From the Department of Physiology and Biophysics,
University of California, Irvine, California 92697-4561, ¶ 4SC
AG Drug Discovery, 82152 Martinsried, Germany,
A. Bernauer
Strasse, 80687 München, Germany, and the ** Department of
Chemistry, University College London, 20 Gordon Street,
London WC1H OAJ, England
Received for publication, December 15, 2000
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ABSTRACT |
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To maintain Ca2+ entry during T
lymphocyte activation, a balancing efflux of cations is necessary.
Using three approaches, we demonstrate that this cation efflux is
mediated by Ca2+-activated K+ (KCa)
channels, hSKCa2 in the human leukemic T cell line Jurkat and hIKCa1 in mitogen-activated human T cells. First,
several recently developed, selective and potent pharmacological
inhibitors of KCa channels but not KV channels
reduce Ca2+ entry in Jurkat and in mitogen-activated human
T cells. Second, dominant-negative suppression of the native
KCa channel in Jurkat T cells by overexpression of a
truncated fragment of the cloned hSKCa2 channel decreases
Ca2+ influx. Finally, introduction of the
hIKCa1 channel into Jurkat T cells maintains rapid
Ca2+ entry despite pharmacological inhibition of the native
small conductance KCa channel. Thus, KCa
channels play a vital role in T cell Ca2+ signaling.
The human leukemic T cell line Jurkat is widely used as a
model system to study intracellular signaling cascades during
lymphocyte activation. These studies have revealed the critical
requirement for two signaling pathways to complete lymphocyte
activation. In the first pathway, activation of protein kinase
C, particularly protein kinase C In human T lymphocytes and in Jurkat T cells, Ca2+ influx
is mediated by the opening of voltage-independent Ca2+
release-activated Ca2+ (CRAC) channels (7-9). Movement of
ions through open channels in the plasma membrane is driven by an
electrochemical gradient. Upon T cell stimulation and opening of CRAC
channels, the electrochemical gradient supporting Ca2+
entry is large, resulting in significant Ca2+ influx.
However, Ca2+ entry could result in depolarization of the
plasma membrane, limiting further influx. Therefore, to maintain
Ca2+ entry over the time scale required for gene
transcription, a balancing cation efflux is necessary. Efflux of
K+ ions through K+ channels is thought to
provide the electrochemical driving force for Ca2+ entry
via regulation of membrane potential (10). We have directly tested this
idea and identified the functionally important K+ channel
subtypes in Jurkat T cells and activated normal human T cells.
Jurkat T cells express two distinct K+ channels. The first
is a voltage-gated K+ (KV) channel encoded by
the Kv1.3 gene, and the second is a small conductance
Ca2+-activated K+ (KCa) channel
recently shown to be encoded by the hSKCa2 gene (11-14).
Human T cells possess a different KCa channel encoded by
hIKCa1, in addition to Kv1.3, but do not express
SKCa2 (15-18). Earlier work investigating the roles of
K+ channels in lymphocyte activation was hampered by the
lack of sufficiently specific blockers, leading to conflicting results and divergent interpretations (see, for example, Refs. 19 and 20).
Expression levels of KV and KCa channels are
similar in Jurkat and in mitogen-activated human T cells, although the
molecular identity of the KCa channel differs in these two
cell types (15). This difference in expression pattern, the advent of
new and highly specific blockers of all three channels, and the
potential for genetic manipulation of functional expression levels
provide an opportunity to examine the contributions of K+
channels in regulating membrane potential and Ca2+
signaling in lymphocytes. Our results emphasize the importance of
KCa channels in the modulation of Ca2+ signaling.
Cell Culture and Chemicals--
Jurkat E6-1 and COS-7 cells
were obtained from ATCC (Manassas, VA). Jurkat E6-1 cells were grown
in RPMI medium supplemented with 10% fetal bovine serum, 2 mM glutamine, and 10 mM HEPES at densities of
1-9 × 105 in a 37 °C humidified incubator
with 5% CO2. COS-7 cells were cultured in Dulbecco's
modified Eagle's medium containing 10% fetal calf serum and 2 mM glutamine and split twice weekly. Human T cells were
isolated and cultured as described (21). T cells were preactivated by
addition of 4 µg/ml phytohemagglutinin or phorbol myristate acetate
(33 nM) + ionomycin (1 µM) (Calbiochem) for 18-72 h prior to use. Unless otherwise specified, all reagents were obtained from Sigma and all optical filters from Chroma
(Brattleboro, VT). The syntheses of bis-quinolinium cyclophane
compounds UCL 1530 (8, 19-diaza-1,7(1,4)-diquinolina-3,5(1,4)-dibenzenacyclononadecanephanedium tetratrifluoroacetate hydrate), UCL 1684 (6, 10-diaza-1,5(1,4)-diquinolina-3(1,3),8(1,4)-dibenzenacyclodecaphanedium tritrifluoroacetate hydrate), UCL 1848 (8,14-diaza-1,7(1,4)-diquinolinacyclotetradecaphanedium ditrifluoroacetate), and UCL 2079 (8, 14-diaza-1,7(1,4)-di(6-trifluoromethylquinolina) cyclotetradecaphanedium ditrifluoroacetate) have been previously described (22-25). UCL 1684 is very stable in aqueous solution, showing no significant degradation after 24 h in culture medium at
37 °C (data not shown). The stability of other drugs was not tested
over long periods of time. ShK-Dap22 and
ChTX-Glu32 were obtained from BACHEM (King of Prussia, PA).
Transfection of Constructs into Mammalian Cells--
In each
electroporation cuvette (gap of 0.4 cm), 107 Jurkat cells
and 10 µg of the DNA of interest were electroporated at 960 µF, 250 V, and then resuspended in 15 ml of fresh culture medium and returned
to the incubator for 36-60 h prior to use. COS-7 cells (5 × 105 cells/chamber) were plated in culture chambers and
transiently transfected using the Lipofectin transfection reagent (Life
Technologies, Inc.) with the DNA of interest following the
manufacturer's recommended protocol in OptiMEM medium (Life
Technologies, Inc.). Following an 8-12-h transfection, the cells were
placed in fresh growth medium in the incubator for 48 h. Typical
transfection efficiencies using this protocol were 18-33%. The DNA
vectors used for transfection were prepared using the Qiagen (Valencia,
CA) endotoxin-free plasmid maxi-prep kit.
DNA Constructs--
The N-terminal GFP fusion protein of
human IKCa1 (GFP-IKCa1) was a gift from J. Aiyar
(AstraZeneca Pharmaceuticals, Wilmington, DE) and was generated by
subcloning hIKCa1 into the pEGFP-C1 vector (CLONTECH, Palo Alto, CA) as a
BamHI/BglII-Xho fragment. This cloning strategy
introduced 12 extra amino acids between GFP and the initiation codon of
IKCa1. The expressed sequence tag clone IMAGE: 2248 (GenBankTM accession number AI810558),
corresponding to nucleotides 491-2193 of the SKCa2 sequence
AF239613, was isolated from the pT7T3 Pac Vector (Amersham Pharmacia
Biotech) using NotI and EcoRI restriction sites and subcloned into pBluescript and from there into the
SacI restriction site of the pGFP-C1 expression vector. The
truncated hSKCa2 dominant negative construct was generated
by removing a 1.24-kilobase pair BclI fragment from
the GFP-SKCa2 construct, leaving a 564-base pair insert
encoding the hSKCa2 N-terminal proximal region terminating
in the S3 transmembrane domain. The human SKCa3 clone
(GenBankTM accession number AJ251016) containing 19 polyglutamines in the N terminus was cloned in frame to GFP in the GFP
vector as an EcoRI/BamI fragment. HEK-293 cells
expressing the skeletal muscle sodium channel hSKM1 (SCN4A)
were a gift from Dr. F. Lehmann-Horn (University of Ulm, Germany) (26).
The murine Kv1.3 channel is stably expressed in L929 cells
as previously described (12). The hSlo construct was the
gift of Dr. Ligia Toro (University of California, Los Angeles, CA) and
is expressed following injection into Xenopus oocytes (27,
28).
Patch Clamp Experiments--
For all KCa
experiments, electrophysiological recordings were made in the
whole-cell mode with a holding potential of -40 mV and an internal
solution consisting of 130 mM potassium aspartate, 10 mM K2EGTA, 8.55 mM
CaCl2, 2.08 mM MgCl2, and 10 mM HEPES, pH 7.2, with a calculated free
[Ca2+] of ~1 µM. In KV
experiments, whole-cell recordings were made with a holding potential
of -80 mV and an internal solution identical except that it contained
2.28 mM CaCl2, resulting in a calculated free
[Ca2+] of ~50 nM. KV
experiments also used a leak subtraction regimen in which the
leak pulse was applied after each voltage pulse. External solutions
consisted of normal Ringer solution (155 mM NaCl, 4.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM
D-glucose, and 5 mM HEPES, pH 7.4),
K+ Ringer solution (with identical ingredients except that
all NaCl was substituted by KCl, resulting in a final KCl concentration of 159.5 mM), or 40 mM K+ Ringer
solution, in which these two solutions were mixed to yield a final
Na+ concentration of 119.5 mM and a final
K+ concentration of 40 mM. Whole-cell patch
clamp recordings were performed using the equipment and techniques
described previously, and all data were corrected for a liquid junction
potential of Cytokine Expression Assays--
Jurkat cells were stimulated as
described above for T cells but in 96-well tissue culture plates with
105 cells in 200 µl of growth medium. Cytokine production
was assayed using the OptEIA enzyme-linked immunosorbent assay kit
(BD-Pharmingen, San Diego, CA) and a fluorescence plate reader
(Molecular Devices, Sunnyvale, CA) to quantify production of IL-2
and IL-8.
Confocal Microscopy--
Confocal fluorescence images were taken
with a Bio-Rad MRC-600 equipped with an argon laser (488 nm) and a
fluorescein isothiocyanate filter set (500-530 nm). All images
were acquired under a 63× oil objective on a Zeiss Axiovert 35 microscope. Z-series sections were captured at 0.5-µm intervals, and
optical section thickness was estimated to be ~0.45-µm.
Ca2+ Imaging and Membrane Potential
Measurements--
Cells were loaded in 1 µM fura-2/AM
ester (Molecular Probes, Eugene, OR) at 21-24 °C for 30 min,
washed, and stored in the dark until use (within 3 h).
Intracellular Ca2+ concentrations were estimated, and
experiments were performed utilizing a complete video microscopic,
ratiometric Ca2+ imaging system (Videoprobe, ETM Systems),
as previously described (30). At the beginning of data collection, the
extracellular solution was normal Ringer solution. Store depletion in
the presence of Tg required perfusion with a 0-Ca2+ version
of normal Ringer solution in which CaCl2 was replaced by
additional MgCl2 to keep divalent concentrations constant
and in which 1 mM EGTA was used to chelate residual
Ca2+. During measurements on transfected cells, individual
GFP-positive, and thus successfully transfected, cells were identified
and marked for analysis using a fluorescein isothiocyanate filter set
consisting of a 480 ± 20 nm exciter, 505 nm dichroic mirror, and 520 nm long-pass emission filter. By comparing Ca2+ responses
of GFP- hSKCa2 Specific K+ Channel Blockers Define the Role of
KV and KCa Channels in Jurkat Ca2+
Signaling--
Using the patch clamp technique, we characterized
highly potent and specific KCa and KV channel
blockers in Jurkat T cells and activated human T lymphocytes. In Jurkat
cells, whole-cell recording with Ca2+ maintained at a low
concentration inside the pipette revealed only KV currents
(Fig. 1A). These currents
inactivated during repetitive pulsing and were blocked by
ShK-Dap22, a sea anemone peptide modified to gain
specificity for the Kv1.3 channel (IC50 = ~25
pM) (31). Application of 10 nM
ShK-Dap22 blocked ~97% of the KV current.
The voltage dependence, inactivation kinetics, and pharmacological
properties of Jurkat KV currents are consistent with
Kv1.3 encoding this channel, as reported previously (32).
Elevation of [Ca2+] inside the pipette to 1 µM activated an additional small, voltage-independent
current. By increasing extracellular K+ concentrations to
40 mM, the inward component of this current could be
observed. The current reversed near the predicted Nernst potential for
a K+-selective current in 40 mM K+
extracellular solution (Fig. 1B). Application of 10 nM apamin, a peptide from bee venom, completely and
irreversibly blocked this KCa current (data not shown), in
agreement with a previous study (11), whereas Kv1.3 blockers
ShK-Dap22 (250 nM, Fig. 1B) and
charybdotoxin (10 nM, data not shown) had little or no
effect. Recently, a group of bis-quinolinium derivatives developed by
the group of C. R. Ganellin and D. H. Jenkinson at University
College London were found to block with high affinity the
apamin-sensitive small conductance KCa channel in rat
superior cervical ganglion cells (33). We tested these same compounds on the apamin-sensitive KCa current in Jurkat T cells. The
bis-quinolinium cyclophane UCL 1684 (see under "Experimental
Procedures" for full name) at a concentration of 10 nM
blocked 95 ± 5% of the KCa current but had no effect
on the KV current even at 250 nM (Fig. 1,
A and B). Blocking was rapid (usually complete
within 1 min) and, in contrast to apamin, rapidly reversible. The
dose-response curve in Fig. 1D shows that UCL 1684 blocks
the Jurkat KCa channel with subnanomolar affinity
(IC50 = 180 pM), making it the most potent inhibitor of this channel yet described. The molecular identity of the
KCa channel in Jurkat T cells was verified by measuring the
efficacy of UCL 1684 in blocking current through the cloned human
SKCa2 and SKCa3 channels expressed in COS-7 cells
(IC50 values of 280 pM and 9.5 nM,
respectively). UCL 1684 selectively blocks Jurkat KCa
channels and SKCa2 channels over the closely related
SKCa3 channel and several other more distantly related channels (Fig. 1D; Table I).
Other bis-quinolinium cyclophanes (UCL 2079, UCL 1848, and UCL 1530;
see under "Experimental Procedures" for full names) also blocked
Jurkat KCa currents with high affinity (Fig.
1C). Table I summarizes the selectivity of all compounds and
demonstrates that UCL 1684, UCL 1848, and UCL 2079 are all ~104-fold more effective at blocking SKCa currents
than any other channels tested. Because these drugs are highly specific
for the KCa channel found in Jurkat T cells and appear
equally potent in blocking the expressed human SKCa2
channel, our results provide a confirmation that the SKCa2
gene encodes the KCa channel in Jurkat T cells (13,
14).
We used a pharmacological approach to discern the contributions of
Kv1.3 and SKCa2 channels to calcium signaling in
Jurkat T-cells. Ca2+ signaling can be induced by
thapsigargin (Tg), a specific inhibitor of the sarco-endoplasmic
reticulum Ca2+ ATPase that enables study of
Ca2+ entry independent of Ca2+ release from
internal stores (34). Depletion of intracellular Ca2+
stores with Tg in the absence of extracellular Ca2+
activates CRAC channels in the plasma membrane, permitting
Ca2+ entry upon reintroduction (35). In Jurkat T cells, the
KCa channel inhibitor UCL 1684 reduced Ca2+
influx, but not the Tg-induced release of intracellular
Ca2+ stores (Fig.
2A), in a
dose-dependent manner that closely paralleled the
dose-dependent block of the native KCa current
(Fig. 2B). Maximal inhibition of the
[Ca2+]i plateau was achieved at 10 nM
UCL 1684, a dosage that blocked 95 ± 5% of Jurkat
KCa channels. This dosage of UCL 1684 had no effect on IL-2
production in Jurkat cells but inhibited IL-8 production by 30% (data
not shown). In contrast, application of the Kv1.3 blocker
ShK-Dap22 (up to 10 nM) did not affect the
Ca2+ response or cytokine production (Fig. 2A
and data not shown). These results demonstrate that KCa
channels but not KV channels help maintain Jurkat cell
Ca2+ entry.
The most likely mechanism by which UCL 1684 inhibits Ca2+
entry is depolarization of the membrane potential resulting from
Ca2+ entry through CRAC channels in the absence of
KCa channel function. To test this possibility, the
membrane potential of Jurkat T cells was monitored using a
voltage-sensitive dye during the same Tg stimulation protocol used in
Fig. 2A. Treatment with Tg in the absence of extracellular
Ca2+ caused a moderate depolarization of most cells,
followed by a marked hyperpolarization upon Ca2+ readdition
(Fig. 2C, solid line). This hyperpolarization must be caused
by the opening of SKCa2 channels because cells treated with
UCL 1684 instead showed profound membrane depolarization following
Ca2+ reintroduction (Fig. 2C, dotted line).
Similar results were observed in perforated-patch current clamp
recordings (data not shown). These results demonstrate the tight link
between KCa channel opening, modulation of membrane
potential, and regulation of Ca2+ entry.
The Role of KCa Channels in Activated Normal Human T
Cells--
Activated human T cells present an excellent system to
further test the role of KCa channels in Ca2+
regulation. Mitogen-activated human T cells and Jurkat cells express
similar numbers of KV and KCa channels,
suggesting that they may regulate calcium signaling in a similar
manner. The KV channel in human lymphocytes and in Jurkat T
cells is encoded by the Kv1.3 gene (32, 36). However, the
KCa channel in human T cells is the product of the
IKCa1 gene that is phylogenetically related to the
SKCa2 gene found in Jurkat cells. Both KCa
channels share a common calmodulin-dependent mechanism for
calcium-dependent gating (29, 37, 38), and both exhibit a
conserved genomic organization (39).
To test whether IKCa1 and SKCa2 serve similar
functions in sustaining Ca2+ signaling, we used agents that
are selective for the block of the KCa channels found in
mitogen-activated human T cells, including the charybdotoxin mutant
ChTX-Glu32 and the clotrimazole analogue TRAM-34 (28, 40).
We first verified that these blockers are selective for
IKCa1 by testing them on this channel in an expressed
system. As shown in Fig. 3A,
IKCa1 channels are blocked by ChTX-Glu32
(IC50 = ~30 nM) (28). Neither the UCL
compounds (10 nM) nor ShK-Dap22 (250 nM) affected the IKCa1 channel (Fig.
3A and data not shown). The triarylmethane blocker of
IKCa1, TRAM-34 (IC50 = ~20 nM), also blocks native or expressed IKCa1 current with high
selectivity (data not shown) (40).
Tg-induced Ca2+ responses in human T cells activated for
48 h with anti-CD3 antibody are partially suppressed by the
IKCa1-specific inhibitors ChTX-Glu32 and
TRAM-34, as shown in Fig. 3B, whereas UCL 1684 (10 nM, Fig. 3C) had little effect. Note that the
absence of any effect of UCL 1684 on Ca2+ influx in T cells
argues that CRAC channels, too, are unaffected by this drug, because
CRAC currents in human T cells and Jurkat cells are identical (8, 9).
In parallel with the inhibition of Ca2+ influx in activated
T lymphocytes achieved by the IKCa1 blockers, TRAM-34 was
previously reported to suppress proliferation of activated T-cells far
more effectively than resting T cells (39, 40). In contrast, the
Kv1.3 blocker ShK-Dap22 (500 nM,
Fig. 3D) did not inhibit Ca2+ influx in
activated T cells (Fig. 3D), consistent with an earlier report that Kv1.3 blockade does not suppress proliferation
of activated cells (39). Taken together with the data on Jurkat cells,
these results further emphasize the role of KCa channels in
cells of the immune system.
Dominant-negative Knockout Strategy--
To verify that UCL 1684 reduces Ca2+ entry in Jurkat cells by specific inhibition
of the KCa channel, we used a dominant-negative suppression
strategy to prevent KCa expression. A functional Jurkat KCa channel is expected to be a homotetramer of the SKCa2
protein. In KV channels, tetramerization is partly
determined by the N-terminal T1 domain, and overexpression of
N-terminal fragments containing the T1 domain results in
dominant-negative suppression of native channels via co-assembly of the
truncated fragments with native subunits in nonfunctional tetramers
(41). Hypothesizing that the N termini of KCa channels
contain a similar tetramerization domain, we transfected COS-7 cells
and Jurkat T-cells with a GFP-tagged N-terminal fragment of the
human SKCa2 gene (GFP-hSKCa2
Recently, it was observed that treatment with the mitogenic lectin
phytohemagglutinin decreases the number of SKCa2 channels expressed in Jurkat cells (14). Corresponding to this decrease, we find
that Tg-induced Ca2+ influx is reduced by ~50% in Jurkat
cells activated with phytohemagglutinin for 48 h (data not shown),
consistent with the suppression of Ca2+ signaling by
pharmacological blockade of KCa channels (Figs. 2 and 3)
and by knocking out native SKCa2 expression using a
dominant-negative construct (Fig. 4).
Compensation with a UCL 1684-resistant KCa Channel,
IKCa1--
We next tested whether the UCL 1684-resistant intermediate
conductance KCa channel, hIKCa1, present in
activated human T cells, can effectively substitute for the Jurkat
SKCa2 channel. Transfection with GFP-tagged
hIKCa1 (GFP-hIKCa1) led to endoplasmic
reticulum and plasma membrane staining of COS-7 and Jurkat cells
(Fig. 5A) and conferred expression of large KCa
currents in both cell types (data not shown). Jurkat cells transfected
with IKCa1 expressed whole-cell KCa currents
>100 times larger than those observed in cells transfected with vector
alone (Fig. 5B). These
currents were Ca2+-activated, insensitive to UCL 1684, and
blocked by the IKCa1-selective peptide
ChTX-Glu32 (Fig. 5B) (28), demonstrating that
the overexpressed GFP-tagged hIKCa1 channel functions as
expected. Cells overexpressing hIKCa1 were rescued from
pharmacological inhibition of Ca2+ influx by UCL 1684 (Fig.
5C). Thus, the hIKCa1 channel can substitute for
the native SKCa2 channel in Jurkat cells and effectively
sustains the Ca2+ response.
Using three independent strategies, we demonstrate a critical role
for KCa channels, but not KV channels, in
regulating the Ca2+ signaling response in T lymphocytes.
First, we used selective and potent blockers of KV
and KCa channels provided by a series of bis-quinolinium
compounds, a modified peptide from sea anemone toxin, and a scorpion
toxin mutant peptide to inhibit SKCa2, Kv1.3 and
IKCa1, respectively. In Jurkat T cells, the bis-quinolinium cyclophane UCL 1684 inhibited Ca2+ influx with a potency
that mirrored its dose-response curve for block of SKCa2
current (Figs. 1 and 2 and Table I), whereas selective blockade of
Kv1.3 had little or no effect. Furthermore, in activated T
cells, blockade of the native IKCa1 channel with the
selective and potent inhibitors, ChTX-Glu32 and TRAM-34,
significantly suppressed Ca2+ influx, whereas UCL 1684 had
no effect in these cells, and Kv1.3 blockers again were
ineffective (Fig. 3). Second, dominant negative suppression of
SKCa2 channel expression in Jurkat cells effectively inhibited Ca2+ entry, confirming the functional importance
of these channels by a completely independent experimental approach
(Fig. 4). Third, overexpression of the pharmacologically distinct
KCa channel, hIKCa1, in Jurkat T cells rescued
Ca2+ entry from inhibition by UCL 1684 (Fig. 5). Thus,
using a pharmacological strategy based on selective blockade of each
channel in conjunction with molecular approaches to vary expression
levels, we demonstrate that the SKCa2 channel in Jurkat T
cells and the IKCa1 channel in human T cells sustain
Ca2+ signaling.
Antigen-dependent activation of T lymphocytes increases
cytoplasmic Ca2+ to micromolar levels. During periods of
Ca2+ entry through CRAC channels, T lymphocytes could
become depolarized because of the inward current carried by
Ca2+ ions. This in turn would lead to the dissipation of
the electrochemical gradient required for continued Ca2+
entry. In resting human T cells that express ~400 Kv1.3
channels along with only ~10 IKCa1 channels and ~10 CRAC
channels (9), the KV channels should be sufficient to
compensate for transient depolarization by opening to allow
K+ efflux, resulting in repolarization of the cell. Indeed,
high K+ extracellular solution or blockade of these
channels by ChTX, correolide, ShK-Dap22, or organic
antagonists such as progesterone suppresses mitogenesis and IL-2
production (20, 39, 43-46). In each case, depolarization of the
membrane potential resulting from K+ channel block or
manipulation of K+ gradients limits Ca2+ entry.
The situation is significantly different in Jurkat T cells and in
previously activated human T lymphocytes, both containing roughly
equivalent numbers of KCa (~300-500/cell) and
KV (300-600/cell) channels (10). It is likely that the
increased number of CRAC channels in these cells (~100-300/cell) (9)
overpowers the ability of Kv1.3 channels to maintain
membrane potential, as evidenced by the fact that KV
channel blockers can no longer suppress proliferation (38, 39). The
up-regulation of KCa channel expression during T cell
activation is sufficient to compensate for increased Ca2+
influx, because T cells have been observed to hyperpolarize during sustained Ca2+ signaling (47). Conversely, T cells
depolarize during blockade of KCa channels, resulting in
reduced Ca2+ entry and emphasizing the importance of
KCa channels in maintaining the membrane potential (Fig. 2,
A and C) (48, 49). This interplay between channel
activation and changes in the membrane potential is likely to result in
the repeated depolarization and repolarization that contributes to
oscillatory Ca2+ entry (21, 50, 51).
Are the roles of the different KCa channels found in T
lymphocytes and in Jurkat cells interchangeable? Both channels are activated by the same mechanism, the binding of Ca2+ to
calmodulin preassociated with the cytoplasmic C terminus of the channel
(29, 37, 38). Both open in response to small changes in intracellular
Ca2+ concentrations, providing the counterbalancing cation
efflux and maintaining the membrane potential required for long lasting Ca2+ entry. Indeed, IKCa1 can effectively
substitute for blocked Jurkat SKCa2 (Fig. 5). Thus, the
KCa channels in Jurkat cells (SKCa2) and in
activated human T lymphocytes (IKCa1) seem to play identical roles in Jurkat T cells and in activated T lymphocytes.
The sustained Ca2+ response of T lymphocytes is necessary
for maintenance of nuclear factor of activated T cells in the nucleus and for numerous other events in T cell activation (4, 6). We are now
beginning to understand the critical role played by KCa
channels in this signaling cascade. In human T cells, KCa channel blockers prevent T cell proliferation and may become
therapeutically useful (38, 40, 52). In Jurkat cells, selective
inhibition by UCL 1684 of IL-8 production over IL-2 production (see
under "Results") hints that reduction of Ca2+ entry may
have complex effects on cytokine gene expression. Indeed, the reduced
Ca2+ entry observed in Th2 cells compared with Th1 cells
results in part from the lower level of KCa channel
expression in Th2 cells (30). Future work should help to clarify the
role that these channels play in cytokine expression and in determining
the cytokine profile of T cells during differentiation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, leads to the
phosphorylation of several cytoplasmic proteins and the triggering of
transcription via the assembly of the Fos/Jun transcription
factor complex on AP1 elements in several genes (1-4). In the second
cascade, the sustained entry of Ca2+ from the external
milieu raises the cytoplasmic Ca2+ concentration, leading
to gene transcription mediated by the nuclear factor of
activated T cells (NF-AT)1 (5, 6). Production of the key
T cell cytokine IL-2 requires the
simultaneous activation of both pathways,
with Ca2+ being absolutely required for the process.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
13mV for aspartate-based solutions (29).
-expressing or
GFP-hIKCa1-expressing cells with GFP-expressing vector
control cells, we eliminated the risk of inaccurate quantification due
to small amounts of contamination of the fura-2 signal by GFP
fluorescence bleed-through. Data processing and statistical analysis
were carried out using IgorPRO (Wavemetrics, Lake Oswego, OR) and Excel
(Microsoft, Redmond, WA) software. The bis-oxonol dye bis-(1,
3-dibutylbarbituric acid)trimethine oxonol (DiBAC4(3), Molecular
Probes) was used as an indicator of membrane potential in imaging
experiments using the same software and hardware described for
Ca2+ imaging, and using the fluorescein, filter set
described above. Cells were preequilibrated with 125 nM
DiBAC4(3) for 5-10 min prior to the start of each experiment, and dye
concentration was maintained in all external solutions throughout the experiment.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Selective K+ channel
blockers. Whole-cell recordings in the presence of 40 mM K+-containing extracellular Ringer solution
to amplify inward K+ currents. A, Jurkat T cells
dialyzed with low Ca2+ internal solution. Current densities
(peak current from each voltage step divided by cell capacitance,
bottom) and raw data from individual steps
(middle) are shown. Repeated stimuli at 35-s intervals using
the protocol shown (top) elicited voltage-gated
K+ currents (1) that were not affected by 250 nM UCL 1684 (2) but were completely blocked by
10 nM ShK-Dap22 (3). KV
data are shown after leak subtraction as described under
"Experimental Procedures." B, KCa current
was observed in isolation by whole-cell recording from a Jurkat T cell
dialyzed with 1 µM Ca2+ during measurement of
slope conductance at ~-80 mV (dashed lines denote region
of measurement). Ramp protocol (top) evoked
Ca2+-activated K+ currents (middle
and bottom, 1) that were unaffected by 250 nM
ShK-Dap22 (2) but completely blocked by 10 nM UCL 1684 (3). Frequent pulses (every 10 s) and depolarized holding potentials (-40 mV) led to inactivation of
KV currents. C and D, dose-response
curves for inhibition of Jurkat or cloned KCa channels by
bis-quinolinium cyclophanes. Each point shown is the mean
percentage of unblocked current of 3-5 cells at a given drug dose ± S.E. Lines illustrate fits to the Hill equation.
KCa current, measured as the slope conductance at ~-80
mV, was compared before and after addition of the blockers shown to
determine percent unblocked current. C, measurements of
efficacy of block of all 4 UCL compounds on Jurkat KCa
currents. D, effect of UCL 1684 in blocking the cloned
channels hSKCa2 and hSKCa3.
Ion channel selectivity of UCL drugs
View larger version (24K):
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Fig. 2.
KCa (SKCa2) but
not KV block reduces Ca2+ influx in Jurkat T
cells. In Ca2+ imaging experiments, fura-2-loaded
Jurkat cells and human T cells were stimulated with 1 µM
Tg in 0-Ca2+ Ringer solution (see bars above
panels) in the presence or absence of UCL 1684 or
ShK-Dap22. After ~8 min, normal Ringer solution (2 mM Ca2+) was reintroduced, causing a rapid,
sustained Ca2+ influx. A, Ca2+
responses of Jurkat cells stimulated with Tg in the presence of varying
doses of UCL 1684 (numbers), of 10 nM
ShK-Dap22, or no drug (control). Each
trace represents the average response of ~100 cells from a
typical experiment. Combined addition of ShK-Dap22 and UCL
1684 had little or no added effect. B, comparison of the
dose-response curve from patch clamp experiments shown in Fig.
1C (solid line) with the plateau Ca2+
values from A (filled circles). C,
membrane potential was monitored during stimulation in the presence
(dotted trace, 64 cells) or in the absence (solid
trace, 45 cells) of UCL 1684 (10 nM). Increasing
DiBAC4(3) fluorescence intensity indicates depolarization of the
membrane potential. Tg (1 µM) was used in a stimulation
protocol identical to that used in A.
View larger version (32K):
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Fig. 3.
KCa (IKCa1) but
not KV block reduces Ca2+ influx in
preactivated human T cells. A, whole-cell
KCa currents recorded from IKCa1-transfected
Jurkat cells with 1 µM Ca2+ internal solution
and 40 mM extracellular K+ Ringer
(1). These currents could be blocked by
ChTX-Glu32 (1 µM, 3) but not UCL
1684 (10 nM, 2). The depolarized holding
potential and rapidity of pulses caused inactivation of the
native KV current. B, in preactivated human T
cells, depletion of intracellular Ca2+ stores with Tg and
0-Ca2+ followed by Ca2+ reintroduction resulted
in sustained Ca2+ entry (solid line), just as it
did in Jurkat cells. Blockade of the intermediate conductance
KCa channel with 1 µM ChTX-Glu32
(dotted line) or 1 µM TRAM-34 (dashed
line) reduces this Ca2+ entry. Shown are 97 control
cells, 108 cells treated with ChTX-Glu32, and 79 cells
treated with TRAM-34. C, the mean Ca2+ response
of 284 preactivated human T cells treated with both Tg and UCL 1684 (10 nM, dotted line) was indistinguishable from that
of 281 preactivated Tg-treated control T cells (solid line).
D, Ca2+ influx in preactivated, untreated human
T cells (solid trace) was not significantly different from
influx in preactivated T cells treated with the KV blocker
ShK-Dap22 (250 nM, dotted trace).
Mean Ca2+ responses of 92 control cells and 81 cells
treated with ShK-Dap22 are shown.
). Confocal microscopy of
transfected COS-7 cells revealed mainly endoplasmic reticulum
localization of GFP-hSKCa2
(Fig.
4A); similar results were
obtained with the smaller Jurkat cells (data not shown). Parallel patch
clamp experiments were performed in control-transfected and
GFP-hSKCa2
-transfected Jurkat cells. Expression of the GFP-hSKCa2
construct abolished whole-cell KCa current in Jurkat cells
(Fig. 4C), compared with vector-transfected control cells
(Fig. 4B). Interestingly, the hSKCa3 N-terminal fragment
also suppressed this current (data not shown), suggesting that these
two channels can heteromultimerize, as has been reported for SKCa2
and SKCa1 (42). These results strongly suggest that SKCa
channels, like KV channels, contain a tetramerization
domain in the N terminus. The GFP-hSKCa2
cells were used to test the
role of the KCa channel in Ca2+ signaling.
Cells expressing the dominant-negative GFP-hSKCa2
construct
exhibited significantly attenuated Ca2+ entry (Fig.
4D, dotted line) compared with vector-control cells, reinforcing the conclusion that functional activity of the
KCa channel sustains normal Ca2+ signals in
Jurkat cells. The decreased level of Tg-induced store release in cells
expressing the GFP-hSKCa2
construct could in principle result from
inhibited release of Ca2+ stores or from a smaller total
store content. By perfusing ionomycin (4 µM) following
depletion of stores with Tg, we found that GFP-hSKCa2
-expressing cells have a larger Tg-nonreleasable store content than vector-control cells (data not shown), suggesting that functional hSKCa2 could be
related to store release in an unknown manner.
View larger version (31K):
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Fig. 4.
Dominant-negative suppression of
SKCa2 expression in Jurkat T cells results in a
decreased Ca2+ response. Jurkat cells and COS-7 cells
were transfected with an otherwise empty vector conferring expression
of GFP (vector control) or with a vector conferring expression of the
N-terminal cytosolic fragment of hSKCa2 conjugated to GFP
(GFP-hSKCa2 ). A, confocal microscopic image of
COS-7 cells transfected with GFP-hSKCa2
. Scale
bar, 1 µm. B, whole-cell recordings from Jurkat cells
dialyzed with 1 µM Ca2+ internal solution.
Control-transfected Jurkat cells with K+ Ringer solution
outside show sizable KCa currents (solid line)
blockable by 10 nM UCL 1684 (dashed line).
C, conversely, expression of GFP-hSKCa2
significantly suppressed KCa currents, leaving little
current (solid line) that was blockable by UCL 1684 (dashed line). Mean current in
GFP-hSKCa2
-expressing cells was ~30% of that observed
in control cells (n = 6 GFP-hSKCa2
cells:
mean slope conductance, 0.15 ± 0.02 nS; n = 7 control cells: mean slope conductance, 0.55 ± 0.08 nS;
p = 0.01). D, Ca2+ responses of
20 Jurkat cells transfected with GFP-hSKCa2
(dashed
line) and of 88 vector control Jurkat cells (solid
line) in a protocol otherwise identical to that shown in Fig.
2A.
View larger version (35K):
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Fig. 5.
The hIKCa1 channel found in
human T cells can substitute effectively for the SKCa2
channel found in Jurkat T cells. COS-7 and Jurkat T cells
were transiently transfected with GFP-IKCa1 or with an
otherwise empty vector conferring expression of GFP (vector control).
A, confocal microscopic image of COS-7 cells transfected
with GFP-IKCa1. Scale bar, 1 µm. B,
in whole-cell recordings from Jurkat T cells with normal Ringer
solution outside and 1 µM Ca2+ internal
solution, transient transfection of GFP-IKCa1 conferred
expression of large KCa currents that were blocked by 1 µM ChTX-Glu32 but not by 10 nM
UCL 1684. Mean current in GFP-IKCa1-transfected cells was
25.7 ± 8.9 nS, compared with 0.23 ± 0.04 nS in control
cells (n = 11 GFP-IKCa1 cells,
n = 6 vector control cells). C, transient
transfection with the hIKCa1 channel rescues Jurkat cells
from suppression of the Ca2+ response by UCL 1684 (10 nM). Traces shown are 41 GFP-hIKCa1-transfected
control cells (solid line, three experiments), 60 GFP-hIKCa1-transfected cells treated with UCL 1684 (dotted line, five experiments), and 65 vector control cells
treated with UCL 1684 (dashed line, three
experiments).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Dr. Luette Forrest for expert technical assistance, Stephan Grissmer for help in early work on the UCL compounds, and C. T. Fun, Dr. D. Yang, and L. Arifhodzic for assistance with strategy and compound synthesis.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants NS14609 and GM41514 (to M. D. C.), National Institutes of Health Grants MH59222 and GM54221 (to K. G. C.), a Feodor Lynen fellowship from the Alexander von Humboldt Foundation (to H. R.), and Deutsche Forschungsgemeinschaft Fellowship Grant WU 320/1-1 and Western States Affiliate of the American Heart Association Grant 9920014Y (to H. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Current address: AstraZeneca R & D Boston, 35 Gatehouse Dr., Waltham, MA 02451.
To whom correspondence should be addressed. Tel.: 949-824-7776;
Fax: 949-824-3143; E-mail: mcahalan@uci.edu.
Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M011342200
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ABBREVIATIONS |
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The abbreviations used are: NF-AT, nuclear factor of activated T cells; IL, interleukin; ChTX, charybdotoxin; CRAC, Ca2+ release-activated Ca2+; Dap, diaminopropionic acid; GFP, green fluorescent protein; IKCa, intermediate conductance KCa; KV, voltage-gated K+; KCa, Ca2+-activated K+; SKCa, small conductance KCa; Tg, thapsigargin.
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