From the Department of Physiology and Biophysics, University of California, Irvine, California 92697
The mechanism by which progesterone causes localized suppression of the immune response
during pregnancy has remained elusive. Using human T lymphocytes and T cell lines, we show
that progesterone, at concentrations found in the placenta, rapidly and reversibly blocks voltage-gated and calcium-activated K+ channels (KV and KCa, respectively), resulting in depolarization of the membrane potential. As a result, Ca2+ signaling and nuclear factor of activated T
cells (NF-AT)-driven gene expression are inhibited. Progesterone acts distally to the initial
steps of T cell receptor (TCR)-mediated signal transduction, since it blocks sustained Ca2+ signals after thapsigargin stimulation, as well as oscillatory Ca2+ signals, but not the Ca2+ transient
after TCR stimulation. K+ channel blockade by progesterone is specific; other steroid hormones had little or no effect, although the progesterone antagonist RU 486 also blocked KV
and KCa channels. Progesterone effectively blocked a broad spectrum of K+ channels, reducing
both Kv1.3 and charybdotoxin-resistant components of KV current and KCa current in T cells,
as well as blocking several cloned KV channels expressed in cell lines. Progesterone had little or
no effect on a cloned voltage-gated Na+ channel, an inward rectifier K+ channel, or on lymphocyte Ca2+ and Cl
channels. We propose that direct inhibition of K+ channels in T cells by
progesterone contributes to progesterone-induced immunosuppression.
Key words:
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Introduction |
Immunosuppression within the uterus is crucial for the
survival of the fetus (1, 2). Although the maternal immune system becomes sensitized to paternal antigens during pregnancy, fetal cells and placental trophoblasts bearing
those antigens do not elicit a cytolytic immune response
(3). High concentrations of progesterone in the placenta
inhibit the maternal immune response against the fetal allograft (6, 7). The immunosuppressive effects of progesterone were demonstrated in vivo by prolonged survival of xenografts near silastic implants containing progesterone at
concentrations typically found in the placenta (3, 6). In vitro
assays have established that progesterone inhibits lymphocyte activation and proliferation in response to allogeneic
cells or mitogens (8). In contrast, progesterone does not
inhibit the effector functions of previously activated cytolytic T cells (11). These data suggest that progesterone
may interfere with the early phases of T cell activation.
Antigen presentation and TCR ligation stimulate tyrosine kinases, leading to the generation of inositol 1,4,5-trisphosphate (IP3)1 and a consequent rise in the cytoplasmic calcium concentration ([Ca2+]i). Elevated [Ca2+]i
activates calcineurin, a phosphatase which then dephosphorylates a cytoplasmic transcription factor, the nuclear factor
of activated T cells (NF-AT). Dephosphorylated NF-AT
moves into the nucleus where it promotes the expression
of the IL-2 gene (12). A sustained elevation in [Ca2+]i requiring Ca2+ influx across the plasma membrane is necessary for the retention of NF-AT in the nucleus and efficient transcription of IL-2 (13). In lymphocytes, the
opening of Ca2+ release-activated Ca2+ (CRAC) channels
initiates Ca2+ influx after the depletion of Ca2+ stores by
IP3 (17). Once the CRAC channels are open, the transmembrane concentration gradient for Ca2+ and the membrane potential (Em) provide the driving force for Ca2+
entry. Em is set by the interplay between several ion channels in the T cell membrane. By itself, the Ca2+ current through
CRAC channels would diminish the driving force for calcium entry by reducing Em. However, currents through
voltage-gated K+ (KV) channels and Ca2+-activated K+ (KCa)
channels enhance Ca2+ entry by driving Em to a negative
voltage. Chloride channels may also play a role in maintaining a negative Em during T cell activation (20, 21). The four
major types of ion channels found in T cells are possible targets for immunosuppressive agents. In particular, the KV
channel encoded by Kv1.3 is required for normal lymphocyte activation both in vitro and in vivo (14, 22).
We have determined the effects of progesterone on lymphocyte ion channels, Ca2+ signaling, and gene expression.
By combining functional assays of gene expression with
patch-clamp and Ca2+-imaging measurements, we demonstrate that progesterone blocks lymphocyte K+ channels,
interferes with TCR-induced [Ca2+]i signaling, and inhibits gene expression. We propose that progesterone acts as
an endogenous immunosuppressant by directly and reversibly blocking K+ channels.
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Materials and Methods |
Chemicals and Solutions.
Salts and other reagents were obtained from Sigma Chemical Co. (St Louis, MO) unless otherwise noted. Thapsigargin (TG) was obtained from Alexis Corp.
(San Diego, CA). Progesterone (10 mM) and PMA (3 mM)
stocks were prepared in DMSO.
Cell Culture.
B3Z cells and K897 cells were provided along
with the antigenic peptide fragment SIINFEKL by Dr. N. Shastri
(University of California, Berkeley, CA). B3Z cells are a murine,
CD8+, T cell hybridoma with a known antigen specificity for
OVA/Kb-MHC and containing a
-galactosidase reporter gene
construct (lacZ) under the control of the NF-AT promoter (26).
The corresponding antigen-presenting K897 cells had been transfected with Kb class I MHC as described (27). The human leukemia T cell line Jurkat E6-1 was obtained from the American
Type Culture Collection (Rockville, MD). Human peripheral T
lymphocytes were collected from venous blood of healthy volunteers and isolated using a Ficoll-Hypaque density gradient as described previously (28). A population of activated T cell blasts was
prepared in culture by treating the resting cells with 10 µg/ml
PHA (PHA-P; Difco Laboratories, Inc., Detroit, MI). Cell lines
expressing the cloned KV channels Kv1.1, Kv1.2, Kv1.3, Kv1.4,
Kv1.5, and Kv3.1 and a voltage-gated Na+ channel hSKM1 were
maintained in culture. All cells were grown in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum, 10 mM
Hepes, 2 mM glutamine, 1 mM pyruvate, 50 µM 2-ME. Cells
were cultured in 25-ml flasks (Costar Corp., Cambridge, MA) at
37°C, 5% CO2 in a humidified incubator. For experiments on inward rectifier K+ channels, rat basophilic leukemia (RBL) cells
were maintained in Eagle's MEM supplemented with 10% FCS
and 1% L-glutamine. RBL cells were plated onto glass coverslips
1-2 d before use.
LacZ Reporter Gene Assay.
The fraction of B3Z cells expressing
-galactosidase was measured using flow cytometry (FACScan®; Becton Dickinson, San Jose, CA) as described previously
(13). In brief, 5 × 104 cells in RPMI without serum were placed
in individual wells of 24-well plates activated either by 1 µM TG
plus 50 nM PMA or by antibodies to the CD3-
subunit of the
TCR complex. In the latter, wells were coated with 10 µg/ml
anti-CD3-
antibodies (PharMingen, San Diego, CA) overnight
and rinsed briefly with PBS before use. Cells were activated in
the incubator for a total of 4 h before being resuspended and
loaded by osmotic shock with the fluorogenic substrate, fluorescein-di-
-galactopyranoside (FDG; Molecular Probes, Inc., Eugene, OR). The fluorescence of lacZ+ cells was at least fivefold
greater than autofluorescence. The effect of progesterone on lacZ
gene expression was quantified using a MUG (4-methylumbelliferyl
-D-galactopyranoside) assay (29). In brief, B3Z cells were
plated at 105 cells per well in 96-well plates, and the fluorescence
produced by cell lysates in a solution containing 3 mM MUG was
measured in a multi-well plate reader (CytoFluor Series 4000;
PerSeptive Biosystems, Framingham, MA).
Patch-clamp Recordings.
Membrane currents were measured
using the whole-cell configuration of the patch-clamp technique
(28, 30), and membrane potential was measured using the perforated patch method in current-clamp mode with nystatin to permeabilize the cells (31). An EPC-9 amplifier (HEKA, Lambrecht/
Pfalz, Germany) interfaced to a Macintosh Quadra 700 computer
was used for pulse application and data recording. Membrane
voltages were corrected for liquid junction potentials, and current
recordings were corrected for leak and capacitative currents.
Patch pipettes were pulled from Accu-fill 90 Micropets (Becton
Dickinson, Parsippany, NJ) using a P87 micropipette puller (Sutter Instruments Co., Novato, CA). Pipettes were coated with Sylgard (Dow Corning Corp., Midland, MI) and heat polished
to final resistances of 2-5 M
. Patch-clamp experiments were
performed at room temperature (20-25°C). Unless otherwise indicated, the membrane currents were filtered at 1.5 kHz. Data
analysis was performed using the program Pulse (HEKA). Mammalian Ringer contained (in mM): 160 NaCl, 4.5 KCl, 2 CaCl2,
1 MgCl2, 10 Hepes (pH 7.4; osmolality 290-310 mosmol/kg).
The ionic composition of the pipette solutions used in the individual experiments is reported in the figure legends.
[Ca2+]i Measurement.
[Ca2+]i was measured ratiometrically
using fura-2, as described previously (13). In brief, cells were
loaded with 3 µM fura-2/AM (Molecular Probes, Inc.) for 30-40
min at room temperature (20-25°C). The cells were then washed
three times with RPMI/10% FCS and stored in the dark. Illumination was provided by a xenon arc-lamp (Carl Zeiss, Jena, Germany) and transmitted through a filter wheel unit (Lambda 10;
Axon Instruments, Inc., Foster City, CA) containing 350- and
385-nm excitation filters. The filtered light was reflected by a
400-nm dichroic mirror through a 63× oil-immersion objective to illuminate cells. Emitted light >480 nm was received by a SIT camera (C2400; Hamamatsu Photonics, Bridgewater, NJ) and the
video information relayed to an image processing system
(Videoprobe; ETM Systems, Petaluma, CA). Full field-of-view
8-bit images, averaged over 16 frames, were collected at 350- and
385-nm wavelengths. Digitally stored 350/385 ratios were constructed from background-corrected 350- and 385-nm images.
Single-cell measurements of [Ca2+]i were calculated from the
350/385 ratios using the equation of Grynkiewicz et al. (32) and
a Kd of 250 nM for fura-2. The minimum 350/385 ratio was
measured in single cells after incubation for 10 min in Ca2+-free
Ringer containing 2 mM EGTA. Maximum ratio values were
obtained after perfusion with Ringer containing 10 mM Ca2+, 1 µM
TG, and 10 µM ionomycin.
Data Analysis.
Numerical values for single-cell [Ca2+]i traces
were analyzed with Origin (Microcal Software, Inc., Northampton, MA). Statistical analysis was performed on data sets using
Excel version 5.0 (Microsoft Corp., Redmond, WA). Data are
reported as mean ± SD. Analysis of variance (ANOVA) or Student's t test was used to compare mean values. Pairs of means
were considered statistically different if P < 0.05.
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Results |
Progesterone Suppresses Gene Expression Driven by NF-AT.
The murine T cell hybridoma, B3Z, recognizes an octapeptide fragment from ovalbumin (SIINFEKL) and expresses the lacZ reporter construct under transcriptional
control of the NF-AT response element of the IL-2 promoter (26, 27). Several treatments that increase [Ca2+]i lead
to NF-AT-driven lacZ expression in B3Z cells (13),
including TCR engagement or stimulation with TG, a
specific inhibitor of the endoplasmic reticulum Ca2+-
ATPase (33). By flow cytometry in the present series of experiments, cross-linking the TCR with anti-CD3-
antibodies or stimulating with TG plus PMA produced lacZ
expression in the majority of B3Z cells (60 ± 4%, n = 7; or
72 ± 10%, n = 3, respectively). Progesterone reduced NF-AT-mediated lacZ gene expression in a concentration- dependent manner, with an IC50 value of 22 ± 2.1 µM in
cells stimulated by TG/PMA (Fig. 1 A, filled circles). The
progesterone antagonist RU 486 also inhibited gene expression with slightly lower potency (Fig. 1 A, open
squares). Progesterone reduced lacZ expression when B3Z
cells were stimulated by TG alone or with PMA, by immobilized anti-CD3-
antibody, or by antigen presentation
(Fig. 1 B). Thus, at concentrations normally obtained in the
placenta (34, 35), progesterone inhibits NF-AT-mediated
gene expression when driven by four treatments that increase [Ca2+]i. Our results with NF-AT-driven lacZ reporter gene expression are consistent with levels of progesterone or RU 486 shown previously to inhibit activation of
human T cells in vitro (8, 9).

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Fig. 1.
Progesterone inhibits NF-AT-
mediated gene expression in B3Z cells. (A)
The concentration-dependent inhibition of
lacZ expression in B3Z cells by progesterone
( ) or RU 486 ( ) was measured in a multiwell fluorescence plate reader using MUG as a
substrate for -galactosidase. The cells were
stimulated for 4 h with 1 µM TG plus 50 nM
PMA. For each experiment, triplicate samples
were corrected for background fluorescence
and normalized for control lacZ expression.
Data are presented as mean ± SD (n = 10),
and were fitted to a Hill equation of the form
where y = the fraction of control lacZ expression with a maximum level represented by ymax, [X] = the concentration of progesterone, IC50 = the dissociation constant, and n = the Hill coefficient. The curve represents a Hill equation with an IC50 value of 22 ± 2.1 µM and n = 1.7 ± 0.3. The effects of
progesterone were not due to nonspecific toxicity, since after treatment with 30 µM progesterone, >95% of the cells stained with vital stain acetoxy-methoxy calcein and <5% stained with propidium iodide, a dye that is excluded from live cells. (B) Application of 30 µM progesterone reduced lacZ expression
when B3Z cells were stimulated for 4 h by 1 µM TG alone, a combination of 1 µM TG plus 50 nM PMA (TG + PMA), immobilized anti-CD3- antibodies, or K897 cells presenting SIINFEKL. Fluorescence readouts from the multiwell plate reader in arbitrary units (a.u.) are presented as mean ± SD (white
bars, stimulation alone; hatched bars, stimulation plus 30 µM progesterone). *Significance was determined with one-tail t tests (P < 0.0001).
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Progesterone Inhibits [Ca2+]i Signals in T Cells after TCR
Engagement or TG Stimulation.
Upon contact with K897
cells preloaded with SIINFEKL, B3Z cells responded with an
initial Ca2+ transient from a resting [Ca2+]i of 180 ± 86 nM
to a peak of 2.5 ± 0.5 µM (n = 20), followed by sustained
Ca2+ oscillations (Fig. 2 A). In the absence of preloaded antigen, K897 cells did not elicit Ca2+ signaling in B3Z cells (data
not shown). Application of 50 µM progesterone reversibly
suppressed antigen-induced Ca2+ oscillations (Fig. 2 A).

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Fig. 2.
Progesterone inhibits [Ca2+]i oscillations induced by TCR ligation. (A) [Ca2+]i responses from four representative B3Z cells activated
by contact with SIINFEKL-presenting K897 cells illustrate that [Ca2+]i
oscillations were reversibly inhibited by the application of 50 µM progesterone to the bath (bar). (B) [Ca2+]i oscillations in four B3Z cells activated
by settling onto coverslips coated with anti-CD3- antibodies in the absence (a) or presence (b) of 50 µM progesterone.
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To determine if progesterone directly inhibits TCR-initiated signaling or interferes with costimulatory pathways,
we activated the TCR complex by cross-linking CD3. Settling B3Z cells onto chambers coated with anti-CD3-
antibodies elicited an initial Ca2+ transient followed by vigorous Ca2+ oscillations that continued for at least 40 min
(Fig. 2 B). In the presence of 50 µM progesterone, most
cells produced only the initial Ca2+ transient lasting 271 ± 178 s, or a transient followed by severely attenuated oscillations (n = 14; Fig. 2 C). These data demonstrate that
progesterone blocks Ca2+ signaling after TCR engagement. Since the initial Ca2+ transient results from IP3-
mediated release of Ca2+ from intracellular stores, and the
sustained Ca2+ signal requires Ca2+ influx, these data also
suggest that progesterone inhibits Ca2+ influx but not the
steps that lead to Ca2+ release from intracellular stores.
TG inhibits the Ca2+ reuptake pump, leading to depletion of the intracellular Ca2+ stores and Ca2+ influx while
bypassing the initial steps of TCR signaling and IP3 generation (36, 37). In resting human T cells, the addition of TG
to the bathing solution increased [Ca2+]i from 72 ± 8 nM
to a stable plateau level of 1.2 ± 0.1 µM (n = 76; Fig. 3
A). Progesterone reversibly inhibited the sustained Ca2+
signal with an IC50 value of 28 ± 2.7 µM (Fig. 3, A and
B). Thus, progesterone blocks Ca2+ influx subsequent to
emptying of the Ca2+ stores.

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Fig. 3.
Progesterone reduces the [Ca2+]i
plateau evoked by TG stimulation in human
T cells. (A) Average [Ca2+]i is plotted against
time (n = 76) before and during stimulation
with 1 µM TG (arrow). TG stimulated Ca2+
influx, resulting in a stable rise in [Ca2+]i. Addition of 30 µM progesterone (bar) reversibly
reduced the [Ca2+]i plateau obtained after TG
stimulation. (B) The concentration dependence of the reduction of [Ca2+]i by progesterone is plotted for TG-stimulated cells. Calcium levels were normalized by subtracting
the resting [Ca2+]i and dividing [Ca2+]i in the
presence of progesterone and TG by [Ca2+]i
in the presence of TG alone. Data are presented as mean ± SD and were fitted to a Hill
equation illustrated by the smooth curve
(IC50 = 28 ± 2.7 µM and n = 1.3 ± 0.2).
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Progesterone Depolarizes the Membrane Potential, Reducing
the Driving Force for Ca2+ Entry.
Ca2+ influx depends upon
the opening of CRAC channels and upon an electrochemical gradient for Ca2+ entry across the cell membrane. The
negative membrane potential supported by K+ channels
provides the electrical component of the driving force favoring Ca2+ entry; membrane depolarization would reduce
this driving force. By direct measurement during perforated-patch current-clamp recording, we found that progesterone
depolarizes the membrane potential, Em. KV channels
normally maintain the resting membrane potential of T
lymphocytes near
60 mV (38). Fig. 4 A illustrates that membrane hyperpolarization accompanies TG stimulation
(from
61 ± 1.2 mV, n = 3; to
78 ± 2.0 mV, n = 11).
The hyperpolarization that follows TG stimulation is produced by activation of KCa channels during the rise in
[Ca2+]i, driving Em towards the K+ equilibrium potential of
~
80 mV. Progesterone (50 µM; application bar, Fig. 4 A)
not only reversed the hyperpolarization, but also resulted in
depolarization to an average of
41 ± 4.3 mV (n = 13, P < 0.0001). These results suggest that progesterone may
block both KV and KCa channels. If the reduction of [Ca2+]i
by progesterone is secondary to decreased K+ current, then
restoration of a K+ flux across the cell membrane should
reverse this effect of progesterone. We tested this hypothesis by using the K+ ionophore, valinomycin, to hyperpolarize Em. In the experiment shown in Fig. 4 B, 30 µM
progesterone reduced the plateau in [Ca2+]i that followed
TG stimulation. The subsequent addition of valinomycin in the continued presence of progesterone resulted in an
increase of [Ca2+]i. Thus, Ca2+-imaging and current-clamp
experiments demonstrate that progesterone reduces the
driving force for Ca2+ entry, an effect that can be reversed
by reestablishing K+ efflux. These data suggest that progesterone may affect [Ca2+]i and gene expression by blocking
K+ channels.

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Fig. 4.
Progesterone reduces the driving force for Ca2+
influx. (A) Current-clamp recordings using the perforated-patch technique were performed
in B3Z cells to determine the
effects of progesterone on Em.
This panel presents a representative single-cell recording. The
addition of 1 µM TG (arrow) hyperpolarized the cell from the
resting potential ( 60 mV) to
near the K+ equilibrium potential (~ 80 mV), enhancing the
driving force for Ca2+ influx.
Subsequent application of 50 µM progesterone depolarized
the cell to 26 mV, resulting in
a reduction of the driving force
for Ca2+ influx. For perforated-patch recordings, the tips of the
pipettes were filled with the following solution (in mM): 120 K2SO4, 16 KCl, 5 MgSO4, 10 Hepes (pH 7.2). A stock solution of nystatin in
DMSO (25 µg/ml) was prepared daily and subsequently diluted in the pipette solution to a final concentration of 100 µg/ml. After sonication, this
solution was used for backfilling the pipettes as described previously (reference 31). (B) Measurements of average [Ca2+]i from B3Z cells (n = 37).
After the addition of 1 µM TG (arrow), [Ca2+]i rose from a resting level of
70 ± 30 nM to a plateau of 1.3 ± 0.4 µM. Progesterone (30 µM) reduced the [Ca2+]i to approximately half of plateau concentration, an effect
that was completely reversed by the addition of 2 µM valinomycin. Application bars, The additions of progesterone and valinomycin to the bath.
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Progesterone Blocks Voltage-gated K+ and Ca2+-activated K+
Channels in T Cells.
Whole-cell recording with the patch-clamp technique permits direct measurement of several
types of channel activity in T cells. A voltage step from a
holding potential of
80 to +30 mV elicits rapidly activating KV currents, which reach a peak and then slowly inactivate. KV currents consist of two components, a predominant fraction encoded by homotetramers of the Shaker-related
Kv1.3 gene and a much smaller fraction of unknown
molecular identity (for a review, see reference 24). The
two components can be readily distinguished by their biophysical and pharmacological properties. Kv1.3 channels
are blocked by nanomolar concentrations of a scorpion
toxin, charybdotoxin (CTX), and undergo cumulative (use-dependent) C-type inactivation during repetitive depolarization. The smaller, CTX-resistant current does not
exhibit use-dependent inactivation. Application of progesterone rapidly and reversibly reduces KV currents in human
or B3Z T cells, with an IC50 of 29 ± 2 µM for the peak
K+ current (Fig. 5). Progesterone accelerates the decline of
K+ current during a depolarizing pulse, and thus the block
is more potent when evaluated at the end of a 200-ms pulse
(Fig. 5 C, filled triangles). The apparent increased rate of
channel inactivation (Fig. 5, A and B) suggests that progesterone may preferentially bind to and block the open or
inactivated Kv1.3 channel. To determine if steady-state inactivation enhances the block by progesterone, we inactivated a significant fraction of Kv1.3 channels by decreasing the holding potential from
80 to
50 mV. As shown in
Fig. 6 A, progesterone (30 µM) reduced the peak Kv1.3
currents more potently when the holding potential was
50 mV (70% block; see open circle in Fig. 5 C) than when
the holding potential was
80 mV (45% block), demonstrating that channel inactivation effectively enhances the block of Kv1.3 currents by progesterone. During antigen-induced oscillations of [Ca2+]i, Kv1.3 channels would undergo repetitive cycles of activation, inactivation, and recovery as the membrane potential fluctuates. Activation cycles
can result in frequency or use-dependent inhibition if the interval between depolarizations is less than the time required
for full recovery from inactivation; normally, pulse intervals
of >20 s allow full recovery. In the presence of progesterone, Kv1.3 currents steadily declined during repetitive
pulsing, because channel inactivation recovers 10-fold
more slowly, resulting in accumulation of channels in the
inactivated state (Fig. 6, B and C).

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Fig. 5.
Progesterone blocks KV channels. Whole-cell currents were measured in human T cells (A) and
B3Z cells (B) during 200-ms voltage pulses from a
holding potential of 80 mV to +30 mV applied every 30 s. The pipette solution contained (in mM):
160 K+ aspartate, 2 MgCl2, 1 CaCl2, 10 EGTA, 10 Hepes (pH 7.2). Currents are shown before and during bath application of 10, 30, or 100 µM progesterone. (C) Concentration dependence for the reduction of KV currents by progesterone. Peak current
amplitudes in human T cells ( ) and B3Z cells ( )
were analyzed and plotted against progesterone concentration. Data points for B3Z and human T cells
overlie each other at most concentrations. For human
T cells, the current at the end of the 200-ms pulse ( ) was also plotted. To determine the effect of depolarization on progesterone block, Em was held at
50 mV ( ). Data were normalized to control currents measured in the absence of progesterone and presented as mean ± SD. The line represents a fit
using a Hill equation with an IC50 of 29 ± 2 µM and n = 2.1 ± 0.4.
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Fig. 6.
Inactivation enhances
KV block by progesterone. (A)
KV currents were elicited by
steps to +30 mV from holding
potential of 80 mV (1, 3) or
50 mV (2, 4) in the absence (1,
2) or presence (3, 4) of 30 µM
progesterone. Solutions used are
as in Fig. 5. (B) Use-dependent
block of voltage-gated K+ currents. Current responses were
elicited by repetitive voltage
pulses from 80 to +30 mV
separated by 20 s. Normalized
peak current amplitudes in the
absence ( ) or presence ( ) of
67 µM progesterone were plotted against time. (C) Time course
of recovery from inactivation in a
B3Z cell before and during application of 60 µM progesterone.
Pairs of 200-ms pulses to +30
mV were applied from a holding
potential of 80 mV. The graph
shows the ratio of peak current
during the second pulse to the
peak current during the first
pulse, plotted as a function of the
time interval between the pulses.
Data were fitted by single exponentials with time constants of 4 s
for control ( ) and 39 s for
progesterone ( ).
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We also examined the effects of progesterone on the
smaller component of KV current by selectively blocking
the Kv1.3 component with 100 nM CTX. At this dose, we
expect a residual Kv1.3 current of only 2% that in the absence of CTX. However, in 11 cells examined in this series
of experiments and in previous work (21), 5-20% of the
KV current remains in the presence of CTX. This residual CTX-resistant current does not undergo use-dependent inactivation during repetitive depolarizing pulses (Fig. 7 A).
Fig. 7 B demonstrates that the larger, use-dependent,
CTX-sensitive component of KV current is blocked either
by progesterone or by RU 486. As shown in Fig. 7 C,
most of the CTX-resistant current is also blocked by 50 µM progesterone, whereas RU 486 at the same concentration is less effective. We conclude that progesterone blocks
both the predominant Kv1.3 component and the CTX-
resistant component of KV current in T cells, and that RU
486 blocks primarily the Kv1.3 component.

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Fig. 7.
Progesterone blocks CTX-resistant KV channels. Pipette solution as in Fig. 5. (A) Current responses of the total KV current ( ) and the
CTX-resistant current ( ) to repetitive voltage pulses from 80 to +30
mV separated by 1 s. At this rate of pulsing, Kv1.3 channels undergo cumulative inactivation, as shown by normalized peak current amplitudes in
the absence of CTX ( ). With 100 nM CTX present ( ), the remaining
current does not decline during repetitive pulsing. (B) Progesterone or
RU 486 (50 µM) blocks the KV component. (C) Progesterone blocks the
CTX-resistant component of KV current more than RU 486. 100 nM
CTX was preapplied in Ringer in order to block Kv1.3 channels.
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KCa channels hyperpolarize the membrane potential of T
cells during the [Ca2+]i signal, effectively counteracting the
depolarizing effects of Ca2+ influx. KCa channels are voltage-independent and highly sensitive to a rise in [Ca2+]i,
with half-activation at ~400 nM and a steep [Ca2+]i dependence, suggesting that at least four Ca2+ ions must bind
to open the channel (for a review, see reference 24). Based
upon biophysical characterization and its expression in T
cells, it is likely that the KCa channel in T cells is encoded by the gene hKCa4 (39). Intracellular dialysis of B3Z or activated human T cells with solutions containing 1 µM
Ca2+ activated a large K+ current that was evident in voltage ramps (Fig. 8 A). Progesterone blocked KCa channels
with an IC50 value of 113 ± 9 µM (Fig. 8 B). Thus,
progesterone blocks the KV currents more potently than the KCa currents in T cells. In contrast, the progesterone
antagonist RU 486 was consistently more potent than
progesterone in blocking KCa current (Fig. 8 C). These experiments demonstrate some degree of selectivity of the
two components of KV current and the KCa current for
progesterone and RU 486.

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Fig. 8.
Concentration-dependent inhibition of KCa current in
B3Z cells by progesterone. (A)
KCa currents were activated by
dialyzing the cell with a solution
containing (in mM) 140 K+ aspartate, 2 MgCl2, 7.8 CaCl2, 10 EGTA, 5 Hepes (pH 7.2). The
nominal free Ca2+ concentration of this solution was 1 µM,
assuming a dissociation constant
for EGTA and Ca2+ of 10 7 at
pH 7.2. Ca2+-activated K+ currents were evoked by voltage
ramps of 200-ms duration from
120 to +50 mV every 30 s.
Application of progesterone at
different concentrations (indicated at the right of each trace)
inhibited the KCa current. (B) Concentration-response curve for progesterone block of KCa currents ( ). The slope conductance between
100 and 60 mV was used as a measure of the KCa conductance to
avoid contamination by KV currents. Data were normalized to the conductance measured in the absence of progesterone and presented as
mean ± SD. The line represents the fit to a Hill equation with IC50 = 113 µM and n = 1.2. Block of KCa channels by 60 µM RU 486 ( ) is shown
for comparison. (C) Comparison of progesterone and RU 486. Slope conductance values at 80 mV illustrate activation and block of the KCa current
by RU 486 and progesterone, each applied at 60 µM.
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Selectivity of Progesterone for KV Channels: Progesterone Does
Not Block CRAC or Chloride Channels in T Cells.
The results from patch-clamp experiments suggested that progesterone, RU 486, and perhaps other steroid hormones
might block several K+ channel types, albeit with rather
low affinity. Therefore, we screened a panel of steroids on
the KV and KCa channels in T lymphocytes. Most of the
compounds tested either had no effect or were less effective
than progesterone in blocking KV or KCa currents (Table 1). In addition, we screened several channel types, including both cloned and native channels expressed in a variety
of cell lines, for block by progesterone, as summarized in
Table 2. Progesterone blocks several Kv1 family members,
including Kv1.3 expressed in lymphocytes as the predominant KV current, as well as Kv3.1 expressed in the brain and
in certain subsets of mouse thymocytes. In contrast, progesterone had very little effect on a cloned voltage-gated Na+
channel found in skeletal muscle, or on a strongly inward
rectifying K+ channel found in RBL cells. We conclude
that progesterone is a broad spectrum, low-affinity K+
channel blocker.

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|
Fig. 9.
Progesterone does not affect CRAC or Cl channels. (A)
Whole-cell recordings of Ca2+ current through CRAC channels measured during voltage ramps from 100 to +40 mV for a duration of 200 ms. The pipette solution contained (in mM) 128 Cs+ aspartate, 12 BAPTA, 0.9 CaCl2, 3.16 MgCl2, Hepes (pH 7.2). The external solution
had the following composition (in mM): 150 NaMeSO3, 20 CaCl2, 10 glucose, 10 Hepes (pH 7.4). (B) Time-dependent changes in the amplitude of the current measured at 80 mV. Arrows, Representative currents
recorded before store depletion (1), and after maximal induction activation of CRAC channels, while superfusing cells with control solution (2)
or with an external solution containing 30 µM progesterone (3). (C)
Whole-cell recordings of swelling-activated Cl current (ICl) measured
during voltage ramps from 120 to +40 mV for a duration of 200 ms.
The pipette solution contained (in mM) 140 Cs+ aspartate, 2 MgCl2, 4 MgATP, 1 CaCl2, 10 EGTA, 10 Hepes (pH 7.2). For the induction of
ICl, the pipette solution was made hypertonic (390-400 mosmol) by the
addition of 100 mM glucose. Currents are shown before the onset of cell
swelling (1), and after maximal induction of ICl, while superfusing cells
with control solution (2) or with an external solution containing 50 µM
progesterone (3). (D) Time-dependent changes in the slope conductance
measured at the reversal potential for Cl .
|
|
In further experiments on Jurkat T lymphocytes, we
evaluated effects of progesterone on CRAC and Cl
channels to determine if modulation of these channel types
might contribute to the inhibition of Ca2+ signaling. During whole-cell patch-clamp recordings, intracellular dialysis
with heavily buffered low-Ca2+ solutions passively depleted the intracellular Ca2+ stores and activated CRAC
channels (Fig. 9 A). After maximal activation, 30 µM
progesterone (Fig. 9 B, application bar) had no effect on the
amplitude or the current-voltage characteristics (n = 6 cells). This experiment rules out direct CRAC channel
block as a possible contributor to the inhibition of Ca2+
signaling by progesterone; instead, it appears that progesterone blocks Ca2+ signaling by inhibiting K+ channels, indirectly reducing the driving force for Ca2+ entry. Cl
channels have also been implicated in lymphocyte signaling
mechanisms by helping to maintain Em during T cell activation (20, 21). However, superfusion of B3Z or Jurkat
cells with 50 µM progesterone did not affect the amplitude
or the current-voltage characteristics of Cl
currents induced by cell swelling (Fig. 9, C and D).
 |
Discussion |
In this report, we demonstrate by patch-clamp measurement that progesterone directly blocks KV and KCa channels, but not Ca2+ or Cl
channels in T lymphocytes. Furthermore, we show that K+ channel blockade is associated
with membrane depolarization, inhibition of Ca2+ signaling, and a reduction of NF-AT-driven gene expression.
Since NF-AT links activation of the TCR to IL-2 production, interruption of these signals would inhibit production
of the major proliferative cytokine for T cells. We propose
that K+ channel blockade provides a mechanism contributing to the immunosuppressive effects of progesterone.
The rapid onset and reversibility of KV channel block by
progesterone is incompatible with changes in mRNA or
protein synthesis, suggesting that these effects are not mediated by the classical steroid receptor pathway (40). The
progesterone antagonist RU 486 is nearly as potent as
progesterone in blocking both KV channels and gene expression, also implicating a nongenomic action of progesterone. Furthermore, channel inactivation enhances block
by progesterone, suggesting that the state of the channel
modulates the affinity of progesterone. In T cells, the direct
block of KV channels by dihydroquinolines and benzhydryl
piperidines is also enhanced by inactivation (41, 42). In addition, progesterone blocks Kv1.3 channels exogenously
expressed in a cell line (Table 2). Progesterone also blocks
other KV and KCa channels with low affinity. These data
suggest that progesterone blocks K+ channels directly,
rather than acting via the classical nuclear progesterone receptor pathway.
Channel blockade by progesterone is not without precedent. Previous studies demonstrated that progesterone
blocks voltage-gated Ca2+ channels in smooth muscle cells
and a variety of K+ channels in MDCK cells and hepatocytes (43). Several transmitter-activated channels are
also suppressed by progesterone in the micromolar concentration range (46). In contrast to its effects on somatic cells, progesterone activates Ca2+ influx in sperm (50, 51).
We found no evidence for progesterone-induced Ca2+ influx in T cells.
Our data provide the first evidence that an endogenous
hormone may act as an immunosuppressant by blocking
K+ channels. Inhibition of K+ channels has been shown to
reduce IL-2 production and T cell activation in vitro (22,
23, 52). Moreover, recent studies demonstrated that the
peptide scorpion toxin margatoxin, a specific blocker of
Kv1.3 channels, inhibits delayed-type hypersensitivity reaction and reduces response to allogeneic challenge in vivo (25). The depolarization and reduction of the driving force for Ca2+ entry resulting from K+ channel inhibition are
sufficient to account for the reduction of Ca2+ signals and
NF-AT-driven gene expression. CRAC channels are inwardly rectifying, and a modest depolarization can reduce
Ca2+ entry significantly, reducing the rise in [Ca2+]i below
the threshold for gene expression (for a review, see reference 24). At high concentrations, progesterone reduces Ca2+ signaling and gene expression almost to control levels,
below a plateau level seen with 100 nM CTX (21, 53, and
data not shown). Progesterone, although acting with low
affinity, may reduce Ca2+ signaling and gene expression to
a greater extent than CTX because progesterone also
inhibits CTX-resistant KV channels. The block of K+
channels by progesterone or RU 486 can also account for
previous results showing that progesterone or RU 486 inhibits activation of human T cells in vitro (8, 9), as well as
the reduction of the number of CD3+ cells in the placenta
compared with maternal blood (11, 54).
During pregnancy, immunoregulatory mechanisms must
operate locally at the placental interface and be readily reversible to preserve the systemic immune competence of
the mother. Several mechanisms involving progesterone
may contribute to fetal-maternal protection, including altered expression of MHC class I proteins in fetal tissue, altered T cell subsets, or elaboration of immunosuppressive factors (2). Biochemical measurements have estimated
progesterone concentrations to be 20 µM within the placenta (34, 35); concentrations in the vicinity of trophoblasts
producing progesterone must be even higher. Average
progesterone levels found in the placenta would be sufficient to block lymphocyte K+ channels and thereby mediate a highly localized and reversible immunosuppression
without compromising the maternal immune system. The
affinity of progesterone for K+ channels ensures that this
mechanism would only be effective in the region of potential contact between allogeneic cells, where progesterone is
present at high concentrations.
Address correspondence to Michael D. Cahalan, Department of Physiology and Biophysics, University of
California, Irvine, CA 92697-4560. Phone: 714-824-7260; Fax: 714-824-3143; E-mail: mcahalan{at}uci.edu
We thank Dr. Patricia Schmidt for helpful discussions at the onset of this project, Dr. K. George Chandy for
providing clones and a panel of stable cell lines, and Dr. Lu Forrest for tissue culture support.
This work is supported by National Institutes of Health grants GM41514 and NS14609 (to M.D. Cahalan),
a Schroedinger Stipendium (to H.H. Kerschbaum), and a fellowship of the Alexander von Humboldt Foundation (to C. Eder).
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