Department of Neuroscience, Ohio State University, College of
Medicine, Columbus, Ohio 43210-1239
Bovine adrenocortical zona fasciculata (AZF)
cells express a novel ATP-dependent K+-permeable channel
(IAC). Whole cell and single-channel recordings were used to characterize IAC channels with
respect to ionic selectivity, conductance, and modulation by
nucleotides, inorganic phosphates, and angiotensin II (ANG II). In
outside-out patch recordings, the activity of unitary
IAC channels is enhanced by ATP in the patch
pipette. These channels were K+ selective with no
measurable Na+ or Ca2+ conductance. In
symmetrical K+ solutions with physiological concentrations
of divalent cations (M2+), IAC
channels were outwardly rectifying with outward and inward chord
conductances of 94.5 and 27.0 pS, respectively. In the absence of
M2+, conductance was nearly ohmic. Hydrolysis-resistant
nucleotides including AMP-PNP and NaUTP were more potent than MgATP as
activators of whole cell IAC currents. Inorganic
polytriphosphate (PPPi) dramatically enhanced
IAC activity. In current-clamp recordings, nucleotides and PPPi produced resting potentials in AZF
cells that correlated with their effectiveness in activating
IAC. ANG II (10 nM) inhibited whole cell
IAC currents when patch pipettes contained 5 mM
MgATP but was ineffective in the presence of 5 mM NaUTP and 1 mM MgATP.
Inhibition by ANG II was not reduced by selective kinase antagonists.
These results demonstrate that IAC is a
distinctive K+-selective channel whose activity is
increased by nucleotide triphosphates and PPPi.
Furthermore, they suggest a model for IAC gating
that is controlled through a cycle of ATP binding and hydrolysis.
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INTRODUCTION |
BOVINE ADRENAL ZONA
FASCICULATA (AZF) cells express a novel K+-permeable
channel (IAC) that functions pivotally in the
regulation of cortisol secretion. IAC channels
may set the resting potential of AZF cells, while inhibition of these
channels by ACTH and angiotensin II (ANG II) is coupled to membrane
depolarization, Ca2+ entry, and cortisol secretion
(15, 39, 40).
Whole cell patch-clamp studies have shown that
IAC combines properties unique among ionic
currents described thus far. Specifically, IAC
is activated when the patch electrode contains ATP at millimolar concentrations (14), while it is inhibited by
cAMP through an A-kinase-independent mechanism
(16). IAC is also inhibited by antagonists of both cyclic nucleotide-gated (CNG) cation channels and
K+ channel blockers (21).
Inhibition of IAC through multiple G
protein-coupled receptors, including those activated by ACTH, ANG II,
and multiple P1 and P2 nucleotide receptors,
requires ATP hydrolysis (16, 39, 40, 47, 48). For at least
one of these receptors and its associated second messenger (ACTH and
cAMP), inhibition of IAC is independent of any
known protein kinases (16).
Together, these results identify IAC as a novel
channel combining properties of K+-selective and CNG cation
channels. The overall sequence similarity between voltage-gated
K+ channels and CNG cation channels suggests a common
origin (26). Several such intermediate forms have been
identified, including the ether-à-go-go
(eag) family of K+ channels, which are modulated
by cAMP. Some eag channels may also display Ca2+
permeability (11, 24).
A large family of K+ channels directly gated by ATP exists.
However, these inwardly rectifying K+ channels are
uniformly inhibited by ATP (5). IAC
channels are the first K+-permeable channel whose activity
depends on the presence of ATP in either hydrolyzable or
nonhydrolyzable forms. In this regard, our results are consistent with
a model in which IAC channel gating is coupled
to a cycle of ATP binding and hydrolysis with similarities to that
proposed for the cystic fibrosis transmembrane conductance regulator
(CFTR) Cl
channel (7). In the proposed
scheme, IAC channel open probability is enhanced
upon ATP binding, while activation of G protein-coupled receptors
promotes channel closing subsequent to ATP hydrolysis.
In the present study, single-channel recording from outside-out patches
was used to characterize these novel K+-permeable channels
with respect to ionic selectivity, conductance, and rectification. In
whole cell recordings, the modulation of IAC by
nucleotides, inorganic phosphates, and ANG II was studied to determine
whether IAC gating might be controlled through
an ATP hydrolysis cycle.
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MATERIALS AND METHODS |
Tissue culture media, antibiotics, fibronectin, and fetal bovine
serum were obtained from GIBCO (Grand Island, NY). Coverslips were from
Bellco Glass (Vineland, NJ). Enzymes, ANG II, MgATP, NaATP, NaUTP,
NaCTP, KATP, 5-adenylylimidodiphosphate (AMP-PNP, lithium salt),
guanosine 5'-O-(2-thiodiphosphate) (GDP
S),
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
(BAPTA), the inorganic phosphates polytriphosphate (PPPi), pyrophosphate (PPi), Pi, and
phosphatidylinositol 4,5-bisphosphate (PIP2), and
staurosporine were obtained from Sigma Chemical (St. Louis, MO).
Penfluridol was purchased from Jansen Pharmaceuticals (Beerse,
Belgium). Calphostin C, AG-490, genistein, herbimycin, and PD-98059
were purchased from Calbiochem (San Diego, CA).
Isolation and culture of AZF cells.
Bovine adrenal glands were obtained from steers (age range: 1-3
yr) within 60 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 (16). 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 cells were added. DMEM/F-12 medium supplemented with 10% fetal
bovine serum, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and the
antioxidants 1 µM tocopherol, 20 nM selenite, and 100 µM ascorbic
acid was used. 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 and outside-out patch configuration. For whole cell recordings, the standard pipette solution consisted of 120 mM KCl, 2 mM
MgCl2, 1 mM CaCl2, 20 mM HEPES, 11 mM BAPTA,
200 µM GTP, and 5 mM MgATP, with pH buffered to 7.2 using KOH.
Titration of pH to 7.2 with KOH raised the total K+
concentration to 160 mM. Pipette solution of this composition yielded a
free Ca2+ concentration of 2.2 × 108 M,
as determined by the Bound and Determined software program (10). In many experiments, MgATP was replaced with other
nucleotides or an inorganic phosphate, as described in the text. The
external solution consisted of 140 mM NaCl, 5 mM KCl, 2 mM
CaCl2, 2 mM MgCl2, 10 mM HEPES, and 5 mM
glucose, with pH adjusted to 7.4 using NaOH.
The standard external and pipette solutions used for single-channel
recording from outside-out patches were identical to those used for
whole cell recordings. A number of other external or pipette solutions
were also used and are described in the text. All solutions were
filtered through 0.22-µm cellulose acetate filters.
AZF cells were used for patch-clamp experiments 2-12 h after they
were plated. Typically, cells with diameters of <15 µm and capacitances of 8-12 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 (World
Precision Instruments, Sarasota, FL). These electrodes 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) according to the procedure of Hamill et al.
(25) with the use of an Axopatch 1-D patch-clamp amplifier.
Pulse generation and data acquisition were performed with the use of a
personal computer and pCLAMP software with a TL-1 interface (Axon
Instruments, Burlingame, CA). Currents were digitized at 5-20 kHz
after filtering with an eight-pole Bessel filter (Frequency Devices,
Haverhill, MA). Linear leak and capacity currents were subtracted from
current records by using scaled hyperpolarizing steps of 1:3-1:4
amplitude. Data were analyzed and plotted with pCLAMP 5.5 and 6.02 (Clampan, Clampfit, Fetchan, and pSTAT) and SigmaPlot 4.0. Drugs were
applied by bath perfusion, controlled manually by a six-way rotary valve.
Calculation of IAC channel activity.
Because of uncertainty about the number of channels in any given patch
(N) and the nonstationary character of
IAC activity in outside-out patches, channel
activity was expressed in terms of NPo rather
than Po (open probability).
NPo was calculated from the expression
I = NPoî,
where I is the measured mean current, N is the
number of active channels in the patch, î is the
single-channel current, and Po is the open
probability for samples of 90-100 consecutive traces, each 400 ms
in duration.
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RESULTS |
ATP-dependent activity of unitary IAC current in
outside-out patches.
Previously, in whole cell recordings from AZF cells, we found that
IAC K+ current is present initially
but grows 10-fold or more to a stable amplitude over a period of
minutes, provided that ATP is present in the recording pipette at
millimolar concentrations (14, 39). In excised outside-out
patch recordings, single IAC channels showed a
similar time- and ATP-dependent increase in channel open probability.
In Fig. 1, unitary currents were recorded
with a pipette containing 2 mM MgATP in response to voltage steps
applied at 4-s intervals from a holding potential of 
40 mV to a test
potential of +30 mV. Under these conditions, a single type of unitary
current was present with a measured mean amplitude of 3.95 ± 0.34 pA. In this experiment, IAC channel activity
(NPo) increased 5.5-fold from an initial value
of 0.17 at time 0 to a value of 0.95 after 7 min of
recording. At this time, it is clear that the membrane patch contains
at least two functioning channels. In contrast to whole cell
recordings, where IAC reaches a stable maximum
amplitude, unitary IAC channel activity
typically increased continuously for the life of the patch.
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Fig. 1.
Time-dependent expression of unitary
K+-permeable channel (IAC) currents
in outside-out patches. Unitary IAC currents
were recorded from outside-out patches with standard external and
pipette solutions supplemented with 2 mM MgATP. Voltage steps of 400-ms
duration were applied at 4-s intervals from a holding potential of 40
mV to a test potential of +30 mV. Records show currents at the times
(t) indicated at which outside-out patch recording was
initiated. Currents were filtered at a cut-off frequency of 1.5 kHz and
sampled at 5 kHz. Channel activity (NPo) was
calculated as described in MATERIALS AND METHODS.
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When outside-out patch recordings were made with pipettes containing
MgATP at concentrations below 1 mM, IAC channel
activity was markedly reduced and NPo failed to
increase even during prolonged recordings. In the experiment shown in
Fig. 2A,
IAC activity in an outside-out patch was
recorded with a pipette containing 0.1 mM MgATP. Analysis of an
amplitude histogram constructed from channel openings indicated the
presence of a single type of channel with a mean amplitude of 3.82 ± 0.28 pA. NPo did not increase from its
initial value of 0.01 during 20 min of recording. Similar results were
obtained from eight separate patches under these conditions.
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Fig. 2.
Activation of unitary IAC currents
by MgATP. Unitary IAC currents were recorded in
the outside-out configuration by using pipettes containing standard
solution supplemented with either 0.1 (A) or 5 mM MgATP
(B). Voltage steps to +30 mV were applied at 4-s intervals
from a holding potential of 40 mV. Top: records show
currents at the times indicated (see time scale) after patch recording
was initiated. Bottom: amplitude histograms were constructed
from idealized channel openings obtained from unitary currents recorded
in response to 96 consecutive voltage steps of 400-ms duration. Unitary
current amplitudes were distributed into bins of 0.15 pA in width.
Continuous lines in the histograms are fits of Gaussian distributions
to the data. NPo was calculated as described in
MATERIALS AND METHODS. Currents were filtered at a cut-off
frequency of 1.5 kHz and sampled at 5 kHz.
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By comparison, when recordings were made with pipettes containing 5 mM
MgATP, NPo increased dramatically during the
course of an experiment. In the experiment shown in Fig. 2B,
amplitude histograms constructed from idealized channel openings showed the presence of three nearly equally spaced peaks indicative of at
least three active channels in the patch. Gaussian fits of these
histograms yielded means of 3.96 ± 0.34, 7.87 ± 0.32, and 11.95 ± 0.42 pA. NPo increased
continuously during this experiment and was estimated to be 0.83 after
6 min of recording from this outside-out patch.
In other experiments, we attempted to study the direct activation of
IAC channels in inside-out patches upon
superfusion of the cytoplasmic membrane surface with internal solution
containing MgATP. Surprisingly, in this configuration, 5 mM MgATP
failed to activate IAC channels in any of nine
cells tested.
K+ selectivity and conductance of
IAC channels.
In whole cell recordings, IAC appears as a
noninactivating, weakly voltage-dependent, ATP-activated K+
current (14, 16, 39). Properties of unitary
IAC current, including ionic selectivity,
conductance, and rectification, in symmetrical K+ solutions
have not been described.
To obtain a measure of the K+ selectivity and conductance
of IAC channels, unitary
IAC currents were recorded from outside-out patches at voltages ranging from 90 to +60 mV by using external solutions containing either Na+(160 mM) or K+
(160 mM) and standard pipette solution supplemented with 5 mM MgATP.
External and pipette solutions also contained divalent cations
(M2+) as in standard solutions. With
Na+-containing external solution,
IAC channels were outwardly rectifying. No
inward Na+ current through IAC
channels was detectable at potentials as negative as 90 mV (Fig.
3, A, left, and
B).
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Fig. 3.
Single-channel current-voltage (I-V) relationships and
ionic selectivity of IAC channels.
I-V relationships for unitary IAC
currents were obtained in external solutions containing 160 mM
Na+ or 160 mM K+ as indicated. Voltage steps of
400-ms duration were applied at 0.1 Hz to test potentials between 90
and +60 mV in 10-mV increments from holding potentials of 40 or 0 mV.
Standard pipette solution (160 mM K+) was supplemented with
5 mM MgATP. A: traces show unitary currents at indicated
voltages in either Na+(Na/K)- or K+-containing
external solution (K/K). B: mean unitary current amplitudes
were plotted against voltage for currents recorded in Na+-
or K+-containing external solutions. Values are mean
amplitudes measured from 3 or 4 cells. Currents were filtered at a
cut-off frequency of 1.5 kHz and sampled at 5 kHz.
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When the external Na+-containing solution was replaced with
K+ solution, IAC channels were still
outwardly rectifying but reversed near 0 mV and became clearly inward
at negative potentials (Fig. 3, A, right, and
B). Although no systematic study of voltage-dependent open
probability was done, IAC channels remained
active at potentials as negative as 80 mV. In these symmetrical
K+ solutions, at test potentials between 0 and +60 mV,
IAC channels had a measured chord conductance of
78.2 pS. By comparison, at potentials between 0 and 60 mV, chord
conductance was 30.1 pS (Fig. 3B).
These results show that, in the presence of physiological
concentrations of M2+, IAC channels
are highly selective for K+ with negligible conductance to
Na+. Furthermore, in symmetrical K+ solutions
containing approximately physiological concentrations of
M2+, IAC channels are outwardly
rectifying with a 2.5-fold ratio of outward relative to inward
K+ conductance.
Effect of M2+ on unitary conductance
and rectification of IAC channels.
The rectifying properties of some K+-selective channels are
caused by the unidirectional block of K+ flux by
M2+. In particular, the rectification of many inward
rectifier K+ channels occurs because intracellular
Mg2+ blocks the outward flow of K+
(37).
In outside-out patch recordings made in symmetrical K+
solutions, it was discovered that the presence of M2+ on
either side of the membrane dramatically altered unidirectional K+ flow through these channels. Specifically, with standard
external and pipette solutions containing Ca2+ and
Mg2+ at approximately physiological concentrations
(external: 2 mM Ca2+, 2 mM Mg2+;
pipette: 22 nM Ca2+, 2 mM Mg2+),
IAC channels were outwardly rectifying in
symmetrical K+ with chord conductance of 94.5 pS measured
between 0 and +80 mV, compared with 27 pS between 0 and 80 mV (Fig.
4, A and E).
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Fig. 4.
Effect of divalent cations on rectifying properties of
IAC K+ channels. Unitary
IAC channel activity was recorded from
outside-out patches in symmetrical 160 mM K+ solutions with
divalent cations (M2+) present on both sides of the
membrane (+/+; A), in the external solution only (/+;
B), in the pipette only (+/; C), or with
M2+ eliminated from both solutions (/; D).
Single-channel I-V relationships were obtained by applying
voltage steps of 400-ms duration at 10-s intervals to test potentials
between 80 and +80 mV. Traces show unitary currents recorded with
pipette solutions supplemented with 5 mM KATP. Currents were filtered
at a cut-off frequency of 1.5 kHz and sampled at 5 kHz. C, closed
state; O, open state. E: effect of M2+ on
single-channel I-V relationship. Single-channel
I-V curves were obtained in the presence (+/+) and absence
(/) of M2+ in external and pipette solutions as
described in A and D. Mean unitary current
amplitudes measured from 3 separate cells were plotted against voltage
for currents recorded in the absence or presence of M2+.
Currents were filtered at 1.5 kHz and sampled at 5 kHz.
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Deletion of M2+ from the external solution markedly
increased the amplitude of inward unitary currents amplitudes measured
at negative potentials but only slightly increased the amplitude of
outward currents measured at positive voltages (Fig. 4B). In the absence of external M2+, the chord conductance between
0 and +80 mV increased only to 98.5 pS, while the chord conductance
measured between 0 and 80 mV was 154.8 pS, a 5.7-fold increase over
that measured in the presence of external Ca2+ and
Mg2+.
Selective removal of internal Mg2+ in the presence of
external M2+ enhanced the amplitude of unitary outward
currents, while inward currents measured at negative potentials were
similar to those recorded with M2+ present on both sides
of the membrane (Fig. 4C). Specifically, the chord
conductance measured between 0 and +80 mV was 143.3 pS, a value 52%
larger than that observed with Mg2+ in the pipette
solution. In contrast, chord conductance between 0 and 80 mV was 26.9 pS, a value nearly identical to that measured with M2+
present on both sides of the membrane.
The results of experiments in which M2+ were selectively
deleted from either the external or pipette solutions indicate that the
rectifying properties of the IAC channel are
caused by bidirectional inhibition of K+ flux by
M2+. When present on the external side of the membrane,
M2+ reduce the influx of K+, and
IAC appears as an outward rectifier. When
M2+ are deleted from the external solution but present
intracellularly, IAC channels become inwardly rectifying.
When M2+ were omitted from the external solution and
pipette solutions contained no added Mg2+ and nominal
Ca2+ (22 nM), the IAC channels
became nearly ohmic. Under these conditions, the chord conductances
measured between 0 and +80 mV and between 0 and 80 were 161 and 175 pS, respectively (Fig. 4, D and E). Although
inward K+ conductance increased dramatically in the absence
of M2+, inward Na+ current remained
undetectable (data not shown).
The inhibition of K+ flux through
IAC channels by M2+ suggests an
interaction in the permeation pathway. CNG cation channels and at least
one K+ channel are permeable to Ca2+ (11,
28). To determine whether IAC channels
display measurable Ca2+ conductance, we measured unitary
IAC currents in the presence of external
solutions containing 107 mM Ca2+ in the absence of
Na+ or K+ at potentials as negative as 100
mV. No inward Ca2+ current through
IAC channels was detected in any of three tested cells (data not shown).
Activation of IAC by poorly hydrolyzable
nucleotides.
Two types of K+ channels expressed by bovine AZF cells are
easily distinguished in whole cell recordings. In addition to the rapidly inactivating A-type K+ current
(IA), the noninactivating
IAC current grows continuously over a period of
minutes in whole cell recordings (14, 16, 39, 41). The
absence of time-dependent inactivation of IAC allows it to be isolated for measurement with the use of either of two
voltage-clamp protocols. When voltage steps of 300-ms duration are
applied from a holding potential of 80 mV to a test potential of +20
mV, IAC can be selectively measured near the end
of a step, at a point where IA has completely
inactivated (Fig. 5A,
left). With the second protocol, IAC
can be selectively activated by an identical voltage step after a 10-s
prepulse to 20 mV has fully inactivated IA
(Fig. 5A, middle).
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Fig. 5.
Activation of whole cell IAC currents by
NaUTP and MgATP. Time-dependent expression of
IAC K+ current was monitored in
whole cell recordings by using patch pipettes containing standard
solutions supplemented with MgATP or NaUTP at various concentrations
between 0.1 and 5 mM. A: K+ currents were
activated at 30-s intervals from a holding potential of 80 mV by
voltage steps to +30 mV. K+ current traces were recorded at
times indicated by numbers on graphs with (middle) and
without inactivating prepulses (left).
IAC current amplitudes with ( )
and without prepulses ( ) are plotted against time
(right). B: effect of MgATP and NaUTP on
IAC current density. Maximum
IAC current density
(IAC max) was obtained by dividing the
maximum IAC current amplitude obtained in
experiments such as those shown in A by the cell capacitance
determined from transient cancellation controls of patch-clamp
amplifier. Values are means ± SE of the number of determinations,
indicated in parentheses.
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Previous studies have led to the hypothesis that
IAC activity is linked to a cycle of ATP-binding
and hydrolysis, wherein IAC open probability
increases with ATP binding while its hydrolysis or dissociation leads
to channel closing (14). If this were the case, then other
poorly hydrolyzable nucleotides might prove to be more potent and/or
effective activators of IAC than ATP itself.
In whole cell patch-clamp recordings, we compared NaUTP with MgATP with
respect to potency and effectiveness as activators of
IAC K+ current. At concentrations
between 0.1 and 5 mM, NaUTP was significantly more potent than MgATP at
enhancing IAC expression (Fig. 5). In the
experiment shown in Fig. 5A, the time-dependent increase in IAC amplitude was monitored by using patch
pipettes containing either 1 mM MgATP or 1 mM NaUTP. With 1 mM MgATP in
the recording pipette, IAC increased <2-fold
from its initial value (trace 1) to a stable maximum
(trace 2) during 14 min of recording (Fig. 5A).
By comparison, with 1 mM NaUTP in the recording pipette, IAC increased >12-fold, reaching a stable
maximum amplitude within 10 min. Overall, with 1 mM NaUTP applied
intracellularly through the pipette, IAC reached
a maximum current density of 50.6 ± 5.5 pA/pF (n = 10), a value nearly fourfold greater than that observed with 1 mM
MgATP in the pipette (13.0 ± 1.0 pA/pF, n = 24)
(Fig. 5B).
At concentrations of 0.1 and 0.4 mM, NaUTP was also significantly more
effective than 1 mM MgATP at activating IAC
(Fig. 5B). Activation of IAC by NaUTP
reached a maximum at concentrations between 2 and 5 mM. At these higher
concentrations, NaUTP was only slightly more effective than 5 mM MgATP
at enhancing IAC activity (Fig. 5B).
In addition to NaUTP, other nucleotides, including the nonhydrolyzable
ATP analog AMP-PNP and the pyrimidines NaTTP and NaCTP, were more
potent activators of IAC than MgATP. With
AMP-PNP, NaTTP, or NaCTP present in the pipette solution at a
concentration of 1 mM, IAC reached maximum
current densities that were 2.5- to 3.5-fold greater than that observed
with 1 mM MgATP (Fig. 6, A and
B).
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Fig. 6.
Activation of IAC currents by
nonhydrolyzable nucleotides. Time-dependent expression of
IAC K+ current was monitored in
whole cell recordings using patch pipettes containing standard solution
supplemented with either MgATP, AMP-PNP, NaTTP, or NaCTP, each at a
concentration of 1 mM. A: K+ currents were
activated at 30-s intervals from a holding potential of 80 mV by
voltage steps to +30 mV. K+ currents were recorded
immediately after whole cell recording was initiated (trace
1) and after IAC had reached a maximum
value (trace 2) with (bottom traces) and without
inactivating prepulses (top traces). B: effect of
nucleotides on IAC current density.
IAC max was obtained by dividing the maximum
IAC current amplitude obtained in experiments
such as those shown in A by the cell capacitance. Values are
means ± SE of the number of determinations, indicated in
parentheses.
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Activation of IAC by inorganic phosphates.
Inorganic phosphates inhibit ATPases by occupying sites on the enzyme
from which phosphate is released after ATP is cleaved, interrupting
further cycles of ATP hydrolysis (7, 22). Inorganic phosphates such as PPi and PPPi may promote the
opening of CFTR Cl channels via this mechanism (7,
22, 23).
The effect of inorganic phosphates, including Pi,
PPi, and PPPi, on IAC
expression was studied in whole cell recordings. Of the three
phosphates tested, PPPi was clearly most effective at enhancing IAC expression. In the experiment
shown in Fig. 7A, K+ currents were recorded with a pipette containing
standard pipette solution supplemented with 5 mM PPPi. In
the presence of PPPi, a noninactivating current resembling
IAC grew continuously over a period of ~10
min, increasing more than 10-fold from an initial value of <100 pA
(Fig. 7A, trace 1) to a stable maximum of
~1,150 pA (trace 2).
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Fig. 7.
Activation of IAC by inorganic
polytriphosphate (PPPi). Time-dependent expression of
IAC K+ current was monitored in
whole cell recordings by using patch pipettes containing standard
solution supplemented with PPPi (2 or 5 mM). A:
K+ current were activated at 30-s intervals from a holding
potential of 80 mV to a test potential of +30 mV with (bottom
traces) or without depolarizing prepulses (top traces).
Bottom: after IAC had reached a
stable amplitude, cells were superfused with saline containing 1 µM
penfluridol (A) or 10 nM ANG II (B).
K+ current traces were recorded immediately after whole
cell recording was initiated (1), after
IAC reached a stable amplitude (2),
and after exposure to penfluridol or ANG II (3).
IAC amplitudes are plotted against times with
() and without depolarizing prepulses
(). Numbers correspond to traces (top).
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To further establish the identity of the PPPi-activated
K+ current, the cell was superfused with the
diphenylbutylpiperidine penfluridol, a selective and potent antagonist
of IAC. Penfluridol inhibits
IAC with an IC50 of 187 nM, while
the transient IA K+ current is
inhibited only at 200-fold higher concentrations (20). Penfluridol (1 µM) inhibited the noninactivating current (Fig. 7A, trace 3) by ~80%, while the inactivating
K+ current IA was not reduced.
When IAC is activated by MgATP, ANG II inhibits
this current with an IC50 of ~150 pM (39,
40). However, when the nonhydrolyzable nucleotide AMP-PNP is
used to activate IAC, ANG II is completely ineffective (40). ANG II was also ineffective as an
inhibitor of IAC when PPPi activated
this current. In the experiment shown in Fig. 7B,
IAC was activated with a pipette solution
containing 2 mM PPPi. Over a 10-min period,
IAC grew from an initial amplitude of <25 pA
(Fig. 7B, trace 1) to a maximum of ~600 pA
(trace 2). The superfusion of 10 nM ANG II failed to
significantly reduce IAC (trace 3).
Overall, at a concentration of 5 mM, PPPi was slightly less
effective than MgATP as an activator of IAC.
With pipette solution containing PPPi,
IAC reached a maximum current density of
44.4 ± 14.7 pA/pF (n = 5) compared with 52.4 ± 7.1 pA/pF (n = 17) obtained in the presence of 5 mM
MgATP (Fig. 8B).
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Fig. 8.
Comparative and additive effects of MgATP and inorganic phosphates
on IAC expression. Effects of inorganic
phosphate (Pi), pyrophosphate (PPi), or
PPPi on IAC expression were
monitored in whole cell recordings by using pipettes containing
standard solution supplemented with MgATP (1 or 5 mM) or inorganic
phosphates (5 mM) either alone or in combination as indicated.
A: K+ currents were activated at 30-s intervals
from a holding potential of 80 mV to a test potential of +30 mV.
K+ current traces were recorded with (right
traces) and without depolarizing prepulses (left
traces) immediately after whole cell recording was initiated
(trace 1), after IAC had reached its
maximum amplitude (trace 2), and after inhibition by 1 µM
penfluridol (trace 3). B: effect of MgATP and
PPPi on IAC current density.
IAC max was obtained from experiments such as
those described in A by dividing the maximum
IAC amplitude by the cell capacitance. Values
are means ± SE for the number of determinations, indicated in
parentheses.
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At concentrations lower than 2 mM, MgATP is relatively ineffective as
an activator of IAC (14). However,
at a concentration of 1 mM, MgATP significantly potentiated activation
of IAC by PPPi. With pipette
solution containing 1 mM MgATP and 5 mM PPPi in
combination, IAC reached a maximum current
density of 80.2 ± 9.6 pA/pF (n = 17) (Fig. 8,
A and B).
PPi and Pi were far less effective than
PPPi as activators of IAC. With
pipette solutions containing 5 mM PPi or Pi in
addition to 1 mM MgATP, IAC reached maximum
current densities of 22.3 ± 7.3 pA/pF (n = 8) and
15.7 ± 3.5 pA/pF (n = 6), respectively, compared
with 13.0 ± 1.0 pA/pF (n = 24) observed in the
presence of 1 mM MgATP alone (Fig. 8, A and B).
Activation of unitary IAC currents by NaUTP and
PPPi.
NaUTP and PPPi both activated IAC
channels in outside-out patches at the same concentrations that were
effective in whole cell recordings. Figure
9 shows representative recordings made with pipettes containing 1 mM NaUTP (Fig. 9A) or 2 mM
PPPi (Fig. 9B). As in outside-out patch
recordings made with MgATP, NPo typically increased with time in the presence of UTP and PPPi.
However, with UTP and PPPi, channel activity was unstable
and sometimes disappeared abruptly.
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Fig. 9.
Activation of unitary IAC currents
by NaUTP and PPPi. Unitary IAC
currents were recorded in outside-out patches with standard external
and pipette solutions supplemented with 1 mM NaUTP (A) or 2 mM PPPi (B). Top: voltage steps to
+30 mV were applied at 10-s intervals from a holding potential of 40
mV. Bottom: amplitude histograms were constructed from
idealized channel openings obtained from unitary currents recorded in
response to 90 consecutive voltage steps of 400-ms duration (2,689 events for NaUTP and 2,732 events for PPPi). Unitary
current amplitudes were distributed into bins of 0.15 pA in width.
Continuous lines in the histograms are fits of Gaussian distributions
to the data. NPo was calculated as described in
MATERIALS AND METHODS. Currents were filtered at a cut-off
frequency of 1.5 kHz and sampled at 5 kHz.
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Amplitude analysis of unitary currents recorded in the presence of
NaUTP and PPPi showed that these two agents activated
identical channels. In Fig. 9A, Gaussian fits of amplitude
histograms generated from idealized openings recorded in the presence
of NaUTP yielded two peaks with mean amplitudes of 3.60 ± 0.39 and 7.13 ± 0.43 pA, indicative of two active channels. Similarly,
Gaussian fits of amplitude histograms generated from recordings with
PPPi in the pipette yielded peaks with means of 3.61 ± 0.36 and 7.05 ± 0.45 pA (Fig. 9B). Unitary currents
activated by UTP and PPPi were blocked nearly completely by
the IAC-selective antagonist penfluridol (500 nM), further establishing the identity of these channels (data not shown).
Effect of nucleotides and PPPi on AZF cell membrane
potential.
IAC channels may set the resting membrane
potential (Vm) of AZF cells. If so, then
Vm should depend on the presence of nucleotides and polyphosphates that have been shown to activate these channels. We
measured Vm of AZF cells in whole cell
current-clamp recordings with patch electrodes containing standard
saline supplemented with MgATP, PPPi, or NaUTP.
The membrane potential of AZF cells was strongly dependent on the
presence of nucleotides or polyphosphates in the recording pipette and
was well correlated with their potency as activators of
IAC. With 1 mM MgATP in the recording pipette,
average Vm reached 25.6 ± 5.3 mV
(n = 8) after 5-20 min of recording. Raising the MgATP concentration to 5 mM increased Vm to
72.7 ± 1.9 mV (n = 3) (Table
1).
At a concentration of 1 mM, UTP was much more effective than ATP at
producing a negative membrane potential in AZF cells. With NaUTP (1 mM)
in the patch electrode, the average Vm was
measured to be 72.0 ± 3.0 mV (n = 7), a value
very similar to that recorded in the presence of 5 mM MgATP (Table 1).
The combination of 5 mM PPPi and 1 mM MgATP was very
effective in activating IAC current in AZF cells
(see Fig. 8). Accordingly, the presence of these two agents in the
patch electrode in current-clamp experiments yielded average
Vm of 67.9 ± 4.2 (n = 8)
in AZF cells (Table 1).
Anionic phospholipid PIP2 does not activate
IAC channels.
The plasmalemmal phospholipid PIP2 activates a number of
ATP-sensitive channels by altering their sensitivity to ATP or by direct interaction with the channels (8, 17, 29, 44). PIP2 failed to enhance the expression of
IAC current in whole cell recordings from AZF
cells. In these experiments, PIP2 was added to pipette
solutions at several different concentrations along with 1 mM MgATP and
200 µM GTP. At PIP2 concentrations of 5, 10, and 50 µM,
IAC reached maximum current densities of
9.2 ± 1.4 (n = 4), 13.6 ± 2.6 (n = 8), and 9.4 ± 2.0 pA/pF (n = 2), respectively, values not significantly different from the control value of 13.0 ± 1.0 pA/pF (n = 24) obtained in
the absence of PIP2. The addition of 10 µM
PIP2 to the patch electrode in addition to 5 mM MgATP also
failed to alter the inhibition of IAC current by
ANG II (10 nM) (data not shown).
ANG II-mediated inhibition of IAC and ATP hydrolysis.
The activity of IAC K+ channels is
promoted by the binding of hydrolyzable and poorly hydrolyzable
nucleotide triphosphates. However, inhibition of
IAC through activation of a number of G protein-coupled receptors, including ANG II receptors, requires the
presence of hydrolyzable ATP (16, 39, 40, 48). These results are consistent with a model in which IAC
opening and closing are controlled through an ATP hydrolysis cycle
involving ATP binding and metabolism by an ATPase. However, the
requirement for hydrolyzable ATP might implicate a protein kinase
rather than an ATPase in IAC inhibition.
Experiments were done to determine whether ATP-dependent inhibition of
IAC by ANG II was mediated through a mechanism
requiring an ATPase or, alternatively, a protein kinase. The inhibition of IAC by ANG II was studied by using pipette
solution containing 5 mM NaUTP and various concentrations of MgATP.
Although UTP is more potent than ATP as an activator of
IAC channels, it is a poor substrate for
phosphate transfer enzymes, including protein kinases and ATPases
(6, 33). ATP, on the other hand, is a substrate for both
protein kinases and ATPases, although typically at different
concentrations. Protein kinases are fully activated by 50 µM ATP,
whereas cellular ATPases frequently display significantly higher
Michaelis-Menten constant (Km) values for ATP
(18, 34).
As previously reported, ANG II effectively inhibits
IAC when the pipette solution contains MgATP (5 mM) and GTP (200 µM) as the only nucleotides. Under these conditions,
ANG II reduced IAC by 82 ± 5%
(n = 6) (Fig. 10,
A and B). When NaUTP replaced ATP in the pipette,
ANG II was much less effective, inhibiting IAC by only 10 ± 5% (n = 8). The addition of 50 µM
or even 1 mM MgATP to pipette solution containing 5 mM NaUTP failed to
restore IAC inhibition by ANG II. Under these
conditions, ANG II inhibited IAC by only 14 ± 4% (n = 3) and 14 ± 7% (n = 3), respectively (Fig. 10, A and B). Raising
intracellular MgATP to 2 mM in the presence of 5 mM NaUTP partially
restored IAC inhibition by ANG II to 57.6%
(n = 5).
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Fig. 10.
Effect of intracellular MgATP and NaUTP on
IAC inhibition by ANG II. K+
currents were recorded by using the two voltage protocols described in
Fig. 5. A: patch pipettes contained standard solution
supplemented with 5 mM MgATP or UTP in addition to 200 µM GTP at
concentrations ranging from 0.05 to 2 mM as indicated. After
IAC reached a maximum value, cells were
superfused with ANG II (10 nM). Top: traces show currents
recorded with (bottom traces) and without depolarizing
prepulses (top traces) to 20 mV immediately after whole
cell recording was initiated (trace 1), after
IAC had reached a maximum (trace 2),
or after superfusion with 10 nM ANG II (trace 3). Pipette
MgATP and NaUTP concentrations are as indicated. Bottom:
IAC amplitudes recorded with ()
or without depolarizing prepulses () to 20 mV are
plotted against time with corresponding traces (top).
B: summary of results from experiments such as those shown
in A. Data indicate fraction of IAC
remaining after inhibition by 10 nM ANG II with pipette solutions
containing ATP and UTP at the indicated concentrations. Values are
means ± SE of the number of determinations, indicated in
parentheses.
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The failure of ATP, at concentrations up to 1 mM, to restore inhibition
of IAC by ANG II suggests that a protein kinase
does not mediate this inhibition. ANG II combines with
losartan-sensitive AT1 receptors to activate a number of
different protein kinases in various cells, including adrenocortical
cells (36, 42, 46, 49). To further explore the possibility
that ANG II-mediated inhibition of IAC involves
a protein kinase, we studied the effect of six different protein kinase
inhibitors on ANG II-mediated inhibition of IAC.
The activation of AT1 receptors on adrenocortical
cells by ANG II leads to the phospholipase C-dependent synthesis of
diacylglycerol, which activates protein kinase C (3, 13).
Calphostin C is a potent and specific protein kinase C antagonist
(IC50 = 50 nM) (45). When applied
directly to the cytoplasm through patch electrodes at a concentration
of 500 nM, calphostin C failed to prevent inhibition of
IAC by ANG II (10 nM). In the presence of this
antagonist, ANG II reduced IAC by 92 ± 2%
(n = 6) (Fig. 11,
A and B).
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Fig. 11.
Effect of protein kinase inhibitors on ANG II-mediated
inhibition of IAC. Time-dependent expression of
IAC K+ current was monitored in
whole cell recordings by using patch pipettes containing standard
saline supplemented with 5 mM MgATP, 200 µM GTP, and 1 of 6 protein
antagonists at the indicated concentration. K+ currents
were activated by voltage steps to +30 mV applied at 30-s intervals
from a holding potential of 80 mV with or without depolarizing
prepulses. A: effect of calphostin C. Current traces were
recorded with (middle) and without depolarizing prepulses
(left) to 20 mV immediately after whole cell recording was
initiated (trace 1), after IAC had
reached a maximum (trace 2), and after steady-state block by
10 nM ANG II (trace 3). IAC
amplitudes recorded with () or without depolarizing
prepulses () to 20 mV are plotted against time
(right). B: summary of results from experiments
such as those shown in A. Data indicate fraction of
IAC remaining after inhibition by 10 nM ANG II
with pipettes containing protein kinase antagonists at the indicated
concentrations. Values are means ± SE of the number of
determinations, indicated in parentheses.
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ANG II activates tyrosine kinases in a number of cells through
interaction with AT1 receptors (42). However,
the tyrosine kinase inhibitors genistein (20 µM) and herbimycin (10 µM) both failed to reduce IAC inhibition by
ANG II, even when used at concentrations at least 10-fold higher than
their reported IC50 values (Fig. 11B) (2,
35).
In at least one type of cell, AT1 receptors are physically
associated with Janus kinase 2 (JAK2) intracellular protein kinases, which are activated by ANG II (36). However, the specific
JAK2 inhibitor AG-490 (5 µM) (38) failed to produce any
inhibition of IAC at a concentration 50 times
its reported IC50 (Fig. 11B).
ANG II also activates the mitogen-activated protein (MAP) kinase
pathway in a variety of cells, including bovine adrenal cortical cells
(46, 49). However, the MAP kinase-selective antagonist PD-98059 (50 µM) was also completely ineffective at suppressing IAC inhibition when applied directly through the
patch pipette at a concentration at least 10 times greater than the
reported IC50 (Fig. 11B).
Staurosporine is a potent nonselective protein kinase antagonist. This
microbial alkaloid inhibits most serine/threonine protein kinases with
IC50 values of <20 nM (45). At a
concentration of 1 µM, staurosporine partially reduced ANG
II-mediated inhibition of IAC from its control
value of 82 ± 5% (n = 6) to 62 ± 11%
(n = 8) (Fig. 11B).
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DISCUSSION |
We have described the properties and regulation of a unique
ATP-activated ion channel in bovine AZF cells. Unitary
IAC channels were shown to be K+
selective with no measurable Na+ or Ca2+
conductance. IAC channels were outwardly
rectifying in the presence of physiological concentrations of
M2+, but conductance became almost ohmic in the absence of
M2+ on either side of the membrane.
The potent activation of IAC channels by poorly
hydrolyzable nucleotides and PPPi and the failure of ANG II
to inhibit these channels when these agents replace ATP in the pipette
solution are consistent with a model for IAC
gating coupled to an ATP hydrolysis cycle. Overall, these results
identify IAC as a distinctive new type of
K+ channel with properties that would allow it to couple
hormonal and metabolic signals to membrane potential and cortisol production.
Nucleotide-dependent activation of IAC channels in
membrane patches.
Nucleotide-dependent activation of unitary IAC
channels in outside-out patch recordings resembled, in time course and
concentration dependence, previous results obtained in whole cell
recordings (14). Specifically, IAC
channels were active in outside-out patches only when ATP or UTP were
present in the patch pipette at millimolar concentrations. These
results are consistent with the idea that nucleotide-dependent
activation of these channels is independent of cytoplasmic proteins
that are absent in outside-out patches and of protein kinases for which
UTP is a poor substrate.
The failure of MgATP to activate IAC channels in
excised inside-out patches suggests that key regulatory factors or
associated membrane proteins are lost during this form of patch
excision. This result is not surprising in view of the rich combination of metabolic factors that control the activity of this K+
channel (14, 16, 19, 39). In particular, because the molecular identity of IAC channels has not been
determined, we do not know whether nucleotide binding sites are located
on the pore-forming proteins or an associated subunit. The inability to
directly activate IAC K+ channels in
inside-out patches limits studies exploring the modulation of these
channels. For example, we have been unable to describe the temporal
pattern and reversibility of ATP-mediated IAC activation.
K+ selectivity, conductance, and
rectification of IAC channels.
Although the molecular structure of IAC and its
relationship to other channels is unknown, the results of the present
study identify it as a true K+-selective channel with no
measurable conductance to Na+ or Ca2+. In
symmetrical K+ solutions, unitary
IAC currents reversed at potentials slightly positive to 0 mV. This probably occurred through a reduction in pipette
K+ activity through binding to ATP or BAPTA.
The identification of IAC as an authentic
K+-selective channel is significant in view of the
aforementioned similarities to CNG cation channels with respect to
pharmacology and gating by cAMP (16, 20, 43). The lack of
measurable Na+ and Ca2+ conductance clearly
distinguishes IAC from CNG cation channels and
Drosophila eag K+ channels (11).
IAC K+ channels may represent a new
intermediate form in a continuum linking true K+-selective
and CNG cation channels.
M2+ and rectification.
Inwardly rectifying ATP-sensitive K+ (KATP)
channels comprise a major class of K+-selective channels
that include two rather than six membrane-spanning regions. Although
KATP channels resemble IAC channels
with respect to gating by ATP, KATP channels are uniformly
inhibited, rather than activated, by the nonhydrolytic binding of ATP
(5). In addition, KATP channels are sensitive
to sulfonylurea agonists and antagonists, whereas
IAC channels are not (1, 5, 20).
In the present study, a third fundamental difference between
IAC K+ channels and KATP
channels was identified. Specifically, in symmetrical solutions,
KATP channels are inwardly rectifying due to the
unidirectional block of K+ efflux by Mg2+ at
physiological concentrations (5, 28). By comparison, in
the presence of physiological concentrations of internal and external
M2+ and symmetrical K+,
IAC channels are outwardly rectifying. Removal
of intracellular Mg2+ showed that outflow of K+
is only weakly blocked by this divalent cation, an effect that is more
than matched by block of K+ influx by Ca2+ and
Mg2+. Accordingly, when these two M2+ are
deleted from external and pipette solutions, IAC
channels conduct K+ almost equally well in either direction.
The relative contributions of Ca2+ and Mg2+ to
block of K+ influx were not determined in our experiments.
K+ efflux through IAC channels is
dramatically inhibited by intracellular Ca2+ at a
concentration of only 2 µM (19). However, this effect appears to occur through an action on IAC gating
rather than permeation.
Overall, although they are both ATP-gated and K+-selective
channels, IAC and KATP channels
differ in several fundamental aspects. It is unlikely that they belong
to the same family of K+ channels.
Activation of IAC channels by nucleotides and inorganic
phosphates.
At concentrations 
1 mM, nucleotides including AMP-PNP, NaUTP, NaCTP,
and NaTTP were much more effective than MgATP as activators of
IAC. In a model for IAC
gating in which nucleotide binding leads to channel opening and
nucleotide hydrolysis or dissociation allows the channel to close,
nonhydrolyzable nucleotides could be more potent through a reduction in
the effective "off rate," lowering the dissociation
constant. Accordingly, NaUTP was at least 10-fold more potent
than MgATP as an activator of IAC.
The activation of IAC channels by
PPPi is also consistent with a model for
IAC gating controlled by an ATP hydrolysis
cycle. ATP hydrolysis by ATPases involves cleavage of the phosphate
bond, followed by liberation of Pi. Inorganic phosphates
bind to sites from which Pi is released after cleavage of
ATP, interrupting further ATP hydrolysis cycles (7, 12,
22). If IAC closing is coupled to the
hydrolysis of ATP and subsequent dissociation of organic phosphate from
its binding site, then these same agents may stabilize
IAC channels in the open or bursting configuration.
IAC K+ channels appear to be the
first K+ channel yet described whose activity is enhanced
by ATP and other nucleotides, as well as inorganic phosphates, through
a mechanism not involving protein kinases. In this regard, the gating
of IAC channels resembles that of the CFTR
Cl channel in that hydrolysis-resistant ATP analogs and
polyphosphates stabilize the open state of the channel, locking it into
a prolonged open or bursting state (7, 22). Thus the
gating of this ATP-activated Cl channel may also be
tightly coupled to an ATP hydrolysis cycle. However, the biochemical
mechanisms controlling the activity of CFTR Cl channels
are quite complex and not well understood. While an ATP hydrolysis
cycle seems to be involved, previous phosphorylation by A-kinase is a
requirement for activation (7, 30). The overall similarity
to IAC gating is not yet clear.
Although the activation of IAC K+
channels by poorly hydrolyzable nucleotides and polytriphosphates is
consistent with a model for IAC gating involving
an ATP hydrolysis cycle, the results raise questions regarding the
nature of the binding sites involved. First, if the nucleotide binding
site is specifically designed to accommodate ATP, it is surprising that
pyrimidine nucleotide triphosphates including UTP, CTP, and TTP also
bind to the site with similar affinity.
Second, if inorganic phosphates bind to a channel-associate site on the
ATPase that is normally occupied by Pi, why is
PPPi much more effective than Pi or
PPi as an activator of IAC channels? The structure-activity results involving nucleotides and inorganic phosphates indicate that it is the triphosphate group itself that is
the critical moiety in the activation of IAC
channels. Currently, there is no satisfactory explanation for this
order of effectiveness. PPPi and PPi have also
been shown to be quite effective in activating CFTR Cl
channels (7, 22).
The failure of the anionic phospholipid PIP2 to activate
IAC channels indicates that the presence of
multiple phosphate groups in a molecule does not ensure that it will
activate this current. It represents a further distinction between
IAC and other ATP-sensitive K+
channels that are uniformly activated by PIP2 (8, 17,
29, 44).
Nucleotides and membrane potential.
Good correlation exists between the activation of
IAC K+ channels by nucleotides and
polyphosphates and the magnitude of the Vm
installed by these same agents in AZF cells. This result provides evidence that IAC channels are primarily
responsible for setting Vm. Furthermore,
single-channel recordings showed that IAC
channels remain active at very negative membrane potentials, as
expected for a channel that sets Vm near the
K+ equilibrium potential.
A direct relationship exists among nucleotide triphosphate
concentration, IAC activity, and membrane
potential. This suggests a specific mechanism whereby membrane
potential, Ca2+ entry, and cortisol secretion could be
linked to the metabolic state of the cell and, therefore, to other
variables such as blood glucose concentration. Cortisol is a glucose
counterregulatory hormone that acts in opposition to insulin in
maintaining blood glucose levels (9).
Mechanism for ANG II-mediated inhibition of IAC.
The failure of ANG II to inhibit IAC when NaUTP
or PPPi replaced ATP in the pipette is consistent with our
previous observation that ANG II was ineffective in the presence of the
nonhydrolyzable ATP analog AMP-PNP (40). However, the
requirement for ATP hydrolysis could signal the involvement of either
an ATPase or a protein kinase. The failure of the addition of 0.05 or 1 mM MgATP to the pipette (in addition to 5 mM UTP) to restore inhibition
by ANG II argues for the involvement of an ATPase rather than a kinase in this response. Nearly all kinases are fully activated by the substrate MgATP at concentrations of 50 µM, whereas cellular ATPases have higher Km values for ATP (18, 27,
34). Thus, if ANG II-mediated inhibition of
IAC occurred through activation of a protein
kinase, low concentrations of ATP should have been sufficient to
restore this effect.
Results of experiments with multiple protein kinase antagonists support
the conclusion that no protein kinase known to be activated by ANG II
mediates inhibition of IAC. In various cells, including those of the adrenal cortex, ANG II acts through
AT1 receptors to activate protein kinase C, tyrosine
kinases, JAK/STAT (signal transducers and activators of transcription)
kinases, and MAP kinases (4, 36, 38, 46, 49). The
failure of specific antagonists of each of these kinases to attenuate
IAC inhibition by ANG II indicates that none of
these is involved in this response.
The nonselective protein kinase antagonist staurosporine (1 µM)
reduced the inhibition of IAC by ANG II from
82% to only 62%. At this concentration, staurosporine completely
inhibits a wide range of protein kinases, including serine/threonine
kinases, and tyrosine kinases (45). It is possible that an
unidentified staurosporine-sensitive protein kinase contributes to
IAC inhibition by ANG II.
Identity and function of IAC
K+ channels.
Although the results of this study identify IAC
as a true K+-selective ion channel, its molecular structure
and relationship to other K+ channel gene families is
unknown. Its weak voltage dependence, insensitivity to sulfonylureas
and PIP2, and lack of inward rectification suggest that it
does not belong to the six-membrane-spanning, voltage-gated channels or
to the two-membrane-spanning inward rectifiers. In this regard, a new
family of K+-selective channels with two pore domains in
tandem has been identified in organisms ranging from yeast to humans
(31, 32). A number of these noninactivating, outwardly
rectifying channels display properties similar to those of
IAC.
Regardless, the convergent inhibition of IAC by
multiple G protein-coupled receptors through second messengers,
including Ca2+ and cAMP, and the activation of these
K+ channels by ATP at physiological concentrations indicate
that IAC is a central control point where
hormonal and metabolic signals are transduced to electrical events
involved in cortisol secretion. In this scheme, the control of
IAC K+ channel activity through a
cycle of ATP binding and hydrolysis may be a fundamental mechanism
linking biochemical signals to AZF cell membrane potential.
In this regard, ATP-activated IAC K+
channels provide an interesting contrast to KATP channels
of insulin-secreting cells, which are inhibited, rather than activated,
by ATP. The opposing actions of ATP on the activity of these two
metabolic sensors are consistent with their function in regulating
insulin and cortisol secretion. In pancreatic -cells, high blood
glucose levels lead to elevated ATP, KATP inhibition,
membrane depolarization, Ca2+ entry, and insulin secretion
(1, 5). In bovine AZF cells, elevated glucose would be
associated with IAC activation, suppressing Ca2+ entry and cortisol secretion. Accordingly, cortisol is
secreted under conditions of metabolic stress, where insulin secretion is suppressed (9).
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-47875 (to J. J. Enyeart).
Address for reprint requests and other correspondence: J. J. Enyeart, Dept. of Neuroscience, The Ohio State Univ., College of
Medicine, 5190 Graves Hall, 333 W. 10th Ave., Columbus, OH 43210-1239 (E-mail: enyeart.1{at}osu.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
TOP
ABSTRACT
INTRODUCTION
