From the * Department of Pharmacology, and The Neuroscience Program, The Ohio State University, College of Medicine, Columbus,
Ohio 43210-1239
Bovine adrenal zona fasciculata (AZF) cells express a noninactivating K+ current (IAC) that is inhibited by adrenocorticotropic hormone and angiotensin II at subnanomolar concentrations. Since IAC appears to set
the membrane potential of AZF cells, these channels may function critically in coupling peptide receptors to
membrane depolarization, Ca2+ entry, and cortisol secretion. IAC channel activity may be tightly linked to the metabolic state of the cell. In whole cell patch clamp recordings, MgATP applied intracellularly through the patch electrode at concentrations above 1 mM dramatically enhanced the expression of IAC K+ current. The maximum IAC
current density varied from a low of 8.45 ± 2.74 pA/pF (n = 17) to a high of 109.2 ± 26.3 pA/pF (n = 6) at pipette MgATP concentrations of 0.1 and 10 mM, respectively. In the presence of 5 mM MgATP, IAC K+ channels
were tonically active over a wide range of membrane potentials, and voltage-dependent open probability increased by only ~30% between 40 and +40 mV. ATP (5 mM) in the absence of Mg2+ and the nonhydrolyzable
ATP analog AMP-PNP (5 mM) were also effective at enhancing the expression of IAC, from a control value of 3.7 ± 0.1 pA/pF (n = 3) to maximum values of 48.5 ± 9.8 pA/pF (n = 11) and 67.3 ± 23.2 pA/pF (n = 6), respectively.
At the single channel level, the unitary IAC current amplitude did not vary with the ATP concentration or substitution with AMP-PNP. In addition to ATP and AMP-PNP, a number of other nucleotides including GTP, UTP, GDP,
and UDP all increased the outwardly rectifying IAC current with an apparent order of effectiveness: MgATP > ATP
= AMP-PNP > GTP = UTP > ADP >> GDP > AMP and ATP-
-S. Although ATP, GTP, and UTP all enhanced IAC
amplitude with similar effectiveness, inhibition of IAC by ACTH (200 pM) occurred only in the presence of ATP.
As little as 50 µM MgATP restored complete inhibition of IAC, which had been activated by 5 mM UTP. Although
the opening of IAC channels may require only ATP binding, its inhibition by ACTH appears to involve a mechanism other than hydrolysis of this nucleotide. These findings describe a novel form of K+ channel modulation by
which IAC channels are activated through the nonhydrolytic binding of ATP. Because they are activated rather than inhibited by ATP binding, IAC K+ channels may represent a distinctive new variety of K+ channel. The combined features of IAC channels that allow it to sense and respond to changing ATP levels and to set the resting potential of AZF cells, suggest a mechanism where membrane potential, Ca2+ entry, and cortisol secretion could be
tightly coupled to the metabolic state of the cell through the activity of IAC K+ channels.
IAC is a novel noninactivating K+ current that may set
the resting potential of bovine adrenal zona fasciculata
(AZF)1 cells. Angiotensin II (AII) and adrenocorticotropic hormone (ACTH) inhibit IAC and depolarize
AZF cells at concentrations identical to those that stimulate cortisol production (Mlinar et al., 1993a). This
K+ channel appears to act pivotally in coupling these
peptide hormone receptors to depolarization-dependent Ca2+ entry and corticosteroid production (Enyeart et al., 1993
).
IAC K+ channel activity may be regulated by the complex interaction of biochemical factors and membrane
voltage. In whole-cell patch clamp recordings from AZF
cells, we found that IAC K+ current measured during depolarizing voltage steps increases dramatically (10-100-fold) over a period of many minutes (Mlinar et al.,
1993a). Inhibitory factors present in the cytoplasm may be diluted during dialysis of the cell by pipette solution,
allowing the functional expression of the IAC current.
In this regard, the time-dependent growth of IAC is suppressed by including the nonhydrolyzable GTP analog
GTP-
-S in the recording pipette, indicating the presence of an inhibitory mechanism requiring a GTP-binding protein (Mlinar et al., 1993a
). Accordingly, inhibition of IAC by both ACTH and AII require G-protein intermediates (Mlinar et al., 1995
; Enyeart et al.,
1996b
).
ACTH receptors are coupled to adenylate cyclase
through GS. Although most cAMP-dependent actions
of ACTH are mediated through cAMP-dependent protein kinase, inhibition of IAC by both ACTH and cAMP
occur through an A-kinase-independent mechanism
requiring ATP hydrolysis (Enyeart et al., 1996b). This
result suggests that opening and closing of IAC K+ channels could be coupled to an ATP hydrolysis cycle, similar to that which controls the activity of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl
channels (Hwang et al., 1994
; Baukrowitz et al., 1994
;
Quinton and Reddy, 1992
). If so, then IAC K+ channels
could be activated by the hydrolytic or nonhydrolytic binding of ATP to these channels or associated proteins.
A large number of ATP-sensitive K+ channels are expressed by a variety of cells ranging from cardiac and
skeletal muscle cells to neurons and insulin-secreting
pancreatic cells (Ashcroft, 1988a
; Hilgemann, 1997
;
Terzic et al., 1994
; Takano and Noma, 1993
). However,
these inwardly rectifying channels are uniformly inhibited by ATP through nonhydrolytic binding to the
channel or an associated protein. In contrast, whole-cell and single channel patch clamp recordings from
bovine AZF cells showed that ATP, over a physiological
range of concentrations, dramatically enhanced IAC K+
current.
Tissue culture media, antibiotics, fibronectin, and fetal bovine
sera were obtained from Gibco Laboratories (Grand Island, NY).
Culture dishes were purchased from Corning Glass Works (Corning, NY). Coverslips were from Bellco Glass, Inc. (Vineland, NJ).
Enzymes, ACTH (1-24), NaATP, MgATP, KATP, KADP, AMP,
5-adenylyl-imidodiphosphate (AMP-PNP, lithium salt), adenosine 5
-O -3-thio-triphosphate (ATP-
-S, tetra-lithium salt), EDTA,
NaGTP, NaGDP, NaUTP, and NaUDP were obtained from Sigma
Chemical Co. (St. Louis, MO). Pinacidil was obtained from Research Biochemicals International (Natick, MA).
Isolation and Culture of AZF Cells
Bovine adrenal glands were obtained from steers (age 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 with some modifications (Gospodarowicz et al., 1977). In a sterile tissue culture
hood, the adrenals were cut in half lengthwise and the lighter
medulla tissue trimmed away from the cortex and discarded. The
capsule with attached glomerulosa and thicker fasciculata-reticularis layer were then dissected into large pieces ~1.0 × 1.0 × 0.5 cm. A Stadie-Riggs tissue slicer (Thomas Scientific, Swedesboro,
NJ) was used to slice fasciculata-reticularis tissue from the glomerulosa layers by slicing 0.3-0.5-mm slices from the larger pieces.
The first medulla/fasciculata slices were discarded. One to two
subsequent fasciculata slices were saved in cold sterile PBS/0.2%
dextrose. Fasciculata tissue slices were then diced into 0.5 mm3
pieces and dissociated with 2 mg/ml (~200-300 U/ml) of Type I collagenase (neutral protease activity not exceeding 100 U/mg of solid), 0.2 mg/ml deoxyribonuclease in DMEM/F12 for ~1 h
at 37°C, triturating after 30 and 45 min with a sterile plastic transfer pipette. After incubating, the 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 hour. Pelleted cells were washed twice with DMEM/0.2% BSA, centrifuging as before. Cells were filtered through 200-µm stainless steel
mesh to remove clumps after resuspending in DMEM. 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 adding cells. Dishes were maintained at 37°C in a humidified atmosphere of 95% air/5% CO2.
To minimize variability in IAC currents, all of the cells used in
experiments measuring current density (~200 cells) were obtained in a single isolation from six bovine adrenal glands and
stored in liquid N2, as described above, in ~70 vials. For each experiment, cells were plated on coverslips from a single vial. Experimental results reported in this paper were obtained in
recordings made over an 8-mo period. Cells stored in liquid N2,
as described above, retained electrophysiological and biochemical properties for at least 1 yr from freezing date. Specifically, AZF cells stored in this way expressed IAC K+ current as well as IA
K+ current with no obvious deterioration during the course of
this study. Further, as previously reported, ACTH and AII both
inhibited IAC K+ current in these cells with a potency not distinguishable from that observed in freshly isolated cells (Enyeart et
al., 1996b; Mlinar et al., 1993a
, 1995
). After months in liquid N2,
cultured AZF cells responded to ACTH and AII with large increases in cortisol secretion, and expression of orphan receptor
mRNAs (Enyeart et al., 1993
, 1996a
, 1996b
; Mlinar et al., 1995
).
Patch Clamp Experiments
Patch clamp recordings of K+ channel currents were made in the
whole-cell and outside-out patch configurations. For both recording configurations, the standard pipette solution was 120 mM
KCl, 2 mM MgCl2, 1 mM CaCl2, 10 mM HEPES, 11 mM BAPTA
(1,2-bis-(2-aminophenoxy)ethane-N,N,N ,N
-tetraacetic acid), and
200 µM GTP, with pH buffered to 7.2 using KOH. Addition of various nucleotides and other deviations from the standard solution
are described in the text. Pipette [Ca2+] was determined using the
"Bound and Determined" program (Brooks and Storey, 1992
).
The external solution consisted of (mM): 140 NaCl, 5 KCl, 2 CaCl,
2 MgCl2, 10 HEPES, and 5 glucose, pH 7.4 using NaOH. All solutions were filtered through 0.22-µm cellulose acetate filters.
AZF cells were used for patch clamp experiments 2-12 h after
plating. Typically, cells with diameters of <15 µm and capacitances of 8-12 picofarads were selected. Coverslips were transferred from 35-mm culture dishes to the recording chamber (1.5 ml vol) that 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 megohms were fabricated from Corning glass
(7052 or 0010; Garner Glass Co., Claremont, CA). These routinely yielded access resistances of 1.5-4 megohms and voltage
clamp time constants of <100 µs. For single channel recordings,
patch electrodes with higher resistances of 3-5 megohms were
used. K+ currents were recorded at room temperature (22-25°C)
following the procedure of Hamill et al. (1981) using an EPC-7
patch clamp amplifier (List Electronic, Darmstadt, Germany).
Pulse generation and data acquisition were done using a personal computer and PCLAMP software with an Axolab interface
(Axon Instruments, Inc., Burlingame, CA). Currents were digitized at 1-20 kHz after filtering with an 8-pole Bessel filter (Frequency Devices Inc., Haverhill, MA). Linear leak and capacity
currents were subtracted from current records using scaled hyperpolarizing steps of 1/3-1/4 amplitude. Data were analyzed
and plotted using PCLAMP 5.5 and 6.02 (CLAMPAN, CLAMPFIT, FETCHAN, and PSTAT) and GraphPAD InPLOT. Drugs were
applied by bath perfusion, controlled manually by a six-way rotary valve. Series resistance compensation was not used in experiments where the IAC currents were <1 nA. A current of this size
in combination with a 4 M access resistance produces a voltage
error of only 4 mV that was not corrected.
Effects of ATP and Analogs on IAC K+ Current
Bovine AZF cells express two types of K+ currents that
are easily distinguished in whole cell recordings. These
include a rapidly inactivating A type K+ current and a
noninactivating K+ current that grows continually over
a period of many minutes in whole cell recordings (Mlinar et al., 1993a; Enyeart et al., 1996b
). The absence of
time-dependent inactivation of the IAC K+ current allowed it to be easily isolated for measurement in whole cell recordings using either of two voltage clamp protocols. When voltage steps of 300-ms duration were applied from a holding potential of
80 mV to a test potential of +20 mV, IAC could be selectively measured
near the end of a step, at a point where the A type K+
current had inactivated entirely (Fig. 1 A, top traces). 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 A type current (Fig. 1
A, bottom traces).
The time-dependent increase in IAC amplitude observed in whole-cell recordings depended on the concentration of ATP in the pipette solution. At concentrations >1 mM, MgATP dramatically increased the maximum IAC amplitude attained (Fig. 1). At lower concentrations, MgATP was much less effective. With MgATP present in the pipette at 0.1 and 0.4 mM, IAC reached maximum current densities of 8.4 ± 2.7 (n = 7) and 12.2 ± 4.9 pA/ pF (n = 10), respectively. In contrast, at concentrations of 5 and 10 mM MgATP, IAC reached, after 10-25 min, maximums of 78.0 ± 8.9 (n = 26) and 109.2 ± 26.3 pA/ pF (n = 6), respectively (Fig. 1 C).
The dramatic enhancement of IAC current by MgATP
observed in whole-cell recordings could have several
explanations. Modulation of ion channel function by
protein kinases is widespread, while ion channel modulation through an ATP hydrolysis cycle involving ATPases
has more recently been reported (Baukrowitz et al.,
1994; Fakler et al., 1994
; Levitan, 1994
). Alternatively,
ATP could modulate IAC activity through nonhydrolytic
binding to the channel or an associated protein, as occurs with ATP-inhibited inward rectifier K+ channels
found in many cells (Ashcroft, 1988a
; Takano and Noma, 1993
). To distinguish between these possibilities, we
eliminated all Mg2+ from the pipette solution by substituting KCl and KATP for MgCl2 and MgATP, respectively. Both protein kinases and ATPases require ATP
complexed with Mg2+ as a substrate (Eckstein, 1985
;
Levitan, 1994
; Hilgemann, 1997
). In the absence of Mg2+,
5 mM ATP promoted large increases in IAC, although
not as large as those observed with MgATP (Fig. 2, A and
C). The nonhydrolyzable ATP analog AMP-PNP (5 mM)
also effectively stimulated IAC expression. At a concentration of 0.1 mM, AMP-PNP and MgATP were equally
ineffective at enhancing IAC activity (Fig. 2 C).
ATP--S is a poorly hydrolyzable ATP analog that is,
like AMP-PNP, a poor substrate for cellular ATPases. In
contrast to AMP-PNP, ATP-
-S is a good substrate for
many protein kinases, while the transferred phosphorothioate group is resistant to hydrolysis by phosphatases (Eckstein, 1985
). ATP-
-S produced effects on K+ currents in AZF cells that were dramatically different from those observed with AMP-PNP. Specifically,
with 2 mM ATP-
-S in the pipette, IAC did not grow in
whole-cell recordings. Instead, any noninactivating IAC
current that was present upon initiating whole-cell recording was inhibited over a period of several minutes
(Fig. 3 A). At the same time, the decay kinetics of the
rapidly inactivating A type K+ current often slowed dramatically in the presence of ATP-
-S. In the experiment
illustrated in Fig. 3 B, 4 min after commencing whole-cell recording, IAC inactivated with a time constant (
i) of 26.1 ms. By 13 min,
i had slowed to 254 ms. In spite
of the slowed inactivation kinetics, a 10-s prepulse to
20 mV was effective at inactivating nearly all of the IA
current (Fig. 3 C). Overall, with 2 mM ATP-
-S in the
recording pipette, IAC was not clearly detectable in any
of 20 cells at any time between 5 and 30 min. In nine of
these cells, IA inactivation kinetics slowed dramatically during the recordings.
ATP and Unitary IAC Currents
Single channel recordings made from AZF cells in the
outside-out configuration showed that, in contrast to
whole-cell currents, unitary IAC current amplitudes
were not increased by raising the "intracellular" MgATP
concentration from 2 to 5 mM. In these experiments, IAC currents were first recorded in the whole-cell configuration to allow IAC to reach a stable amplitude. After obtaining an outside-out patch, the holding potential was set to 40 mV, a potential where nearly all IA
channels are inactivated (Mlinar and Enyeart, 1993b
). Under these conditions, a single type of K+ channel
was typically present in the patch membrane. Fig. 4 A shows unitary currents activated by voltage steps to +30
mV from a holding potential of
40 mV in the presence of either 2 or 5 mM MgATP. Histogram analysis of
unitary current amplitudes showed a single major peak
for the lower and higher MgATP concentrations with
respective means of 3.81 ± 0.62 pA and 3.92 ± 0.81 pA
(Fig. 4 A). A second minor peak, with a mean of approximately twice the unitary amplitude, was also present
(data not shown).
In whole-cell recordings, MgATP and AMP-PNP both
induced expression of noninactivating outward currents presumed to be IAC. Single channel current-voltage (IV) relationships recorded in the presence of the
hydrolyzable and nonhydrolyzable nucleotides showed
that the corresponding unitary K+ currents were identical. Single channel IVs for IAC K+ channels were obtained by applying voltage steps in 10-mV increments to
outside-out patches from a holding potential of 40
mV. The single channel IV relationship obtained with 5 mM MgATP or 2 mM AMP-PNP in the pipette were
nearly identical (Fig. 4 B). In each case, with otherwise
standard pipette and external solutions, IAC channel
currents displayed a unitary conductance of ~70 pS, estimated between potentials of 0 and +40 mV. Thus, the
unitary currents activated by low and high concentrations of MgATP or a nonhydrolyzable ATP analog, appear to flow through the same IAC channel.
IAC Increase by Other Nucleotides
Experiments with KATP and AMP-PNP indicated that
nonhydrolytic binding of ATP was sufficient to convert
IAC channels to an active or activatable form. To determine whether other adenine nucleotides would increase IAC amplitude in whole cell recordings, we compared MgATP with ADP and AMP. A time-dependent
increase in IAC amplitude occurred in the presence of
ADP but not AMP (Fig. 5, A and C). When the pipette
contained 5 mM AMP, IAC failed to grow above the control amplitude measured immediately after initiating whole-cell recording (Fig. 5, B and C). Further, in each
of five cells, the rapidly inactivating A type K+ current
was observed to decrease with time in the presence of AMP (Fig. 5 B). Overall, when the pipette contained 5 mM ADP, IAC grew to a maximum current density of
36.1 ± 10.8 pA/pF (n = 12), compared with 78.0 ± 8.9 pA/pF (n = 26) observed with 5 mM MgATP in the
patch electrode (Fig. 5 D). When the patch electrode contained 0.1 mM ADP, IAC reached a maximum of
only 10.7 ± 2.4 pA/pF (n = 4), a value not significantly
different from that observed with 0.1 mM MgATP.
In addition to ATP, we found that other nucleotide
triphosphates, including GTP and UTP, were also effective in promoting IAC activity in whole cell recordings.
In these experiments, sodium salts of ATP, UTP, or
GTP were added to the pipette solution at a concentration of 5 mM. With each of these agents, a time-dependent increase in IAC amplitude, which usually reached a
maximum value in 10-25 min, was observed (Fig. 6).
Overall, these three nucleotides produced similar effects on IAC current densities with maximum values of
72.0 ± 27.5 (n = 5), 57.7 ± 23.8 (n = 7), and 53.7 ± 18.0 pA/pF (n = 8), for ATP, GTP, and UTP respectively (Fig. 6 B). The nucleotide diphosphates GDP and
UDP were much less effective than the nucleotide
triphosphates at enhancing IAC, but both did significantly increase IAC over the control value observed in the absence of nucleotides (Fig. 6 B).
Current-Voltage Characteristics of Nucleotide-activated K+ Current
The IV relationships for the noninactivating K+ currents induced by ATP, GTP, and UTP were similar and
indicated that, regardless of the nucleotide, a large
fraction of IAC channels are open at membrane potentials at least as negative as 40 mV. In these experiments, IAC was selectively activated by applying voltage
steps of varying size from a holding potential of
40
mV. Fig. 7, A and B illustrates typical IV relationships
obtained with 5 mM ATP and 5 mM UTP. Sustained
outward currents were present at the holding potential. The K+ current observed in response to depolarizing
steps consisted of an apparently instantaneous component and a time-dependent fraction that became more
prominent with stronger depolarizations (Fig. 7 A).
Similar outwardly rectifying currents were observed with all three nucleotide triphosphates.
The characteristics of IAC currents observed in current-voltage relationships suggested that IAC K+ channels are at most weakly voltage dependent over potentials ranging from 40 to +40 mV. In an effort to
determine to what extent the outwardly rectifying properties of IAC were due to the conductance properties of
open channels as opposed to voltage-dependent activation, we compared the steady state IV for the IAC current to the instantaneous current-voltage relationship.
The open channel IV (IIV) provides a measure of the
open channel conductance properties.
The IIV for IAC was obtained by selectively activating
this current from a holding potential of 40 mV with
150-s depolarizing steps to +50 mV, after which the
membrane potential was stepped to new levels between
+40 and
120 mV. The IAC "tail current" was measured
after 1 ms, before a significant change in the number of open channels occurred (Fig. 7 C).
An estimate of the voltage dependence of IAC activation over the range of potentials from 40 to +40 mV
was obtained by dividing current amplitudes taken
from the steady state IV relationship by corresponding
amplitudes from the IIVs. Dividing the steady state IV
values obtained with ATP in Fig. 7 B by the IIV values from Fig. 7 C indicated that the open probability of IAC
channels increased by only 30% over this 80-mV range
of potentials.
The weak voltage dependence of IAC open probability
indicated that the current-voltage characteristics of IAC
are due to the conductance properties of open channels. Accordingly, in a previous study using elevated external K+, we have shown IAC to be an outwardly rectifying current (Enyeart et al., 1996b).
Effect of Other Agents on IAC
Each of the nucleotides that activated IAC is a polyvalent
anion that can bind polyvalent metal cations. Nucleotide binding of polyvalent trace metals may activate a
calcium-activated chloride current in Xenopus oocytes
(Hilgemann, 1997). It is unlikely that nucleotide chelation of metals activates IAC in AZF cells since EDTA, a
nonspecific metal ion chelator, failed to activate this K+ current when included in the pipette solution.
When whole cell recordings were made with standard
pipette solution supplemented with 0.1 mM MgATP,
IAC reached a maximum density of 8.45 ± 2.74 pA/pF
(n = 17). The addition of EDTA (1 mM) to this pipette
solution did not increase IAC. In the presence of 1 mM
EDTA, IAC reached a maximum density of 6.24 ± 4.56 pA/pF (n = 6).
Although IAC channels are activated rather than inhibited by ATP, modulation by this nucleotide could
indicate that IAC channels are structurally similar to
ATP-sensitive K+ channels. Inwardly rectifying ATP-sensitive K+ channels such as those found in pancreatic cells display a distinctive pharmacology. K+ channel activators such as pinacidil dramatically enhance the activity of these channels (Takano and Noma, 1993
).
However, pinacidil at concentrations of 30 (n = 4) and
100 (n = 2) µM failed to measurably increase IAC amplitude in whole-cell recordings.
ACTH and ATP Hydrolysis
The activity of IAC K+ channels is promoted by a number of nucleotide triphosphates including the nonhydrolyzable ATP analog AMP-PNP. Apparently, activation of IAC channels by these nucleotides requires only
binding to the channel or an associated protein. Relatedly, ACTH and its primary intracellular messenger,
cAMP, can both inhibit IAC by an A-kinase-independent mechanism requiring ATP hydrolysis (Enyeart et
al., 1996b). 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 via an ATPase.
To test this model and clarify the mechanism of
ACTH, we took advantage of our finding that UTP
(and GTP) could activate IAC almost as effectively as
ATP. However, unlike ATP, UTP is not a substrate for
enzymes including adenylate cyclase, protein kinases, and most ATPases. As previously reported (Mlinar et
al., 1993a; Enyeart et al., 1996b
), when the pipette contains standard solution supplemented with 5 mM ATP
and 200 µM GTP, ACTH inhibits IAC almost completely
within 3-5 min (n > 50) (Fig. 8 A). When UTP replaced ATP in the recording pipette, ACTH was totally
ineffective (n = 3) (Fig. 8 B). ACTH was also ineffective
when GTP replaced ATP (n = 3) (data not shown).
The addition of only 50 µM ATP to a pipette solution
containing 5 mM UTP restored the characteristic near
complete inhibition of IAC by ACTH (Fig. 8 C). In each
of three cells, IAC was inhibited by >90% under these
conditions.
These results indicate that ACTH does not close IAC channels through ATPase-catalyzed hydrolysis of ATP at the nucleotide binding site since the activating site presumably remained occupied by UTP. The findings also convincingly demonstrate that the outwardly rectifying, noninactivating K+ current activated by ATP and UTP in AZF cells are the identical ACTH-inhibited IAC current.
The central finding of this study is that over a physiological range of concentrations, ATP, in hydrolyzable
or nonhydrolyzable forms, dramatically increases IAC
K+ current in bovine AZF cells through a mechanism
that presumably requires only the binding of this nucleotide to the channel or a related protein. A number of
other nucleotides, including ADP, UTP, and GTP were
also effective at enhancing IAC activity. With any of
these nucleotides in the pipette, a significant fraction of IAC channels remained tonically active at membrane
potentials at least as negative as 40 mV and the open
probability increased little between
40 and +40 mV.
Although the opening of IAC K+ channels under physiological conditions may require only the binding of ATP
to the IAC channel, its inhibition by ACTH appears to
proceed through a mechanism other than the hydrolysis of this nucleotide. Due to their capacity to sense cellular ATP levels and set the membrane potential of AZF
cells, IAC K+ channels may act as transducers that couple metabolic signals to membrane depolarization and
cortisol secretion.
Modulation of IAC K+ Current by Adenine Nucleotides
The activation of IAC K+ channels by both hydrolyzable
and nonhydrolyzable forms of ATP clearly distinguishes
these channels from the ATP-sensitive K+ channels described in many other cells. These inwardly rectifying channels are uniformly inhibited by the nonhydrolytic
binding of ATP and analogs (Ashcroft, 1988a). However, in a number of ATP-inhibited K+ channels, low
concentrations of MgATP (<100 µM) are actually required to maintain K+ channel activity. Phosphorylation of the channel by a protein kinase is the mechanism
involved (Lederer and Nichols, 1989
; Terzic et al., 1994
;
Levitan, 1994
). Regardless, when ATP concentrations are raised to the millimolar range, inwardly rectifying
ATP-sensitive K+ channels are uniformly inhibited. In
contrast, outwardly rectifying IAC channels are activated.
There are very few reports of ion channels that are directly activated by ATP binding. In human sweat glands,
the CFTR Cl channel has been reported to be activated
by both hydrolyzable and nonhydrolyzable forms of
ATP at millimolar concentrations (Quinton and Reddy,
1992
). However, this channel appears to differ from
the IAC channel in that the CFTR channel requires
A-kinase-dependent phosphorylation, as well as ATP
binding, for activity. Enhanced activity of voltage-gated
L-type Ca2+ channels in heart cells by nonhydrolyzable
ATP analogs has also been reported (O'Rourke et al.,
1992
). IAC may be the first example of a K+-selective
channel that is directly activated by nonhydrolytic binding of ATP.
In a variety of cells that express ATP-sensitive K+
channels, including myocytes, pancreatic cells, and
neurons, the inhibitory actions of ATP are antagonized
by ADP, thereby tightly coupling channel activity to the
energetic state of the cell (Ashcroft, 1988a
; Takano and
Noma, 1993
; Terzic et al., 1994
). In AZF cells, ADP and
ATP each enhance the IAC K+ current, although ADP is
less effective. Thus, in contrast to many cells where ATP
and ADP exert opposing actions on ATP-sensitive K+
channels, in bovine AZF cells both nucleotides are agonists. It is possible that at some [ATP]/[ADP] ratios,
the less effective ADP might antagonize the stimulatory
action of ATP on IAC channels.
In contrast to ATP and AMP-PNP, the poorly hydrolyzable ATP analog ATP--S failed to enhance IAC current and was actually inhibitory. While ATP-
-S is a
poor substrate for ATPases and phosphatases, it is a surprisingly good substrate for most kinases (Eckstein,
1985
). Further, the phosphorothioate that is transferred from ATP-
-S is poorly hydrolyzed. Proteins normally regulated by a phosphorylation/dephosphorylation cycle end up highly thiophosphorylated. This
would suggest that phosphorylation of IAC channels by
an unidentified protein kinase inhibits IAC and overrides enhancement of channel activity by ATP. The reduction, rather than increase, in IAC K+ current amplitude
observed in the first few minutes of whole-cell recording with pipettes containing ATP-
-S is consistent with this model. Presumably, ATP-
-S binds to the nucleotide binding site associated with enhanced IAC activity.
However, this action is negated by a slowly reversible
phosphorylation. This model would also account for
the dramatically different effects of ATP-
-S and AMP-PNP on K+ current in AZF cells.
The slowing of IA inactivation kinetics induced by
ATP--S may also be due to the slowly hydrolyzable
thiophosphorylation of the normally rapidly inactivating IA K+ channels. The inactivation kinetics of several
A type K+ channels are regulated by protein kinase-mediated phosphorylation that can either accelerate or
slow inactivation (Covarrubias et al., 1994
; Drain et al.,
1994
).
Activation of IAC by Other Nucleotides
Other nucleotide triphosphates including GTP and
UTP increased IAC K+ current in a manner similar to
ATP. Accordingly, ATP-sensitive K+ channels in skeletal
muscle and ventricular myocytes are inhibited by GTP
and UTP, although less effectively than by ATP itself
(Spruce et al., 1987; Lederer and Nichols, 1989
). In
contrast to the inhibitory effects of nucleotide triphosphates on many ATP-sensitive channels, various nucleotide diphosphates including UDP and GDP activate these same channels (Takano and Noma, 1993
; Terzic
et al., 1994
). Thus, while nucleotide di- and triphosphates exert opposing effects on the activity of classic
ATP-sensitive channels, these nucleotides have qualitatively similar effects on IAC channel activity.
Our results indicate that a diverse group of nucleotides, including purine and pyrimidine triphosphates
and diphosphates, alone or complexed with Mg2+, can
activate IAC K+ channels through nonhydrolytic binding. Two nucleotide binding domains are present on
ATP-sensitive K+ channel-associated sulfonylurea receptors that form the subunit of these channels in
various cells (Aguilar-Bryan et al., 1995
). Although the
nucleotide binding site(s) on IAC channels seem to be
similar with regard to lack of nucleotide specificity, the number and location of these sites on IAC channels remain to be identified.
The sulfonylurea receptor-coupled ATP-sensitive K+
channels are inwardly rectifying and, unlike most K+
channels, include two rather than six membrane-spanning domains (Ho et al., 1993; Aguilar-Bryan et al.,
1995
). The structure of the novel outwardly rectifying
IAC channel has not been determined. However, the ineffectiveness of pinacidil in enhancing IAC current does
not suggest a similarity between IAC and ATP-inhibited
channels. Further, in other experiments, we have found
that the sulfonylurea glibenclamide, which potently inhibits inwardly rectifying ATP-sensitive K+ channels, is
much less effective at inhibiting IAC K+ channels (our
unpublished observations).
ATP and IAC Channel Gating
Gating of IAC channels appears to be controlled by the
complex interaction of metabolic factors in conjunction with a weak voltage dependence. In spite of the
dramatic enhancement of IAC K+ current by ATP, the
mechanism involved is not yet clear. Perhaps the binding of ATP alone is sufficient to activate the channel, regardless of membrane potential. Whole-cell and single-channel recordings did indicate that, with ATP and
other nucleotides in the patch electrode, a large fraction of IAC channels are open at 40 mV. Channel
open probability increased by only ~0.3 between
40
and +40 mV. In this regard, when ATP is excluded
from the pipette in whole-cell recordings, IAC current
cannot be activated at test potentials as positive as +70
mV. Thus, if the binding of ATP shifts the voltage dependence of the channel such that IAC channels open
at more negative potentials, then this shift must be very
large (i.e., >100 mV). A quantitative study of IAC activation at more negative potentials and the possible effects of ATP on the process is hampered by the copresence
of the IA K+ current as well as the outwardly rectifying
nature of IAC itself.
It may be possible to study the modulation of single
IAC channels by ATP in excised inside-out patches.
However, in excised patches, the activity of IAC channels
is quite variable and unstable. ACTH and membrane-permeable forms of cAMP do not reliably inhibit single
IAC channels in outside-out patches as they do in whole-cell recordings (our unpublished observations). Likewise, in an extensive study of ATP-sensitive K+ channels
in rat heart, a large variability in ATP sensitivity and
Hill coefficients was observed. Apparent Kds for ATP
ranged from 9-580 µM. ATP sensitivity of the cardiac
channel also decreased during the course of an experiment (Findlay and Faivre, 1991). Overall, in excised
patches from a variety of cells, ATP inhibits these inward rectifier K+ channels at concentrations far below
physiological concentrations of this nucleotide (Ashcroft, 1988a
; Terzic et al., 1994
; Takano and Noma,
1993
). In contrast, it is clear from our studies using whole-cell recording in AZF cells that only physiological concentrations of ATP enhance the activity of IAC
channels over a wide range of potentials.
ACTH and ATP Hydrolysis
In the present study, we have demonstrated that the
binding of ATP dramatically enhances the activity of IAC
channels measured in response to membrane depolarization. Previously, we had demonstrated that ACTH
and cAMP can each inhibit IAC K+ current by a mechanism that is independent of A kinase activation while
requiring ATP hydrolysis. Taken together, these results suggested that the gating of IAC could be controlled
through an ATP hydrolysis cycle, as has been observed
for CFTR Cl channels of the heart (Baukrowitz et al.,
1994
). If ACTH-mediated hydrolysis of ATP at its binding site on the IAC channel resulted in channel closing,
then it is unlikely that channels activated by UTP would
be effected since UTP is not a substrate for most ATPases (Azhar and Menon, 1975
; Krebs and Beavo, 1979
;
Edelman et al., 1987
). Therefore, the effective inhibition of UTP-activated IAC channels by ACTH in the
presence of only 50 µM ATP argues strongly against a
model for channel closing requiring hydrolysis of this
nucleotide by an ATPase. However, an unusual class of ATPases that hydrolyze a variety of nucleotides including UTP has been described (Beukers et al., 1993
).
In contrast to most ATPases that have Kms of one to
several millimolar, protein kinases are typically activated by ATP at 100-1,000-fold lower concentrations
(Krebs and Beavo, 1979). ACTH may inhibit IAC channels through activation of an unidentified protein kinase. The suppression of IAC by ATP-
-S is consistent with this model.
ATP Sensing, Membrane Potential, and Cortisol Secretion
The resting potential of bovine AZF cells approaches
the Nernst equilibrium potential for K+ (Mlinar et al.,
1993a). Of the two detectable K+ currents expressed by
these cells, IAC channels display properties consistent
with one that would contribute strongly to the resting potential. The activation of IAC K+ channels by physiological levels of ATP suggests that these membrane proteins could act as sensors coupling the metabolic state
of the cell to membrane potential and ultimately, cortisol secretion (Enyeart et al., 1993
).
In insulin-secreting cells of the pancreas, ATP-sensitive K+ channels play a key role in excitation-secretion
coupling. High blood glucose levels are associated with
elevated ATP, K+ channel inhibition, and membrane
depolarization leading to Ca2+ entry and insulin secretion (Ashcroft, 1988a, 1988b). In bovine AZF cells
where IAC channels are activated rather than inhibited by ATP, elevated glucose would be associated with IAC
activation and membrane hyperpolarization, thereby
suppressing Ca2+ entry and cortisol secretion (Enyeart
et al., 1993
). Thus, because ATP-sensitive K+ channel
activity in AZF cells and pancreatic
cells is modulated in opposite directions by ATP, metabolic conditions inducing insulin secretion might be expected to suppress
cortisol production. In this regard, cortisol is secreted
under conditions of metabolic stress such as starvation,
whereas insulin secretion is suppressed (Bondy, 1985
).
Metabolically, the glucose-conserving hormone cortisol has effects opposing those of insulin. Cortisol stimulates gluconeogenesis and inhibits glucose uptake and
use in many tissues (Bondy, 1985). Because of the antagonistic actions of these two hormones on glucose metabolism, it may then be appropriate that the cells
that secrete them express ATP-sensitive K+ channels
whose activity is regulated by ATP in opposite directions.
Although the above scheme linking cellular ATP levels to K+ channels and secretion of two opposing hormones is attractive, cortisol secretion occurs primarily
under the control of ACTH released by the pituitary.
However, in bovine AZF cells, ACTH triggers depolarization-dependent Ca2+ entry and cortisol secretion
through inhibition of IAC (Enyeart et al., 1993). It
would be interesting to determine whether ACTH- mediated IAC inhibition could be modulated by altering
external glucose concentration. Irrespective of its function in cortisol secretion, IAC is a distinctive new type of
K+ channel that is activated rather than inhibited by
ATP. Perhaps other cells that secrete hormones with
antiinsulin effects (e.g., glucagon, catecholamines) also
express ATP-activated K+ channels.
Address correspondence to Dr. John J. Enyeart, Department of Pharmacology, The Ohio State University, College of Medicine, 5188 Graves Hall, 333 W. 10th Avenue, Columbus, OH 43210-1239. Fax: 614-292-7232; E-mail: enyeart.1{at}osu.edu
Received for publication 23 July 1997 and accepted in revised form 1 October 1997.
1 Abbreviations used in this paper: ACTH, adrenocorticotropic hormone; AII, angiotensin II; AZF, bovine adrenal fasciculata; CFTR, cystic fibrosis transmembrane conductance regulator; IV, current-voltage relationship.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.
1. |
Aguilar-Bryan, L.,
C.G. Nichols,
S.W. Wechsler,
J.P.I. Clement,
A.E. Boyd III,
G. Gonzalez,
H. Herrera-Sosa,
K. Nguy,
J. Bryan, and
D.A. Nelson.
1995.
Cloning of the ![]() |
2. |
Ashcroft, F.M..
1988a.
Adenosine 5![]() |
3. |
Ashcroft, F.M.,
S.J.H. Ashcroft, and
D.E. Harrison.
1988b.
Properties of single potassium channels modulated by glucose in rat
pancreatic ![]() |
4. |
Azhar, S., and
K.M. Menon.
1975.
Adenosine 3![]() ![]() |
5. |
Baukrowitz, T.,
T.-C. Hwang,
A.C. Nairn, and
D.C. Gadsby.
1994.
Coupling of CFTR Cl![]() |
6. | Beukers, M.W., I.M. Pirovano, A. Van Weert, C.J. Kerkhof, A.P. Ijzerman, and W. Soudijn. 1993. Characterization of ecto-ATPase on human blood cells. A physiological role in platelet aggregation? Biochem. Pharmacol. 46: 1959-1966 [Medline]. |
7. | Bondy, P.K. 1985. Diseases of the adrenal gland. In Williams Textbook of Endocrinology. W.B. Saunders Company, Philadelphia, PA. 816-890. |
8. | Brooks, S.P.J., and K.B. Storey. 1992. Bound and determined: a computer program for making buffers of defined ion concentrations. Anal. Biochem 201: 119-126 [Medline]. |
9. | Covarrubias, M., A. Wei, L. Salkoff, and T.B. Vyas. 1994. Elimination of rapid potassium channel inactivation by phosphorylation of the inactivation gate. Neuron. 13: 1403-1412 [Medline]. |
10. | Drain, P., A.E. Dubin, and R.W. Aldrich. 1994. Regulation of shaker K+ channel inactivation gating by the cAMP-dependent protein kinase. Neuron. 12: 1097-1109 [Medline]. |
11. | Eckstein, F.. 1985. Nucleoside phosphorothiates. Annu. Rev. Biochem 54: 367-402 [Medline]. |
12. | Edelman, A.M., D.K. Blumenthal, and E.G. Krebs. 1987. Protein serine/threonine kinases. Annu. Rev. Biochem 56: 567-613 [Medline]. |
13. | Enyeart, J.J., R.T. Boyd, and J.A. Enyeart. 1996a. ACTH and AII differentially stimulate steroid hormone orphan receptor mRNAs in adrenal cortical cells. Mol. Cell. Endocrinol. 124: 97-110 [Medline]. |
14. | Enyeart, J.J., B. Mlinar, and J.A. Enyeart. 1993. T-type Ca2+ are required for ACTH-stimulated cortisol synthesis by bovine adrenal zona fasciculata cells. Mol. Endocrinol. 7: 1031-1040 [Abstract]. |
15. | Enyeart, J.J., B. Mlinar, and J.A. Enyeart. 1996b. Adrenocorticotropic hormone and cAMP inhibit noninactivating K+ current in adrenocortical cells by an A-kinase-independent mechanism requiring ATP hydrolysis. J. Gen. Physiol 108: 251-264 [Abstract]. |
16. | Fakler, B., U. Brandle, E. Glowaatzki, H.-P. Zenner, and J.P. Ruppersberg. 1994. Kir2.1 inward rectifier K+ channels are regulated independently by protein kinases and ATP hydrolysis. Neuron 13: 1413-1420 [Medline]. |
17. | Findlay, I., and J.F. Faivre. 1991. ATP-sensitive K+ channels in heart muscle: spare channels. FEBS Lett 279: 95-97 [Medline]. |
18. | Gospodarowicz, D., C.R. Ill, P.J. Hornsby, and G.N. Gill. 1977. Control of bovine adrenal cortical cell proliferation by fibroblast growth factor. Lack of effect of epidermal growth factor. Endocrinology 100: 1080-1089 [Abstract]. |
19. | Hamill, O.P., A. Marty, E. Neher, B. Sakmann, and F.J. Sigworth. 1981. Improved patch clamp techniques for high resolution current recording from cells and cell-free membrane patches. Pflügers Arch 391: 85-100 [Medline]. |
20. | Hilgemann, D.W.. 1997. Cytoplasmic ATP-dependent regulation of ion transporters and channels: mechanisms and messsengers. Annu. Rev. Physiol. 59: 193-220 [Medline]. |
21. | Ho, K., C.G. Nichols, W.J. Lederer, J. Lytton, P.M. Vassilev, M.V. Kanazirska, and S.C. Hebert. 1993. Cloning and expression of an inwardly rectifying ATP-regulated potassium channel. Nature. 362: 31-37 [Medline]. |
22. | Hwang, T., G. Nagel, A.C. Nairn, and D.C. Gadsby. 1994. Regulation of the gating of cystic fibrosis transmembrane conductance regulator Cl channels by phosphorylation and ATP hydrolysis. Proc. Natl. Acad. Sci. USA. 91: 4698-4702 [Abstract]. |
23. | Krebs, E.G., and J.A. Beavo. 1979. Phosphorylation-dephosphorylation of enzymes. Annu. Rev. Biochem. 48: 923-959 [Medline]. |
24. | Lederer, W.J., and C.G. Nichols. 1989. Nucleotide modulation of the activity of rat heart ATP-sensitive K+ channels in isolated membrane patches. J. Physiol. (Camb.) 419: 193-211 [Abstract]. |
25. | Levitan, I.B.. 1994. Modulation of ion channels by protein phosphorylation and dephosphorylation. Annu. Rev. Physiol. 56: 193-212 [Medline]. |
26. |
Mlinar, B.,
B.A. Biagi, and
J.J. Enyeart.
1993a.
A novel K+ current
inhibited by ACTH and Angiotensin II in adrenal cortical cells.
J.
Biol. Chem
268:
8640-8644
|
27. |
Mlinar, B.,
B.A. Biagi, and
J.J. Enyeart.
1995.
Losartan-sensitive AII
receptors linked to depolarization-dependent cortisol secretion
through a novel signaling pathway.
J. Biol. Chem.
270:
20942-20951
|
28. | Mlinar, B., and J.J. Enyeart. 1993b. Voltage-gated transient currents in bovine adrenal fasciculata cells II: A-type K+ current. J. Gen. Physiol. 102: 239-255 [Abstract]. |
29. | O'Rourke, B., P.H. Backx, and E. Marban. 1992. Phosphorylation-independent modulation of L-type calcium channels by magnesium-nucleotide complexes. Science. 257: 245-248 [Medline]. |
30. | Quinton, P.M., and M.M. Reddy. 1992. Control of CFTR chloride conductance by ATP levels through non-hydrolytic binding. Nature. 360: 79-81 [Medline]. |
31. |
Spruce, A.E.,
N.B. Standen, and
P.R. Stanfield.
1987.
Studies of the
unitary properties of adenosine-5![]() |
32. | Takano, M., and A. Noma. 1993. The ATP-sensitive K+ channel. Prog. Neurobiol. (Oxf.). 41: 21-30 [Medline]. |
33. | Terzic, A., R.T. Tung, and Y. Kurachi. 1994. Nucleotide regulation of ATP sensitive potassium channels. Cardiovasc. Res. 28: 746-753 [Medline]. |