Properties of ATP-dependent K+ channels in adrenocortical cells

Lin Xu and John J. Enyeart

Department of Neuroscience, Ohio State University, College of Medicine, Columbus, Ohio 43210-1239


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

potassium channel; adenosine 5'-triphosphate; nucleotide; angiotensin II


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
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INTRODUCTION
MATERIALS AND METHODS
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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) (GDPbeta 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 × 10-8 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 MOmega were fabricated from Corning 0010 glass (World Precision Instruments, Sarasota, FL). These electrodes routinely yielded access resistances of 1.5-4 MOmega and voltage-clamp time constants of <100 µs. For single-channel recordings, patch electrodes with higher resistances of 3-5 MOmega 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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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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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.

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.

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.

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.

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 (open circle ) 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.

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.

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 (open circle ) and without depolarizing prepulses (). Numbers correspond to traces (top).

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.

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.

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).

                              
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Table 1.   Effect of MgATP, NaUTP, and PPPi on membrane potential

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 (open circle ) 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.

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 (open circle ) 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.

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).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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).


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-47875 (to J. J. Enyeart).


    FOOTNOTES

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.

Received 13 April 2000; accepted in final form 28 August 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aguilar-Bryan, L, Nichols CG, Wechsler SW, Clement JPI, Boyd AE, III, Gonzalez G, Herrera-Sosa H, Nguy K, Bryan J, and Nelson DA. Cloning of the beta  cell high-affinity sulfonylurea receptor: a regulator of insulin secretion. Science 268: 423-429, 1995[ISI][Medline].

2.   Akiyama, T, and Ogawara H. Use and specificity of genistein as inhibitor of protein-tyrosine kinases. In: Methods in Enzymology, edited by Hunter T, and Sefton BM.. New York: Academic, 1991, vol. 201, p. 362-369.

3.   Ambroz, C, and Catt KJ. Angiotensin II receptor-mediated calcium influx in bovine adrenal glomerulosa cells. Endocrinology 131: 408-413, 1992[Abstract].

4.   Anderson, MP, Berger HA, Rich DP, Gregory RJ, Smith AE, and Welsh MJ. Nucleoside triphosphates are required to open the CFTR chloride channel. Cell 67: 775-784, 1991[ISI][Medline].

5.   Ashcroft, FM. Adenosine 5'-triphosphate-sensitive potassium channels. Annu Rev Physiol 11: 97-118, 1988.

6.   Azhar, S, and Menon KM. Adenosine 3':5'-cyclic monophosphate-dependent and plasma-associated protein kinase(s) form bovine corpus luteum. Biochem J 151: 23-36, 1975[ISI][Medline].

7.   Baukrowitz, T, Hwang T-C, Nairn AC, and Gadsby DC. Coupling of CFTR Cl- channel gating to an ATP hydrolysis cycle. Neuron 12: 473-482, 1994[ISI][Medline].

8.   Baukrowitz, T, Schulte U, Oliver D, Herlitz S, Krauter T, Tucker SJ, Ruppersberg JP, and Fakler B. PIP2 and PIP as determinants for ATP inhibition of KATP channels. Science 282: 1141-1144, 1998[Abstract/Free Full Text].

9.   Bondy, PK. Diseases of the Adrenal Gland. In: Williams Textbook of Endocrinology. Philadelphia: Saunders, 1985, p. 816-890.

10.   Brooks, SPJ, and Storey KB. Bound and determined: a computer program for making buffers of defined ion concentrations. Anal Biochem 201: 119-126, 1992[ISI][Medline].

11.   Bruggemann, A, Pardo LA, Stuhmer W, and Pongs O. Ether-a-go-go encodes a voltage-gated channel permeable to K+ and Ca2+ and modulated by cAMP. Nature 365: 445-448, 1993[ISI][Medline].

12.   Combeau, C, and Carlier M-F. Probing the mechanism of ATP hydrolysis on F-actin using vanadate and the structural analogs of phosphate BeF3- and AlF4-. J Biol Chem 263: 17429-17436, 1988[Abstract/Free Full Text].

13.   Dudley, DT, Panek RL, Major TC, Lu GH, Bruns RF, Klinkefus BA, Hodges JC, and Weishaar RE. Subclasses of angiotensin II binding sites and their functional significance. Mol Pharmacol 38: 370-377, 1990[Abstract].

14.   Enyeart, JJ, Gomora JC, Xu L, and Enyeart JA. Adenosine triphosphate activates a noninactivating K+ current in adrenal cortical cells through nonhydrolytic binding. J Gen Physiol 110: 679-692, 1997[Abstract/Free Full Text].

15.   Enyeart, JJ, Mlinar B, and Enyeart JA. T-type Ca2+ are required for ACTH-stimulated cortisol synthesis by bovine adrenal zona fasciculata cells. Mol Endocrinol 7: 1031-1040, 1993[Abstract].

16.   Enyeart, JJ, Mlinar B, and Enyeart JA. 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, 1996[Abstract].

17.   Fan, Z, and Makielski JC. Anionic phospholipids activate ATP-sensitive potassium channels. J Biol Chem 272: 5388-5395, 1997[Abstract/Free Full Text].

18.   Glynn, IM, and Hoffman JF. Nucleotide requirements for sodium-sodium exchange catalysed by the sodium pump in human red cells. J Physiol (Lond) 218: 239-256, 1971[ISI][Medline].

19.   Gomora, JC, and Enyeart JJ. Ca2+ depolarizes adrenal cortical cells through selective inhibition of an ATP-activated K+ current. Am J Physiol Cell Physiol 275: C1526-C1537, 1998[Abstract/Free Full Text].

20.   Gomora, JC, and Enyeart JJ. Dual pharmacological properties of a cyclic AMP-sensitive potassium channel. J Pharmacol Exp Ther 290: 266-275, 1999[Abstract/Free Full Text].

21.   Gomora, JC, Xu L, Enyeart JA, and Enyeart JJ. Effect of mibefradil on voltage-dependent gating and kinetics of T-type Ca2+ channels in cortisol-secreting cells. J Pharmacol Exp Ther 292: 96-103, 2000[Abstract/Free Full Text].

22.   Gunderson, KL, and Kopito RR. Effects of pyrophosphate and nucleotide analogs suggest a role for ATP hydrolysis in cystic fibrosis transmembrane regulator channel gating. J Biol Chem 269: 19349-19353, 1994[Abstract/Free Full Text].

23.   Gunderson, KL, and Kopito RR. Conformational states of CFTR associated with channel gating: the role of ATP binding and hydrolysis. Cell 82: 231-239, 1995[ISI][Medline].

24.   Guy, HR, and Durell SR. Similarities in amino acid sequences of Drosophila eag and cyclic nucleotide-gated channels. Science 254: 730-730, 1991[ISI][Medline].

25.   Hamill, OP, Marty A, Neher E, Sakmann B, and Sigworth FJ. Improved patch clamp techniques for high resolution current recording from cells and cell-free membrane patches. Pflügers Arch 391: 85-100, 1981[ISI][Medline].

26.   Heginbotham, L, Abramason T, and MacKinnon R. A functional connection between the pores of distantly related ion channels as revealed by mutant K+ channels. Science 258: 1152-1155, 1992[ISI][Medline].

27.   Hilgemann, DW. Cytoplasmic ATP-dependent regulation of ion transporters and channels: mechanisms and messengers. Annu Rev Physiol 59: 193-220, 1997[ISI][Medline].

28.   Hille, B. Ionic Channels of Excitable Membranes. Sunderland, MA: Sinauer, 1992.

29.   Huang, C-L, Feng S, and Hilgemann DW. Direct activation of inward rectifier potassium channels by PIP2 and its stabilization by Gbg. Nature 391: 803-806, 1998[ISI][Medline].

30.   Hwang, T-C, Nagel G, Nairn AC, and Gadsby DC. 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, 1994[Abstract].

31.   Jan, LY, and Jan YN. Cloned potassium channels from eukaryotes and prokaryotes. Annu Rev Neurosci 20: 91-123, 1997[ISI][Medline].

32.   Ketchum, KA, Joiner WJ, Sellers AJ, Kaczmarek LK, and Goldstein SA. A new family of outwardly rectifying potassium channel proteins with two pore domains in tandem. Nature 376: 690-695, 1995[ISI][Medline].

33.   Krebs, EG, and Beavo JA. Phosphorylation-dephosphorylation of enzymes. Annu Rev Biochem 48: 923-959, 1979[ISI][Medline].

34.   Lemaire, S, Labrie F, and Gauthier M. Adenosine-3',5'-monophosphate-dependent protein kinase from bovine anterior pituitary gland. Can J Biochem 52: 137-141, 1974[ISI][Medline].

35.   Levitzki, A, and Gazit A. Tyrosine kinase inhibition: an approach to drug development. Science 267: 1782-1788, 1995[ISI][Medline].

36.   Marrero, MB, Schieffer B, Paxton WG, Heerdt L, Berk BC, Delafontaine P, and Bernstein KE. Direct stimulation of Jak/STAT pathway by the angiotensin II AT1 receptor. Nature 375: 247-250, 1995[ISI][Medline].

37.   Matsuda, H, Saigusa A, and Irisawa H. Ohmic conductance through the inwardly rectifying K channel and blocking by internal Mg2+. Nature 325: 156-159, 1987[ISI][Medline].

38.   Meydan, N, Grunberger T, Dadi H, Shahar M, Arpaia E, Lapidot Z, Leeder JS, Freedman M, Cohen A, Gazit A, Levitzki A, and Roifman CM. Inhibition of a acute lymphoblastic leukaemia by a Jak-2 inhibitor. Nature 379: 645-648, 1996[ISI][Medline].

39.   Mlinar, B, Biagi BA, and Enyeart JJ. A novel K+ current inhibited by ACTH and angiotensin II in adrenal cortical cells. J Biol Chem 268: 8640-8644, 1993[Abstract/Free Full Text].

40.   Mlinar, B, Biagi BA, and Enyeart JJ. Losartan-sensitive AII receptors linked to depolarization-dependent cortisol secretion through a novel signaling pathway. J Biol Chem 270: 20942-20951, 1995[Abstract/Free Full Text].

41.   Mlinar, B, and Enyeart JJ. Voltage-gated transient currents in bovine adrenal fasciculata cells II: A-type K+ current. J Gen Physiol 102: 239-255, 1993[Abstract].

42.   Molloy, CJ, Taylor DS, and Weber H. Angiotensin II stimulation of rapid protein tyrosine phosphorylation and protein kinase activation in rat aortic smooth muscle cells. J Biol Chem 268: 7338-7345, 1993[Abstract/Free Full Text].

43.   Nicol, GD. The calcium channel antagonist, pimozide, blocks the cyclic GMP-activated current in rod photoreceptors. J Pharmacol Exp Ther 265: 626-632, 1993[Abstract].

44.   Shyng, S-L, and Nichols CG. Membrane phospholipid control of nucleotide sensitivity of KATP channels. Science 282: 1138-1141, 1998[Abstract/Free Full Text].

45.   Tamaoki, T. Use and specificity of staurosporine, UCN-01 and calphostin C as protein kinase inhibitors. In: Methods in Enzymology, edited by Hunter T, and Sefton BM.. New York: Academic, 1991, vol. 201, p. 340-347.

46.   Tian, Y, Smith RD, Balla T, and Catt KJ. Angiotensin II activates mitogen-activated protein kinase via protein kinase C and Ras/Raf-1 kinase in bovine adrenal glomerulosa cells. Endocrinology 139: 1801-1809, 1998[Abstract/Free Full Text].

47.   Xu, L, and Enyeart JJ. Adenosine inhibits a noninactivating K+ current in adrenal cortical cells by activation of multiple P1 receptors. J Physiol (Lond) 521: 81-97, 1999[Abstract/Free Full Text].

48.   Xu, L, and Enyeart JJ. Purines and pyrimidine nucleotides inhibit a noninactivating K+ current and depolarize adrenal cortical cells through a G protein-coupled receptor. Mol Pharmacol 55: 364-376, 1999[Abstract/Free Full Text].

49.   Yang, H, Yu K, and Raizada MK. Regulation of neuromodulatory actions of angiotensin II in the brain neurons by the Ras-dependent mitogen-activated protein kinase pathway. J Neurosci 16: 4047-4058, 1996[Abstract/Free Full Text].


Am J Physiol Cell Physiol 280(1):C199-C215
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