Department of Neuroscience, The Ohio State University, College of
Medicine, Columbus, Ohio
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Introduction |
Several
large families of K+-selective ion channels have
been identified that are expressed throughout the plant and animal kingdoms. These include voltage-gated and metabolically regulated "background" K+ channels that regulate the
frequency and duration of action potentials and set the resting
membrane potential in various cells (Chandy and Gutman, 1995
; Goldstein
et al., 1998). Consequently, K+ channels function
critically in regulating cellular functions, including hormone
secretion, muscle contraction, and neural conduction and transmission.
Although many cells express multiple K+-channel
subtypes that are modulated through a variety of signaling pathways,
functional coupling between these K+ channels
under physiological conditions has not been described.
Bovine adrenocortical cells express two distinct types of
K+ selective channels. These include a
voltage-gated, rapidly inactivating A-type channel
(IA) and a noninactivating background
K+ channel (IAC) that set
the resting membrane potential (Mlinar et al., 1993; Mlinar and
Enyeart, 1993; Enyeart et al., 1996, 1997). IAC
is unique among K+ channels described thus far
and seems to act pivotally in the physiology of cortisol secretion. In
whole-cell, patch-clamp recordings from AZF cells,
IAC grows continuously over many minutes when ATP
is present in the patch electrode at concentrations greater than 1 mM
(Mlinar et al., 1993; Enyeart et al., 1996, 1997; Gomora and Enyeart,
1998).
IAC channels are potently inhibited by
corticotropin (IC50 = 5.4 pM) at concentrations
identical with those that depolarize AZF cells and stimulate cortisol
secretion (Mlinar et al., 1993; Enyeart et al., 1996). The inhibition
of IAC by corticotropin is independent of
A-kinase but requires the presence of hydrolyzable ATP, suggesting that
the gating of IAC channels could be coupled to an
ATP hydrolysis cycle (Enyeart et al., 1996). Regardless, IAC channels function as sensors, coupling
hormonal and metabolic signals to membrane potential,
Ca2+ entry, and cortisol secretion (Enyeart et
al., 1993).
Molecular cloning of the rapidly inactivating IA
K+-channel cDNA shows that it belongs to the
bKv1.4 K+-channel family (Enyeart et al., 2000).
Although this voltage-gated channel is prominently expressed in
virtually every AZF cell, its function has not been determined (Mlinar
and Enyeart, 1993). IA channels have not been
shown to be modulated by corticotropin.
Previous studies examining the activation of IAC
by nucleotides and its inhibition by corticotropin have not uncovered a
link between the activity of IA and
IAC K+ channels in AZF
cells (Enyeart et al., 1996, 1997). However, in recent experiments, we
have discovered compelling evidence for a unique form of channel
regulation in which the gating of IA and
IAC K+ channels are
reciprocally controlled in tandem by nucleotides and corticotropin receptors.
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Materials and Methods |
Tissue culture media, antibiotics, fibronectin, and fetal bovine
sera were obtained from Life Technologies (Grand Island, NY).
Coverslips were purchased from Bellco Glass, Inc. (Vineland, NJ).
Enzymes, corticotropin(1-24), MgATP, NaATP, NaUTP,
5-adenylylimido-diphosphate (AMP-PNP, lithium salt), NaGTP,
guanosine-5'-O-(2-thio)diphosphate, BAPTA, and pimozide were
obtained from Sigma Chemical Company (St. Louis, MO). Penfluridol and
fluspirilene were obtained from Janssen Pharmaceuticals (Beerse, Belgium).
Isolation and Culture of AZF Cells.
Bovine adrenal glands
were obtained from steers (age range, 1 to 3 years) within 30 min of
slaughter at a local slaughterhouse. Fatty tissue was removed
immediately and the glands were transported to the laboratory in
ice-cold phosphate-buffered saline containing 0.2% dextrose. Isolated
AZF cells were prepared as described previously (Enyeart et al., 1996).
After isolation, cells were either resuspended in Dulbecco's modified
Eagle's medium/Ham's F12 medium (1:1) with 10% fetal bovine serum,
100 U/ml penicillin, 0.1 mg/ml streptomycin, and antioxidants 1 µM
tocopherol, 20 nM selenite, and 100 µM ascorbic acid and
plated for immediate use, or resuspended in fetal bovine serum/5%
dimethyl sulfoxide, divided into 1-ml aliquots, each containing about
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
10 µg/ml fibronectin at 37°C for 30 min then rinsed with warm,
sterile phosphate-buffered saline immediately before adding cells.
Dishes were maintained at 37°C in a humidified atmosphere of 95%
air/5% CO2.
Patch-Clamp Experiments.
Patch-clamp recordings of
K+-channel currents were made in the whole-cell
configuration. The standard pipette solution was 120 mM KCl, 2 mM
MgCl2, 1 mM CaCl2, 20 mM
HEPES, 11 mM BAPTA, 200 µM GTP, and 5 mM MgATP, pH buffered to 7.2 using KOH. Deviations from the standard solution are described in the
text. The external solution consisted of 140 mM NaCl, 5 mM KCl, 2 mM
CaCl, 2 mM MgCl2, 10 mM HEPES, and 5 mM glucose,
pH buffered to 7.4 using NaOH. All solutions were filtered through
0.22-µm cellulose acetate filters. Drugs were applied externally by
bath perfusion controlled manually by a six-way rotary valve.
AZF cells were used for patch-clamp experiments 2 to 12 h after
plating. Typically, cells with diameters of <15 µm and capacitances of 8 to 15 pF were selected. Coverslips were transferred from 35-mm
culture dishes to the recording chamber (volume, 1.5 ml), which was
continuously gravity-perfused at a rate of 3 to 5 ml/min. To minimize
series resistance errors, patch electrodes with resistances of <1.5
M
were fabricated from Corning 0010 glass (Garner Glass Co.,
Claremont, CA). These routinely yielded access resistances of <3 M.
K+ currents were recorded at room temperature
(22-25°C) following the procedure of Hamill et al. (1981) using an
Axopatch 1D patch-clamp amplifier (Axon Instruments, Inc., Burlingame, CA).
Pulse generation and data acquisition were done using a personal
computer and PCLAMP software with a TL-1 interface (Axon Instruments).
Currents were digitized at 5 to 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-third to one-fourth amplitude. Data were analyzed and plotted using pCLAMP 5.5 and 6.04 (Clampan and Clampfit) and SigmaPlot (ver 4.0; SPSS, Chicago, IL).
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Results |
Reciprocal Effects of ATP on IA and
IAC.
Differences in the nucleotide dependence and the
voltage-dependent gating and kinetics of IA and
IAC K+ channels allow them
to be isolated and measured in whole-cell recordings. In this study,
K+ currents from AZF cells were elicited using
either of two voltage clamp protocols. Voltage steps to +20 mV, applied
from a holding potential of 
80 mV, elicited combined
IA and IAC currents (Fig. 1, left voltage protocol). With this
protocol, IAC could be measured near the end of
the voltage step at a time when IA current had completely inactivated. Identical voltage steps preceded by a 10 s
prepulse to 20 mV inactivate IA channels,
allowing IAC current to be recorded and measured
in isolation (Fig. 1, right voltage protocol).
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Fig. 1.
Effect of ATP on the time-dependent expression of
IA and IAC K+ currents. Whole-cell
K+ currents were recorded from bovine AZF cells at 30-s
intervals with pipettes containing 1 or 5 mM MgATP in response to
voltage steps to +20 mV, applied from a holding potential of 80 mV,
with (right traces) or without (left traces) 10-s prepulses to 20 mV.
A, 1 mM ATP: K+ currents were activated with either of the
illustrated voltage protocols over 19 min. Left and middle traces show
currents at indicated times. IAC amplitudes recorded with
( ) and without ( ) depolarizing prepulses are plotted at right. B,
5 mM ATP: K+ currents were activated with either of the
voltage protocols over 17 min. Left and middle traces show currents at
indicated times. Shown at right are IAC amplitudes recorded
with () and without () depolarizing prepulses.
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When pipette solutions contained ATP at concentrations 
1 mM,
IAC was poorly expressed, as reported previously
(Enyeart et al., 1997), and voltage steps to +20 mV primarily activated
the rapidly inactivating bKv1.4 current (IA), the
amplitude of which remained nearly constant over many minutes of
recording (Fig. 1, left traces). In these experiments, isolation of
IAC with a depolarizing prepulse confirmed that
this K+ current was very small (<50 pA) and did
not increase during the course of the experiment (Fig. 1A, right traces
and graph). Overall, with pipettes containing 1 mM MgATP,
IA showed no measurable decrease in whole-cell
recordings lasting from 15 to 25 min (n = 6).
In recordings made with pipettes containing ATP at concentrations >1
mM, IAC typically increases dramatically over a
period of minutes, as reported (Enyeart et al., 1997; Xu and Enyeart, 2001). In the experiment illustrated in Fig. 1B,
IAC increased more than 20-fold to a maximum of
>2630 pA during 17 min of recording with a patch pipette containing 5 mM MgATP (middle trace). In whole-cell recordings,
IAC appears as a noninactivating current composed
of an instantaneous and a smaller, time-dependent component (Enyeart et
al., 1997).
Furthermore, in recordings such as those shown in Fig. 1B, left traces,
the combined IA plus IAC
K+ current recorded when
IAC had reached a maximum value was less than
predicted by the simple addition of the initial
IA current to the maximum
IAC current. Specifically, it appeared as if
IA amplitude had been reduced.
Digital subtraction of IAC currents from combined
IA plus IAC currents
demonstrated that the development of IAC current
is accompanied by a decrease in the amplitude of
IA current. In the experiment illustrated in Fig.
2A, combined currents
(IA + IAC) and isolated
IAC current are shown immediately after
initiating whole-cell recording (To) and then
approximately 20 min later, when IAC had reached
a stable maximum value (TMAX). During this interval, IAC grew from its initial value of 120 pA to a maximum amplitude of 2451 pA, while peak
IA current decreased from 2159 pA to 1081 pA
during the same interval. IA currents at
To and TMAX are
superimposed for comparison in Fig. 2, top left traces. The difference
between IA and IAC is shown
as 
IA (1078 pA) and represents the
quantity of IA current that was lost between
To and TMAX. In 27 experiments, IAC amplitude increased by an
average of 1208 ± 117 pA during whole-cell recordings, whereas
peak IA currents were reduced by 507 ± 58 pA (Fig. 2B). The magnitude of IAC increase was
positively correlated with IA decrease with a
correlation coefficient of 0.548 and a slope factor of 2.05 ± 0.20 (n = 27) (Fig. 2C).
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Fig. 2.
Reciprocal relationship between IA and
IAC K+ currents. Combined IA and
IAC or isolated IAC K+ currents
were recorded using the two voltage protocols shown in the legend of
Fig. 1. IA current traces were determined initially
(To) and after IAC had reached a stable maximum
amplitude (TMAX) by point-for-point digital subtraction of
IAC current traces from combined IA + IAC current traces. A, combined K+ currents
(IA + IAC) and isolated IAC
currents were recorded at To and TMAX.
IA traces were obtained as described above by digital
subtraction. B, IA was determined from digital
subtraction of IA (TMAX) from IA
(To). C, comparison of time-dependent increases in
IAC versus decreases in IA K+
current in AZF cells obtained from current measurements at
To and TMAX. Maximum decreases in
IA (IA) are plotted versus corresponding
increases in IAC (41 IAC) for each of 27 cells.
Linear regression analysis yielded a slope factor of 2.05 ± 0.20 and a correlation coefficient of 0.548. Changes in K+
currents IK) are plotted at right as mean ± S.E.M.
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Higher ATP concentration alone was insufficient to induce a
time-dependent rundown of IA. In some cells,
IAC K+ current fails to
grow dramatically even when pipettes contain ATP at concentrations
greater than 1 mM. IA current also showed little
time-dependent decrease in these recordings. In a total of seven cells
in which IAC reached a maximum of <50 pA with 2 or 5 mM MgATP in the pipette, peak IA current
decreased only 2.5 ± 1.7% during recordings lasting at least 15 min.
Time Dependence of Reciprocal Changes in K+-Current
Amplitudes.
If the development of IAC in
whole-cell recordings is coupled to a decrease in the number of
functional IA channels, then the temporal pattern
for these reciprocal changes should be similar. Figure
3 shows that the time-dependent
development of IAC was paralleled by a
corresponding decrease in IA. Over a 15-min
period, IAC grew gradually from an initial value
of 150 pA to a maximum amplitude of 1110 pA. Over this same time
interval, IA decreased monotonically from its
initial value of 2375 pA to a final value of 1848 pA. Once
IAC reached a stable maximum amplitude, no
further decrease in IA was observed. Similar
results were obtained in each of nine experiments.
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Fig. 3.
Temporal pattern for reciprocal changes in
IA and IAC K+ currents. The
time-dependent changes in IA and IAC currents
were monitored at seven time points during a 15-min recording. At each
time point, K+ currents were recorded in the absence
(IA + IAC) and presence (IAC) of
10-s depolarizing steps to 20 mV as described in the legend of Fig.
1. IA current at each time was determined by digital
subtraction of IAC from combined IA + IAC currents. Traces show measured and calculated currents
immediately after initiating recording and at t = 2 and 14.5 min.
Measured IAC and calculated IA currents are
plotted against time at bottom.
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Reciprocal Modulation of IAC by Other Nucleotides.
Other nucleotides, including the poorly hydrolyzable ATP analog
AMP-PNP, UTP, and GTP, each activate IAC when
present in the recording pipette at millimolar concentrations (Enyeart
et al., 1997; Xu and Enyeart, 2001). The time-dependent increases in
IAC amplitude observed with these nucleotides in
the pipette were also accompanied by a corresponding decrease in
IA current. In the experiment illustrated in Fig.
4, increases in IAC
current observed with AMP-PNP (1 mM) and GTP (5 mM) resulted in
reductions of IA of 599 pA and 362 pA,
respectively, at TMAX. Similar results were
obtained in each of eight experiments with pipettes containing AMP-PNP,
GTP, or UTP.
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Fig. 4.
Reciprocal effects of AMP-PNP and GTP on
IA and IAC currents. Patch pipettes containing
AMP-PNP (2 mM) or GTP (5 mM) were used to record combined
(IA + IAC) and isolated IAC
K+ currents at To and TMAX with the
two voltage protocols described in the legend of Fig. 1. IA
traces were obtained by digital subtraction of IAC current
traces from combined current traces.
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Selective Block of IAC Reveals Reduction of
IA.
Digital subtraction of IAC
from combined (IA+ IAC)
currents indicated that the growth of IAC in
whole-cell recordings was accompanied by a simultaneous decrease in
IA current. This point was demonstrated by a
second method using diphenylbutylpiperidine (DPBP) antipsychotics that
potently and selectively block IAC channels in
AZF cells. The DPBPs pimozide, penfluridol, and fluspirilene inhibit
IAC channels with IC50
values of 0.35, 0.19, and 0.23 µM, respectively, while 
200-fold
higher concentrations are required to inhibit IA
channels (Gomora and Enyeart, 1999).
In the experiment shown in Fig. 5,
IAC was allowed to grow to a maximum value before
superfusing the cell with pimozide (2.5 µM) to selectively inhibit
IAC. Digital subtraction of
IAC from combined K+
currents indicated that IA decreased by 509 pA,
or 26%, between To and
TMAX. Measurement of IA
after this cell was superfused with pimozide (2.5 µM) at a
concentration that inhibits IAC almost completely
and reduces IA by approximately 5% showed a
reduction in IA at TMAX by
an amount similar to that calculated by digital subtraction of
IAC from the combined current. The additional
small reduction of IA current from 1433 to 1322 pA would probably occur through a direct action of pimozide on
IA channels. In a total of nine cells, determined
from digital subtraction, IA at
TMAX was reduced to 63.3 ± 5.4% of its
original amplitude. By comparison, direct measurement of
IA in these same cells after preferential block
of IAC with DBPBs at 2.5 µM showed that
IA was reduced to 52.4 ± 4.3%
(n = 9) of its original value. Most of this difference can be attributed to a direct inhibition of a small fraction of IA channels by the DPBPs.
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Fig. 5.
Reciprocal relationship between IA and
IAC K+ channels revealed by pimozide. Combined
(IA + IAC) and isolated IAC
K+ currents were recorded using the two voltage protocols
described in the legend of Fig. 1 initially (To), when
IAC reached a maximum value (TMAX) and after
steady-state block of IAC K+ current by 2.5 µM pimozide. IA current traces were obtained by digital
subtraction of isolated IAC currents from combined
K+ currents.
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Inhibition of IAC by Corticotropin is Coincident with
Recovery of IA.
Although IAC is
activated in the presence of intracellular nucleotides, this current is
potently inhibited (IC50 = 5.4 pM) by
corticotropin (Mlinar et al., 1993). Interestingly, the inhibition of
IAC by corticotropin appeared to differ
significantly from that observed with the DPBPs. Specifically, the
corticotropin-mediated inhibition of IAC was
accompanied by recovery of IA current that was
lost during the development of IAC.
In the experiment illustrated in Fig. 6A,
IAC grew to a maximum value of nearly 1600 pA
after 16 min of whole-cell recording (TMAX).
During this same interval, IA decreased by 766 pA
to 70% of its initial value. Superfusion of this cell with
corticotropin (200 pM) inhibited IAC almost
completely, and IA amplitude increased by 346 pA
to 84% of its initial value. Similar results were obtained in each of
10 experiments, where inhibition of IAC by
corticotropin was associated with an increase in
IA from 71 ± 4% to 89 ± 4% of its
original value (Table 1).
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Fig. 6.
Corticotropin-stimulated inhibition of
IAC and recovery of IA K+ current.
Combined (IA + IAC) and isolated
IAC K+ currents were recorded using the two
voltage protocols described in the legend of Fig. 1 with pipettes
containing 5 mM (A) or 1 mM ATP (B). Traces show combined and isolated
currents recorded immediately after initiating recording
(To), after IAC reached a maximum value
(TMAX), and after steady-state inhibition of
IAC by corticotropin. IA current traces were
obtained by digital subtraction of isolated IAC currents
from combined K+ currents.
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TABLE 1
Effect of DPBPs and corticotropin on IA K+ current
IA K+ current amplitudes are expressed as a percentage
of its initial value (To), after IAC had reached a
maximum value (TMAX) and after superfusion of a DPBP or
corticotropin (Treatment). Values are expressed as mean ± S.E.M.
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The corticotropin-stimulated increase in IA
current seemed to be tightly linked to the inhibition of
IAC current. When whole-cell recordings were made
with pipette solutions containing low ATP ( 1 mM) to retard the
development of IAC current, subsequent superfusion of corticotropin did not significantly increase the amplitude of IA K+ current
in any of the eight cells tested (Fig. 6B).
Reciprocal Changes in IA K+ Current are
Independent of Voltage.
The decrease in IA
current amplitude associated with development of
IAC and the corticotropin-stimulated increase in
IA current associated with
IAC inhibition were found to be present over a wide range of test potentials. In the experiments illustrated in Fig.
7A, current-voltage relationships were
first obtained in control saline immediately after initiating
whole-cell recording (left traces). After IAC had
grown to a stable maximum value of at least 500 pA, cells were
superfused with corticotropin (200 pM) or penfluridol (2.5 µM),
producing nearly complete inhibition of IAC
(middle traces), and current-voltage relationships were again
recorded (right traces).
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Fig. 7.
Reciprocal relationship between IA and
IAC K+ currents is voltage-independent. The
current-voltage (I-V) relationship was obtained immediately after
initiating whole-cell recording by applying voltage steps to test
potentials between 30 and +50 mV. IAC was then allowed to
reach a maximum amplitude before superfusing the cell with penfluridol
(2.5 µM) or corticotropin (200 pM). I-V relationships were again
obtained after steady-state block was reached. A, traces show initial
I-versus (left), combined IA + IAC currents
before (1) and after (2) superfusion of corticotropin or penfluridol as
indicated (middle traces), and I-V after steady-state block by
corticotropin or penfluridol (right traces). B, I-V plots: maximum peak
currents are plotted against test potential before and after
superfusion of penfluridol (left) or corticotropin (right). Results are
mean ± S.E.M. for four separate determinations.
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Penfluridol inhibits IAC and
IA with respective IC50
values of 187 nM and 42 µM (Gomora and Enyeart, 1999). At a
concentration of 2.5 µM, penfluridol (2.5 µM) produces nearly
complete inhibition of IAC and reduces
IA by approximately 5%. Current-voltage
relationships obtained after block of IAC
by penfluridol showed that IA had decreased by 39 to 45% at potentials between 10 and +50 mV, compared with respective
control values (n = 4) (Fig. 7). Besides demonstrating that the reduction of IA current coincident with
IAC development is not voltage dependent, these
results also indicate that it is not an artifact attributable to a
spontaneous rightward shift in the voltage dependence of
IA activation. Accordingly, when the
IA current values shown in Fig. 7B were
normalized against the maximum, both before and after penfluridol, and
then plotted on the same graph, these values were nearly identical,
indicating no shift in voltage dependence (data not shown).
In contrast to the voltage-independent reduction of
IA current associated with
IAC development that was unmasked by selective inhibition of IAC current with penfluridol, the
nearly complete inhibition of IAC current with
corticotropin (200 pM) was not accompanied by a decrease in
IA current over the entire range of test
voltages. In four experiments, inhibition of IAC
current with 200 pM corticotropin produced IA
currents that did not differ significantly from control currents over
the entire range of test potentials. This result demonstrates that
IA current lost during the development of
IAC is restored over a range of test potentials when corticotropin inhibits IAC.
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Discussion |
In this study, we discovered that the activity of voltage-gated
rapidly inactivating bKv1.4 A-type K+ channels
and noninactivating "background" K+ channels
in AZF cells were reciprocally modulated through intracellular nucleotides and G-protein-coupled corticotropin receptors.
Specifically, in whole-cell recordings, the nucleotide-dependent
increase of IAC K+ channel
activity was accompanied by a coincident decrease in the number of
functional IA channels. Conversely, the nearly
complete inhibition of IAC current by
corticotropin was associated with the reappearance of functional
IA channels. Regardless of the mechanism that may
couple these two K+ channels, this is the first
report demonstrating the modulation of IA
activity by nucleotides and corticotropin.
Model for IA-IAC Coupling.
Overall, the results of the current study in combination with our
previous work on IAC suggest a novel form of
channel modulation in which the activity of IA
and IAC K+ channels is
reciprocally coupled in a dynamic equilibrium. In this model, shown in
Fig. 8, the binding of ATP or other nucleotides to the
IAC channel, or associated protein, increases the
number of active IAC channels and reduces the
number of functional IA channels. In contrast,
the activation of corticotropin receptors shifts the equilibrium in the
reverse direction, reducing the number of active
IAC channels and increasing the pool of
IA channels.
Considerable evidence supporting the model for tight reciprocal
coupling between the two different K+ channels
was presented. The nucleotide-induced increase in
IAC channel activity was consistently paralleled
in time by a decrease in IA current. Conversely,
in the absence of IAC growth,
IA amplitude remained nearly constant over many
minutes. Furthermore, the inhibition of IAC
current by corticotropin was always accompanied by the recovery of
IA current in whole-cell recordings. In the
absence of IAC current, corticotropin has no
effect on IA K+ current.
Despite these findings, questions remain regarding the nature of the
relationship that links these two K+ channels.
Molecular Basis of K+-Channel Coupling.
Our
findings, as depicted in Fig. 8, suggest
that IA and IAC
K+ channels are physically linked by signaling
pathways involving nucleotide binding and the G-protein-coupled
corticotropin receptor. In previous studies, we showed that
IAC channels' activity was enhanced by
hydrolyzable and nonhydrolyzable nucleotides, as well as
polytriphosphates (Enyeart et al., 1997; Xu and Enyeart, 2001). Furthermore, inhibition of IAC channels by
corticotropin was independent of A-kinase but required hydrolyzable
forms of ATP (Enyeart et al., 1996). Taken together, these results
suggest a model for IAC gating that involves an
ATP hydrolysis cycle: channel activity is enhanced by the binding of
ATP and inhibited through corticotropin-stimulated ATP hydrolysis.
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Fig. 8.
Model for reciprocal modulation of IAC by
nucleotides and corticotropin. Schematic depicts the reciprocal
coupling of IA and IAC K+ channels
controlled by nucleotides and corticotropin. Nucleotides, including
ATP, AMP-PNP, and GTP increase the number of active IAC
channels and simultaneously decrease the number of active
IA channels. Conversely, corticotropin reduces the number
of functional IAC channels and increases the pool of
available IA channels.
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In the present study, we found that in addition to enhancing
IAC channel activity, hydrolyzable and
nonhydrolyzable nucleotides also promoted the "rundown" of
IA K+ currents.
Furthermore, corticotropin-mediated inhibition of
IAC was accompanied by an increase in
IA. Taken together, these results suggest that
the reciprocal, coupled gating of both of these
K+ channels could be mediated through a cycle of
ATP binding and hydrolysis involving G-protein-coupled receptors.
The molecular basis for functional coupling between
IA and IAC
K+ channels is unknown. Perhaps these two
K+ channels exist within a protein complex in the
plasma membrane in close association with corticotropin receptors. The
binding and hydrolysis of ATP might be linked to the shuttling of a
common, shared subunit, leading to the activation of one
K+ channel and the coincident inactivation of the
coupled channel.
In this regard, auxiliary subunits that could modulate the function of
IA and IAC
K+ channels are yet to be identified. The
molecular identity of the primary ± subunit of
IAC channels is also unknown. Unlike other
ATP-gated K+ channels, IAC
channels are insensitive to sulfonylureas (Gomora and Enyeart, 1999).
Thus, it is unlikely that these channels include a sulfonylurea
receptor as the 
subunit. No subunit common to voltage-gated and
background K+ channels has yet been identified.
In this regard, most of the background K+
channels that set the resting membrane potential in mammalian cells
belong to a large family of two-pore, four-membrane-spanning channels
for which no auxiliary subunits have been described (Goldstein et al.,
1998).
Stoichiometry of K+-Channel Coupling.
If
IA and IAC
K+ channels are in close proximity within
functional complexes of the AZF cell membrane, the channel number and stoichiometric ratio will be an important consideration. In this regard, the average increase in macroscopic IAC
current was 2.38 times larger than the corresponding mean decrease in
IA K+ current in the same
experiments. This data might suggest that the stoichiometry involved in
IA-IAC channel coupling
could be calculated by comparing the observed changes in the two
macroscopic currents to the relative unitary conductances measured
under similar conditions. For example, if the two
K+ channels are functionally coupled in a
one-to-one stoichiometry, the ratio of the measured changes in the
macroscopic K+ currents might be equal to the
ratio of unitary current amplitudes for IA and
IAC channels.
However, macroscopic currents are a product of
NPoî, where N is the number of
functioning channels, Po is the channel open probability, and î is the unitary current. Therefore, even if the activity of IA and IAC
channels were tightly coupled in a 1:1 reciprocal relationship, this
would not be evident from macroscopic recordings unless
Po was identical for the two channels. Because Po is generally quite variable, it is unlikely
that averaged Po values for
IA and IAC channels would
be equal in a single cell. Accordingly, although they were positively
correlated, considerable variability was present in the ratios of
measured IAC increases to
IA decreases measured from cell to cell.
Nevertheless, the average ratio of 2.38 for IAC
increase compared with IA decrease is consistent
with the fact that the unitary IAC current
amplitude is severalfold larger than unitary IA
currents measured under similar conditions (Latorre and Miller, 1983;
Xu and Enyeart, 2001).
Although corticotropin (200 pM) inhibited IAC
almost completely, the corresponding recovery of
IA was often less efficient. Again, this could be
caused by time-dependent decreases in Po for
IA channels, as a result of rightward shifts in
the voltage dependence of activation that can occur with cell dialysis.
Alternatively, it is possible that the inhibition of an
IAC channel by corticotropin is not absolutely
tied to the activation of a coupled IA channel.
Functional Significance.
In addition to corticotropin
receptors, bovine AZF cells express several other receptors, the
activation of which is coupled to IAC inhibition
and membrane depolarization (Mlinar et al., 1993; Mlinar et al., 1995;
Xu and Enyeart, 1999a,b). It will be interesting to determine
whether the inhibition of IAC by angiotensin II, external ATP, and adenosine is also linked to an increase in
the number of available IA channels.
Regardless, the results of this study describe a novel form of channel
modulation where the activity of voltage-gated and metabolically
regulated background K+ channels are reciprocally
controlled in tandem by intracellular ATP and a G-protein-coupled
peptide hormone receptor. How this unique form of ion channel
modulation functions in the physiology of cortisol secretion has not
been determined.
In a previous study, we showed that corticotropin-stimulated increases
in cortisol secretion from bovine AZF cells require Ca2+ entry through low-voltage activated T-type
Ca2+ channels (Enyeart et al., 1993). Perhaps,
the opposing action of corticotropin on IA and
IAC K+ channels promotes
electrical activity such as an oscillating membrane potential that
maximizes Ca2+ entry through the rapidly
inactivating T-type channels.
It is not known whether K+ channels in other
types of cells might be functionally linked as they are in AZF cells.
In cell-attached patch recordings from dendrites of rat hippocampal
neurons, arachidonic acid was found to reduce the amplitude of a
transient K+ current and increase that of a
sustained K+ current (Colbert and Pan, 1999).
Perhaps the activity of these two K+ channels
could also be linked in a reciprocal relationship. Coupled modulation
of voltage- and non-voltage-gated K+ channels
could represent a new form of modulation operating in a wide range of cells.
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases-National Institutes of Health Grant
DK47875 (to J.J.E.).
Dr. John J. Enyeart, Department
of Neuroscience, The Ohio State University College of Medicine, 5190 Graves Hall, 333 W. 10th Avenue, Columbus, OH 43210-1239. E-mail:
enyeart.1{at}osu.edu
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Abstract
Introduction
