Vol. 275, Issue 6, C1526-C1537, December 1998
Ca2+ depolarizes adrenal
cortical cells through selective inhibition of an ATP-activated
K+ current
Juan Carlos
Gomora1 and
John J.
Enyeart1,2
1 Department of Pharmacology
and 2 Neuroscience Program,
Ohio State University College of Medicine, Columbus, Ohio 43210-1239
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ABSTRACT |
Bovine adrenal
zona fasciculata cells (AZF) express a noninactivating
K+ current
(IAC) whose
inhibition by adrenocorticotropic hormone and ANG II may be coupled to
membrane depolarization and
Ca2+-dependent
cortisol secretion. We studied
IAC
inhibition by
Ca2+ and the
Ca2+
ionophore ionomycin in whole cell and single-channel patch-clamp recordings of AZF. In whole cell recordings with intracellular (pipette)
Ca2+
concentration
([Ca2+]i)
buffered to 0.02 µM,
IAC reached
maximum current density of 25.0 ± 5.1 pA/pF
(n = 16); raising
[Ca2+]i
to 2.0 µM reduced it 76%. In inside-out patches, elevated
[Ca2+]i
dramatically reduced
IAC channel
activity. Ionomycin inhibited IAC by 88 ± 4% (n = 14) without altering rapidly
inactivating A-type K+ current.
Inhibition of IAC
by ionomycin was unaltered by adding calmodulin inhibitory peptide to
the pipette or replacing ATP with its nonhydrolyzable analog
5'-adenylylimidodiphosphate.
IAC inhibition by
ionomycin was associated with membrane depolarization. When
[Ca2+]i
was buffered to 0.02 µM with 2 and 11 mM
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), ionomycin inhibited
IAC by 89.6 ± 3.5 and 25.6 ± 14.6% and depolarized the same AZF by 47 ± 8 and 8 ± 3 mV, respectively (n = 4). ANG II inhibited
IAC significantly
more effectively when pipette BAPTA was reduced from 11 to 2 mM. Raising
[Ca2+]i
inhibits IAC
through a mechanism not requiring calmodulin or protein kinases,
suggesting direct interaction with
IAC channels. ANG
II may inhibit
IAC and
depolarize AZF by activating parallel signaling pathways, one of which
uses Ca2+ as
a mediator.
adrenal cortex; potassium channel; angiotensin II; membrane
depolarization; cortisol secretion
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INTRODUCTION |
BOVINE ADRENAL ZONA FASCICULATA cells (AZF cells)
express a noninactivating K+
current (IAC)
that appears to set the resting potential of these cells and to
function critically in the regulation of cortisol secretion (20). These
IAC channels show
little voltage dependence and are open at negative membrane potentials
(6, 8, 20). IAC
channel activity is regulated by diverse hormonal and metabolic factors. Specifically, these channels are activated by hydrolyzable and
nonhydrolyzable forms of ATP over a physiological range of concentrations, suggesting that
IAC channels may function as
metabolic sensors linking the metabolic state of the cell to membrane
potential (6).
IAC is inhibited
by adrenocorticotropic hormone (ACTH) and ANG II at concentrations
identical to those that trigger membrane depolarization and cortisol
secretion (7, 20). ACTH and ANG II each depolarize AZF cells by a
maximum of >50 mV (20). The signaling pathways that link ACTH and ANG
II receptors to
IAC inhibition
have been partially characterized. ACTH inhibits
IAC completely by
a cAMP-dependent mechanism that appears to be independent of increases
in intracellular Ca2+
concentration
([Ca2+]i)
(8, 20). ANG II inhibits
IAC through
activation of a losartan-sensitive
AT1 receptor that is coupled to
activation of phospholipase C (PLC) and the release of
Ca2+. ANG II produces only partial
inhibition (~75%) of
IAC under conditions in which
[Ca2+]i
is strongly buffered by 11 or 20 mM
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) (20, 22). Although this partial inhibition of IAC by ANG II may
be independent of Ca2+, a
Ca2+-dependent mechanism may be
necessary for complete inhibition leading to membrane depolarization.
Ca2+ modulates the activity of a
number of different K+ channels.
Several types of Ca2+-activated
K+ channels are expressed by a
variety of excitable and nonexcitable cells (12). Recently,
K+ channels whose activity is
inhibited by Ca2+ have been
identified in cells, including neurons and lymphocytes (1, 29, 31). The
modulation of K+ channel activity
by Ca2+ may occur through one of
many different mechanisms. These include allosteric modulation of
K+ channel gating through a direct
interaction of Ca2+ with the
channels, as occurs with both
Ca2+-activated and
Ca2+-inhibited
K+ channels (5, 27, 28).
Alternatively, Ca2+ might function
through activation of any of several
Ca2+-dependent enzymes, including
protein kinase C (PKC), or
Ca2+/calmodulin-activated enzymes,
including calmodulin kinase II and the
Ca2+-activated phosphatase
calcineurin (16, 18). Direct modulation of cyclic nucleotide-gated,
nonselective cation channels by
Ca2+/calmodulin has also been
reported (17).
We have studied the inhibition of
IAC by
Ca2+ in whole cell patch-clamp
recordings from bovine AZF cells.
Ca2+ inhibits
IAC channels by a
mechanism that is independent of calmodulin and protein kinases but is
tightly linked to membrane depolarization.
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METHODS |
Materials.
Tissue culture media, antibiotics, fibronectin, and fetal bovine serum
(FBS) were obtained from GIBCO Laboratories (Grand Island, NY).
Coverslips were from Bellco Glass (Vineland, NJ). Enzymes, ACTH
(1
24), ANG II, MgATP, 5'-adenylylimidodiphosphate (AMP-PNP;
lithium salt), NaGTP, BAPTA, and ionomycin were obtained from Sigma
Chemical (St. Louis, MO). Calmodulin inhibitory peptide (residues
290-309 of calmodulin kinase II) was obtained from Biomol (Plymouth Meeting, PA).
Isolation and culture of AZF cells.
Bovine adrenal glands were obtained from steers (age range 1-3 yr)
within 15 min of slaughter at a local slaughterhouse. Fatty tissue was
removed immediately, and the glands were transported to the laboratory
in ice-cold PBS containing 0.2% dextrose. Isolated AZF cells were
prepared as previously described (9), with some modifications. In a
sterile tissue culture hood, the adrenals were cut in half lengthwise,
and the lighter medulla tissue was trimmed away from the cortex and
discarded. The capsule with attached glomerulosa and thicker
fasciculata layer were then dissected into large pieces of ~1.0 × 1.0 × 0.5 cm. A Stadie-Riggs tissue slicer
(Thomas Scientific) was used to separate fasciculata tissue from the
glomerulosa layers by slicing 0.3- to 0.5-mm slices from the larger
pieces. The first medulla/fasciculata slices were discarded. One or two
subsequent fasciculata slices were saved in cold sterile PBS-0.2%
dextrose. The fasciculata/glomerulosa margin (~0.5 mm) and capsule
with attached glomerulosa were discarded. Fasciculata tissue slices
were then diced into 0.5-mm3
pieces and dissociated with 2 mg/ml (~200-300 U/ml) type I
collagenase (neutral protease activity not exceeding 100 U/mg of solid)
and 0.2 mg/ml deoxyribonuclease in DMEM-F12 for ~1 h at 37°C,
with trituration after 30 and 45 min with a sterile, plastic transfer pipette. The tissue-cell suspension was filtered through two layers of
sterile cheesecloth and then centrifuged to pellet cells at 100 g for 5 min. Undigested tissue
remaining in the cheesecloth was collagenase treated for an additional
1 h. Pelleted cells were washed twice with DMEM-0.2% BSA and
centrifuged as before. After resuspension in DMEM, cells were filtered
through 200-µm stainless steel mesh to remove clumps. Dispersed cells
were again centrifuged and either resuspended in DMEM-F12 (1:1) with
10% FBS, 100 U/ml penicillin, and 0.1 mg/ml streptomycin and plated for immediate use or resuspended in FBS-5% DMSO, divided into 1-ml
aliquots each containing ~2 × 106 cells, and stored in liquid
nitrogen for future use. Cells were plated in 35-mm dishes containing
9-mm2 glass coverslips that had
been treated with fibronectin (10 µg/ml) at 37°C for 30 min and
then rinsed with warm, sterile PBS immediately before addition of
cells. Dishes were maintained at 37°C in a humidified atmosphere of
95% air and 5% CO2.
Patch-clamp experiments.
Patch-clamp recordings of K+
channel currents were made in the whole cell and inside-out patch
configurations. For whole cell recordings, the standard external
solution consisted of (in mM) 140 NaCl, 5 KCl, 2 MgCl2, 2 CaCl2, 10 HEPES, and 5 glucose,
with pH buffered to 7.4 using NaOH. The standard pipette solution was (in mM) 120 KCl, 2 MgCl2, 10 HEPES, and 5 MgATP, as well as 200 µM GTP, with pH buffered to 7.2 using KOH. The buffering capacity and
Ca2+ concentration of the pipette
solutions was varied by adding combinations of BAPTA and
CaCl2 using the Bound and
Determined program (2). Low- and high-capacity
Ca2+-buffering solutions contained
2 and 11 mM BAPTA, respectively. In most experiments,
[Ca2+]i
was buffered to 0.02 µM. Variations are noted in the text. All
solutions were filtered through 0.22-µm cellulose acetate filters.
For inside-out patch recordings, the standard external and pipette
solutions used in whole cell recordings were switched.
AZF cells were used for patch-clamp experiments 2-12 h after
plating. Typically, cells with diameters of 10-15 µm and
capacitances of 8-15 pF were selected. Coverslips were transferred
from 35-mm culture dishes to the recording chamber (volume 1.5 ml),
which was continuously perfused by gravity at a rate of 3-5
ml/min. For whole cell recordings, patch electrodes with resistances of 1.0-2.0 M
were fabricated from Corning 0010 glass (Garner
Glass, Claremont, CA). These routinely yielded access resistances of 1.5-4 M and voltage-clamp time constants of <100 µs. For
single-channel recordings, patch electrodes with higher resistances of
3-5 M were used. K+
currents were recorded at room temperature (22-25°C),
following the procedure of Hamill et al. (10) and using a List EPC-7
patch-clamp amplifier.
Pulse generation and data acquisition were done using a personal
computer and pCLAMP software with a TL-1 interface (Axon Instruments,
Burlingame, CA). Currents were digitized at 1-20 kHz after
filtering with an eight-pole Bessel filter (Frequency Devices,
Haverhill, MA). Linear leak and capacity currents were subtracted from
current records using scaled hyperpolarizing steps of one-half to
one-fourth amplitude. Data were analyzed and plotted using pCLAMP 5.5 and 6.02 (Clampan, Clampfit, Fetchan, and Pstat), SigmaPlot 3.0, and
GraphPad InPlot 4.03. Drugs were applied by bath perfusion that was
controlled manually by a six-way rotary valve.
Series resistance compensation was not used in most experiments. The
mean amplitude of
IAC in AZF cells
was <500 pA. A current of this size in combination with a 4-M
access resistance produces a voltage error of only 2 mV, which was not corrected.
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RESULTS |
[Ca2+]i
and IAC expression.
As previously reported, bovine AZF cells express two types of
K+ currents: a rapidly
inactivating A-type K+ current
(IA) (23) and a
noninactivating K+ current
(IAC) whose
amplitude increases continuously over many minutes in whole cell
recordings (8, 20). The expression of
IAC requires the
presence of ATP at millimolar concentrations in the recording pipette
(6). The absence of time-dependent inactivation allows the
IAC to be easily
isolated for measurement in whole cell recordings, using either of two
voltage-clamp protocols. When voltage steps 300 ms in duration were
applied from a holding potential of 
80 mV to a test potential of
+20 mV or +30 mV,
IAC could be
selectively measured near the end of a step, at a point at which the
IA had
inactivated entirely (Fig.
1A,
left). Using the second protocol,
IAC was
selectively activated with an identical voltage step after a 10-s
prepulse to 20 mV had fully inactivated the
IA (Fig.
1A,
middle).
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Fig. 1.
Effect of intracellular Ca2+
concentration
([Ca2+]i)
on noninactivating K+ current
(IAC)
expression. K+ currents were
activated at 30-s intervals by voltage steps to +20 mV from a holding
potential of 80 mV with or without a 10-s prepulse to 20
mV that inactivated A-type K+
current. A:
K+ currents were recorded with
patch pipettes containing standard solution (see
METHODS) with
Ca2+ buffered to 0.02 µM
(top) or 2 µM
[Ca2+]i
(bottom) using 11 mM
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid (BAPTA), with or without inactivating prepulses, as indicated by
voltage protocol diagrams. Left and
middle: traces show currents recorded
at indicated times after initiation of whole cell recording.
Right:
IAC current
amplitudes (measured near end of voltage step) were plotted against
time;  , with depolarizing prepulse;  , without prepulse.
B:
IAC current
density (expressed as pA/pF) was obtained by dividing maximum
IAC current
amplitude obtained in experiments such as those illustrated in
A by cell capacitance determined from
transient cancellation controls of patch-clamp amplifier. Results are
means ± SE for indicated no. of determinations at
[Ca2+]i
of 0.02, 2, and 10 µM.
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The time-dependent increase in
IAC amplitude
observed in whole cell recordings was markedly inhibited when
[Ca2+]i
was raised from 0.02 to 2.0 µM with
Ca2+ buffered by 11 mM BAPTA (Fig.
1, A and
B). Overall, with 0.02 µM
[Ca2+]i,
after 10-20 min,
IAC reached a
maximum current density of 25.0 ± 5.1 pA/pF
(n = 16). By comparison, with 2 µM
[Ca2+]i,
IAC reached a
maximum density of 5.9 ± 3.0 pA/pF
(n = 11), a reduction of ~76%.
Increasing
[Ca2+]i
from 2.0 to 10 µM did not further suppress
IAC expression, which grew to a maximum of 6.4 ± 2.0 pA/pF
(n = 13; Fig.
1B).
Inhibition of IAC by
ionomycin.
The time-dependent expression of
IAC typically
observed in whole cell recordings from AZF cells was suppressed by
increasing [Ca2+]i
to 2 and 10 µM. To determine whether
IAC, once
expressed, could be inhibited by
Ca2+ influx across the plasma
membrane, cells were superfused with the
Ca2+ ionophore ionomycin after
IAC had reached a
stable maximum amplitude.
In this series of experiments, ionomycin was superfused at a
concentration of 10 µM, and
[Ca2+]i
was buffered to 0.02 µM with 2 mM BAPTA. Reducing BAPTA from 11 to 2 mM suppressed maximum
IAC expression by
~60%, although [Ca2+]i
was maintained at 0.02 µM in each case. With 2 mM BAPTA in the
pipette, ionomycin was less effective at concentrations <10 µM. The
combination of 2 mM BAPTA and 10 µM ionomycin proved to be best for
this study.
As illustrated in Fig. 2, ionomycin (10 µM) selectively inhibited the
IAC. Inhibition
was detectable after a delay of 2-3 min and progressed to near
completion after 10 min (Fig. 2, A and
B). In contrast to
IAC, the
IA current
remained after
IAC was nearly
completely inhibited by ionomycin (Fig.
2A). Overall, ionomycin (10 µM)
inhibited IAC by
88 ± 4% (n = 14).
Ionomycin-mediated inhibition of
IAC was only
partially reversible. The current was restored to 44 ± 11%
(n = 8) of its maximum value with
prolonged washing.
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Fig. 2.
Effect of ionomycin on
IAC.
K+ currents were activated at 30-s
intervals by voltage steps to +30 mV from a holding potential of
80 mV. After
IAC had reached a
maximum value, cell was superfused with ionomycin (10 µM).
A: current records after
IAC had reached a
maximum value (top) and 12 min after
superfusion of 10 µM ionomycin
(bottom).
B:
IAC current
amplitudes are plotted against time for same cell as in
A.
[Ca2+] of external
solution was 2 mM.
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Current-voltage relationships recorded before and after superfusion of
ionomycin clearly showed that this ionophore selectively inhibited
IAC. Furthermore,
this inhibition was independent of voltage over a wide range of test
potentials. In the experiment illustrated in Fig.
3, once
IAC reached a
stable amplitude, K+ currents were
activated by voltage steps to test potentials between 50 and +60
mV before and after superfusion of ionomycin (10 µM). As shown in
Fig. 3A, ionomycin selectively
inhibited the IAC
almost completely at each test potential, leaving only the
IA (Fig.
3A, left and
right). The effective inhibition of
IAC by ionomycin at each test potential (Fig. 3B)
indicated that ionomycin-stimulated Ca2+ influx did not merely shift
the voltage dependence of
IAC open probability to the right along the voltage axis.
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Fig. 3.
Voltage-independent inhibition of
IAC by ionomycin.
K+ currents were activated at 30-s
intervals from a holding potential of 80 mV by voltage steps of
various sizes before and after superfusion of cell with 10 µM
ionomycin. A: current records at test
potentials between 50 and +60 mV (in 10-mV increments) before
(left) and after
(right) superfusion of ionomycin.
B: current-voltage relationships.
IAC current
amplitudes from experiment in A are
plotted against test voltage [membrane potential
(Vm)].
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Experiments with ionomycin indicated that
IAC could be
specifically inhibited by raising
[Ca2+]i
through enhanced Ca2+ influx
across the cell membrane. To further characterize the specific
Ca2+ dependence of the response,
we examined the effect of ionomycin on
IAC when the
Ca2+-buffering capacity of the
pipette solution was increased by raising BAPTA in the pipette from 2 to 11 mM.
With the high-capacity
Ca2+-buffering pipette solution,
ionomycin was much less effective at inhibiting
IAC. As
illustrated in Fig.
4A,
superfusion of this cell with ionomycin (10 µM) failed to reduce
IAC, whereas ACTH
(200 pM) completely inhibited this current within 3 min. ACTH inhibits
IAC through a
cAMP-dependent mechanism that appears to be independent of changes in
[Ca2+]i
(8). Overall, in four cells, ionomycin (10 µM) inhibited IAC by 25 ± 14% when the pipette contained 11 mM BAPTA, compared with 88 ± 4%
(n = 14) inhibition observed with 2 mM
BAPTA. In contrast, ACTH inhibition of
IAC was
insensitive to changes in pipette BAPTA concentration.
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Fig. 4.
Effect of intracellular Ca2+
buffering and external Ca2+
removal on ionomycin-mediated
IAC inhibition.
Inhibition of IAC
by ionomycin (10 µM) was measured using high-capacity
Ca2+-buffering pipette solution
(11 mM BAPTA) and standard external solution (2 mM
CaCl2;
A) or external solution with no
added Ca2+
(Ca2+ free) and pipette solution
containing 2 mM BAPTA (B).
Left: after
IAC reached a
stable amplitude, cells were sequentially superfused with ionomycin (10 µM) and adrenocorticotropic hormone (ACTH; 200 pM) while
K+ currents were recorded.
Middle: A-type
K+ currents were inactivated
either by a depolarizing prepulse
(A) or by holding cell at 40
mV (B).
Right:
IAC current
amplitudes are plotted against time.
1, maximum
IAC;
2,
IAC after
ionomycin (10 µM); 3,
IAC after ACTH
(200 pM).
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In other experiments, we found that with standard pipette solution (2 mM BAPTA) ionomycin failed to inhibit
IAC when
Ca2+ was omitted from the external
solution. For Fig. 4B, the cell was
superfused with external solution containing no added
Ca2+ followed by a switch to
solutions containing ionomycin (10 µM), followed by ACTH (200 pM).
Ionomycin was ineffective in the absence of added
Ca2+, whereas ACTH completely
inhibited IAC.
Calmodulin and ionomycin inhibition of
IAC.
Many Ca2+-mediated processes,
including the modulation of some ion channels, require calmodulin as an
intermediate. Once activated by
Ca2+,
Ca2+/calmodulin may interact
directly with the channel or indirectly through activation of a
calmodulin-dependent enzyme. Calmodulin inhibitory peptide (residues
290-309 of calmodulin kinase II) potently inhibits calmodulin
kinase II (IC50 50 nM) as well as other calmodulin-dependent processes (25). Calmodulin inhibitory peptide (2.5 µM), applied intracellularly through the recording pipette solution, failed to significantly alter ionomycin-mediated inhibition of
IAC.
Figure 5 illustrates an experiment in which
IAC was allowed
to grow to a stable value in the presence of calmodulin inhibitory peptide before superfusion of ionomycin (10 µM). The inhibitory peptide had no apparent effect on
IAC growth or
inhibition by ionomycin. In this experiment,
IAC was inhibited
almost completely, but the
IA was
unaffected. Overall, with 2.5 µM calmodulin kinase II inhibitory
peptide in the pipette solution, ionomycin (10 µM) inhibited
IAC by 83 ± 7% (n = 8), compared with
88 ± 4% (n = 14) under control
conditions. These results indicate that
Ca2+ does not inhibit
IAC through
calmodulin acting either directly or through activation of a
calmodulin-dependent enzyme.
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Fig. 5.
Effect of calmodulin (CaM) inhibitory peptide on ionomycin inhibition
of IAC. Adrenal
zona fasciculata cells (AZF cells) were voltage clamped in whole cell
configuration using standard pipette solution supplemented with 2.5 µM CaM inhibitory peptide. After
IAC had reached a
stable amplitude, cells were superfused with ionomycin (10 µM) while
IAC was recorded
at 30-s intervals. A:
K+ current records with
(bottom) and without
(top) depolarizing prepulses before
(control) and after superfusion of ionomycin (10 µM).
B:
IAC amplitudes
are plotted against time for experiment in
A; , with depolarizing prepulse;
, without prepulse.
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AMP-PNP and ionomycin inhibition of
IAC.
The growth of IAC
in whole cell recordings requires the presence of ATP at millimolar
concentrations (6). Inhibition of this current by either ACTH or ANG II
requires hydrolyzable forms of ATP, suggesting the involvement of a
kinase or ATPase (6, 8, 22). To determine whether ATP hydrolysis is
required for Ca2+-mediated
inhibition of
IAC, the
nonhydrolyzable ATP analog AMP-PNP was substituted for ATP in the
pipette solution. Figure 6 shows that, with
2 mM AMP-PNP in the pipette, ionomycin (10 µM) reversibly inhibited
IAC by ~70%.
Overall, ionomycin inhibited
IAC by 75.4 ± 6% (n = 5) when AMP-PNP replaced ATP
in the pipette. In each of three experiments, ACTH (200 pM) failed to
produce any measurable inhibition of
IAC when
superfused before ionomycin, thus ensuring that AMP-PNP was present
intracellularly at effective concentrations. The consistent inhibition
of IAC by
ionomycin with AMP-PNP in the pipette demonstrated that
Ca2+ does not inhibit
IAC by activation
of an ATPase or Ca2+-dependent
enzymes such as PKC.
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Fig. 6.
Effect of 5'-adenylylimidodiphosphate (AMP-PNP) on
ionomycin-mediated inhibition of
IAC. AZF cell was
voltage clamped in whole cell configuration with pipette containing
standard solution in which ATP was replaced with 2 mM AMP-PNP. After
IAC had reached a
stable amplitude, cell was superfused with ionomycin (10 µM) while
K+ currents were recorded at 30-s
intervals. A:
K+ current records in response to
voltage steps with (bottom) and
without (top) depolarizing prepulses
before (2) and after
(3) superfusion of ionomycin.
B:
IAC amplitudes
are plotted against time; , with depolarizing prepulse; , without
prepulse. 1, immediately after
initiation of whole cell recording; 2,
after IAC had
reached a maximum amplitude; 3, after
inhibition by 10 µM ionomycin.
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Effect of
Ca2+ on unitary
current.
Whole cell patch-clamp experiments indicated that
Ca2+-mediated inhibition of
IAC might occur
through a direct interaction of Ca2+ with the channel.
Single-channel recording experiments in which Ca2+ was directly applied to the
cytoplasmic surface of excised inside-out patches were consistent with
this model of inhibition. In these experiments, the membrane patch was
excised into an internal solution containing 22 nM
Ca2+, and the holding potential
was set to 40 mV (inside negative), a potential at which all
IA channels are
inactivated. Under these conditions, a single type of
K+ current was typically present
in the membrane patch. Figure
7A shows
unitary currents recorded in response to voltage steps to +20 mV
from a holding potential of 40 mV in control saline (20 nM
Ca2+). Under these conditions,
IAC channel
activity increased spontaneously and continuously during
prolonged recordings. Histogram analysis of unitary current amplitudes
showed a major peak with a mean ± SE of 2.41 ± 0.82 pA, and two other peaks with means that were approximately
twice (4.82 ± 0.73 pA) and three times (7.36 ± 0.67 pA) the unitary amplitude. When the cytoplasmic membrane surface was
superfused with saline containing 35 µM
Ca2+,
IAC channel
activity was dramatically reduced (Fig.
7B). Inhibition was evident after
30-60 s and reached a maximum within 2 min. When solution was
switched back to low-Ca2+ saline,
channel activity was restored to a level greater than control.
Histogram analysis showed four separate peaks, each a multiple of the
unitary amplitude (Fig. 7C). Unitary
IAC were also
inhibited by
[Ca2+]i
of 10 and 20 µM (data not shown).
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Fig. 7.
Inhibition of unitary
IAC by
Ca2+. Unitary
IAC were recorded
in inside-out configuration using pipettes containing standard external
solution used in whole cell recordings (see
METHODS). Patches were excised into
internal solution containing 22 nM
Ca2+. Voltage steps to +20 mV were
applied at 30-s intervals from a holding potential of 40 mV.
Amplitude histograms were constructed from unitary currents recorded in
response to voltage steps 300 ms in duration. Current amplitudes were
distributed into bins 0.150 pA in width.
A-C:
unitary currents (top) and
corresponding amplitude histograms
(bottom) from recordings in control
solution (22 mM Ca2+), in
presence of 35 µM Ca2+, and
after return to control (wash), respectively. Currents were sampled at
5 kHz and filtered at a cutoff frequency of 2 kHz.
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Effect of ionomycin on membrane potential.
Results presented to this point indicate that
IAC channels are
inhibited by Ca2+ through a
mechanism that is independent of calmodulin and protein kinases.
Because IAC
channels appear to set the membrane potential of AZF cells (8, 20), our
findings suggest that increasing [Ca2+]i
could trigger membrane depolarization through inhibition of these
channels. To explore this possibility, we examined the effects of
ionomycin on membrane potential and
IAC in combined
whole cell voltage-clamp and current-clamp recordings.
In the experiment illustrated in Fig.
8A,
IAC was monitored
in whole cell voltage clamp until it reached a stable value of ~325
pA (Fig. 8A,
right). Membrane potential was then
recorded after switching to current clamp. In control saline, the
resting potential of the cell was 63 mV. The superfusion of
ionomycin (10 µM) produced, after a delay of several minutes, a
progressive depolarization that reached a maximum of ~45 mV during
the next 2 min (Fig. 8A,
left). At this time, voltage-clamp
recordings showed that
IAC had been
inhibited by 90% but the
IA was not
affected (Fig. 8A,
right). In a total of four similar
experiments, ionomycin inhibited
IAC by 89.6 ± 3.5% while depolarizing cells by an average of 47 ± 8 mV.
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Fig. 8.
Depolarization of AZF cells by ionomycin in low and high BAPTA.
K+ currents were recorded at 30-s
intervals with or without depolarizing prepulses. When
IAC reached a
stable maximum value, patch-clamp amplifier was switched to current
clamp to record
Vm. Patch
pipettes contained either 2 mM (A)
or 11 mM (B) BAPTA.
Left: while
Vm was recorded
(sampling rate 10 s 1),
cells were superfused with ionomycin or ACTH at indicated times.
Right:
K+ currents were recorded at times
indicated on record of
Vm by switching
to voltage clamp. Traces were recorded with or without depolarizing
prepulses as indicated by voltage protocol diagrams.
|
|
Ionomycin-mediated membrane depolarization was markedly reduced when
the pipette contained the high-capacity
Ca2+-buffering solution (11 mM
BAPTA). In the experiment illustrated in Fig.
8B, ionomycin (10 µM) depolarized
this cell by a maximum of <5 mV and failed to significantly inhibit
IAC. When this
same cell was then superfused with ACTH (200 pM),
IAC was inhibited nearly completely and the cell was depolarized by ~40 mV. Overall, in
recordings made with 11 mM BAPTA in the pipette, ionomycin (10 µM)
inhibited IAC by
25.3 ± 14.6% (n = 4)
and depolarized cells by only 8 ± 3 mV
(n = 4).
Ca2+ and ANG
II inhibition of IAC.
In previous studies, we have shown that ANG II inhibits
IAC by a maximum
of ~75% under conditions in which
[Ca2+]i
was strongly buffered by 10 or 20 mM BAPTA (20, 22). Because ANG II
inhibits IAC
through activation of an AT1
receptor that is coupled to inositol 1,4,5-trisphosphate
(IP3)-stimulated release of
intracellular Ca2+, we tested the
possibility that ANG II-mediated inhibition of IAC would be
enhanced by reducing BAPTA in the patch pipette.
ANG II was significantly more effective at inhibiting
IAC when
[Ca2+]i
was buffered with 2 mM rather than 11 mM BAPTA (Fig.
9). Overall, ANG II (10 nM) inhibited
IAC by 76.5 ± 4.5% (n = 6) when
[Ca2+]i
was buffered to 0.02 µM with 11 mM BAPTA. In comparison, at the same
concentration, ANG II inhibited
IAC by 97.8 ± 3.4% (n = 4) when
[Ca2+]i
was buffered by 2 mM BAPTA (Fig.
9B).
View larger version (20K):
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|
Fig. 9.
Effect of BAPTA on ANG II-mediated inhibition of
IAC. Inhibition
of IAC by ANG II
(10 nM) was measured with pipette solutions containing 11 mM
(A) or 2 mM
(B) BAPTA.
Top:
K+ currents were activated by
voltage steps to +20 mV, applied at 30-s intervals from a holding
potential of 80 mV. 1, initial
K+ current;
2, maximum current in control saline;
3, current after steady-state block by
ANG II (10 nM). Bottom:
IAC amplitude is
plotted against time. Numbers correspond to traces at
top.
|
|
| |
DISCUSSION |
Measures that increased
[Ca2+]i
in bovine AZF cells specifically inhibited
IAC channel
activity by a mechanism that appears to be independent of calmodulin,
protein kinases, and ATP hydrolysis. The inhibition of
IAC by the
Ca2+ ionophore ionomycin was
tightly linked to membrane depolarization. ANG II-mediated inhibition
of IAC was
blunted by strongly buffering [Ca2+]i
with BAPTA. These results suggest a positive feedback mechanism by
which ANG II-stimulated release of intracellular
Ca2+ triggers
IAC inhibition,
membrane depolarization, and Ca2+
entry through voltage-gated Ca2+ channels.
Specificity and mechanism of
IAC inhibition by
Ca2+.
The inhibition of
IAC produced by
raising
[Ca2+]i,
either by addition of Ca2+ to the
patch electrode or through bath perfusion of ionomycin, was specific.
IA was not
altered. A direct action of ionomycin on
IAC channels
seems unlikely, since the effect of this ionophore was blunted or
eliminated by removing external
Ca2+ or by buffering
[Ca2+]i
strongly with 11 mM BAPTA.
Activation of IAC
channels is weakly voltage dependent (6, 8). Ionomycin-mediated
inhibition of IAC
was independent of voltage over a wide range of test potentials.
Presumably, Ca2+-mediated
inhibition of IAC
did not result from a rightward shift in the voltage dependence of
IAC activation.
In contrast, Ca2+ appears to act
directly on Ca2+-activated
K+ channels, producing a leftward
shift in the voltage dependence of activation such that these
K+ channels are activated at
less-depolarized potentials (5). Apparently,
Ca2+ modulation of these two
K+ channel subtypes occurs through
fundamentally different mechanisms.
Because the calmodulin inhibitory peptide, applied at 40 times its
IC50, failed to alter
ionomycin-mediated inhibition of IAC, it is
unlikely that Ca2+-mediated
inhibition occurs through activation of a calmodulin-dependent enzyme,
such as calmodulin kinase or the
Ca2+-dependent phosphatase
calcineurin. Because this inhibitory peptide binds calmodulin with high
affinity, it is also unlikely that Ca2+/calmodulin directly inhibits
IAC channels
(25).
The ability of ionomycin to inhibit
IAC when ATP is
replaced with the nonhydrolyzable ATP analog AMP-PNP indicates that
Ca2+-mediated inhibition of
IAC does not
require the activation of any kinase or ATPase. Protein kinases
typically require hydrolyzable forms of ATP at concentrations of <100
µM, whereas ATPases often display dissociation constants
for this nucleotide in the millimolar range (11, 26).
The inability of calmodulin inhibitory peptide and AMP-PNP to prevent
IAC inhibition by
ionomycin suggests a direct interaction between
Ca2+ and
IAC channels. In
this regard, IAC
inhibition by ionomycin begins only after a delay of up to several
minutes and requires additional minutes to reach a maximum. Although
this would seem excessively long for a process requiring a direct
interaction of Ca2+ with the
channel, the impact of the combination of 10 µM ionomycin and 2 mM
BAPTA on
[Ca2+]i
dynamics is unknown. It is clear that BAPTA is a rapidly acting Ca2+ buffer that can, at high
concentrations, suppress
[Ca2+]i
increases induced by influx during action potentials or release from
ligand-induced IP3 (14, 19, 30,
31). Furthermore, both ANG II- and ACTH-stimulated inhibition of
IAC and membrane depolarization occur after a delay of one to several minutes, even in
intact cells (20, 22). Thus the slow
Ca2+-mediated inhibition could
occur through a physiological mechanism instead of one generated as an
artifact of the Ca2+-buffering
system. In excised inside-out patches, inhibition of unitary current
activity was somewhat faster but still required at least 2 min to reach
a maximum.
Comparison with other
Ca2+-inhibited
ion channels.
Ca2+ inhibits
K+ channels in neurons and
lymphocytes (1, 14, 15, 18, 29, 31). The mechanisms and signaling
pathway that underlie
Ca2+-mediated inhibition are
varied. Im is a
voltage- and time-dependent K+
current in neuronal cells whose inhibition by
Ca2+ has been extensively studied.
Raising
[Ca2+]i
to values >450 nM suppresses the expression of
Im (31).
Likewise, Im is
inhibited by Ca2+ increases
induced by muscarinic receptor activation or photolysis of a caged
Ca2+ chelator, whereas inhibition
is blunted by buffering Ca2+ with
20 mM BAPTA (14, 19, 31).
The inhibition of
Im by
Ca2+ may occur through a direct
action on the K+ channel, since
Ca2+ inhibits these channels in
excised inside-out patches even in the absence of ATP (27, 28).
However, it has been reported that
Ca2+-dependent inhibition of
Im in sympathetic
neurons is mediated through activation of the
Ca2+-dependent phosphatase
calcineurin (18). It is unlikely that activation of a similar
phosphatase is responsible for
IAC inhibition, since calcineurin activation requires a calmodulin intermediate.
Other ion channels, including K+
channels in the kidney and cyclic nucleotide-gated channels of the
olfactory epithelium, are modulated by
Ca2+ through calmodulin-dependent
mechanisms (4, 15). Inhibition of the cyclic nucleotide-gated channel
by Ca2+ requires binding of the
Ca2+/calmodulin complex to a
specific site on the amino-terminal end of the channel (4, 17).
IAC channels
resemble cyclic nucleotide-gated cation channels in that the gating of
each is regulated by cAMP (8, 13). However,
Ca2+ modulation of
IAC channels does
not appear to occur through a calmodulin-dependent mechanism.
Physiological significance.
Our findings indicate that membrane potential and
[Ca2+]i
are tightly coupled through the activity of
IAC channels in
AZF cells. Both ACTH and ANG II inhibit
IAC, depolarize
AZF cells and stimulate cortisol secretion. However, the importance of
Ca2+ as an intracellular messenger
linking peptide receptor activation to
IAC inhibition
has not been established.
ANG II stimulates cortisol secretion through the activation of a
losartan-sensitive AT1 receptor.
Although these AT1 receptors are
coupled to PLC activation and
IP3-stimulated release of
Ca2+, ANG II-mediated inhibition
of IAC may be
mediated in part by a distinct
Ca2+-independent signaling pathway
(22). ANG II inhibits
IAC by ~75%,
even when
[Ca2+]i
is strongly buffered with 11 or 20 mM BAPTA (20, 22). Furthermore, under these conditions, ANG II-mediated inhibition of
IAC requires the
presence of hydrolyzable ATP (22). This result contrasts with
ionomycin-mediated inhibition of
IAC, which is not
altered by substituting AMP-PNP for ATP.
The nearly complete inhibition of
IAC by ANG II
observed in the present study when pipette BAPTA was reduced to 2 mM is
consistent with a model that includes activation of parallel signaling
pathways, one of which utilizes
Ca2+ as a mediator. Because nearly
complete inhibition of
IAC channels may
be required to effectively depolarize AZF cells, separate inhibitory
mechanisms that converge on
IAC channels
could provide an efficient mechanism for membrane depolarization.
In a previous study, we found that ANG II and ACTH produce nearly
identical maximal depolarization of AZF cells as measured by
high-resistance (100-150 M) intracellular electrodes (20). By
comparison, ACTH was significantly more effective than ANG II at
inhibiting IAC in
whole cell patch-clamp experiments in which
[Ca2+]i
was strongly buffered with 11 mM BAPTA. This apparent discrepancy can
now be explained if we assume that ANG II was more effective at
increasing
[Ca2+]i
and inhibiting
IAC in cells in
which membrane potential was measured with sharp electrodes. In these
intact cells, ANG II-mediated IAC inhibition
would not be diminished by artificial
Ca2+ buffering. Presumably, under
these conditions, both ACTH and ANG II produce similar
IAC inhibition
and membrane depolarization.
cAMP is the primary intracellular messenger coupling ACTH receptor
activation to membrane depolarization (8). Accordingly, ACTH produces
nearly complete inhibition of
IAC even when
[Ca2+]i
is strongly buffered with 11 mM BAPTA. However,
Ca2+ entering through
low-voltage-activated T-type channels in these AZF cells could
contribute to IAC
inhibition. Specifically, at low ACTH concentrations, partial
inhibition of IAC
channels mediated by cAMP could produce sufficient depolarization to
activate T-type Ca2+ channels,
producing further
IAC inhibition
and membrane depolarization (21).
The inhibition of
IAC channels by
Ca2+ at physiological
concentrations identifies a novel mechanism whereby AZF cell membrane potential is tightly linked in a reciprocal relationship to
[Ca2+]i.
Agents that trigger Ca2+ increases
of sufficient magnitude by influx across the cell membrane or release
from intracellular stores would inevitably depolarize AZF cells. This
system incorporates the elements of a positive feedback mechanism
driven by a regenerative Ca2+
signal. In addition to inducing the synthesis of corticosteroids in
these secretory cells,
[Ca2+]i
would, at the same time, regulate its own entry across the plasma
membrane (3, 7, 24).
The IAC channel
is a distinctive new type of K+
channel with properties that identify it as a central control point for
the regulation of cortisol secretion. In addition to setting the
resting potential of AZF cells,
IAC channel
activity is regulated by a variety of metabolic factors and hormonally
induced second messengers. Although
IAC open
probability is greatly enhanced by ATP at physiological concentrations,
it is inhibited by peptide hormone-generated second messengers
including cAMP and Ca2+. Thus
IAC channels are
intracellular sensors that integrate complex metabolic and hormonal
signals, coupling the metabolic state of the cell to membrane potential
and cortisol secretion.
| |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-47875 and by National American
Heart Association Grant-in-Aid 94011740 to J. J. Enyeart.
| |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests: J. J. Enyeart, Dept. of Pharmacology,
Ohio State University College of Medicine, 5188 Graves Hall, 333 W. 10th Ave., Columbus, OH 43210-1239.
Received 13 April 1998; accepted in final form 19 August 1998.
| |
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Abstract
Introduction
