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J. Biol. Chem., Vol. 280, Issue 35, 30814-30828, September 2, 2005

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Angiotensin II Inhibits bTREK-1 K⁺ Channels in Adrenocortical Cells by Separate Ca²⁺- and ATP Hydrolysis-dependent Mechanisms^*

John J. Enyeart, Sanjay J. Danthi¶||, Haiyan Liu, and Judith A. Enyeart

From the Department of Neuroscience, The Ohio State University College of Medicine and Public Health, Columbus, Ohio 43210-1239 and ^¶The Mathematical Biosciences Institute, The Ohio State University, Columbus, Ohio 43210

Received for publication, April 19, 2005 , and in revised form, June 30, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Bovine adrenocortical cells express bTREK-1 K⁺ channels that set the resting membrane potential (V_m) and couple angiotensin II (AngII) and adrenocorticotropic hormone (ACTH) receptors to membrane depolarization and corticosteroid secretion. In this study, it was discovered that AngII inhibits bTREK-1 by separate Ca²⁺- and ATP hydrolysis-dependent signaling pathways. When whole cell patch clamp recordings were made with pipette solutions that support activation of both Ca²⁺- and ATP-dependent pathways, AngII was significantly more potent and effective at inhibiting bTREK-1 and depolarizing adrenal zona fasciculata cells, than when either pathway is activated separately. External ATP also inhibited bTREK-1 through these two pathways, but ACTH displayed no Ca²⁺-dependent inhibition. AngII-mediated inhibition of bTREK-1 through the novel Ca²⁺-dependent pathway was blocked by the AT₁ receptor antagonist losartan, or by including guanosine-5'-O-(2-thiodiphosphate) in the pipette solution. The Ca²⁺-dependent inhibition of bTREK-1 by AngII was blunted in the absence of external Ca²⁺ or by including the phospholipase C antagonist U73122 [GenBank] , the inositol 1,4,5-trisphosphate receptor antagonist 2-amino-ethoxydiphenyl borate, or a calmodulin inhibitory peptide in the pipette solution. The activity of unitary bTREK-1 channels in inside-out patches from adrenal zona fasciculata cells was inhibited by application of Ca²⁺ (5 or 10 µM) to the cytoplasmic membrane surface. The Ca²⁺ ionophore ionomycin also inhibited bTREK-1 currents through channels expressed in CHO-K1 cells. These results demonstrate that AngII and selected paracrine factors that act through phospholipase C inhibit bTREK-1 in adrenocortical cells through simultaneous activation of separate Ca²⁺- and ATP hydrolysis-dependent signaling pathways, providing for efficient membrane depolarization. The novel Ca²⁺-dependent pathway is distinctive in its lack of ATP dependence, and is clearly different from the calmodulin kinase-dependent mechanism by which AngII modulates T-type Ca²⁺ channels in these cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Bovine adrenocortical cells, including cortisol-secreting AZF¹ cells and aldosterone-secreting AZG cells express bTREK-1 leak-type K⁺ channels that function pivotally in the physiology of corticosteroid secretion (1, 2). bTREK-1 belongs to the mechanogated, thermo- and fatty acid-sensitive subgroup of two-pore/four-transmembrane fragment family of K⁺ channels (3–9). In the adrenal cortex, bTREK-1 channels couple hormonal signals originating at the cell membrane to depolarization-dependent Ca²⁺ entry (1, 2, 10, 11). bTREK-1 channels are inhibited by AngII and ACTH at concentrations identical to those that trigger membrane depolarization and corticosteroid secretion (1, 12). Other paracrine factors, including ATP, which stimulates cortisol secretion through a G protein-coupled P2Y₃ receptor, also inhibit bTREK-1 and depolarize AZF cells with similar potency (13, 14).

The signaling pathways that link the peptide hormones and paracrine factors to bTREK-1 inhibition are only partially understood. In particular, adrenocortical cells express two pharmacologically distinct types of AngII receptors (15–17). Losartan-sensitive AT₁ receptors are coupled to multiple signaling pathways (17–19). Most physiological responses, including AngII-stimulated corticosteroid secretion, are mediated through AT₁ receptor-dependent activation of PLC, which catalyzes the synthesis of inositol trisphosphates (IP₃) and diacylglycerol from phosphatidylinositol 4,5-bisphosphate (17, 20).

Adrenocortical cells also express losartan-insensitive AT₂ receptors, which comprise about 20% of the AngII receptors in these cells (15, 17). The signaling pathways and function of these receptors in the physiology of corticosteroid-secreting cells is unknown.

In whole cell patch clamp recordings from bovine AZF and AZG cells, AngII maximally inhibited bTREK-1 by 72–77% with an IC₅₀ of 145 pM, provided that ATP was present in the recording pipette at millimolar concentrations (1, 11, 12). AngII-mediated inhibition of bTREK-1 was eliminated when ATP in the pipette solution was replaced by the non-hydrolyzable ATP analog AMP-PNP, or UTP (12, 21). The kinase or ATPase that mediates AngII inhibition through this ATP hydrolysis-dependent pathway has not been identified.

In these whole cell recording studies, [Ca²⁺]_i was buffered to 22 nM with 11 mM BAPTA. In a separate study, we found that bTREK-1 in AZF cells was inhibited by raising the [Ca²⁺]_i in the pipette solution from 22 nM to 2 µM (22). bTREK-1 was also inhibited by superfusing cells with the Ca²⁺ ionophore ionomycin, provided that [Ca²⁺]_i was buffered by 2 mM, rather than 11 mM BAPTA. At the single channel level, in excised inside-out patch recordings, the application of saline containing 35 µM Ca²⁺ to the cytoplasmic face of the membrane markedly inhibited bTREK-1 channel activity. In these experiments, 5 mM ATP was present at the cytoplasmic face of the membrane.

Taken together, the results of these studies could suggest that AngII inhibits bTREK-1 through a single Ca²⁺- and ATP hydrolysis-dependent signaling pathway that requires the activation of a Ca²⁺-dependent kinase. In bovine AZG cells, AngII enhances the activity of T-type Ca²⁺ channels through the activation of Ca²⁺/calmodulin-dependent kinase II (23–25). Alternatively, the effective inhibition of bTREK-1 by AngII in whole cell recordings with [Ca²⁺]_i strongly buffered to 22 nM using 11 mM BAPTA suggests that AngII may inhibit bTREK-1 by multiple signaling pathways, only one of which is Ca²⁺-dependent.

In whole cell and single channel patch clamp recordings from bovine adrenocortical cells, we discovered that AngII does inhibit bTREK-1 through separate Ca²⁺ and ATP hydrolysis-dependent signaling pathways. Simultaneous activation of both pathways by AngII leads to near complete inhibition of bTREK-1 current and pronounced depolarization of bovine adrenocortical cells. Molecular features of the new Ca²⁺-dependent mechanism were identified.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Tissue culture media, antibiotics, fibronectin, and fetal bovine sera were obtained from Invitrogen. Coverslips were from Bellco (Vineland, NJ). Enzymes, BAPTA, MgATP, Na₂ATP, GDPS, ACTH-(1–24), AngII, 8-pcpt-cAMP, ionomycin, and EGTA were obtained from Sigma. U73122 [GenBank] , U73343 [GenBank] , and 2-aminoethoxydiphenyl borate (2-APB) were purchased from Biomol (Plymouth Meeting, PA). The calmodulin inhibitory peptide (CIP) was obtained from Calbiochem (La Jolla, CA). AngII receptor antagonists, losartan and PD123319, were kindly provided by Dr. Ronald Smith (Merck Pharmaceutical Co.) and Dr. Joan Keiser (Parke-Davis), respectively.

Isolation and Culture of AZF Cells—Bovine adrenal glands were obtained from steers (age 2–3 years) at a local slaughterhouse. Isolated AZF and AZG cells were obtained and prepared as previously described (26). After isolation, cells were either resuspended in Dulbecco's modified Eagle's medium/F-12 (1:1) with 10% fetal bovine serum, 100 units/ml penicillin, 0.1 mg/ml streptomycin, and the antioxidants 1 µM tocopherol, 20 nM selenite, and 100 µM ascorbic acid (Dulbecco's modified Eagle's medium/F-12+) and plated for immediate use, or resuspended in fetal bovine serum, 5% Me₂SO, divided into 1-ml aliquots, and stored in liquid nitrogen for future use. For patch clamp experiments, cells were plated in Dulbecco's modified Eagle's medium/F-12+ in 35-mm dishes containing 9-mm² glass coverslips. Coverslips were treated with fibronectin (10 µg/ml) at 37 °C for 30 min then rinsed with warm, sterile phosphate-buffered saline immediately before adding cells. Cells were maintained at 37 °C in a humidified atmosphere of 95% air, 5% CO₂.

Transient Transfection and Visual Identification of CHO-K1 or COS-7 Cells Expressing bTREK-1—For whole cell patch clamp recording of cloned bTREK-1 K⁺ currents, CHO-K1 or COS-7 cells were co-transfected with a mixture of pCR3.1 Uni-bTREK-1 and an expression plasmid (p3-CD8) for the -subunit of the human CD8 lymphocyte surface antigen at a 5:1 ratio using Lipofectamine (Invitrogen). Cells were visualized 1–2 days post-transfection after a 15-min incubation with anti-CD8 antibody-coated beads (Dynal Biotech Inc., Lake Success, NY) as described (27).

Cells were used for patch clamping 24–48 h after transfection with bTREK-1. Transfected cells were plated on 9-mm glass coverslips as described above. Fifteen min before initiating a patch clamp experiment, anti-CD8 antibody-coated beads were added to the culture dish. Upon transferring coverslips to the recording chamber, transfected cells were identified based on decoration with the beads. Whole cell and single channel bTREK-1 currents were recorded from transfected cells as described below for AZF cells.

Patch Clamp Experiments—Patch clamp recordings of K⁺ channel currents were made in the whole cell and inside-out patch configuration from bovine AZF cells. Although the results reported in this study were obtained by recording currents from AZF cells, preliminary recordings from AZG cells showed no difference in bTREK-1 currents with respect to modulation by AngII. For whole cell recordings, the standard external solution consisted of 140 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, 10mM HEPES, and 5 mM glucose, with pH adjusted to 7.3 using NaOH. The standard pipette solution consisted of (in mM): 120 KCl, 2 MgCl₂, 10 HEPES, and 0.2 GTP, with pH titrated to 6.8 using KOH. The buffering capacity of pipette solutions was varied by adding combinations of Ca²⁺ and BAPTA or EGTA using the Bound and Determined software program (28). Low and high capacity Ca²⁺ buffering solutions contained 0.5 mM EGTA and 11 mM BAPTA, respectively. The low capacity Ca²⁺ buffering solution was normally Ca²⁺-free. [Ca²⁺]_i was buffered to 22 nM in the high capacity buffering solution. Nucleotides, including MgATP, NaUTP, and AMP-PNP were added to pipette or bath solutions as noted in the text. For inside-out patch recordings, the standard external and pipette solutions used for whole cell recordings were switched.

Patch clamp recordings of I_T-Ca were made in the whole cell configuration. The standard pipette solution was in mM: 120 CsCl, 2 MgCl₂, 2 NaUTP, 0.5 EGTA, 0.2 GTP, 10 HEPES with pH titrated to 7.2 using CsOH. The external solution contained (in mM): 117 tetraethylammonium, 5 CsCl, 20 CaCl₂, 2 MgCl₂, 5 HEPES, with pH adjusted to 7.3 using tetraethylammonium-OH. All solutions were filtered through 0.22-µm cellulose acetate filters.

Recording Conditions and Electronics—AZF cells were used for patch clamp experiments 2–12 h after plating. Typically, cells with diameters <15 µm and capacitances of 10–15 pF were selected. Coverslips were transferred from 35-mm culture dishes to the recording chamber (volume: 1.5 ml) 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 M were fabricated from Corning 0010 glass (World Precision Instruments, Sarasota, FL). These electrodes routinely yielded access resistances of 1.5–4.0 M and voltage-clamp time constants of <100 µs. For single channel recordings, patch electrodes with higher resistances (3–5 M) were used. K⁺ currents were recorded at room temperature (22–25 °C) according to the procedure of Hamill et al. (29) using a List EPC-7 patch clamp amplifier.

Pulse generation and data acquisition were done using a personal computer and PCLAMP software with TL-1 interface (Axon Instruments, Inc., Burlingame, CA). Currents were digitized at 2–10 KHz after filtering with an 8-pole Bessel filter (Frequency Devices, Haverhill, MA). Linear leak and capacity currents were subtracted from current records using summed scaled hyperpolarizing steps of to pulse amplitude. Data were analyzed using PCLAMP (CLAMPFIT 9.2, FETCHAN 6.04, and PSAT 6.04) and SigmaPlot (version 8.0) software. Drugs were applied by bath perfusion, controlled manually by a six-way rotary valve. p values were calculated using Student's t test.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
AngII Inhibits bTREK-1 by Separate Ca²⁺ and ATP-dependent Mechanisms—Bovine AZF cells express two types of K⁺ channels, voltage gated, rapidly inactivating Kv1.4 channels, and leak-type bTREK-1 channels (2, 30, 31). In whole cell recordings, bTREK-1 amplitude often increases spontaneously over a period of minutes, provided that the recording pipette contains ATP or other nucleotide triphosphates at millimolar concentrations (1, 2, 26).

The absence of time- and voltage-dependent bTREK-1 inactivation allows the corresponding membrane current to be easily isolated in whole cell recordings, using either of two voltage clamp protocols. When voltage steps of 300 ms duration are applied from a holding potential of –80 mV, bTREK-1 can be measured near the end of a voltage step when the transient Kv1.4 current has inactivated (Fig. 1, A–C, left traces). Alternatively, bTREK-1 can be selectively activated by an identical voltage step, after a 10-s prepulse to –20 mV has fully inactivated Kv1.4 channels (Fig. 1, A–C, right traces). Measurement of bTREK-1 by either method yielded nearly identical results.

In previous whole cell recording studies on AZF cells using patch electrodes containing 2–5 mM MgATP and [Ca²⁺]_i strongly buffered to 22 nM with 11 mM BAPTA, AngII inhibited bTREK-1 by a maximum of 77–82% with an IC₅₀ of 145 pM. However, when MgATP in the pipette was replaced with UTP, which is not a substrate for kinases or ATPases, AngII was ineffective (1, 12, 21).

View larger version (23K): [in this window] [in a new window]   FIG. 1. AngII inhibits bTREK-1 through separate Ca2+- and ATP-dependent pathways. The inhibition of bTREK-1 in bovine AZF cells by AngII was measured in whole cell patch clamp recordings using pipette solutions that permitted or blocked activation of Ca2+- and ATP-dependent signaling. K+ currents were recorded from bovine AZF cells at 30-s intervals in response to voltage steps to +20 mV, applied from a holding potential of –80 mV with or without 10-s prepulses to –20 mV. After bTREK-1 reached a stable maximum amplitude, cells were superfused with saline containing AngII (2 or 10 nM). A–C, K+ currents were recorded with (right traces) or without (left traces) depolarizing prepulses. bTREK-1 amplitudes recorded with (open circles) or without (closed circles) prepulses are plotted against time. Numbers on traces correspond to those on the plot. Pipette solution containing: A, 2 mM UTP, 11 mM BAPTA; B, 2 mM UTP, 0.5 mM EGTA; or C, 5 mM MgATP, 0.5 mM EGTA. D, summary of experiments as in A–C. Bars indicate percent of bTREK-1 remaining after steady state block by AngII (2 or 10 nM). Values are mean ± S.E. for the indicated number of determinations.

These results suggest that AngII-mediated inhibition of bTREK-1 may occur through separate Ca²⁺ and ATP hydrolysis-dependent signaling pathways. If AngII modulates bTREK-1 by parallel mechanisms, each of which produces only partial inhibition, it is likely that simultaneous activation of both pathways would more effectively suppress this current. Accordingly, when bTREK-1 currents were recorded with a pipette solution whose composition supported activation of both ATP- and Ca²⁺-dependent pathways (0.5 mM EGTA, 5 mM MgATP), AngII (2 nM) was significantly more effective, inhibiting bTREK-1 by 92.9 ± 0.7% (n = 8) (Fig. 1, C and D).

Not only was AngII more effective at inhibiting bTREK-1 when both the Ca²⁺ and ATP pathways were available, it was also more potent. To compare the potency of AngII as an inhibitor of bTREK-1 through Ca²⁺ and combined pathways, AZF cells were superfused with AngII at various concentrations and bTREK-1 currents were recorded with pipette solutions containing 0.5 mM EGTA and either 2 nM NaUTP or 5 mM MgATP. Inhibition curves constructed from data obtained in these experiments clearly showed that AngII was more potent, as well as more effective, at inhibiting bTREK-1 when Ca²⁺- and ATP-dependent pathways were available (Fig. 2A, left).

With both pathways available for activation, AngII inhibited bTREK-1 almost completely, with an IC₅₀ of 63 pM. By comparison, with only the Ca²⁺ pathway available for activation, AngII inhibited bTREK-1 by a maximum of 70%, with an IC₅₀ of 149 pM (Fig. 2A). The marked enhancement of bTREK-1 inhibition by AngII when both Ca²⁺- and ATP-dependent pathways were activated was most pronounced at lower AngII concentrations. At low AngII concentrations, bTREK-1 inhibition was partially reversible with washing (Fig. 2A).

Activation of Ca²⁺- and ATP-dependent Pathways Correlate with Membrane Depolarization—bTREK-1 channels are largely responsible for setting the resting potential of bovine adrenocortical cells (1, 12). In the absence of a significant inward current, the Goldman-Hodgkin-Katz voltage equation predicts that a large fraction of the bTREK-1 channels would have to be inhibited to effectively depolarize these cells. The simultaneous activation of both the Ca²⁺- and ATP-dependent pathways should therefore provide a more efficient means of depolarizing adrenocortical cells. Accordingly, in combined current and voltage clamp experiments, AngII inhibited bTREK-1 and strongly depolarized AZF cells only when both the Ca²⁺- and ATP-dependent pathways were available for activation.

When recordings were made with pipette solutions that sustained only activation of the Ca²⁺-dependent pathway, AngII (2 nM) inhibited bTREK-1 current by 62.2 ± 5.7%, whereas these cells were depolarized by an average of 11.9 ± 2.4 mV (n = 5) from their resting potential of –68.8 ± 4.4 mV (Fig. 2B). By comparison, when recordings were made with pipette solutions that allowed activation of both Ca²⁺- and ATP-dependent pathways, AngII (2 nM) inhibited bTREK-1 almost completely by 96.8 ± 1.0%, and depolarized AZF cells by an average of 44.3 ± 4.3 mV (n = 4) from their resting potential of –63.3 ± 3.2 mV (Fig. 2C).

ATP, but Not ACTH or cAMP, Inhibits bTREK-1 through Both Ca²⁺- and ATP-dependent Pathways—Previously, we demonstrated that externally applied ATP and UTP inhibited bTREK-1 channels through activation of a G protein-coupled purinergic receptor with a P2Y₃ agonist profile (32). In that study where [Ca²⁺]_i was strongly buffered with 11 mM BAPTA, ATP (100 µM) inhibited bTREK-1 by a maximum of 71.3 ± 3.2% and this inhibition was eliminated by substituting AMP-PNP for MgATP in the pipette solution. Thus, similar to AngII, externally applied ATP inhibits bTREK-1 through a ATP hydrolysis-dependent mechanism.

P2Y₃ receptors are coupled to PLC activation and the release of [Ca²⁺]_i (33, 34). To determine whether externally applied ATP also inhibited bTREK-1 through a Ca²⁺-dependent mechanism, inhibition was studied with a pipette solution designed to permit activation of the Ca²⁺, but not the ATP-dependent pathway. With [Ca²⁺]_i weakly buffered by 0.5 mM EGTA, ATP at 10 and 100 µM inhibited bTREK-1 by 26.2 ± 9.3 (n = 7) and 42.8 ± 4.4% (n = 6), respectively (Fig. 3, A and D).

When considered in conjunction with the previously mentioned study (32), these results indicate that similar to AngII, external ATP inhibits bTREK-1 through separate Ca²⁺- and ATP hydrolysis-dependent mechanisms. Accordingly, when AZF cells were superfused with ATP under conditions where Ca²⁺- and ATP-dependent pathways could be activated, bTREK-1 was inhibited by 85.6 ± 5.8% (n = 4) (Fig. 3D).

ACTH also potently inhibits bTREK-1 in bovine AZF cells with an IC₅₀ of 5pM (1). Although the signaling pathways are only partially understood, inhibition likely involves both protein kinase A-dependent and -independent actions of cAMP (5, 35). Inhibition by ACTH or cAMP is eliminated when AMP-PNP or UTP is substituted for ATP in the pipette solution with [Ca²⁺]_i strongly buffered by 11 mM BAPTA (26, 35). Experiments were done to determine whether, in addition to the ATP-dependent mechanism, ACTH or cAMP could also inhibit bTREK-1 by a Ca²⁺-dependent process.

In contrast to AngII, ACTH failed to inhibit bTREK-1 in whole cell recordings made with pipette solutions containing 2 mM UTP and 0.5 mM EGTA. In the experiment illustrated in Fig. 3B, bTREK-1 grew to a stable maximum amplitude before the cell was superfused with 200 pM ACTH. ACTH produced no measurable inhibition of bTREK-1. When this same cell was superfused with 2 nM AngII, bTREK-1 was inhibited by 72.7% within 3 min. Overall, in similar experiments, ACTH inhibited bTREK-1 by 9.5 ± 5.3% (n = 9) (Fig. 3D). Subsequent superfusion of AngII (2 nM) in 7 of these cells inhibited bTREK-1 by 72.1 ± 7.6%.

Because cAMP is the principle intracellular messenger for ACTH, the failure of ACTH to inhibit bTREK-1 in the above experiments could be because of a requirement for cAMP synthesis, previous to inhibition through a Ca²⁺-dependent pathway. However, when AZF cells were superfused with the membrane-permeable cAMP analog 8-pcpt-cAMP (250 µM) while recording bTREK-1 with pipettes containing 0.5 mM EGTA and 2 mM UTP, no significant inhibition of bTREK-1 was observed (Fig. 3, C and D). Pre-exposure to 8-pcpt-cAMP also did not affect inhibition of bTREK-1 by AngII (Fig. 3D).

Properties of the Ca²⁺-dependent Inhibitory Pathway—Results to this point have identified a new Ca²⁺-dependent pathway by which hormones and paracrine factors inhibit a specific background K⁺ current and depolarize bovine adrenocortical cells. Because AngII physiologically regulates the secretion of aldosterone and cortisol, it is important to identify the molecular components of the signaling pathway that links AngII receptors to TREK-1 channels and membrane depolarization.

The Ca²⁺-dependent Inhibition of bTREK-1 Is Voltage-independent and -specific—Although bTREK-1 K⁺ channels are active at negative membrane potentials, they display weak voltage dependence (35). The Ca²⁺-dependent inhibition of bTREK-1 by AngII was independent of voltage over a wide range of test potentials. The experiment illustrated in Fig. 4 shows current-voltage relationships recorded before and after superfusing the cell with AngII (2 nM) under conditions where only the Ca²⁺-dependent pathway could be activated. In this experiment, bTREK-1 was inhibited by greater than 90% over the range of test potentials where it could be accurately measured (–20 to +40 mV). Inhibition through the Ca²⁺-dependent pathway was entirely selective for bTREK-1. The voltage-gated Kv1.4 current was not altered in this and three similar experiments.

View larger version (22K): [in this window] [in a new window]   FIG. 2. Properties of AngII-mediated inhibition of bTREK-1 and membrane depolarization by Ca2+- and combined Ca2+/ATP-dependent pathways. A, potency and effectiveness. The inhibition of bTREK-1 by AngII at various concentrations was determined with pipette solutions designed to allow activation of only the Ca2+-dependent pathway (2 mM NaUTP, 0.5 mM EGTA) or both Ca2+ and ATP-dependent pathways (5 mM MgATP, 0.5 mM EGTA). Left, time-dependent inhibition of bTREK-1 by 200 pM AngII through combined pathways. Right, inhibition curves. Unblocked bTREK-1, expressed as percent of control, is plotted against the AngII concentration under conditions where only the Ca2+-dependent or both the Ca2+- and ATP-dependent pathways were available for activation as indicated. Data were fit with an equation of the form: I/Imax = 1/[1 + (B/Kd)x], where B is the AngII concentration, Kd is the equilibrium dissociation constant, and X is the Hill coefficient. Values are mean ± S.E. of from 4 to 23 determinations. B and C, AngII-mediated bTREK-1 inhibition and membrane depolarization. K+ currents were recorded from AZF cells using the voltage protocols described in the legend of Fig. 1 with pipette solutions designed to allow activation of the Ca2+ (B), or the combined Ca2+-/ATP-dependent pathways (C). When bTREK-1 reached a stable maximum value, membrane potential was then recorded after switching to current clamp, and AngII (2 nM) was superfused as indicated. When membrane potential reached a stable value, K+ currents were recorded to determine the extent of bTREK-1 inhibition. Membrane potential was plotted against time at the right. Numbers on traces correspond to currents recorded before (1) and after (2) superfusing AngII.

View larger version (20K): [in this window] [in a new window]   FIG. 3. ATP but not ACTH or 8-pcpt-cAMP displays Ca2+-dependent inhibition of bTREK-1. Whole cell K+ currents were recorded from AZF cells at 30-s intervals in response to voltage steps to +20 mV applied from a holding potential of –80 mV with or without a 10-s prepulse to –20 mV. Standard pipette solution was supplemented with 0.5 mM EGTA, 2 mM UTP, or 5 mM MgATP, and 200 µM GTP. After bTREK-1 reached a stable maximum amplitude, cells were superfused with saline containing NaATP (10 or 100 µM), ACTH (200 pM), or 8-pcpt-cAMP (250 µM). Cells treated with ACTH or 8-pcpt-cAMP were then superfused with AngII (2 nM). A–C, K+ currents recorded with (right traces) or without (left traces) depolarizing prepulses and associated plots of current amplitudes for cells treated with NaATP (A), ACTH followed by AngII (B), or 8-pcpt-cAMP followed by AngII (C). Numbers on the traces correspond to those on plots at right. D, summary of experiments as in A–C. Bars indicate fraction of bTREK-1 remaining after the steady-state block was achieved. White bar shows block by external ATP (100 µM*) through combined Ca2+- and ATP hydrolysis-dependent pathways. Values are mean ± S.E. for the indicated number of determinations.

Overall, in a total of nine experiments, losartan (500 nM) alone inhibited bTREK-1 by 12.2 ± 4.9%. In the presence of 500 nM losartan, 2 nM AngII inhibited bTREK-1 by only 13.8 ± 5.3%. In five of these experiments, subsequent superfusion of AngII alone inhibited bTREK-1 by 77.1 ± 5.9% (Fig. 5, A and D).

The specific AT₂ antagonist PD123319 failed to blunt AngII-mediated inhibition of bTREK-1 (Fig. 5, B and D). In the presence of 500 nM PD123319, AngII (2 nM) inhibited bTREK-1 by 65.3 ± 1.6% (n = 10), a value not significantly different from that produced by AngII alone at this concentration. By itself, PD123319 (500 nM) had no effect on bTREK-1 current.

View larger version (15K): [in this window] [in a new window]   FIG. 4. Ca2+-dependent inhibition is voltage-independent and specific. K+ currents were activated at 30-s intervals by voltage steps of varying size from a holding potential of –80 mV, before and after superfusing the AZF cell with AngII (2 nM). Top, current records at test potentials between –60 and +40 mV (in 10-mV increments) before and after AngII as indicated. Bottom, bTREK-1 current amplitudes from experiments shown are plotted against test voltage.

PLC and Ca²⁺ Dependence of AngII-mediated Inhibition of bTREK-1—Activation of AT₁ receptors on adrenocortical cells is coupled to PLC activation and the synthesis of IP₃ and diacylglycerol (37–39). PLC itself displays a dependence on Ca²⁺ for full activity (40, 41). The suppression of AngII-mediated inhibition of bTREK-1 by strongly buffering [Ca²⁺]_i to 22 nM with 11 mM BAPTA could have resulted from a failure to activate PLC rather than inhibition of an IP₃-stimulated Ca²⁺ transient. If so, then inhibition through this pathway could be mediated through a PLC-dependent second messenger, such as diacylglycerol rather than by a direct effect of Ca²⁺ on the channel.

However, when whole cell recordings were made with [Ca²⁺]_i buffered to a resting physiological concentration of 100 nM with 11 mM BAPTA, AngII (2 nM) remained ineffective, inhibiting bTREK-1 by 10.6 ± 5.9% (n = 4). Thus, at physiological [Ca²⁺]_i, which supports the activation of PLC, AngII does not inhibit bTREK-1 in the presence of strong Ca²⁺ buffering.

In addition, it was observed that raising [Ca²⁺]_i in the pipette to 100 nM did not alter the expression of bTREK-1. At [Ca²⁺]_i of 22 and 100 nM, maximum bTREK-1 current densities were 50.8 ± 7.9 and 57.1 ± 8.9 pA/pF (n = 5), respectively. Apparently, Ca²⁺ does not affect bTREK-1 activity at concentrations up to 100 nM.

To further determine whether the Ca²⁺-dependent inhibition of bTREK-1 by AngII is mediated through PLC, whole cell recordings were made with pipette solutions containing the PLC antagonist U73122 [GenBank] (42). U73122 [GenBank] (3 µM) suppressed inhibition of bTREK-1 by AngII (2 nM) from the control value of 66.0 ± 2.9 (n = 22) to 48.2 ± 7.3% (n = 16) (Fig. 6, A and C). In contrast, the inactive analog of U73122 [GenBank] , U73343 [GenBank] , failed to blunt inhibition by AngII. With U73343 [GenBank] (3 µM) in the patch pipette, AngII inhibited bTREK-1 by 73.2 ± 2.6% (n = 6) (Fig. 6C). At concentrations of 5 µM and higher, U73122 [GenBank] was found to markedly inhibit the expression of the bTREK-1 current in whole cell recordings.

To determine whether AngII-mediated inhibition of bTREK-1 through the Ca²⁺ pathway is dependent on [Ca²⁺]_ext, AZF cells were superfused with saline containing AngII and no added Ca²⁺. In the absence of added Ca²⁺, AngII-mediated inhibition of bTREK-1 was blunted, but not eliminated (Fig. 6, B and C).

In the experiment illustrated in Fig. 6B, currents were initially recorded in control saline, after which the cell was sequentially superfused with saline without added Ca²⁺, followed by this "zero Ca²⁺" solution containing 2 nM AngII, and finally by one containing AngII as well as 2 mM Ca²⁺. The amplitude of the bTREK-1 current rapidly increased in the absence of external Ca²⁺, and then decreased by 30% upon superfusion of AngII. Superfusion of saline containing 2 nM AngII and 2 mM Ca²⁺ further reduced bTREK-1 current amplitude to 29% of its maximum value within 10 min.

Overall, in 6 cells, AngII (2 nM) inhibited bTREK-1 by an average of 45.1 ± 8.7% (n = 6) in the absence of added external Ca²⁺, compared with 66.0 ± 2.9% (n = 22) in control saline (Fig. 6C). In three cells where inhibition of bTREK-1 by AngII was first measured in the absence, and then in the presence of external Ca²⁺, inhibition increased from 28.4 to 66.2%.

The Ca²⁺-dependent inhibition of bTREK-1 by AngII may be mediated in part by the IP₃-stimulated release of Ca²⁺ by intracellular stores. 2-APB inhibits IP₃-stimulated release of Ca²⁺ from intracellular stores with an IC₅₀ of 40 µM (43). Including 2-APB (80 µM) in the recording pipette significantly reduced the inhibition of bTREK-1 by AngII (2 nM) to 32.9 ± 11.3% (n = 7) (Fig. 6C).

Mechanism of Ca²⁺-dependent Inhibition of bTREK-1—The results presented thus far indicate that Ca²⁺ entering through the plasma membrane and released from intracellular stores may both contribute to AngII-mediated inhibition of bTREK-1. The molecular mechanism by which Ca²⁺ inhibits the activity of bTREK-1 channels is not known. Because the Ca²⁺ pathway functions in the absence of hydrolyzable ATP, it is likely that Ca²⁺ interacts directly with the bTREK-1 channel or an associated subunit or protein. If Ca²⁺ interacts directly with the bTREK-1 channel protein, then agents that increase intracellular Ca²⁺ should effectively inhibit the activity of cloned bTREK-1 channels expressed in a cell line. The Ca²⁺ ionophore ionomycin (1 and 5 µM) inhibited the activity of bTREK-1 channels expressed in CHO-K1 cells by 54.5 ± 18.6 and 75.6 ± 5.6%, respectively (Fig. 7, A and C).

The inhibition of bTREK-1 channels expressed in CHO-K1 cells by ionomycin suggests a direct interaction with the channel or a Ca²⁺-dependent protein expressed in a wide range of cells. The Ca²⁺-dependent modulation of a number of ion channels requires a calmodulin intermediate. In some instances Ca²⁺-free apocalmodulin is constitutively bound to the channel, whereas in others, only the Ca²⁺-calmodulin complex binds to the channel with high affinity (44, 45). To determine whether the Ca²⁺-dependent inhibition of bTREK-1 is calmodulin dependent, the inhibition of bTREK-1 by AngII was studied in whole cell recordings with pipettes containing a CIP. This 17-residue peptide mimics the calmodulin-binding domain of myosin light chain kinase and binds to the calmodulin with a K_d of 6 pM (46).

View larger version (20K): [in this window] [in a new window]   FIG. 5. Ca2+-dependent inhibition of bTREK-1 by AngII through G protein-dependent AT1 receptor. A, receptor subtype: whole cell K+ currents were recorded from AZF cells at 30-s intervals in response to voltage steps to +20 mV applied from a holding potential of –80 mV with or without depolarizing prepulses. After bTREK-1 current reached a stable maximum amplitude, cells were superfused with losartan (500 nM) or PD123319 (500 nM), followed by saline containing the antagonist and AngII (2 nM). Current traces are shown at the left. Numbers on traces correspond to those on plot of current amplitudes at right. B, G protein dependence: bTREK-1 reached a stable value, cell was superfused with AngII (2 nM). Numbers on current traces at the left correspond to those on the plot of bTREK-1 amplitudes at right. C, summary of results of experiments as in A and B. Bars indicate fraction of bTREK-1 remaining after steady state block by AngII (2 nM) in the presence or absence of losartan, PD123319, or GDPS. Inhibition of bTREK-1 by ionomycin (5 µM) with GDPS in the pipette is also shown. Values are mean ± S.E.

Effect of Ca²⁺ on Unitary bTREK-1 Currents—Whole cell patch clamp experiments indicated that Ca²⁺-mediated inhibition of bTREK-1 might occur through a direct interaction of Ca²⁺ with the channel or a channel-associated protein. Single channel recording experiments in which Ca²⁺ was applied directly to the cytoplasmic surface of excised inside-out patches in the absence of ATP were consistent with this model for inhibition. In these experiments, AZF cell membrane patches were excised into an internal solution where [Ca²⁺] was buffered to 22 nM with 11 mM EGTA at a test potential of +40 mV (inside positive). At this potential, all Kv1.4 K⁺ channels are inactivated, leaving only bTREK-1 channels active in the membrane patch. With 2 mM UTP applied to the cytoplasmic face of the patch, bTREK-1 activity typically increased spontaneously and continuously during prolonged recordings.

View larger version (20K): [in this window] [in a new window]   FIG. 6. Phospholipase C and Ca2+ dependence of AngII inhibition of bTREK-1. A, phospholipase C dependence: whole cell K+ currents were recorded with standard pipette solution supplemented with 0.5 mM EGTA and 2 mM UTP, to which the phospholipase C antagonist U73122 [GenBank] (3 µM) was added. Whole cell K+ currents were recorded at 30-s intervals with (right traces) and without (left traces) depolarizing prepulses. bTREK-1 amplitudes recorded with (open circles) or without (closed circles) prepulses are plotted against time. Numbers on traces correspond to those on the plot at the right. B, external Ca2+ dependence: whole cell K+ currents were recorded with standard pipette solution supplemented with 0.5 mM EGTA and 2 mM UTP. After recording in standard saline, external solution was sequentially switched to Ca2+-free saline, Ca2+-free saline plus AngII (2 nM), and standard saline plus AngII (2 nM), as indicated. Currents were recorded with and without depolarizing prepulses as described in A. Numbers on traces at the left correspond to those on the plot at the right. C, Summary of experiments as in A and B. Bars indicate the fraction of bTREK-1 remaining after steady state block by AngII (2 nM) under control conditions, with U73122 [GenBank] (3 µM), U73343 [GenBank] (3 µM), or 2-APB (80 µM) in the pipette solution, or in the absence of external Ca2+, as indicated. Values are mean ± S.E. of the indicated number of separate determinations. p values for bars with asterisk: p = 0.0154 for AngII + U73122 [GenBank] , 0.0057 for AngII ([Ca2+]ext = 0), and 0.0003 for 2-APB.

View larger version (17K): [in this window] [in a new window]   FIG. 7. Effect of ionomycin and CIP on cloned and native bTREK-1 channels. A, inhibition of cloned bTREK-1 channels by ionomycin. K+ currents were recorded from bTREK-1-transfected CHO-K1 cells in response to voltage steps to +20 mV applied at 30-s intervals from a holding potential of –80 mV. After recording currents in control saline, cell was superfused with ionomycin (5 µM). Numbers on current traces correspond to those on the plot of bTREK-1 amplitudes at the right. B, effect of CIP on bTREK-1 inhibition by AngII. Whole cell K+ currents were recorded from an AZF cell at 30-s intervals in response to voltage steps to +20 mV with or without depolarizing prepulses using a pipette solution that contained 500 nM CIP. After bTREK-1 reached a stable maximum, cell was superfused with 2 nM AngII. Numbers on current traces correspond to those on the plot of bTREK-1 amplitudes at the right. C, summary of experiments as in A and B. Bars indicate fraction of bTREK-1 remaining after steady state block by ionomycin (1 or 5 µM) or AngII (2 nM). Values are mean ± S.E. for the indicated number of separate determinations. *, p value for AngII + CIP = 0.0046.

View larger version (34K): [in this window] [in a new window]   FIG. 8. Inhibition of unitary bTREK-1 currents by Ca2+. Unitary bTREK-1 currents were recorded from inside-out AZF cell membrane patches excised from AZF cells (A and B) or COS-7 cells transfected with bTREK-1 (C) using pipette and bath solutions described under "Materials and Methods." Patches were excised into solution containing 22 nM Ca2+. Unitary bTREK-1 currents were continuously recorded at +30 mV in control solution and after switching to saline containing 5 (A) or 10 µM (B) free Ca2+. Amplitude histograms were constructed from unitary currents recorded at +30 mV over a 50-s period. Current amplitudes were distributed into bins 0.2 pA in width. Currents were sampled at 5 KHz and filtered at a cutoff frequency of 2 KHz. A, unitary currents (top) and corresponding amplitude histograms (bottom) from recordings in control solution (22 nM Ca2+), in 5 µM Ca2+, and after returning to control (wash), as indicated. B, unitary currents and amplitude histograms in control solution (22 nM Ca2+) and after superfusion of 10 µM Ca2+, as indicated. C, unitary currents and amplitude histograms from COS-7 patch recordings in control saline (22 nM Ca2+), 10 µM Ca2+, and again in control saline (wash).

Nearly all freshly plated bovine AZF cells express only low voltage-activated, slowly deactivating T-type Ca²⁺ currents (47). In the experiment illustrated in Fig. 9A, T-type deactivating "tail" currents were recorded with pipette solutions containing 2 mM NaUTP in place of MgATP, with Ca²⁺ weakly buffered by 0.5 mM EGTA. Currents were recorded at –80 mV after activation by 10-ms depolarizing pulses to 0 mV. AngII (2 nM) failed to increase the amplitude of the T-type Ca²⁺ current in this or any of six similar experiments. AngII also failed to significantly shift the voltage dependence of T channel activation under similar conditions (Fig. 9B).

View larger version (21K): [in this window] [in a new window]   FIG. 9. AngII does not modulate T-type Ca2+ current through the Ca2+-dependent pathway. The effect of AngII on T-type Ca2+ current amplitude and voltage-dependent activation were measured in AZF cells. A, Ca2+ currents were activated by 10-ms voltage steps to –5 mV applied at 30-s intervals from a holding potential of –80 mV. After recording Ca2+ currents in standard saline, cell was superfused with saline containing 2 nM AngII. "Tail current" amplitudes recorded at –80 mV are plotted at the right. B, activation curves. The effect of AngII on the voltage dependence of T-type Ca2+ channel activation was measured by recording tail currents at a constant potential (–80 mV) after applying activating voltage steps of 10-ms duration in 10-mV increments to potentials between –60 and +30 mV before (•) and after () superfusing cells with 2 nM AngII. Tail current amplitudes were normalized to the maximum value and plotted as the fraction of open channels against test potential. Data points were fit with a Boltzmann function of the form: Fraction open = 1/[1 + exp(v – v)/k)], where v is the voltage at which one-half of the channels are in the open conformation and k is the slope factor.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In addition to AngII, ATP also inhibited bTREK-1 through the Ca²⁺-dependent pathway at concentrations that stimulate large increases in cortisol production from bovine AZF cells (13). In a previous study, we showed that ATP also inhibited bTREK-1 by an ATP hydrolysis-dependent mechanism through a nucleotide receptor with a P2Y₃ agonist profile (14). These two results indicate that activation of the PLC-coupled P2Y₃ receptor in AZF cells inhibits bTREK-1 by the same two pathways utilized by AngII. Accordingly, externally applied ATP was significantly more effective at inhibiting bTREK-1 when recordings were made under conditions that supported activation of both the Ca²⁺- and ATP-dependent paths.

In contrast to AngII and external ATP, ACTH and cAMP failed to inhibit bTREK-1 when only the Ca²⁺-dependent mechanism was available for activation. Thus, although ACTH and cAMP both inhibit bTREK-1 by protein kinase A-dependent and -independent mechanisms, neither of these is mediated by Ca²⁺ (35).

The Ca²⁺-dependent Signaling Pathway—The effective inhibition of bTREK-1 by AngII in whole cell recordings with pipettes containing UTP or AMP-PNP in place of MgATP precluded the involvement of a kinase or ATPase in the Ca²⁺-dependent response, because UTP and AMP-PNP are not substrates for these enzymes. However, the molecular components of the Ca²⁺-dependent signaling pathway remained to be identified.

View larger version (15K): [in this window] [in a new window]   FIG. 10. Model for Ca2+-dependent inhibition of bTREK-1 by AngII.

AT₁ receptors in many cells, including adrenocortical cells, are coupled to PLC through the GTP-binding protein G_q (17). U73122 [GenBank] (3 µM) significantly reduced, but did not eliminate, AngII-mediated inhibition of bTREK-1. This result is consistent with a model where the Ca²⁺-dependent inhibition of bTREK-1 depends on PLC activation. U73122 [GenBank] inhibits agonist-induced PLC activation in intact cells with IC₅₀ values in the micromolar range (42). At concentrations above 3 µM, U73122 [GenBank] significantly inhibited the expression of bTREK-1 in whole cell recordings as has been previously reported for other K⁺ channels, precluding the use of higher concentrations in our experiments (49). In this regard, it is significant that the inactive U73122 [GenBank] analog U73343 [GenBank] (3 µM) had no effect on AngII-mediated inhibition of bTREK-1.

PLC activation in adrenocortical cells leads to IP₃-induced release of intracellular Ca²⁺, as well as Ca²⁺ influx through plasma membrane channels (20, 37, 39). Superfusing AZF cells with Ca²⁺-free saline initially increased bTREK-1 current and significantly reduced the inhibition of this current by AngII, indicating that Ca²⁺ influx through plasma membrane channels contributes to the regulation of channel activity. Bovine adrenocortical cells express TRPC4 non-selective cation channels that may be activated by AngII (37, 38). Ca²⁺ entering through these TRPC4 channels could be linked to the inhibition of nearby bTREK-1 channels.

The rapid increase in bTREK-1 current observed upon superfusion of Ca²⁺-free saline could result from a reduction in the basal Ca²⁺ influx through the plasma membrane or by a change in the effective transmembrane voltage. Removal of extracellular Ca²⁺ shifts the voltage dependence of channel activation in the hyperpolarizing direction by effectively reducing the electric field strength across the membrane (50). Consequently, because bTREK-1 channels display weak voltage dependence, the absence of [Ca²⁺]_o could enhance current by increasing channel open probability (26).

Bovine adrenocortical cells also express T-type voltage-gated Ca²⁺ channels that may represent the other major pathway for Ca²⁺ entry in these cells (10, 47, 51). However, because AngII failed to increase Ca²⁺ current through T-type Ca²⁺ channels under the conditions of our experiment, it is unlikely that Ca²⁺ entering through these channels contributed to inhibition of bTREK-1.

The reduction in AngII-stimulated inhibition of bTREK-1 current produced by 2-APB (80 µM) is consistent with a model wherein inhibition is mediated in part by IP₃-stimulated release of Ca²⁺ from intracellular stores. In this regard, 2-APB inhibits IP₃-stimulated Ca²⁺ release with an IC₅₀ of 42 µM, but increases Ca²⁺ release from microsomes at concentrations above 90 µM (43). Therefore, 2-APB was not used at concentrations above 80 µM in our studies. Overall, the results indicate that the Ca²⁺-dependent inhibition of bTREK-1 by AngII involves both PLC-dependent Ca²⁺ influx and release from intracellular stores.

Molecular Mechanism for Ca²⁺ Inhibition of bTREK-1—The results of experiments using ionomycin, CIP, and Ca²⁺ applied directly to excised membrane patches provide insight into the molecular mechanism by which Ca²⁺ inhibits bTREK-1. The absence of MgATP in all of these experiments ruled out the participation of protein kinases, simplifying the problem. However, Ca²⁺ modulates the gating of a number of K⁺ channels by multiple kinase-independent mechanisms. Several Ca²⁺-actvated K⁺ channels are gated by Ca²⁺ binding directly to the channel (52, 53). For other K⁺ channels, modulation by Ca²⁺ proceeds through a calmodulin intermediate. Calmodulin can be constitutively associated with the channel, or it may bind to the channel only after first binding Ca²⁺ (44, 45, 54, 55).

Ionomycin inhibited bTREK-1 K⁺ current in transfected cells by a maximum of 75.6 ± 5.6%, indicating that as many as three-fourths of these channels retain their Ca²⁺ sensitivity even when expressed in a foreign cell type. Similarly, Ca²⁺ (10 µM) inhibited the activity of bTREK-1 channels expressed in COS-7 cells in half of the patches tested. If Ca²⁺-mediated inhibition of bTREK-1 required an AZF cell-specific subunit or other protein not expressed in other eukaryotic cells, ionomycin and Ca²⁺ would have been ineffective as inhibitors of the transfected channels. However, if the Ca²⁺-dependent inhibition of bTREK-1 were mediated through a ubiquitously expressed protein such as calmodulin, ionomycin and Ca²⁺ would likely retain their effectiveness in transfected cells. The incomplete inhibition of transfected bTREK-1 channels by ionomycin and Ca²⁺ in whole cell and single channel recordings may suggest that not all bTREK-1 channels become associated with the Ca²⁺-dependent protein in CHO-K1 or COS-7 cells.

The reduced inhibition of bTREK-1 observed when the patch pipette contained CIP supports a role for calmodulin in the inhibition of this channel by AngII. However, because this peptide binds to calmodulin with extremely high affinity (K_d = 6pM), it is perhaps surprising that CIP did not more effectively suppress the AngII response. This may suggest that apocalmodulin is constitutively and tightly bound to the native bTREK-1 channel in a Ca²⁺-independent fashion. Near its carboxyl-terminal end (amino acids 340–354), bTREK-1 protein contains a sequence that conforms partially to the consensus "IQ" motif calmodulin-binding site found in several other K⁺ channels where calmodulin is constitutively associated (54, 56). The inhibition of bTREK-1 by Ca²⁺ in excised inside-out AZF cell patches indicates that if Ca²⁺-dependent inhibition of bTREK-1 is mediated through calmodulin, then calmodulin must be constitutively associated with the bTREK-1 channel.

Comparison of Signaling Pathways for AngII Inhibition of bTREK-1 and Activation of T-type Ca²⁺ Channels—In bovine AZG cells, AngII enhances current through Ca_v3.2 T-type Ca²⁺ channels by activation of Ca²⁺/calmodulin-dependent kinase II, which phosphorylates these channels at a specific site (25, 57). Interestingly, we have shown that AngII inhibits bTREK-1 channels in bovine adrenocortical cells by two entirely different pathways. Furthermore, AngII does not modulate Ca_v3.2 T-type Ca²⁺ channels in bovine adrenocortical cells by either of the two mechanisms that inhibit bTREK-1 channels. In a previous study, we showed that AngII has no effect on the T-type Ca²⁺ current in bovine AZG cells through the ATP-dependent pathway (11). Finally, the results of the current study do not rule out the possibility that AngII may also inhibit bTREK-1 by a third pathway involving a Ca²⁺-dependent kinase such as Ca²⁺/calmodulin kinase or kinase C.

Modulation of Cloned Neuronal TREK-1 Channels—The inhibition of rat brain TREK-1 channels by G_q-coupled metabotropic glutamate receptors has also been reported, and the signaling pathways mediating rat brain TREK-1 regulation have been explored in transfected cell lines (58). Glutamate-mediated inhibition of TREK-1 was blocked by U73122 [GenBank] , indicating the involvement of PLC. However, in contrast with our studies, these whole cell experiments were done with [Ca²⁺]_i buffered by 5 mM EGTA and without ATP in the pipette solution. In this system, evidence is presented indicating that inhibition occurred through a direct effect of the PLC-generated second messenger diacylglycerol, rather than through Ca²⁺-or ATP-dependent pathways. It appears that TREK-1 K⁺ channels in various cell types are modulated by a range of signaling pathways, involving kinases, diacylglycerol, cAMP, and Ca²⁺.

The differences that we have observed in the signaling pathways that modulate native bTREK-1 channels compared with cloned TREK-1 channels from other species could arise through differences in channel-associated proteins, or signaling pathways present in the native or host cells. For example, the availability of calmodulin for interaction with TREK-1 may be quite different in a transfected cell, when compared with a native cell.

Physiological Significance—bTREK-1 channels function as a central control point in bovine adrenocortical cells where hormonal and paracrine signals originating at the cell membrane are coupled to depolarization-dependent Ca²⁺ entry. We have shown that bovine AZF cells have developed a fail-safe mechanism for potent and effective inhibition of bTREK-1 current and membrane depolarization by AngII through the activation of parallel Ca²⁺- and ATP hydrolysis-dependent signaling pathways (Fig. 10).

In neurons and other excitable cells, bTREK-1 and other four transmembrane/two-pore K⁺ channels act as a brake on electrical activity (5, 59). It remains to be seen whether, under physiological conditions, bTREK-1 inhibition by AngII triggers Ca²⁺-dependent action potentials in bovine adrenocortical cells. Action potentials have been recorded in adrenocortical cells from several species (60–62). In the absence of bTREK-1, opposing T-type Ca²⁺ currents and A-type K⁺ currents could give rise to action potentials.

In addition to AngII, external ATP and perhaps other paracrine factors inhibit bTREK-1 through these same two pathways. Inhibition of specific ion channels by simultaneous activation of multiple signaling pathways may be a common theme when near total inactivation is required. Accordingly, ACTH- and cAMP-mediated inhibition of bTREK-1 by protein kinase A-dependent and -independent mechanisms may be yet another example (5, 35). Overall, ACTH and AngII inhibit bTREK-1 through at least four different signaling pathways.

Although it is clear that AngII acts through an AT₁ receptor by a G protein-dependent mechanism to inhibit bTREK-1 via separate Ca²⁺- and ATP-dependent pathways, the underlying molecular events are incompletely understood. Specifically, our results do not demonstrate that the inhibition of bTREK-1 by Ca²⁺ is entirely dependent on PLC. Furthermore, although Ca²⁺ entry through the plasma membrane contributes to bTREK-1 inhibition, the molecular identity of the plasma membrane channel has not been determined. Finally, the specific role of calmodulin in TREK-1 inhibition by Ca²⁺ remains to be defined.

	FOOTNOTES

 
^* This work was supported in part by National Institutes of Health Grant R01-DK47875 (to J. J. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| Supported in part by the National Science Foundation under agreement 0112050.

To whom correspondence should be addressed: Dept. of Neuroscience, Ohio State University College of Medicine and Public Health, 5196 Graves Hall, 333 W. 10th Ave., Columbus, OH 43210-1239. Tel.: 614-292-3511; E-mail: enyeart.1{at}osu.edu.

¹ The abbreviations used are: AZF, bovine adrenal fasciculata; AZG, bovine adrenal glomerulosa; V_m, resting membrane potential; AngII, angiotensin II; ACTH, adrenocorticotropic hormone; PLC, phospholipase C; 2-APB, 2-aminoethoxydiphenyl borate; CIP, calmodulin inhibitory peptide; CHO, Chinese hamster ovary; BAPTA, 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N''-tetraacetic acid; IP₃, inositol 1,4,5-trisphosphate; AMP-PNP, 5'-adenylyl-,-imidodiphosphate; GDPS, guanosine-5'-O-(2-thiodiphosphate); 8-pcpt-cAMP, 8-(4-chlorophenylthio) adenosine 3',5'-cyclic monophosphate.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Mlinar, B., Biagi, B. A., and Enyeart, J. J. (1993) J. Biol. Chem. 268, 8640–8644[Abstract/Free Full Text]
2. Enyeart, J. J., Xu, L., Danthi, S., and Enyeart, J. A. (2002) J. Biol. Chem. 277, 49186–49199[Abstract/Free Full Text]
3. Goldstein, S. A., Bockenhauer, D., O'Kelly, I., and Zilberberg, N. (2001) Nat. Rev. Neurosci. 2, 175–184[Medline] [Order article via Infotrieve]
4. Maingret, F., Lauritzen, I., Patel, A. J., Heurteaux, C., Reyes, R., Lesage, F., Lazdunski, M., and Honore, E. (2000) EMBO J. 19, 2483–2491[CrossRef][Medline] [Order article via Infotrieve]
5. Patel, A. J., and Honore, E. (2001) Trends Neurosci. 24, 339–346[CrossRef][Medline] [Order article via Infotrieve]
6. Maingret, F., Patel, A. J., Lesage, F., Lazdunski, M., and Honore, E. (2000) J. Biol. Chem. 275, 10128–10133[Abstract/Free Full Text]
7. Bang, H., Kim, Y., and Kim, D. (2000) J. Biol. Chem. 275, 17412–17419[Abstract/Free Full Text]
8. Kang, D., Choe, C., and Kim, D. (2005) J. Physiol. (Lond.) 564, 103–116[Abstract/Free Full Text]
9. Kim, D. (2003) Trends Pharmacol. Sci. 24, 648–654[CrossRef][Medline] [Order article via Infotrieve]
10. Enyeart, J. J., Mlinar, B., and Enyeart, J. A. (1993) Mol. Endocrinol. 7, 1031–1040[Abstract/Free Full Text]
11. Enyeart, J. A., Danthi, S. J., and Enyeart, J. J. (2004) Am. J. Physiol. 287, E1154–E1165
12. Mlinar, B., Biagi, B. A., and Enyeart, J. J. (1995) J. Biol. Chem. 270, 20942–20951[Abstract/Free Full Text]
13. Hoey, E. D., Nicol, M., Williams, B. C., and Walker, S. W. (1994) Endocrinology 134, 1553–1560[Abstract/Free Full Text]
14. Xu, L., and Enyeart, J. J. (1999) Mol. Pharmacol. 55, 364–376[Abstract/Free Full Text]
15. Dudley, D. T., Panek, R. L., Major, T. C., Lu, G. H., Bruns, R. F., Klinkefus, B. A., Hodges, J. C., and Weishaar, R. E. (1990) Mol. Pharmacol. 38, 370–377[Abstract]
16. Smith, R. D., Chiu, A. T., Wong, P. C., Herblin, W. F., and Timmermans, P. B. (1992) Annu. Rev. Pharmacol. Toxicol. 32, 135–165[Medline] [Order article via Infotrieve]
17. De Gasparo, M., Catt, K. J., Inagami, T., Wright, J. W., and Unger, Th. (2000) Pharmacol. Rev. 52, 415–472[Abstract/Free Full Text]
18. Smith, R. D., Baukal, A. J., Dent, P., and Catt, K. J. (1999) Endocrinology 140, 1385–1391[Abstract/Free Full Text]
19. Nadler, J. L., Natarajan, R., and Stern, N. (1987) J. Clin. Investig. 80, 1763–1769[Medline] [Order article via Infotrieve]
20. Spat, A., and Hunyady, L. (2004) Physiol. Rev. 84, 489–539[Abstract/Free Full Text]
21. Xu, L., and Enyeart, J. J. (2001) Am. J. Physiol. 280, C199–C215
22. Gomora, J. C., and Enyeart, J. J. (1998) Am. J. Physiol. 275, C1526–C1537[Medline] [Order article via Infotrieve]
23. Lu, H. K., Fern, R. J., Nee, J. J., and Barrett, P. Q. (1994) Am. J. Physiol. 267, F183–F189[Medline] [Order article via Infotrieve]
24. Chen, X. L., Bayliss, D. A., Fern, R. J., and Barrett, P. Q. (1999) Am. J. Physiol. 276, F674–F683[Medline] [Order article via Infotrieve]
25. Wolfe, J. T., Wang, H., Perez-Reyes, E., and Barrett, P. Q. (2002) J. Physiol. (Lond.) 538, 343–355[Abstract/Free Full Text]
26. Enyeart, J. J., Gomora, J. C., Xu, L., and Enyeart, J. A. (1997) J. Gen. Physiol. 110, 679–692[Abstract/Free Full Text]
27. Jurman, M. E., Boland, L. M., Liu, Y., and Yellen, G. (1994) BioTechniques 17, 876–881[Medline] [Order article via Infotrieve]
28. Brooks, S. P., and Storey, K. B. (1992) Anal. Biochem. 201, 119–126[CrossRef][Medline] [Order article via Infotrieve]
29. Hamill, O. P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F. J. (1981) Pflügers Arch. 391, 85–100[CrossRef][Medline] [Order article via Infotrieve]
30. Mlinar, B., and Enyeart, J. J. (1993) J. Gen. Physiol. 102, 239–255[Abstract/Free Full Text]
31. Enyeart, J. A., Xu, L., and Enyeart, J. J. (2000) J. Biol. Chem. 275, 34640–34649[Abstract/Free Full Text]
32. Xu, L., and Enyeart, J. J. (1999) J. Physiol. (Lond.) 521, 81–97[Abstract/Free Full Text]
33. Webb, T. E., Henderson, D., King, B. F., Wang, S., Simon, J., Bateson, A. N., Burnstock, G., and Barnard, E. A. (1996) Mol. Pharmacol. 50, 258–265[Abstract]
34. Williams, M., and Burnstock, G. (1997) in Purinergic Approaches in Experimental Therapeutics (Jacobson, K. A., and Jarvis, M. F., eds) pp. 3–26, Wiley-Liss, Inc., New York
35. Enyeart, J. J., Mlinar, B., and Enyeart, J. A. (1996) J. Gen. Physiol. 108, 251–264[Abstract/Free Full Text]
36. Venema, R. C., Ju, H., Venema, V. J., Schieffer, B., Harp, J. B., Ling, B. N., Eaton, D. C., and Marrero, M. B. (1998) J. Biol. Chem. 273, 7703–7708[Abstract/Free Full Text]
37. Burnay, M. M., Python, C. P., Vallotton, M. B., Capponi, A. M., and Rossier, M. F. (1994) Endocrinology 135, 751–758[Abstract]
38. Philipp, S., Trost, C., Warnat, J., Rautmann, J., Himmerkus, N., Schroth, G., Kretz, O., Nastainczyk, W., Cavalie, A., Hoth, M., and Flockerzi, V. (2000) J. Biol. Chem. 275, 23965–23972[Abstract/Free Full Text]
39. Rossier, M. F., and Capponi, A. M. (2000) Vitam. Horm. 60, 229–284[Medline] [Order article via Infotrieve]
40. Rhee, S. G. (2001) Annu. Rev. Biochem. 70, 281–312[CrossRef][Medline] [Order article via Infotrieve]
41. Ellis, M. V., James, S. R., Perisic, O., Downes, C. P., Williams, R. L., and Katan, M. (1998) J. Biol. Chem. 273, 11650–11659[Abstract/Free Full Text]
42. Thompson, A. K., Mostafapour, S. P., Denlinger, L. C., Bleasdale, J. E., and Fisher, S. K. (1991) J. Biol. Chem. 266, 23856–23862[Abstract/Free Full Text]
43. Maruyama, T., Kanaji, T., Nakade, S., Kanno, T., and Mikoshiba, K. (1997) J. Biochem. (Tokyo) 122, 498–505[Abstract/Free Full Text]
44. Saimi, Y., and Kung, C. (2002) Annu. Rev. Physiol. 64, 289–311[CrossRef][Medline] [Order article via Infotrieve]
45. Schonherr, R., Lober, K., and Heinemann, S. H. (2000) EMBO J. 19, 3263–3271[CrossRef][Medline] [Order article via Infotrieve]
46. Tokok, K., and Trentham, D. R. (1994) Biochemistry 33, 12807–12820[CrossRef][Medline] [Order article via Infotrieve]
47. Mlinar, B., Biagi, B. A., and Enyeart, J. J. (1993) J. Gen. Physiol. 102, 217–237[Abstract/Free Full Text]
48. Doronin, S. V., Potapova, I. A., Lu, Z., and Cohen, I. S. (2004) J. Biol. Chem. 279, 48231–48237[Abstract/Free Full Text]
49. Suh, B. C., and Hille, B. (2002) Neuron 35, 507–520[CrossRef][Medline] [Order article via Infotrieve]
50. Frankenhaeuser, B., and Hodgkin, A. L. (1957) J. Physiol. (Lond.) 137, 218–244[Free Full Text]
51. Capponi, A. M., Lew, P. D., Jornot, L., and Valloton, M. B. (1984) J. Biol. Chem. 259, 8863–8869[Abstract/Free Full Text]
52. Niu, X., Qian, X., and Magleby, K. L. (2004) Neuron 42, 745–756[CrossRef][Medline] [Order article via Infotrieve]
53. Jiang, Y., Lee, A., Chen, J., Cadene, M., Chait, B. T., and MacKinnon, R. (2002) Nature 417, 515–522[CrossRef][Medline] [Order article via Infotrieve]
54. Wen, H., and Levitan, I. B. (2002) J. Neurosci. 22, 7991–8001[Abstract/Free Full Text]
55. Xia, X. M., Fakler, B., Rivard, A., Wayman, G., Johnson-Pais, T., Keen, J. E., Ishii, T., Hirschberg, B., Bond, C. T., Lutsenko, S., Maylie, J., and Adelman, J. P. (1998) Nature 395, 503–507[CrossRef][Medline] [Order article via Infotrieve]
56. Bradley, J., Bonigk, W., Yau, K. W., and Frings, S. (2004) Nature Neurosci. 7, 705–710[CrossRef][Medline] [Order article via Infotrieve]
57. Welsby, P. J., Wang, H., Wolfe, J. T., Colbran, R. J., Johnson, M. L., and Barrett, P. Q. (2003) J. Neurosci. 23, 10116–10121[Abstract/Free Full Text]
58. Chemin, J., Girard, C., Duprat, F., Lesage, F., Romey, G., and Lazdunski, M. (2003) EMBO J. 22, 5403–5411[CrossRef][Medline] [Order article via Infotrieve]
59. Fink, M., Duprat, F., Lesage, F., Reyes, R., Romey, G., Heurteaux, C., and Lazdunski, M. (1996) EMBO J. 15, 6854–6862[Medline] [Order article via Infotrieve]
60. Lymangrover, J. R., Matthews, E. K., and Saffran, M. (1982) Endocrinology 110, 462–468[Abstract/Free Full Text]
61. Matthews, E. K. (1967) J. Physiol. (Lond.) 189, 139–148[Abstract/Free Full Text]
62. Matthews, E. K., and Saffran, M. (1973) J. Physiol. (Lond.) 234, 43–64[Abstract/Free Full Text]

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