time course
of the effect indistinguishable from wild-type Kv1.3 channels. In fact, the proproliferative effect of Kv1.3WF was also
sensitive to 10 nmol/L MgTx. These results strongly suggest that the only requirement to induce the proproliferative
effect is the presence of the voltage sensor in the membrane.
However, they open the question of how MgTx (an open channel blocker that inhibits channel permeation23) inhibits Kv1.3
induced proliferation. The characterization of the effects of
MgTx and PAP-1 on Kv1.3WF gating currents (Figure 6)
indicated that these 2 blockers are able to interfere with the
conformational changes associated with the movement of the
voltage sensor. Figure 6A shows an example of the reversible
effect of PAP-1 on the gating currents of a Kv1.3WF transfected cell. MgTx and PAP-1 significantly reduced the maximal charge movement for the Qon (Figure 6B) and the Qoff
(Figure 6C), and PAP-1 produced a significant leftward shift
in the voltage dependence of the Qon movement. No significant changes in the slope of the Boltzmann fits were observed
(data not shown). Therefore, MgTx and PAP-1 may inhibit
Kv1.3 by producing some charge immobilization, in addition
to their effects blocking K+ fluxes.
Discussion
Many novel targets involved in VSMC phenotypic switch
have been investigated in several in vitro and animal models
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Figure 4. A, Representative current traces obtained from the indicated constructs with Standarde and High-Ki solutions (bath and pipette,
respectively) with the voltage protocol shown at the bottom. B, Whole-cell current traces from a Kv1.3-transfected cell illustrating K+ currents washout on intracellular dialysis with a solution free of K+ (replaced by N-methyl--glucamide). Trace labeled as t=0 was obtained
immediately after breaking the patch. A family of current traces after complete dialysis is shown on the right panel (the thicker trace
corresponds to the pulse to +40 mV). Recording solutions: N-methyl-d-glucamine (NMDG)i/NMDGe. C, Resting membrane potential (VM)
was measured with the perforated-patch configuration in all the conditions studied, including nontransfected HEK cells (HEK). Data are
mean±SEM, n=8 to 15. D, Effect of voltage-dependent potassium channel (Kv)1.3 blockers on VM. The bars plot shows the change in VM
(mean±SEM) on application of PAP-1, in control cells or cells transfected with Kv1.3 or Kv1.3WF channels (n=8–10). Sample traces are
shown in the inset.
of VSMC proliferation. Among these, ion channels offer
new venues for alternative treatments of restenosis and
hypertension,2,5–7,24,25 but additional efforts are required to validate these targets and to develop optimized modulatory drugs
that could be used as cardiovascular protective agents.10 Here,
we endeavored to characterize the role of Kv1.3 channels
upregulation in VSMC proliferation. We have explored if our
initial findings, pointing to the upregulation of Kv1.3 currents
in mouse femoral VSMC as an essential component of the phenotypic switch,6 could be reproduced in other vascular beds
such as conduit (aorta) and resistance vessels (mesenteric).
In both vessels we observed a consistent change in the Kv1.3
to Kv1.5 ratio, with a clear-cut functional correlate: Kv1.5
(diphenyl phosphine oxide-1-sensitive) currents represented
the largest fraction of total Kv1 currents in contractile VSMC,
being nearly absent in proliferating VSMC, where Kv1 currents were mediated by Kv1.3 channels (MgTx- or PAP-1sensitive). The specificity and potency of diphenyl phosphine
oxide-1 blocking Kv1.5 currents was determined in parallel
experiments using heterologously expressed Kv1.5 channels
(data not shown), and the fraction of Kv1 current in both mesenteric and femoral VSMC was previously defined by using
the correolide-sensitive fraction of the outward K+ current.6,18
The fact that a proproliferative role of Kv1.3 channels
was described in VSMC from several human vessels6,7 could
reflect an obligatory association of Kv1.3 channels with vascular remodeling, increasing its value as a new therapeutical
target. Although there are several other ion channels (including TRPC1,2 T-type calcium channels,1 Kv3.4 channels,3,4
and IK1 channels5,25) whose expression and/or function has
been linked to VSMC proliferation, the information regarding conservation across different vascular beds and/or species
is missed for most of these candidates. Besides, involvement of Kv1.3 channels in proliferation has been described
in a substantial number of cell types (see introduction).
In this work, we add another preparation to this long list,
as this effect of Kv1.3 could be reproduced by its heterologous expression in HEK cells. The antagonistic effect
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Figure 5. A, Cell counting was used to determine proliferation rate of the different voltage-dependent potassium channel (Kv)1.3 mutants
in parallel with proliferation of HEK cells transfected with the empty vector (control) and with the wild-type Kv1.3 channel (positive control). Each data point is the mean±SEM of 3 to 5 independent experiments. The inset shows the effect of 10 nmol/L MgTx treatment on
proliferation (measured also by cell counting) of control cells or cells expressing Kv1.3 or Kv1.3WF mutant at 24 hours post transfection.
B, Percentage of cells incorporating EdU at 24 h posttransfection when transfected with the indicated constructs. Cells were maintained
in 5% FBS (control) or in control media containing 10 nmol/L MgTx (MgTx). Data are mean±SEM of 4 independent experiments with internal controls in all cases (empty vector and Kv1.3 transfected cells).
of Kv1.5 and Kv1.3 channels in this system points to concurrent changes in Kv1.5 and Kv1.3 channels expression in
native systems as a requirement for the functional changes
associated with phenotypic switch. The effects on HEK cells
were not very large but very consistent; moreover, considering our transfection efficiencies (42.58±1.75%, mean±SEM),
they were probably underestimated. Finally, cotransfection of
Kv1.3+Kv2, led to an increased HEK cell proliferation that
could be simply explained by its role increasing functional
expression of Kv1.3 channels (Figure 2B).
Kv channels may modulate proliferation by functioning
either as VM modulators, so that proliferation is affected by
the change in VM induced by ion fluxes through the channels or as VM sensors, so that VM induced conformational
changes could affect associated proteins that participate in
proliferation pathways. The former possibility underlies the
current hypothesis for K+ channels-mediated proliferation,
stating that cell hyperpolarization by K+ channels activation will increase the driving force for Ca2+ entry. However,
our work suggests that membrane Kv1.3 channels only
need their voltage-sensing capability to impact proliferation, being their effect on VM irrelevant. This finding could
accommodate the data obtained in native tissue, where we
observed that although several Kv channel blockers depolarize VSMC, only the Kv1.3 channels blockade affected
proliferation.6 Moreover, in femoral arteries, the phenotypic
switch associates with a shift toward a more depolarized resting membrane potential (from –50.33±2.85 in freshly dissociated to –40.34±1.66 mV in cultured VSMC; mean±SEM
of 12–20 cells), stressing the fact that the physiological relevance of the change in the Kv1.3:Kv1.5 ratio is not related
to the hyperpolarizing role of K+ channels. Furthermore, the
absolute value of VM seems to be irrelevant for HEK cells,
because Kv1.3 and Kv1.3WF have similar effects on proliferation with a very different effect on VM, whereas Kv1.3
and Kv1.5 have similar effects on VM and opposite effects
on proliferation (Figure 4B). Therefore, we propose that
Kv1.3 behaves as a sensor of VM changes occurring during the cell cycle, which translate into a proliferative signal through coupling to some yet unknown proproliferative
pathway (Figure IV in the online-only Data Supplement).
This role of voltage-dependent ion channels as voltage sensors is reminiscent of other well characterized physiological
processes such as the conformational coupling between the
L-type calcium channel and the ryanodine receptor in the
skeletal muscle.26 In a more related scenario, similar mechanisms have been described for EAG-induced proliferation
of fibroblasts and myoblasts, where the voltage-dependent
conformational change of the channel seems to be the signaling switch that modulates cell proliferation.27 Similarly, K+
efflux with the consequent hyperpolarization and enhanced
Ca2+ entry are not necessary for IK1-induced HEK cell proliferation.28 In this case IK1 does not even seem to behave
as a voltage sensor because expression at the cell membrane
is not required to potentiate proliferation.
The effect on proliferation of Kv1.3 and the nonpermeant Kv1.3WF is inhibited by selective Kv1.3 blockers
(MgTx and PAP-1), that have been previously considered
as open channel blockers with no effects on the kinetics of
the channel.10,29,30 Their inhibitory effect on proliferation
seems to be in open contradiction with the observation that
ion flux through the channels is not required to induce proliferation. Characterization of the gating charge movements
in the Kv1.3WF mutant shows that PAP-1 clearly affects
the voltage-sensing mechanism. This effect could explain
its inhibition of Kv1.3-induced proliferation. However, the
1306 Arterioscler Thromb Vasc Biol May 2012
Downloaded from http://atvb.ahajournals.org/ by guest on October 2, 2016
Figure 6. A, Time course of the effect of PAP-1 100 nmol/L on gating charge movement of voltage-dependent potassium channel
(Kv)1.3WF transfected cells on depolarization to +40 mV (Qon, white dots) and repolarization to –170 mV (Qoff, black squares). Representative gating current traces at the indicated times1,2,3 are shown on the right, with the voltage protocol depicted below the current
traces. Shaded areas in the control trace represent the gating charge movement (current integral) elicited on depolarization (Qon) and
repolarization (Qoff). B, Qon voltage dependence of Kv1.3WF mutants obtained with the voltage protocol depicted in Figure 4A in N-methyld-glucamine (NMDG)e solution alone or with 10 nmol/L MgTx or 100 nmol/L PAP-1. Data are mean±SEM of 4 to 6 different cells. Solid
lines represent the data fit to a Boltzmann function. Insets represent the average±SEM of the fit parameters (Normalized Qon(max) and V0.5)
obtained in each individual cell. C, As in panel B, but exploring the Qoff voltage dependence. Qoff was obtained with a family of pulses like
that shown in panel A with repolarization from –40 to –200 mV.
surprisingly small effect of MgTx does not seem to fit our
hypothesis. Nevertheless, since the effect of MgTx appears to
be voltage-independent (a decrease of Qmax without changes
in the V0.5 or the slope of the Boltzmann fits), this small fraction of channels could also be immobilized at resting VM.
Future studies will explore whether this effect suffices to
explain proliferation inhibition, or if alternative mechanisms
of action for MgTx are involved. In this regard, characterization of the effects of Kv1.3 blockers on the functional expression of the channels along the time scale used in proliferation
studies (24–48 hours) may contribute to solve the puzzle.
In summary, the data presented here show that the change
in the expression ratio of Kv1.3 and Kv1.5 channels could be
a general requirement to facilitate VSMC phenotype modulation, due to differential effects of both channels on the proliferative response. Besides, we propose that conformational
changes of Kv1.3 in response to VM, but not changes in ion
fluxes, are the key element in the proliferation signaling pathway. Further studies in this direction will provide additional
components of the cascade, offering new possibilities for the
control and modulation of cell proliferation.
Acknowledgments
We thank Esperanza Alonso for excellent technical assistance.
Sources of Funding
Supported by Ministerio de Sanidad, ISCIII grant R006/009 (Red
Heracles), Ministerio de Ciencia e Innovación grant BFU201015898 (M.T.P.G.) and Junta de Castilla y León grant VA094A11-2
(J.R.L.L.). L.J.M. and S.T. are fellows of the Spanish MICINN and
C.R.M. is a fellow of CSIC (JAE-predoc program).
Disclosures
None.
Cidad et al Mechanisms of Kv1.3 Channel-Induced Proliferation 1307
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Kv1.3 Channels Can Modulate Cell Proliferation During Phenotypic Switch by an
Ion-Flux Independent Mechanism
Pilar Cidad, Laura Jiménez-Pérez, Daniel García-Arribas, Eduardo Miguel-Velado, Sendoa
Tajada, Christian Ruiz-McDavitt, José R. López-López and M. Teresa Pérez-García
Arterioscler Thromb Vasc Biol. 2012;32:1299-1307; originally published online March 1, 2012;
doi: 10.1161/ATVBAHA.111.242727
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
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SUPPLEMENT MATERIAL
Materials
Cell culture media and media supplements were obtained from Lonza. Unless otherwise stated,
all solution reagents were from Sigma. The Kv1.3 selective inhibitors Margatoxin (MgTx)1 and 5-(4Phenoxybutoxy)psoralen (PAP-1)2 were obtained from Alomone labs and Sigma respectively.
MgTx was prepared as a 1 µM stock solution in water, stored at -20º C, and diluted to the final
concentration of 10 nM prior to use. PAP-1 was prepared as a 10 mM stock in DMSO and used at
final concentrations ranging between 0.1 and 1µM. The selective Kv1.5 blocker diphenyl phosphine
oxide-1 (DPO)3 was purchased from Tocris. Stock solutions at 10 mM were prepared in ethanol
and stored frozen (-20ºC). The solutions were diluted at the final working concentration of 5 µM just
prior to use, as we found lost of potency within days even in frozen stocks in aqueous media.
Paxilline was obtained from Alomone labs and used at a final concentration of 500 nM to block
maxi-K currents in electrophysiological experiments.
Animals and tissues
BPN strain mice were purchased from The Jackson Laboratories (Maine, USA) and maintained
with inbred crossing in the animal facilities of the School of Medicine of Valladolid. Animals were
housed under temperature-controlled conditions (21° C) with free access to water and food. Animal
protocols were approved by the Institutional Care and Use Committee of the University of
Valladolid and are in accordance with the European Community guiding principles in the care and
use of animals.
To collect arterial samples mice were killed by decapitation after isofluorane anesthesia. VSMC
from femoral, mesenteric and aorta arteries were obtained after carefully dissection and cleaning of
connective and endothelial tissues, and then arteries were either frozen in liquid nitrogen and
stored at -80º C for ulterior RNA extraction, or used directly to obtain tissue explants or fresh
dispersed VSMC as previously described4;5.
VSMC isolation and culture
For acutely dispersed cells, small pieces of endothelium-free artery were placed in SMDS Ca2+free solution containing 0.4 mg/ml Papain (Worthington), 1 mg/ml BSA (Sigma) and 1 mg/ml DTT
1
(Sigma) and incubated at 37º C for 20 min in a shaking water bath. After two washings in SMDS10μM Ca2+, another 25 min incubation was performed with SMDS-10 μM Ca2+, 0.28 mg/ml
collagenase F, 0.12 mg/ml collagenase H (Sigma) and 1 mg/ml BSA solution. Single cells were
obtained by gentle trituration with a wide-bore glass pipette, stored at 4º C and used within the
same day.
The composition of SMDS was (mM): NaCl 145; KCl 4.2; KH2PO4 0.6; MgCl2 1.2; Hepes 10;
Glucose 11. pH was adjusted to 7.4 with NaOH. Electrophysiological experiments were performed
within 2-8 h after isolation.
The detailed protocol for obtaining cultured VSMC has been previously described4;6. Briefly,
small pieces of arteries were placed in a 35 mm culture dish covered with 2 % gelatine (Type B
from bovine skin, Sigma) in DMEM supplemented with 20 % SFB, penicillin-streptomycin (100 U/ml
each), 5 μg/ml fungizone, and 2 mM L-glutamine at 37º C in a 5% CO2 humidified atmosphere.
Migration and proliferation of VSMC from the explants was evident within 6-10 days. When cells
reached confluence, they were detached by exposure to trypsin-EDTA during 2-6 minutes followed
by mechanical scraping with a rubber spatula, and seeded in a new culture plate at a 1/3 density in
SMC-P-STIM medium: D-MEM medium supplemented with 5% FBS, penicillin-streptomycin (100
U/ml each), 5 μg/ml fungizone, L-glutamine (2 mM), Insulin (5 μg/ml), bFGF (2ng/ml) and EGF (0.5
ng/ml). VSMC were subjected to several (3-4) passages without showing morphological changes.
RNA isolation, RT and real time PCR
Total RNA from arteries and cultured VSMC was isolated with Trizol Reagent (Invitrogen). 6-10
femoral arteries, 20-30 mesenteric arteries, 3-5 aorta arteries or 3-5 35 mm culture dishes with a
cell density close to confluence were employed for each determination. For cultured VSMC, cells
originated from several of different animals were pooled in each experiment, to eliminate individual
variations. Quality of the RNA was determined by OD measurement at 260 and 280 nm and by
agarose gels electrophoresis. After DNAse I (Ambion) treatment, 500-750 ng of RNA were used for
each reverse transcription reaction as previously described4.
The mRNA levels of the ribosomal protein 18S (RP18S) and Kv1.x potassium channels were
determined by real-time qPCR with TaqMan assays (Applied Biosystems) on a Rotor-Gene 3000
instrument (Corbett Research) using the 2-∆∆Ct relative quantification method7. Initially, a small
fraction of the obtained cDNAs was used for real-time amplifications of a control gene
2
(Glyceraldehyde 3-phosphate dehydrogenase, mGapdh) to compare the different samples, and
also, given the intronless nature of the mGapdh gene, to check the efficiency of the DNAse
treatment by comparing expression levels of the gene between RT+ and RT- samples. mRNA
expression levels were normalized to RP18S as internal control, so that the relative abundance of
the gene of interest was calculated as 2(-∆Ct), where ∆Ct=Ctgene-Ct18S.
Changes in the mRNA expression levels between Kv1.3 and Kv1.5 in each phenotype
(contractile and proliferative) and in each vascular bed studied were calculated with the expression
2(-∆∆Ct), where ∆∆Ct = ∆Ct(Kv1.3)–∆Ct(Kv1.5). In this way, data in the different experimental conditions
are presented as the fold change in gene expression of Kv1.3 versus Kv1.5. For the representation
of these data, the logarithm of 2-∆∆Ct was used, so that a value of 0 means equal relative expression
of both genes (i.e., a Kv1.3:Kv1.5 ratio of 1), positive values represent increased expression of
Kv1.3 and negative values decreased expression. In order to perform the comparisons, a Ct value
of 40 was assigned when the expression of a gene was not detected in one of the conditions.
Primers,TaqMan probes and TaqMan® Gene Expression Assays (Applied Biosystems) used
are listed below:








mGapdh: 5’-TGTGTCCGTCGTGGATCTG-3’, 5’- GATGCCTGCTTCACCACCTT-3’ and
5’-FAMTGGAGAAACCTGCCAAGTATGATGACATCA-BHQ2-3’;
18S rRNA: Hs99999901_s1
Kcna1: Mm00439977_s1
Kcna2: Mm00434584_s1
Kcna3: Mm00434599_s1
Kcna4: Mm00445241_s1
Kcna5: Mm00524346_s1
Kcna6: Mm00496625_s1
Plasmid construction
A complete mouse Kv1.3 coding sequence into pCR4-TOPO plasmid (TOPO-Kv1.3) was
purchased from Geneservice Ltd. The full Kv1.3 cDNA sequence was subcloned into different
vectors, such as the dsRed2ire bicistronic vector (kindly provided by Dr. Kääb, LMU, Munchen) and
the pEGFP-N3 vector, (a gift of Dr A. Alonso, IBGM, Valladolid) in which a fusion protein was
generated. The mouse Kv1.3 coding sequence was subcloned into the dsRed2ire vector using the
EcoRI sites flanking the sequence in the TOPO-Kv1.3 plasmid.
A Kv1.3 cDNA flanked by EcoRI sites was generated by PCR and cloned into the MCS of the
pEGFP-N3 vector, between the CMV promoter and the EGFP coding sequence, allowing the
3
EGFP to be in frame with the channel, as such creating C-terminal GFP-tagged channels (Kv1.3GFP). Chimeric constructs were developed using a loop-in PCR strategy. Site-directed
mutagenesis was carried out using the QuikChange method (Stratagene, La Jolla, CA, USA). All
the obtained constructs were verified by DNA sequencing. The Kv1.3 mutants we used were: a
dominant-negative pore mutant Kv1.3AYA (G399A/G401A)8, a non-conducting point mutant with
intact gating properties Kv1.3WF (W389F)9 and a voltage-insensitive KV1.3WF channel generated
by means of a triple mutation in the S4 region, Kv1.3WF3X (R320N/L321A/R326I). This triple
mutation shifts channel activation to potentials bellow -170 mV, so that the channel remains
inactivated along the physiological range of voltages, behaving as an inward rectifier10. As in the
case of Kv1.3, all these mutants were fusion proteins with a C-terminal GFP tag.
The pCMS-EGFP-Kv1.5 vector was provided by Dr. JX Yuan (UCSD). In this vector, the coding
sequence of the human Kv1.5 gene was subcloned into EcoRI and XbaI sites of the pCMS-EGFP
mammalian expression vector (Clontech), creating also a Kv1.5-GFP fusion protein11. The
pcDNA3-Kvβ2 vector was provided by Dr. WC Cole (University of Calgary, Canada).
HEK293 cell maintenance and transfection
HEK293 cells were maintained in DMEM medium supplemented with 5% FBS, penicillinstreptomycin (100 U/ml each), 5 μg/ml fungizone, and 2 mM L-glutamine at 37º C in a 5% CO2
humidified atmosphere.
HEK cells were transiently transfected with the expression constructs by using LipofectamineTM
2000 reagent (Invitrogen) according to the manufacturer's instruction. Briefly, transfection was
performed on 80–90% confluent cells at 37° C in serum-free Opti-MEM I medium (Invitrogen) with
0.1 to 1 µg DNA/ml transfection media and Lipofectamine reagent in a ratio 1:3 (µg DNA: µl
Lipofectamine). After overnight exposure to the transfection medium, cells were harvested, counted
with hemocytometer and seed at the adequate density (see below) and fed with serum-containing
medium for the indicated times before experiments. The transfection efficiency was consistently
>35% (35-60%) and was quantified in each experiment by fluorescence microscopy. Mock
transfected cells underwent the same protocol in the absence of plasmid DNA.
Kv1.3:Kvβ2 ratio in cotransfections ranged from 1:10 to 1:100, to ensure maximal functional
effect of Kvβ212. From low to high amounts of Kv1.3 HEK cells were cotransfected with the
following amounts of Kv1.3 and Kvβ2 (in ng/35 mm dish): 1:100, 5:200, 50:500 and 250:2500.
4
Current densities were tested through these ratios and maximal functional effect was observed in
all cases (data not shown).
Immunocytochemistry
HEK 293 cells transfected with vectors expressing Kv1.3-GFP or the different K1.3-GFP mutant
channels, were seeded in coverslips placed in the bottom of petri dishes 12-24 h before the
immunoflourescence assay. Cells were fixed with 4% paraformaldehyde (PF) in phosphate buffer,
pH 7.5, for 15 min at 20º C, washed in PBS and blocked with PBS with 2% of normal goat serum
for 10 min. Primary antibody (anti-Kv1.3 extracellular, APC101, Alomone Labs) was diluted 1:50 in
blocking solution and incubated with the cells for 60 min at RT. Then cells were incubated with the
fluorescently labelled secondary Alexa 594 goat anti-rabbit (Molecular Probes) for 60 min at a final
dilution of 1:1000. Finally, the cells were incubated with Hoechst 33342 (1:2000 in PBS) for 30 min
at RT. After each incubation and before mounting the cells, they were washed several times in
PBS. Coverslips were mounted with Vectashield (Vector Labs), and photomicrographs were
acquired with a LEICA SP5 confocal microscope.
Image analysis
Confocal images obtained from HEK cells transfected with different Kv1.3-GFP constructs and
labelled with anti-Kv1.3 antibody (see above) were used to assess the relative expression of Kv1.3
constructs at the plasma membrane (Supplemental figure I). Briefly, 40x (1.25NA) or 63x (1.4NA)
objectives were used to capture Z-stacks with a pinhole aperture of 1 Airy and a voxel size
compatible with the Nyquist criteria. GFP fluorescence (488ex/499-528em) was taken as a
measure of total expression of the fusion proteins, whilst anti-Kv1.3 antibody fluorescence
(585ex/608-651em) was taken as a measure of membrane expression, since the antibody
recognize an extracellular epitope and the immunocytochemical staining was performed in non
permeabilized cells. Both images were binarized using a threshold automatically defined with the
ImageJ software (http://rsb.info.nih.gov/ij/), and the number of pixels with a value of 1 was taken as
an estimation of the image area filled with the Kv1.3-construct labelling: GFP image define the total
area (ATotal), anti-Kv1.3 image define the membrane area (AMembrane) and both values are used to
compute the % of membrane expression. An example of this procedure is illustrated in
supplemental figure I.
5
Western blot
Total protein from HEK 293 cells transfected with vectors expressing Kv1.3-GFP or the different
K1.3-GFP mutant channels, was isolated using RIPA buffer (150 mM NaCl, 1% NP-40, 0.5%
sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 8 and 1x protease inhibitor cocktail; Roche).
Protein content was determined by using the BCA Protein Assay Kit (Pierce). Samples containing
50 g of protein were heated for 5 min at 95 ºC and the proteins were separated by SDS-PAGE in
10 % Bis-Tris precast gels (Criterion™ XT precast gel, BioRad) and transferred to a PVDF
membrane. After blockade of the membrane with 5 % non-fat dry milk in PBST (PBS with 0.1 %
Tween 20), membranes were incubated with either mouse monoclonal anti-Kv1.3 (clone 23/27,
Antibody Inc., USA) or mouse monoclonal anti-β-actin (ab8226, Abcam, Cambridge, UK) in
blocking solution at a final concentration of 1:1000 for 1 h. Then the membranes were washed with
PBST, incubated with horseradish peroxidase conjugated secondary antibody (donkey anti-mouse,
BioRad) at final concentration 1:10000 for 1 h and developed with VersaDocTM 4000 Image System
(BioRad) using chemiluminescence reagents (SuperSignal West Femto Chemiluminescent
Substrate, Pierce Biotechnology).
Digestion of glycoproteins with N-glycosidase F. HEK 293 cells transfected with the vector
expressing Kv1.3-GFP channels, were resuspended in N-Glycosidase F incubation buffer (12.5
mM Na2HPO4, 1% Triton X-100, 0.1% SDS, 2 mM EDTA, 150 mM NaCl, 1% (w/v) 2mercaptoethanol and protease inhibitor cocktail). 40-50 µg of total protein were incubated with or
without 0.7 units of N-glycosidase F (Roche) at 37 ºC for 18h with shaking. The digested samples
were resolved by SDS/PAGE and subjected to anti-Kv1.3 western blotting as described above.
Proliferation assays
The rate of proliferation of HEK cells transfected with the different plasmids was determined in
all cases by two alternative, complementary methods: Cell counting and EdU incorporation assays.
Proliferation assays were always designed with parallel intra-experimental controls, using HEK
cells transfected with the corresponding empty vector (dsRed-IRES, pEGFP-N3) at the same
concentration that the channel-expressing vector. For cell counting assays, transfected cells were
harvested, and seeded in 24 well plates at a density of 40,000 cells/well. In all cases, each data
point was the mean of 3 independent determinations in each experiment. Cells were allowed to
6
attach for five hours; after that, triplicate wells for each condition were harvested and the averaged
number of cell counted was taken as the t=0 for each condition. Cells were harvested and counted
at the indicated times, up to 96h in some experiments. When the effect of Kv1.3 specific blockers
(MgTx or PAP-1) was explored, the drugs were added to the media when seeding the cells.
The percentage of cells entering the S-phase of the cell cycle was determined by means of EdU
incorporation assays using a commercial kit (Click-iT® EdU Imaging Cell proliferation assay,
Invitrogen) following manufacturer specifications. Transfected cells were harvested and plated onto
poli-L-lysine coated 12 mm coverslips at a density of 40,000 cells/coverslip, and incubated for 2448 h in 5% FBS supplemented DMEM media with or without additional treatment. At the end of
these periods, cells were incubated with a modified nucleoside, EdU, (5-ethynyl-2´-deoxyuridine),
that is incorporated into DNA during active DNA synthesis, for 45 min at 37º C in a 5% CO2
humidified atmosphere, fixed with formaldehyde and permeabilized with Triton X-100. Incorporated
EdU was detected by incubation with a reaction buffer containing Alexa fluor reagent for
fluorescence detection of EdU-incorporating cells. Cells were incubated with Hoechst 33342
(1:2000 in PBS) for 30 min at RT, for counting nuclei. Coverslips were mounted in Vectashield and
proliferation rate was estimated as the percentage of EdU positive cells (EdU+) from the total cell
number stained with Hoechst. In each experiment, EdU incorporation was obtained from the
average of at least 10 different fields for each condition.
Apoptosis assays
HEK transfected with 1 µg or Kv1.3-GFP, Kv1.5-GFP or the empty vector pEGFP-N3 were
plated on poli-L-lysine coated 12mm glass coverslips at a density of 40000 cells/well and incubated
for 24-48h. After this time, apoptosis was detected by TUNEL method (In Situ Cell Death Detection
Kit, Roche Applied Science, Germany), that identifies DNA strand breaks by enzymatic labelling
with terminal deoxynucleotidyl transferase (TdT). This enzyme catalyzes polymerization of labelled
nucleotides (fluorescein –dUTP) to the free 3’-OH termini. The assay was performed following
manufacturer instructions. The positive and negative controls were cells treated with DNAse I
(3000 U/ml, Ambion) before labelling and cells incubated without TdT respectively. Cells were
incubated with Hoechst 33342 as described previously and mounted with Vectashield®. Positive
(apoptotic) cells were expressed as the percentage of the total cell number. In each experiment, at
least 10 different fields were averaged for each condition
7
Electrophysiological studies
Ionic currents were recorded at RT using the whole-cell configuration of the patch-clamp
technique as previously described4;5. Patch pipettes were made from borosilicate glass (2.0 mm
O.D., WPI) and double pulled (Narishige PP-83) to resistances ranging from 4 to 8 MΩ when filled
with an internal solution (High-Ki), containing (mM): 125 KCl, 4 MgCl2, 10 HEPES, 10 EGTA, 5
MgATP; (pH 7.2 with KOH). The composition of the bath solution (Standarde) was (mM): 141 NaCl,
4.7 KCl, 1.2 MgCl2, 1.8 CaCl2, 10 glucose and 10 HEPES (pH 7.4 with NaOH). Whole-cell currents
were recorded using an Axopatch 200 patch-clamp amplifier, filtered at 2 kHz (-3 dB, 4-pole Bessel
filter), and sampled at 10 kHz. When leak-subtraction was performed, an online P/4 protocol was
used. Recordings were digitized with a Digidata 1200 A/D interface, driven by CLAMPEX 8
software (Axon Instruments). Outward K+ currents were elicited by depolarizing pulses from a
holding potential of -80 mV to +40 mV applied in 10 s intervals. In some cells, full current/voltage
curves were constructed from potentials ranging from -60 to +100mV in 10 mV steps. The study of
the amplitude of the different Kv currents was performed in the presence of 500 nM paxilline in the
bath solution to block MaxiK currents. Kv1.3 component was defined as the PAP-1 (1µM) or
Margatoxin (10 nM) –sensitive current and Kv1.5 currents were dissected with their sensitivity to
DPO (5µM). In all cases, the effects of the drugs were calculated taken as control the averaged
current amplitude before and after drug application.
Membrane potential (VM) measurements were performed with the perforated-patch technique to
avoid dialysis of intracellular medium. For these experiments, recordings were obtained with an
Axopatch 700A patch-clamp amplifier. Pipette tips were briefly dipped into a solution containing (in
mM): 40 KCl, 95 KGlutamate, 8 CaCl2, 10 HEPES, pH 7.2 with KOH, and backfilled with the same
solution containing amphotericin B (480 μg/ml). After obtaining a high-resistance seal, electrical
access to cell cytoplasm was assessed by monitoring the increase in cell capacitance. At this point,
the amplifier was switched to current-clamp mode and membrane potential was continuously
recorded. The high Ca2+ content of the pipette solution ensures the correct performance of the
perforate-patch technique, as accidental rupture of the patch (changing to whole-cell configuration)
would lead to a sudden Ca2+ load and cell death. The composition of the bath solution was the
same as the indicated for the voltage-clamp experiments.
8
The solutions and protocols used for gating current recordings are a modification of previous
reports describing the gating charge movement in Kv channels9;13;14. The composition of the
internal solution (NMDGi) was (mM): 140 N-methyl-D-glucamide (NMDG), 1 MgCl2, 10 EGTA and
10 HEPES (pH 7.2 with ClH). The transfected HEK cells were bathed in a solution (NMDGe)
containing (in mM): 140 NMDG, 1 MgCl2, 1 CaCl2, 10 HEPES, 10 glucose (pH 7.4 with ClH). Patch
pipettes had resistances ranging from 1 to 3 MΩ when filling with the internal solution. Capacity
compensation was routinely used, and series resistance was between 2 and 8 MΩ for all
recordings. For some experiments, currents were recording using 60-75% series resistance
compensation.
The voltage-dependence of Qon, was studied with a series of depolarizing pulses of 10 ms
duration from a holding potential of -120, ranging from -100 to +100 mV in 10 mV intervals. For the
study of the voltage dependence of Qoff, after a 10 ms pulse from -100 to +40 mV to activate the
channels, repolarizing pulses of 30 ms from -40 to -200 mV in 10 mV intervals were applied.
Charge measures (Qon, and Qoff) were obtained by integrating the on and off gating currents over
sufficient time to allow the currents to return to baseline. Charge-Voltage (Q/V) relationships were
constructed from the Qon, or Qoff elicited to variable test potentials with the above detailed
protocols, and normalized to the maximum Qon, or Qoff respectively. The voltage dependence of
gating charge movement was obtained from the fit of the normalized Q/V curves to following
Boltzmann functions
eV V0.5  / k
Qon(V )  Qonmax
1  eV V0.5  / k
Qoff (V ) 
Qoff max
1  eV V0.5  / k
Were Qmax is the maximum gating charge, V is the membrane potential, V0.5 is the voltage for
half charge movement and k the slope factor.
Data analysis
Electrophysiological analyses were performed with both the CLAMPFIT subroutine of the
PCLAMP software (Axon) and ORIGIN 7.5 software. Pooled data are expressed as mean ±
standard error of the mean (SEM).
9
In all cases, statistical comparisons between groups of data were carried out with the two-tailed
Student t test for paired or unpaired data, as indicated, and values of p<0.05 were considered
statistically different.
Figure legends
Supplemental Figure I. Protocol of image analysis carried out to estimate the membrane
expression (%) of the different Kv1.3-GFP contructs used in this work. Color image illustrates a
HEK cell transfected with Kv1.3-GFP (green) and labelled with anti-Kv1.3 (red). The nucleus was
stainned with Hoechst and appears in blue. Binary images obtained as described above are
depicted in the right. Bars represent the average results (mean ± SEM) obtained in different
experiments. Each value was obtained by averaging data from several confocal planes of 4 to 7
different cells in each condition.
Supplemental Figure II. TUNEL assays in cells transfected with the maximal dose of the
plasmid used in our studies (1 µg in a 35 mm petri dish). The positive and negative controls were
cells treated with DNAse and untreated with the terminal deoxynucleotidyl transferase,
respectively. Each bar is the mean ± SEM of 3-5 independent experiments.
Supplemental Figure III. Inmunoblot of cell lysates of Kv1.3 transfected cells treated overnight
with N-glycosidase F.
Supplemental Figure IV. Proposed model to frame the results obtained in this work. Kv
channels are voltage sensors which respond to changes in membrane potential (VM) with a
conformational change. It is well known that this conformational change translates into a change in
the potassium flux through the channel that aims to recover the original potential (Conventional
loop). In fact, if the resting VM is above the activation threshold, a fraction of the channels are
constitutively open and both depolarizing and hyperpolarizing stimuli could translate into
conformational changes of the channels. We propose that in addition to determine ion flux, this
conformational change could promote the association and/or the activation of intracellular proteins
that modulate cell progression through the cell-cycle (Moonlighting loop). Since the conventional
loop does not seem to be required to modulate proliferation, we propose that Kv1.3 channels
behave as sensors of changes in the VM that occur during the cell cycle15, and contrary to Kv1.5
channels, they interact with the appropriate set of intracellular effectors to facilitate cell cycle
progression. The molecular nature of the channel partners that close the “moonlighting” loop is an
10
issue that needs to be addressed with future experiments. Similarly, a thorough knowledge of the
VM changes associated with cell-cycle progression is required to properly address the role of Kv1.3
channels in cell proliferation from a mechanistic point of view.
References
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ML. Purification, characterization, and biosynthesis of margatoxin, a component of Centruroides
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1993;268:18866-18874.
2. Schmitz A, Sankaranarayanan A, Azam P, Schmidt-Lassen K, Homerick D, Hansel W, Wulff H.
Design of PAP-1, a selective small molecule Kv1.3 blocker, for the suppression of effector memory
T cells in autoimmune diseases. Mol Pharmacol. 2005;68:1254-1270.
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4. Cidad P, Moreno-Dominguez A, Novensa L, Roque M, Barquin L, Heras M, Perez-Garcia MT,
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6. Miguel-Velado E, Moreno-Dominguez A, Colinas O, Cidad P, Heras M, Perez-Garcia MT, LópezLópez JR. Contribution of Kv Channels to Phenotypic Remodeling of Human Uterine Artery
Smooth Muscle Cells. Circ Res. 2005;97:1280-1287.
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8. Pérez-García MT, López-López JR, Riesco AM, Hoppe UC, Marban E, Gonzalez C, Johns DC.
Viral gene transfer of dominant-negative Kv4 construct suppresses an O2-sensitive K+ current in
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9. Perozo E, MacKinnon R, Bezanilla F, Stefani E. Gating currents from a nonconducting mutant
reveal open-closed conformations in Shaker K+ channels. Neuron. 1993;11:353-358.
10. Miller AG, Aldrich RW. Conversion of a delayed rectifier K+ channel to a voltage-gated inward
rectifier K+ channel by three amino acid substitutions. Neuron. 1996;16:853-858.
11. Brevnova EE, Platoshyn O, Zhang S, Yuan JX. Overexpression of human KCNA5 increases IK
V and enhances apoptosis. Am J Physiol Cell Physiol. 2004;287:C715-C722.
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beta-subunits on the human lymphocyte K+ channel Kv1.3. J Biol Chem. 1999;274:20123-20126.
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immobilization in Shaker potassium channels. Science. 1991;254:679-683.
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11
Kv1.3(GFP)
Ab‐antiKv1.3
ATotal
AMembrane
Kv1.3(GFP)
Ab‐antiKv1.3
 A m ·1 0 0 


A
T


Memb
brane expresssion (%)
50
40
30
20
10
0
Kv1.3
Supplemental Figure I, Cidad et al.2012
AYA
WF
WF3X
Kv1.5
Kv1.3
GFP
Control +
Control 0
5 10 15 20 80 90 100
Apoptotic cells (%)
Supplemental Figure II, Cidad et al.2012
100
K 13
Kv1.3
75
50
N-Glyc
N
Glyc
Supplemental Figure III, Cidad et al.2012
-
+
K+
K+
Conventional
loop
Kv1.3
N-terminal
Effector
domain
Sensor
Domain
∆Vm
(S4)
Sensor
Domain
(S4)
Ct
C-terminal
i l
Ct
C-terminal
i l
Effector
domain
Effector
domain
Cellll
C
cycle
“Moonlighting”
loop
Supplemental Figure IV, Cidad et al.2012
Kv1.3WF
∆Vm
N-terminal
Effector
domain
Cell
y
cycle
Kv1.5