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Departments of
* Internal Medicine, and
Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, OH, 45267; and
Division of Hematology/Oncology, Cincinnati Children’s Hospital, Cincinnati, OH 45229
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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2 and SK2 Ca-activated K+
channel subunit levels. The decrease in Kv1.3 protein levels occurs
with no change in Kv1.3 mRNA expression and is associated with a
significant decrease in K+ current density. A decrease in
Kv1.3 polypeptide levels similar to that obtained during hypoxia is
produced by Kv1.3 channel blockage. Our results indicate that hypoxia
produces acute and long-term inhibition of Kv1.3 channels in T
lymphocytes. This effect could account for the inhibition of lymphocyte
proliferation during hypoxia. Indeed, we herein present evidence
showing that hypoxia selectively inhibits TCR-mediated proliferation
and that this inhibition is associated with a decrease in Kv1.3
proteins. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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90–100 mmHg and normal
tissues typically have a PO2 in the range of
40–60 mmHg, oxygen tensions as low as 4 mmHg have been measured in
lymphoid organs (1). Moreover, immune cells reach
pathological sites such as tumors, wounds, and inflammated areas where
severe hypoxia (PO2 < 10 mmHg) has been reported
(2, 3, 4). Thus, hypoxia might affect normal immune cell
development as well as immune cell activity at hypoxic pathological
sites. Indeed, various in vitro studies have indicated that hypoxia can
affect the function of the host immune cells (5, 6, 7, 8, 9).
Hypoxia has been shown to markedly diminish lymphocyte expression of
IL-2, proliferation, and to impair the activity of NK cells (7, 8, 10). Recently, Caldwell et al. (1) has shown
that hypoxia can alter lymphokine secretion pattern and CTL
development. Still, although hypoxia can have such profound effects on
immune cell function, the mechanisms by which immune cells respond and
adapt to hypoxia are poorly understood (11). However, the
downstream effects of hypoxia on nonimmune cells, in particular
chemosensitive cells, have been extensively studied (12).
It is now well established that one of the immediate cellular responses
to hypoxia is inhibition of O2-sensitive
K+
(Ko2)3
currents. This inhibition leads to membrane depolarization, changes in
intracellular Ca2+ concentration
([Ca2+]), and ultimately, it affects cell
function (12). In many O2-sensitive
cells such as carotid body type I cells, pulmonary artery smooth muscle
cells, and pulmonary neuroepithelial body cells, the
Ko2 channel appears to belong to the family of
voltage-dependent K+ (Kv) channels
(13). Kv channels are tetrameric arrangements of four
separate pore-forming 
proteins and auxiliary Kv subunits
(14). The genes that encode the Kv subunits have been
classified in four major subfamilies: Kv1–Kv4. New subfamilies
(Kv5–Kv10) have been recently added. The Kv subunits implicated in
forming Ko2 channels are: Kv2.1 (homomultimer or
heteromultimer in combination with the silent Kv9.3 subunit), Kv1.5,
Kv1.2, Kv3.1b, and Kv4.2 (12). Hypoxia exerts two effects
on Ko2 channels: an acute effect occurs within
minutes, consisting of inhibition of the channel activity; a chronic
effect occurs after prolonged exposure to hypoxia, which is associated
with down-regulation of the channel itself (12). Although
much progress has been made in identifying the different
Ko2 channels in different chemosensitive cells,
the mechanisms mediating their oxygen sensitivity are not fully
understood. Moreover, the expression of these channels in other cell
types is still to be determined. Lymphocytes express voltage-dependent (Kv) and Ca-activated K (K(Ca)) channels and their activity is essential in T cell activation. In fact, K+ channels indirectly regulate the Ca2+ signal necessary for cell proliferation and cytokine production by modulating the resting potential of T cell membrane (15). The Kv channel expressed in lymphocytes is a member of the Kv1 family of Kv channels, specifically Kv1.3. These channels are highly expressed in resting human T lymphocytes and blockade of Kv1.3 channels suppresses cytokine production and cell proliferation (15, 16). Kv1.3 channels are also important in the regulation of cell volume and lymphocyte adhesion and migration (17, 18).
Although various studies have been performed to study the mechanisms of lymphocyte adaptation to hypoxia (11, 19), no information is available as to whether other elements important in sensing changes in O2 availability, such as the O2-sensitive K (Ko2) channels, are expressed in immune cells. The present study was undertaken to investigate the sensitivity to hypoxia of Kv1.3 channels in human T lymphocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Jurkat T cells (clone E6-1) were obtained from American Tissue
Culture Collection (Rockville, MD). PBMC were obtained from consenting
healthy adult donors. Venous blood was collected in heparinized tubes
and diluted 50% with balanced salt solution (in millimolars: Tris 145,
CaCl2 0.05, MgCl2 1 mM, KCl
5.4, NaCl 140, glucose 0.1% (pH 7.6)). The suspension was centrifuged
at 400 x g through a Ficoll-Paque (Amersham
Biosciences, Uppsala, Sweden) density gradient for 30 min at
room temperature. The interface was removed, washed with balanced salt
solution, and resuspended in RPMI medium (10% FBS and 100 U/ml
penicillin, and 100 µg/ml streptomycin). Adherent cells were removed
by incubation in petri dishes for 2 h at 37°C. PBMC were
cultured at an initial density of 1 x 106
cells/ml. FACS analysis of PBMC showed that CD3+
lymphocytes constitute 80% of the peripheral blood lymphocytes in
the PBMC population (data not shown). Resting and activated freshly
isolated lymphocytes were used in our experiments. Activation was
accomplished by incubating the lymphocytes with PHA (4 µg/ml) for at
least 24 h. In some experiments, T cells were separated from PBMC
using the E-rosette technique (StemCell Technologies, Vancouver,
Canada). FACS analysis showed that 98% of the cells isolated by
E-rosetting were CD3+ (data not shown). Jurkat
and freshly isolated lymphocytes were grown in RPMI (supplemented as
above) and maintained in a humidified incubator at 37°C with 5%
CO2.
Electrophysiology
K+ currents were recorded in either
whole-cell or perforated patch configurations. In these experiments,
the external solution had the following composition (in millimolars):
150 NaCl, 5 KCl, 2.5 CaCl2, 1.0
MgCl2, 10 glucose, and 10 HEPES, pH 7.4. The
pipette solution was composed of (millimolars): 134 KCl, 1
CaCl2, 10 EGTA, 2 MgCl2, 5
ATP-sodium, and 10 HEPES, pH 7.4, with an estimated free
[Ca2+] of 10 nM (20). Recordings
were performed on resting and activated freshly isolated lymphocytes
with a membrane capacitance of 2.0 ± 0.6 pF (n =
14) and 3.4 ± 0.3 pF (n = 15; p
< 0.05), respectively. Similar capacitance has been reported for
resting and activated human T cells (21). During our
electrophysiological experiments, we visually excluded monocytes and
phagocytic cells and selectively chose lymphocytes by their appearance
and small diameter (6–8 µm, 2 pF) (22). Resting
CD3+ cells isolated by E-rosetting have similar
capacitance (1.9 ± 0.4 pF n = 7;
p = 0.8) and current density (Id;
300 ± 99 pA/pF for CD3+ obtained by
E-rosetting, n = 7, and 208 ± 74 pA/pF,
n = 14, in resting lymphocytes visually selected from
the PBMC population, p = 0.5). Jurkat cells had a
membrane capacitance of 11.7 ± 1.0 pF (n = 31).
K+ currents were recorded in voltage-clamp mode
by depolarizing voltage steps to +50 mV (800 ms duration, every 30
s) from a holding potential of -80 mV. The temperature in the
patch-clamp chamber was stable at 23 ± 0.1°C (n
= 6) both in normoxic and hypoxic solutions, as it is known that Kv1.3
channels are highly sensitive to changes in temperature
(23). To obtain the perforated patch configuration,
nystatin (Sigma-Aldrich, St. Louis, MO) was dissolved before use in
DMSO (Fisher Scientific, Fair Lawn, NJ) at a concentration of 50
mg/ml and added to the pipette solution to a final concentration of
150–200 µg/ml. Nystatin solutions were used within 2 h after
preparation. The pipette tip was dipped in the intracellular solution,
then backfilled with nystatin solution. Experiments were performed
using Axopatch 200A amplifier (Axon Instruments, Foster City, CA). The
digitized signals were stored and analyzed using pClamp 8 software
(Axon Instruments).
Exposure of cells to hypoxia
During electrophysiological experiments, the effect of acute
hypoxia was studied by switching from a perfusion medium bubbled with
air (21% O2) to a medium equilibrated with 100%
N2. A polarographic oxygen electrode (World
Precision Instruments, Sarasota, FL) placed in the perfusion chamber
was used to monitor the PO2 in the bath
solutions. A complete exchange of PO2 in the
perfusion chamber was reached within 60 s. To study the effect of
prolonged hypoxia on protein and gene expression, cells were maintained
in 1–5% O2 (8–40 mmHg) for the entire
course of an experiment (24 h) in an incubator with controlled
PO2 (Thermo Forma, Marietta, OH). To study the
effect of hypoxia on T cell proliferation, cells were maintained in a
modular incubator chamber (Billups-Rothenberg, Del Mar, CA) at 37°C,
in an atmosphere saturated with 1% O2.
Cell viability assay
At the end of chronic hypoxia experiments, cell viability was determined by trypan blue exclusion. Cells were then pelleted by centrifugation (1,400 rpm for 2 min) and resuspended in normal Tyrode solution (in millimolars: NaCl 143, KCl 5.4, MgCl2 0.5, CaCl2 1.8, HEPES 5, glucose 5.5, pH 7.4). Equal volumes of cell suspension and 0.4% trypan blue (Sigma-Aldrich) were mixed and incubated at room temperature for 5 min. Cells were then counted using a hemocytometer. Cell viability (percentage) was determined as the ratio of total viable cells (unstained)/total cells (stained and unstained). The data reported are the average of separate experiments, each in quadruplicate.
Western blotting
Jurkat T cell total lysate was prepared according to standard
procedures. Human fresh lymphocytes total lysate was prepared from
Buffy Coat. Cells were harvested by centrifugation (1,400 rpm for 2
min) and resuspended in lysis buffer containing (in millimolars): 10
Tris, 1 EDTA, 1 PMSF, and 1% Triton X-100, 2 µg/ml leupeptin, and 2
µg/ml aprotinin. After sonication and centrifugation, the protein
content was measured using the BCA kit (Pierce, Rockford, IL). Aliquots
of cell proteins were fractionated on 6% SDS-polyacrylamide gels and
transferred to nitrocellulose membranes. Nonspecific protein-binding
sites were blocked by incubation in TBST with 5% nonfat dry milk for
1 h at room temperature. The blots were incubated with the primary
Abs (1/200 dilution for anti-Kv1.3 Ab, 1/400 for anti-SK2, and
1/1600 for anti-2 Abs) overnight at 4°C. After washing three
to four times, the membranes were incubated for 1 h at room
temperature with affinity-purified HRP-conjugated secondary Abs (Pierce
and Alphadiagnostic International, San Antonio, TX). Bands were
visualized using SuperSignal Chemiluminescence (Pierce) and quantitated
by densitometric analysis using UN-SCAN-IT gel software (Silk
Scientific, Orem, UT). Prestained m.w. standards were used to assess
the apparent m.w. Anti-Kv1.3 and anti-SK2 Abs were obtained from
Alomone Labs (Jerusalem, Israel). Anti-Kv shaker -2
(anti-Kv2) and anti--actin Abs were obtained from
BioSource International (Camarillo, CA) and Alphadiagnostic
International, respectively.
Northern blot hybridization
Total RNA was isolated from Jurkat cells with TRI REAGENT (Molecular Research Center, Cincinnati, OH), according to the manufacturer’s protocol (24). Total RNA samples (30 µg/lane) were fractionated on a 1.2% agarose-formaldehyde gel and transferred to nylon membranes using 10x sodium chloride-sodium phosphate-EDTA as transfer buffer. Membranes were cross-linked by UV light and baked for 1 h. Hybridization was performed according to standard procedures. Briefly, membranes were placed for 1 h in 0.1x sodium chloride-sodium phosphate-EDTA/1% SDS solution at 65°C. The membranes were then prehybridized for 10 min at 65°C with 0.5 M sodium phosphate buffer, pH 7.2, 7% SDS, 1% BSA, and 1 mM EDTA. Thereafter, the membranes were hybridized overnight in the above solution with 30–50 x 106 cpm of 32P-labeled Kv1.3 cDNA probe obtained by PCR of the full-length cDNA clone. The membranes were washed twice in 40 mM sodium phosphate buffer, pH 7.2, 5% SDS, 0.5% BSA, and 1 mM EDTA for 10 min at 65°C, washed four times in 40 mM sodium phosphate buffer, pH 7.2, 1% SDS, and 1 mM EDTA for 10 min at 65°C, exposed to PhosphorImager cassettes at room temperature for 24–72 h, and read by a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Bands were quantitated by densitometric analysis (Image-Quant 5.0; Molecular Dynamics).
Proliferation assay
Resting PBMC were seeded at 2 x 105 cells/well in culture medium in flat-bottom 96-well plates (final volume 200 µl). Cells were cultured for 2 h in normoxia or hypoxia (1% O2), after which the following proliferating agents were added: 25 µg/ml PHA, 167 µg/ml Con A, 25 µg/ml OKT3 (immobilized anti-CD3 Ab), 0.5–2 µg/ml ionomycin (Ionom) (with or without PMA (PMA/Ionom = 10 ng/ml/0.5 µg/ml), 20 µg/ml candida (Greer Labs, Lenoir, NC), and 0.025–0.1 U/ml tetanus (Aventis Pasteur, Lyon, France). The cells were returned to either the normoxic or hypoxic atmosphere for the remaining of the experiment. Fourteen hours before harvesting, [3H]thymidine (2 µCi/well) was added. The cells were harvested onto glass fiber filters, [3H]thymidine incorporation was measured in a scintillation counter.
Data analysis
All data are presented as means ± SEM. Statistical
analyses were performed using Student’s t test (paired or
unpaired); p 
0.05 was defined as significant.
Chemicals
4-Amino pyridine (4-AP), PHA, Con A, Ionom, and PMA were obtained from Sigma-Aldrich. OKT3 was obtained from Ortho Biotech (Toronto, Ontario, Canada). Margatoxin (MgTX) was purchased from Alomone Labs.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Electrophysiological experiments were performed to study the
effect of acute hypoxia on Kv1.3 channel activity in lymphocytes. Acute
hypoxia was produced by switching from a perfusion medium bubbled with
air (21% O2) to a medium equilibrated with 100%
N2. The mean PO2 values
measured in the recording chamber were 133 ± 1.5 mmHg (air,
n = 8) and 20 ± 1 mmHg (100%
N2, n = 8; p <
0.0001). A representative actual PO2 measurement
in the recording chamber is Fig. 1A, inset. Outward
K+ currents (IK) were
recorded in lymphocytes in whole-cell voltage-clamp experiments. Kv1.3
currents were isolated by dialyzing the cells with a nominally
Ca2+-free pipette solution which allows
minimization of the contribution of
Ca2+-activated K+
(K(Ca)) channels to the total current
(25). The pipette solution also contained ATP to
compensate for reduced cellular energy metabolism during hypoxia. Acute
hypoxia (20 mmHg) inhibits Kv1.3 currents in fresh human lymphocytes
(Fig. 1A). Introduction of hypoxia rapidly inhibited
IK peak amplitude by 20.1 ± 3.0% from
690.8 ± 145.6 pA in normoxia to 554.2 ± 113.6 pA in hypoxia
(n = 11, p < 0.005; Fig. 1, A, B, and D). The inhibitory effect of
hypoxia was displayed by 80% of the cells tested. A similar degree of
inhibition was observed in resting and activated lymphocytes: 19.7
± 6.5% (n = 4) in resting and 20.4 ± 3.3%
(n = 7) in activated (p = 0.9).
The inhibition of Kv1.3 peak current amplitude by hypoxia was
immediate, reversible, and reproducible (Fig. 1B).
Inhibition of similar amplitude was produced by a second exposure to
hypoxia (21.7 ± 6.2%, n = 3; p =
0.8 vs first exposure). This response was confirmed in human
CD3+ cells separated from PBMC by E-rosetting. In
these cells, hypoxia inhibited IK peak amplitude
by 19.3 ± 8.4% (n = 3). Furthermore, a similar
response was produced in Jurkat T cells. Hypoxia inhibited peak
IK of 19.7 ± 5.4% from 1027.4 ±
292.5 pA in normoxia to 864.9 ± 275.6 pA after 2 min in hypoxia
(n = 7; p < 0.05). Hypoxia inhibition
was observed in 64% of the cells tested and it was observed in both
whole-cell and nystatin patch experiments: 19.0 ± 3.4%
(n = 3) and 20.2 ± 9.8% (n = 4),
respectively.
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T cells respond immediately to acute hypoxia with inhibition of
the Kv1.3 current. This response is characteristic of
O2-sensitive K channels (12). There
are many examples that the expression of these
Ko2 channels in other cell types is regulated
during prolonged exposure to hypoxia (26, 27, 28). Therefore,
we evaluated the effect of chronic hypoxia on Kv1.3 expression in
Jurkat cells maintained in 1% O2 (8 mmHg) for
24 h. The Kv1.3 protein levels in hypoxic cells were compared with
those measured in cells maintained in normoxia and cells exposed to 8.6
mM 4-AP (a Kv channel blocker) in normoxia for 24 h. This
concentration of 4-AP has previously been shown to inhibit Kv current
and mitogenesis in human T lymphocytes without affecting cell viability
(29). The Kv1.3 polypeptide in total lysate of Jurkat
cells was detected with the affinity-purified Ab anti-Kv1.3 (Fig. 2). Western blot analysis revealed a
single band of 58 kDa. This band disappeared when the Ab was
preabsorbed to the corresponding Ag (data not shown), thus indicating
specificity of the anti-Kv1.3 Ab. A band of similar size has been
identified as an subunit for Kv1.3 (30). Kv1.3 protein
expression significantly decreased after 24 h of exposure to 1%
O2 (H; Fig. 2). No decrease in cell viability was
observed in Jurkat cells maintained 24 h in hypoxia: the
percentage viability, as determined by trypan blue exclusion, was
86 ± 1% in normoxia and 93 ± 1.4% in hypoxia
(n = 2). The Kv channel inhibitor 4-AP under normoxic
conditions also produced a decrease in Kv1.3 protein levels. The
hypoxic down-regulation of Kv1.3 protein expression depended on the
severity of hypoxia (Fig. 2C). Exposure to 5%
O2 (40 mmHg) produced a significant 10%
decrease in protein expression from 100.0 ± 1.3% in normoxia
(n = 4) to 89.6 ± 1.9% in hypoxia
(n = 4; p = 0.004), while a 47%
inhibition was obtained with more severe hypoxia (8 mmHg, see
above). Neither hypoxia nor MgTX (a specific blocker of Kv1.3)
(31) regulated Kv1.3 gene expression (Fig. 3).
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subunit expression.
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Whole-cell voltage-clamp experiments were performed to determine
whether the decrease in Kv1.3 subunit expression produced by
chronic hypoxia translated into an actual decrease in functional Kv1.3
channels. Consistent with the Western blot experiments, chronic hypoxia
significantly reduced Kv1.3 Id in Jurkat cells.
Whole-cell voltage-clamp experiments indicated that chronic hypoxia
(1% O2, 24 h) caused a 42% decrease in
Id as compared with that observed in Jurkat cells
maintained in normoxia (Fig. 5). It is
possible that this decreased expression of functional Kv1.3 channels by
chronic hypoxia may produce long-term effects on T cell activity.
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subunit expression can contribute to the
decreased Kv1.3 Id produced by chronic hypoxia.
Functional Kv channels are heteromultimeric complexes of the
pore-forming subunits and auxiliary Kv subunits
(14). The latter subunits are important to promote Kv
channel surface expression and to stabilize the channel complexes
(33). Thus, to establish a possible contribution of Kv
subunits in the decreased Kv1.3 Id during hypoxia
the expression of Kv2 subunits was determined in normoxic and
chronically hypoxic T lymphocytes. Anti-Kv2 polyclonal Ab recognized
a band of the predicted molecular size (39 kDa) in Jurkat cell total
lysate (Fig. 6). This 39-kDa protein is
identical in size to the Kv2 channel subunit purified from bovine
brain (34) and the Kv2 subunit detected in human T
lymphocytes (35). A faint band just below the 39-kDa band
is likely to correspond to a previously reported splice variant of
Kv2 (35). In contrast to the Kv1.3 subunits, the
levels of Kv2 polypeptide do not change during hypoxia.
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It is well established that blockers of Kv channels are able to
inhibit T cell proliferation (15). Hypoxia was also
shown to inhibit lymphocyte proliferation (1, 10). We have
studied the effect of hypoxia on T cell proliferation and we have
compared its effect on proliferation resulting from different mitogenic
stimuli. Proliferating agents used were: either 1) agents that induce
cell activation by cross-linking the TCR, such as PHA, Con A, OKT3, and
Ags such as candida and tetanus presented by APCs; or 2) agents that
induce proliferation bypassing the membrane such as Ionom (a calcium
ionophore), with and without PMA. The difference between these two
groups is that the former relies on the influx of
Ca2+ through the calcium release-activated
channel (CRAC) channels (whose activity depends on the Kv1.3 channels)
while the latter produces an increase in intracellular
[Ca2+] independently of membrane
channels. Viability of the cells was estimated at the end of each
experiment. No change in viability was observed between normoxic and
hypoxic cells (95 ± 2 and 96 ± 1%, n = 14,
after up to 7 days in normoxic and hypoxic conditions, respectively).
Our data show that hypoxia selectively inhibits T cell proliferation
induced by agents that cross-link the TCR (Fig. 7A). Proliferation induced by
stimuli that bypassed the membrane was not affected by hypoxia. This is
expected if the decrease in proliferation during hypoxia is mediated by
the hypoxic inhibition of the Kv1.3 channels. Thus, we have measured
the levels of Kv1.3 subunits in the protein lysates obtained from
hypoxic and normoxic T lymphocytes after 48–72 h stimulation with PHA.
We found that the levels of Kv1.3 subunit proteins were
significantly decreased during hypoxia by 46 ± 13%
(n = 4, p = 0.01) (Fig. 7B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Inhibition of Kv1.3 channel activity by acute hypoxia
The acute effect of hypoxia on Kv1.3 channels in T lymphocytes resembles the effect described for other Ko2 channels in other cell types (24, 36, 37, 38). Ko2 channels are rapidly inhibited by hypoxia and their hypoxic inhibition occurs without known modifications in cytosolic pH, [Ca2+], and ATP (12). Our experiments have shown that exposure to decreased PO2 produces an immediate inhibition of Kv1.3 current in T cells, reaching steady state within 2 min from exposure. The inhibitory effect of hypoxia is observed under conditions of constant intracellular ATP levels, pH, and [Ca2+] as they are maintained by dialyzing the cell with a pipette solution of known composition during whole-cell voltage-clamp experiments.
The ability of Ko2 channels to immediately respond to hypoxia had placed them in a key position in the process of O2 sensing, and it was even speculated that the Ko2 channel itself could function as O2 sensor (39). The mechanisms by which mammalian cells sense changes in O2 availability and the nature of the O2 sensor are still under debate (12, 39). Changes in intracellular redox state as well as membrane-associated events have been implicated in mediating the hypoxic response (12). It is possible that distinct molecular mechanisms may mediate the regulation of different Kv channels by hypoxia in different cells. Recently, Cayabyab et al. (30) have shown that anoxia together with glucose deprivation inhibits a Kv1.3 current in rat microglia (immune-competent cells present in the central nervous system). This inhibition was mediated by src tyrosine kinases. It has also been shown that recombinant and native Kv1.3 channels are inhibited by hydrogen peroxide (H2O2) and it was proposed that this effect is due to direct oxidation of these channels as it was independent of src-like tyrosine kinase phosphorylation and was mimicked by thiol group-oxidizing agents (20, 40). In contrast to the hypoxic inhibition of Kv1.3 channels, the effect of H2O2 occurs over a much longer time frame and is irreversible. Therefore, it appears that different mechanisms underlie hypoxia- and H2O2-mediated Kv1.3 inhibition. Additional experiments are underway to determine the mechanisms mediating Kv1.3 current inhibition by hypoxia in T cells.
Selective down-regulation of functional Kv1.3 channels during chronic hypoxia
Chronic hypoxia reduces the expression of functional Kv1.3
channels in T cells by down-regulating the protein expression of the
pore-forming Kv1.3 subunit. The inhibitory effect of hypoxia is
specific to this channel since the expression of SK2 channel subunit or
the Kv channel regulatory subunit Kv2 is not affected. The
expression of functional Ko2 channels in other
cell types is regulated by chronic hypoxia (24, 28). In
particular, chronic exposure to hypoxia reduces
Ko2 current in pulmonary artery smooth muscle
cells by down-regulating the mRNA and protein expression of Kv1.5 and
Kv2.1 subunits (28). Interestingly, we have observed
that hypoxia does not induce a decrease in steady-state levels of Kv1.3
mRNA in Jurkat T cells, suggesting that different mechanisms are likely
to mediate the down-regulation of Ko2 channels in
different cell types. There is evidence that regulation of Kv1.3
protein occurs posttranscriptionally, independent of changes in RNA
expression (41). In fact, the increase in Kv1.3 channels
that occurs in activated T cells is not accompanied by an increase in
its mRNA; on the contrary, recent gene array data have shown that
activation produces a decrease in Kv1.3 mRNA (42). Our
data suggest that down-regulation of Kv1.3 channel expression by
hypoxia also occurs posttranscriptionally. There are other examples of
Kv channel regulation occurring at the protein level: in PC12 cells,
Kv2.1 protein levels are increased by exposure to nerve growth factor,
with no change in steady-state levels of Kv2.1 mRNA (43).
It has been proposed that phosphorylation mechanisms, known to affect
protein synthesis at the initiation or elongation phase
(44), could be responsible. It is possible that changes in
channel protein synthesis or degradation might be also responsible for
the decreased Kv1.3 levels in T cells during hypoxia.
Similar to pulmonary artery smooth muscle cells, prolonged hypoxia does
not alter the expression of auxiliary Kv subunits in T cells. Kv1.3
channels in T lymphocytes exist in association with Kv2 subunits
(45). These subunits promote Kv channel surface expression
and stabilize the channel complexes (33). It has been
reported that during T cell mitogenesis, Kv2 gene expression is
enhanced and this increase might account for the increase in functional
Kv1.3 channels (33). Therefore, it is possible that a
decrease in expression of Kv subunits could contribute to the
observed decrease in functional Kv1.3 channels during hypoxia.
Moreover, the Kv subunits are of particular interest in hypoxia
studies because they have been proposed as possible
O2 sensors due to their similarity to
NADPH-oxidoreductase enzymes (46, 47, 48). Our results
indicate that Kv2 subunit expression is not regulated by hypoxia,
thus suggesting that the expression of Kv1.3 channels during hypoxia is
independent of the Kv2 subunit levels.
Although both acute and chronic effects of hypoxia on
K+ channels have been reported, the mechanisms
underlying these responses are not fully understood. Furthermore, it is
not clear if these two effects are directly correlated or if they occur
independently of each other. Our results indicate that a decrease in
Kv1.3 levels similar to that observed in hypoxia is produced by
exposing the cells in normoxia to a blocker of Kv channels (4-AP). Kv
channel blockade, like hypoxia, decreases Kv1.3 protein levels but not
mRNA. The down-regulation of the Kv1.3 subunit is selective, as it
is not observed for other K+ channels such as SK2
or other Kv channel subunits. This similarity between hypoxic and
4-AP-mediated responses suggests that the long-term consequences of
hypoxia on Kv1.3 channel expression might be linked to the initial
Kv1.3 channel blockade. It has been previously shown that 4-AP can
mimic the acute hypoxic response in other cell types. Exposure to
either 4-AP or hypoxia inhibits the Ko2 current
and induces membrane depolarization in pulmonary artery smooth muscle
cells (36). It is possible that the membrane
depolarization or other events triggered by the inhibition of the
activity of Kv1.3 channels in T cells can ultimately regulate its
protein expression. Expression of another member of the Kv1 family,
Kv1.5, was shown to be down-regulated by membrane depolarization in
pituitary cells (49).
Functional implications of Ko2 channels in T cells
The ability of Kv1.3 channels to sense changes in O2 availability could have important implication in T cell function. Kv1.3 channels play an important role in lymphocyte activation by providing the driving force to maintain the influx of Ca2+ from the CRAC. A sustained increase in intracellular Ca2+ is necessary to drive many events in T cell activation, such as cytokine production and proliferation (2, 15, 50). It is well known that inhibition of Kv1.3 channels induces membrane depolarization and suppresses the activation response of human T cells (18, 31, 51, 52). It has been shown that hypoxia inhibits T cell proliferation and affects cytokine release (1, 5, 6, 7, 8, 10, 53). Our data support the notion that hypoxia inhibits T cell proliferation. Furthermore, we showed that the effect of hypoxia involves membrane-delimited mechanisms as it was only observed when the mitogenic stimuli used involved the TCR complex and thus activation of the CRAC channels. Proliferation induced by stimuli that bypassed the membrane was not affected by hypoxia. This is expected if the decrease in proliferation during hypoxia is mediated by the hypoxic inhibition of the Kv1.3 channels. Still, the functional consequences of hypoxia may be different depending on the T cell activation state. In fact, Kv1.3 channels constitute the dominant conductance in resting and chronically activated T cells while K(Ca) channels are instead important in controlling T cell activation in acutely activated cells (31, 51, 52).
In conclusion, our data indicate that the Kv1.3 channels expressed in T lymphocytes are O2 sensitive. Inhibition of Kv1.3 channel expression and function by hypoxia could explain the changes in cellular proliferation that occurs in T cells exposed to hypoxia.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Laura Conforti, Department of Internal Medicine, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, OH 45267-0585. E-mail address: Laura.Conforti{at}uc.edu
3 Abbreviations used in this paper: Ko2, O2-sensitive K+; [Ca2+], Ca2+ concentration; Ionom, ionomycin; 4-AP, 4-amino pyridine; MgTX, margatoxin; CRAC, calcium release-activated channel; OKT3, immobilized anti-CD3 Ab; Kv, voltage-dependent K+; K(Ca), Ca-activated K; IK, K+ current; Id, current density.
Received for publication May 23, 2002. Accepted for publication November 7, 2002.
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1.2 subunit coexpression in HEK293 cells confers O2 sensitivity to kv4.2 but not to Shaker channels. J. Gen. Physiol. 113:897.This article has been cited by other articles:
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