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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
Department of Neuroscience, The Ohio State University, College of Medicine and Public Health, Columbus, Ohio
Submitted 16 January 2007 ; accepted in final form 4 May 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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25%) by the PKC activator phorbol 12,13 dibutyrate (PDBu) through an ATP-dependent mechanism that was blocked by BIM. Addition of Phosphatidylinositol(4,5) bisphosphate diC8 [DiC8PI(4,5)P2], a water-soluble derivative of phosphotidyl inositol 4,5 bisphosphate (PIP2) to the pipette solution failed to alter inhibition by ANG II. bTREK-1 inhibition by ANG II was also insensitive to antagonists of other protein kinases activated by ANG II in adrenocortical cells but was completely blocked by inorganic polytriphosphate PPPi. DiC8PI(4,5)P2 was a weak activator of bTREK-1 channels, compared with the high-affinity ATP analog N6-(2-phenylethyl)adenosine-5'-O-triphosphate (6-PhEt-ATP). These results demonstrate that the modulation of bTREK-1 channels in bovine AZF cells is distinctive with respect to activation by phosphoinositides and nucleotides and inhibition by Gq-coupled receptors. Importantly, ANG II inhibits bTREK-1 channels through a novel pathway that is different from that described for inhibition of native TREK-1 channels in neurons, or cloned channels expressed in cell lines. They also indicate that, under physiological conditions, ANG II inhibits bTREK-1 and depolarizes AZF cells by two, novel, independent pathways that diverge proximal to the activation of PLC. adrenal cortex; ion channels; patch clamp
The metabolic and ionic signaling mechanisms that regulate bTREK-1 activity and couple ACTH, ANG II, and nucleotide receptors to bTREK-1 channel gating are partially understood. In early studies, TREK-1 channels were shown to be activated by membrane stretch, high temperatures, and low pH (33, 34). More recently, TREK-1 has been reported to be activated by membrane phospholipids, including PIP2 (7, 31). Native bTREK-1 channels in bovine AZF cells are activated by intracellular ATP, and other nucleotide triphosphates through nonhydrolytic binding (15, 60). ATP has also been reported to activate TREK-1 channels in rat ventricular myocytes (53). Overall, the gating of bTREK-1 channels by PIP2 and ATP may resemble that of several other channels, including KATP and M-type K+ channels, and cystic fibrosis transmembrane conductance regulator (CFTR) Cl– channels. The activity of each of these channels can be regulated by PIP2 and ATP at physiological concentrations by binding to identical or related sites on the channel (23, 32, 39, 47).
The signaling pathways that couple receptors for ANG II to bTREK-1 inhibition are also complex and incompletely understood. ANG II inhibits bTREK-1 K+ current and depolarizes bovine AZF cells through activation of a losartan-sensitive AT1 receptor (14, 36). Although AT1 receptors in adrenocortical cells activate multiple signaling pathways (28, 49, 55), the primary transduction mechanism is the Gq-mediated activation of PLC
, leading to the production of inositol trisphosphate (IP3) and diacylglycerol (DAG) from PIP2. These PIP2-derived second messengers activate effectors, including PKC and Ca2+ channels of the endoplasmic reticulum (28, 50). Further, the PLC-modulated hydrolysis of PIP2 leads to the depletion of this membrane-associated phosphoinositide (59). It is not clear which of these PLC-dependent signaling mechanisms mediates bTREK-1 inhibition by ANG II in bovine adrenocortical cells.
In this regard, studies describing the modulation of native or cloned neuronal TREK-1 channels by Gq-coupled receptors have implicated PKC, DAG, and PIP2 as mediators, but the results are conflicting. Specifically, although the reports indicate that TREK-1 channels can be activated by PIP2, the importance of PLC-mediated PIP2 degradation to TREK-1 inhibition is controversial. Although one study reported that the inhibition of TREK-1 through PLC-coupled receptors is mediated through the hydrolysis-dependent depletion of PIP2, a second study found that Gq-coupled receptors inhibit these K+ channels through the activation of PKC, independently of PIP2 hydrolysis (31, 38). A third study reported that TREK-1 inhibition through PLC-coupled receptors was mediated directly by DAG independently of PKC activation or PIP2 depletion (6).
Nearly all of the studies of TREK-1 modulation by Gq-coupled receptors were done using cloned channels expressed in cell lines. The relevance of these studies to modulation of native TREK-1 channels is uncertain. Recently, we reported that ANG II inhibited native bTREK-1 K+ channels in AZF cells by separate Ca2+- and ATP hydrolysis-dependent mechanisms (14). The Ca2+-dependent inhibition of bTREK-1 requires activation of PLC. The signaling mechanism for the ATP hydrolysis-dependent inhibition of bTREK-1 by ANG II has not been determined.
In this study, we discovered that ATP-dependent inhibition of bTREK-1 by ANG II occurs by a mechanism that is different from that described for TREK-1 inhibition through Gq-coupled receptors in other cells or expression systems. Specifically, this novel pathway is independent of PLC, PKC, PIP2 hydrolysis, and DAG. Overall, these results indicate that, under physiological conditions, ANG II inhibits bTREK-1 K+ channels and depolarizes bovine adrenocortical cells by parallel ATP- and Ca2+-dependent mechanisms that have not been described for TREK-1 inhibition in other cells.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-S, ACTH (1-24), ANG II, PPPi, and EGTA were obtained from Sigma (St. Louis. MO). U73122
[GenBank]
and U0126 were purchased from Tocris Bioscience (Ellisville, MO). Edelfosine, wortmannin, SB203580, phorbol-12,13-dibutyrate (PDBu), bisindolylmaleimide I (BIM), and calphostin C were obtained from EMD Biosciences (San Diego, CA). DiC8PI(4,5)P2 was purchased from Echelon Biosciences (Salt Lake City, UT), curcumin was from Biomol (Plymouth Meeting, PA). N6-(2-Phenylethyl)adenosine-5'-O-triphosphate, sodium salt (6-PhEt-ATP, Biolog #P-012) was purchased from Axxora, LLC (San Diego, CA). Isolation and culture of AZF cells. Bovine adrenal glands were obtained from steers (age 2–3 yr) at a local slaughterhouse. Isolated AZF cells were obtained and prepared as previously described (15). After isolation, cells were either resuspended in DMEM/F12 (1:1) with 10% FBS, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and the antioxidants 1 µM tocopherol, 20 nM selenite, and 100 µM ascorbic acid (DMEM/F12+) were plated for immediate use, or resuspended in FBS/5% DMSO, divided into 1-ml aliquots and stored in liquid nitrogen for future use. For patch-clamp experiments, cells were plated in DMEM/F12+ in 35-mm dishes containing 9 mm2 glass coverslips. To ensure cell attachment, coverslips were treated with fibronectin (10 µg/ml) at 37°C for 30 min then rinsed with warm, sterile PBS immediately before adding cells. Cells were maintained at 37°C in a humidified atmosphere of 95% air-5% CO2.
Patch-clamp experiments. Patch-clamp recordings of K+ channel currents were made in the whole cell configuration from bovine AZF cells. The standard 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 7.3 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 patch pipette solution was maintained at pH 6.8 to enhance the expression of bTREK-1. The buffering capacity of the pipette solutions was varied by adding combinations of Ca2+ and BAPTA or EGTA using the Bound and Determined software program (2). Low- and high-capacity Ca2+-buffering solutions contained 0.5 mM EGTA and 11 mM BAPTA, respectively. The low -capacity Ca2+-buffering solution was nominally 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.
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 8–15 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. Drugs were applied externally by bath perfusion controlled manually by a six-way rotary valve. Patch electrodes with resistances of 1.0–2.0 M
were fabricated from Corning 0010 glass (World Precision Instruments, Sarasota, FL). K+ currents were recorded at room temperature (22–24°C) following the procedure of Hamill et al. (22) 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, 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 1/2 to 1/4 pulse amplitude. Data were analyzed using PCLAMP (CLAMPFIT 9.2) and SigmaPlot (ver. 10.0) software.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The absence of time- and voltage-dependent inactivation allows bTREK-1 K+ current to be isolated in whole cell recordings using either of two voltage clamp protocols. When voltage steps of several hundred millisecond duration are applied from a holding potential of –80 mV, bTREK-1 K+ current can be measured near the end of a voltage step when the transient Kv1.4 current has fully inactivated (Fig. 1, A–C, left traces). Alternatively, bTREK-1 K+ current can be selectively activated by an identical voltage step applied immediately after a 10-s prepulse to –20 mV has fully inactivated Kv1.4 channels (Fig. 1, A–C, right traces).
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PLC and ANG II Inhibition of bTREK-1. The ineffectiveness of ANG II in the absence of hydrolyzable ATP could suggest that activation of a PLC-dependent kinase is required for bTREK-1 inhibition. Experiments were done to characterize the ATP-dependent pathway for ANG II inhibition of bTREK-1. Both the Ca2+- and ATP hydrolysis-dependent inhibition of bTREK-1 are mediated through an AT1-type ANG II receptor (14, 35, 36). Activation of AT1 receptors in AZF cells is coupled to PLC activation through the GTP binding protein Gq (50). ANG II-dependent inhibition of bTREK-1 through the Ca2+-dependent pathway is blunted by the PLC antagonist U73122 [GenBank] (14, 54). To determine whether the ATP-dependent inhibition of bTREK-1 required the activation of PLC, whole cell recordings were made in the presence of either U73122 [GenBank] or a second PLC antagonist, the ether lipid analog edelfosine (42). When K+ currents were recorded with pipette solutions containing U73122 [GenBank] (3 µM), inhibition of bTREK-1 by ANG II (10 nM) was not reduced. With standard pipette solutions, ANG II (10 nM) inhibited bTREK-1 by 78.6 ± 5.2% (n = 12), compared with 79.7 ± 6.9% (n = 4) in the presence of U73122 [GenBank] (Fig. 1, B and D).
In a second series of experiments, AZF cells were preincubated for 30 min with edelfosine (10 µM) before recording bTREK-1 currents with standard pipette solution. Preincubation of cells with edelfosine failed to reduce bTREK-1 inhibition by ANG II (Fig. 1, C and D). In other experiments, K+ currents were recorded from cells that had been preincubated with edelfosine using pipettes containing edelfosine (10 µM). Under these conditions, ANG II inhibited bTREK-1 by 84.5 ± 4.4% (n = 4), a value not significantly different from control (Fig. 1D).
PKC and ANG II inhibition of bTREK-1. In AZF cells, ANG II-mediated activation of PLC leads to the synthesis of DAG, which, in turn, activates PKC (28, 50). The failure of U73122 [GenBank] and edelfosine to suppress ATP-dependent inhibition of bTREK-1 suggests that neither PLC nor PKC is necessary for ANG II-mediated inhibition of bTREK-1 through this pathway. However, these results do not exclude the possibility that activation of PKC alone would be sufficient to inhibit bTREK-1.
Phorbol esters such as PDBu activate PKC at nanomolar concentrations (3). It was discovered that PDBu (100 nM) inhibited bTREK-1 by 31.7 ± 9.0% (n = 10) with pipette solutions containing 5 mM MgATP (Fig. 2, A and C). The activation of PKC by DAG is facilitated by Ca2+ (40). However, increasing [Ca2+] in the pipette solution from 20 nM to 200 nM failed to enhance the inhibition of bTREK-1 by PDBu (100 nM) (Fig. 2C).
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At higher concentrations, PDBu inhibited bTREK-1 more effectively, but a large fraction of this inhibition was independent of PKC. As shown in Fig. 2C, PDBu (1 µM) inhibited bTREK-1 by 67 ± 4.4% (n = 3) with MgATP in the pipette solution, compared with 51.3 ± 7.4% (n = 3) in the presence of UTP. Thus, depending on its concentration, PDBu inhibits bTREK-1 by PKC-dependent and -independent mechanisms.
Experiments with PDBu showed that native bTREK-1 could be partially inhibited by activation of PKC. If the ATP-dependent actions of ANG II on bTREK-1 were mediated solely by PKC, then inhibition of this current by PDBu and ANG II should be similar and less than additive. However, when AZF cells were sequentially superfused with PDBu (100 nM), followed by ANG II (10 nM), inhibition by these two agents was additive, and ANG II was far more effective (Fig. 3A). In these experiments, PDBu inhibited bTREK-1 by 21.0 ± 4.9% (n = 3), while ANG II (10 nM) inhibited the remaining current by 80.5 ± 7.0% (n = 3).
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The PKC-independent inhibition of bTREK-1 by ANG II was confirmed in additional experiments in which either PDBu or ANG II were applied to cells while recording bTREK-1 with pipettes containing BIM (1 or 5 µM). With 5 µM BIM in the pipette, the inhibition of bTREK-1 by PDBu was reduced from its control value of 31.7 ± 9.0% (n = 10) to 12.4 ± 6.2% (n = 9) (Fig. 3D). In contrast, with pipette solutions containing 1 or 5 µM BIM, ANG II inhibited bTREK-1 by 76.9 ± 7.4% (n = 7) and 67.7 ± 5.5% (n = 13), respectively (Fig. 3, C and D). Thus, even at the higher concentration, BIM only slightly blunted ANG II inhibition of bTREK-1.
TREK-1 resembles M-type (KCNQ2/3) K+ channels in that both can be associated with an AKAP150 anchoring protein within a signaling complex (27, 45). The PKC-dependent inhibition of KCNQ channels observed upon activation of a muscarinic receptor is suppressed by calphostin C, which acts at the DAG binding site of the enzyme, but not by BIM, which acts at the catalytic site (27, 46). In this regard, calphostin C was particularly effective at suppressing KCNQ1 inhibition at low agonist concentrations, thereby effecting a pronounced rightward shift in the dose-response curve (27). This raised the possibility that at low concentrations, ANG II inhibits bTREK-1 through activation of PKC, while at higher concentrations, the activation of additional pathways masked this effect.
We found that calphostin C was ineffective at suppressing ANG II-mediated inhibition of bTREK-1, even at low ANG II concentrations (Fig. 4). With pipettes containing calphostin C (1 µM), ANG II inhibited bTREK-1 with an IC50 of 95 pM, a value slightly lower than that previously observed under the control condition (35). Overall, these results provide convincing evidence that over a wide range of concentrations, ANG II effectively inhibits bTREK-1 K+ channels through an ATP-dependent pathway that does not involve activation of PKC.
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20 nM (51). Application of SB203580 (10 µM) to the cytoplasm directly through the pipette also failed to blunt ANG II-mediated inhibition of bTREK-1 (Fig. 5, A and B). With SB203580 in the pipette solution, ANG II inhibited bTREK-1 by 90.4 ± 3.13% (n = 4), compared with the control value of 78.6 ± 5.2% (n = 12).
Activation of PI3 kinase in bovine adrenocortical cells by ANG II is linked to the activation of other kinases, including Raf-1 (49). PI3 kinase is selectively inhibited by wortmannin with an IC50 of 
3 nM (58). However, wortmannin failed to blunt ATP-dependent inhibition of bTREK-1 by ANG II (Fig. 5, A and B). In the presence of wortmannin (100 nM), ANG II (10 nM) inhibited bTREK-1 by 79.4 ± 8.3% (n = 3).
ANG II stimulates cortisol and aldosterone secretion from H295R human adrenocortical cells by activation of PKD (44). PKD is inhibited by curcumin with an IC50 of 4.1 µM (57). The addition of curcumin (20 µM) to the pipette solution also failed to suppress the inhibition of bTREK-1 by ANG II (Fig. 5, A and B).
At 10 nM, ANG II activates multiple kinases in bovine adrenal cortical cells, including PKC, MAP42, and PI3 kinase (27). The failure of individual kinase antagonists to suppress inhibition of bTREK-1 could indicate that inhibition by ANG II is mediated through multiple kinases, which act synergistically. However, the addition of multiple kinase antagonists including U0126, BIM, and wortmannin to patch electrodes failed to significantly affect inhibition of bTREK-1 by ANG II (Fig. 5B).
Inorganic polyphosphates block ATP-dependent bTREK-1 inhibition. The bTREK-1 K+ channel resembles the CFTR Cl– channel in its complex regulation by protein kinases, nucleotides, and polyphosphates (1, 8, 60). The gating of CFTR channels is coupled to an ATP hydrolysis cycle, which is blocked by inorganic phosphates, locking CFTR in the open state (1, 4, 21).
Native bTREK-1 channels are activated by the polytriphosphate PPPi (60). In the present study, we found that PPPi blocked ATP- but not Ca2+-dependent inhibition of bTREK-1. When K+ currents were recorded with pipette solutions containing PPPi (2 mM) and MgATP (1 mM), ANG II was ineffective as an inhibitor of bTREK-1, reducing this current by only 10.0 ± 5% (n = 3) (Fig. 5, A and B). The suppression of ATP-dependent inhibition of bTREK-1 by PPPi was specific. When K+ currents were recorded with PPPi containing pipette solutions that supported activation of the Ca2+ but not the ATP-dependent pathway, ANG II inhibited bTREK-1 by 46.7 ± 7.8% (n = 6), a value similar to that obtained previously in the absence of PPPi (14) (Fig. 5B).
PIP2 and bTREK-1 gating. The membrane phosphoinositide PIP2 regulates the gating of a number of K+ channels, including members of the 2P/4TMS family of leak channels (7, 31, 32, 39). While several studies have reported that TREK-1 activity is increased by PIP2, only one of these has found that receptor-mediated PIP2-depletion mediates inhibition of TREK-1 K+ channels (7, 31, 38). Nearly all of the conflicting results were obtained in studies of cloned TREK-1 channels expressed in cell lines.
We did experiments to determine whether PIP2 could modulate the activity of native bTREK-1 in AZF cells. At concentrations as low as 1 µM, the water-soluble PIP2 derivative DiC8PI(4,5)P2 has been reported to markedly activate neuronal TREK-1 channels (7). At 40 µM, DiC8PI(4,5)P2 increased bTREK-1 current density modestly from a control value of 35.2 ± 6.0 pA/pF (n = 9) to 50.6 ± 11.7 pA/pF (n = 11) (Fig. 6A).
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In these experiments, recordings were made with pipette solutions containing DiC8PI(4,5)P2 (40 µM). When the bTREK-1 amplitude reached a stable maximum, AZF cells were superfused with AA (10 µM). AA proved to be far more effective than DiC8PI(4,5)P2 as an activator of bTREK-1 (Fig. 6B). In four experiments, bTREK-1 current density reached a maximum of 23.1 ± 3.4 pA/pF (n = 4) in the presence of DiC8PIP2. Subsequent superfusion of AA increased bTREK-1 density nearly 10-fold to 209 ± 47 pA/pF (n = 4) (Fig. 6B). These results show that DiC8PIP2 activates a very small percentage of available bTREK-1 channels in AZF cells.
Activation of bTREK-1 by a high-affinity ATP analog. ATP and other nucleotide triphosphates activate native bTREK-1 K+ channels at millimolar concentrations. ATP analogs have been synthesized that bind with high affinity to ATP-binding proteins. One of these is 50 times more potent than ATP as an activator of CFTR chloride channels (62). We found that this same ATP analog, 6-PhEt-ATP, was significantly more effective than DiC8PI(4,5)P2 as an activator of bTREK-1. Specifically, with pipettes containing 6-PhEt-ATP (50 µM), bTREK-1 current density reached a value more than twice that observed with DiC8PI(4,5)P2 (Fig. 6A). At much higher concentrations, UTP and PPPi were both more effective than DiC8PI(4,5)P2 as an activator of bTREK-1.
PIP2 and voltage-dependent bTREK-1 gating. Native bTREK-1 K+ channels display limited voltage-dependent gating in whole-cell recordings (15). PIP2 has been reported to enhance the activity of cloned bTREK-1 channels by shifting the voltage-dependent activation in the hyperpolarizing direction so that channel open probability is increased at more negative potentials (31).
Experiments were done to determine whether PIP2 altered the voltage-dependent gating of native bTREK-1 channels in AZF cells. In whole cell recordings, ramp voltage steps were applied between test potentials of +60 and –100 mV, with or without DiC8PI(4,5)P2 (40 µM) in the pipette solution. Fig. 6C shows averaged, normalized current traces derived from control and DiC8PI(4,5)P2-treated cells. The currents were nearly superimposable over the entire range of potentials. Importantly, no significant difference in the relative amplitude of bTREK-1 currents was observed at more negative potentials where a hyperpolarizing shift would be most evident.
PIP2 and ANG II-dependent inhibition of bTREK-1 K+ channels. Experiments were done to determine whether the ATP-dependent inhibition of bTREK-1 involved the depletion of membrane-associated PIP2. To explore this possibility, cells were superfused with ANG II (10 nM) while recording K+ currents with pipette solutions containing DiC8PI(4,5)P2 (40 µM), thereby delivering an unlimited supply of this phosphoinositide directly to the cytoplasm. DiC8PI(4,5)P2 failed to reduce ANG II-dependent inhibition of bTREK-1 (Fig. 7A). With DiC8PI(4,5)P2 in the pipette solution, ANG II inhibited bTREK-1 by 86.1 ± 3.6% (n = 3), compared with the control value of 78.6 ± 5.2% (n = 12).
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To determine whether ANG II-dependent inhibition of bTREK-1 is voltage dependent, bTREK-1 currents were activated by voltage ramps between +100 and –100 mV, before and after superfusing cells with ANG II (10 nM). In these experiments, bTREK-1 was inhibited equivalently over the range of voltages where it could be accurately measured (Fig. 7B). In particular, ANG II effectively inhibited bTREK-1 at +100 mV. If ANG II functioned through a depolarizing shift in voltage-dependent activation, it would have been much less effective at reducing bTREK-1 at this positive potential. Thus, bTREK-1 inhibition by ANG II through the ATP-dependent pathway is apparently not mediated by modulating voltage-dependent gating.
PIP2 and Ca2+-dependent regulation of bTREK-1. In contrast to the ATP-dependent inhibition of bTREK-1, Ca2+-dependent inhibition of this channel by ANG II is suppressed by the PLC antagonist U73122 [GenBank] (14). Experiments were done to determine whether the Ca2+- and PLC-dependent inhibition of bTREK-1 by ANG II required PIP2 depletion. When K+ currents were recorded with pipette solutions designed to allow the selective activation of the Ca2+-dependent pathway (2 mM UTP, 0.5 mM EGTA), inclusion of DiC8PI(4,5)P2 in the pipette again failed to blunt ANG II-dependent inhibition of bTREK-1 (Fig. 8, A and B). In the presence of DiC8PI(4,5)P2 (40 µM), ANG II inhibited bTREK-1 by 67.2 ± 5.0% (n = 6), compared with the control value of 57.8 ± 4.0% (n = 4). The failure of DiC8PI(4,5)P2 to alter Ca2+-dependent inhibition of bTREK-1 indicates that endogenous PIP2 depletion does not contribute to this response. In these same experiments with pipette solutions containing 2 mM UTP, DiC8PI(4,5)P2 also failed to increase bTREK-1 current density compared with the control value (Fig. 8C).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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When combined with the findings of a previous study (14), the results reported here demonstrate that the modulation of AZF bTREK-1 K+ channels, including their activation by phosphoinositides and nucleotide triphosphates and inhibition through Gq-coupled receptors, is distinctly different from that reported for native and cloned TREK-1 channels from other species and organs. With regard to inhibition, the novel ATP- and Ca2+-dependent mechanisms allow for complete inhibition of bTREK-1 in nearly every cell, providing an effective fail-safe means for membrane depolarization (Fig. 9). Our results do not, however, rule out the possibility of a contribution from PKC in bTREK-1 inhibition by ANG II. Overall, they demonstrate that the modulation of native bTREK-1 channel activity in AZF cells is complex, and mediated through multiple signaling pathways.
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even in the absence of the PLC antagonists. Activation of PCL is strictly Ca2+-dependent and is blocked, provided that [Ca2+]i is maintained below resting levels (25, 43). Overall, we have provided convincing evidence that the ATP-dependent inhibition of bTREK-1 by ANG II bypasses PLC. ANG II-mediated activation of PLC is the major mechanism for PKC activation in adrenocortical cells. Because ANG II inhibited bTREK-1 in the apparent absence of PLC activation, it was not surprising that inhibition was not blocked by the PKC antagonist BIM. BIM is among the most potent and specific PKC antagonists available, with a reported IC50 as low as 10 nM (56). However, because BIM is a competitive inhibitor of ATP-binding to PKC, its potency varies inversely with the ATP concentration. When ATP is present at millimolar concentrations, the IC50 for BIM increases to concentrations as high as 1 µM or more (56). Therefore, it was critical to establish that BIM effectively inhibited PKC when 5 mM ATP was present in the pipette. Accordingly, experiments with PDBu showed that this phorbol ester significantly inhibited bTREK-1 and that this response was completely eliminated by BIM at concentrations that did not affect the inhibition by ANG II. Overall, these results indicate that BIM effectively suppressed PKC-dependent bTREK-1 inhibition and that inhibition by ANG II, at least at this high concentration (10 nM), occurred through a separate mechanism.
Experiments with calphostin C were done to address the possibility that bTREK-1 channels in AZF cells are bound, along with PKC, to AKAP150-anchoring protein, an association that renders an antagonist like BIM, but not calphostin C, less effective at suppressing PKC activation (27, 45). Calphostin C produced a distinct rightward shift in the dose-response curve for Gq-coupled receptor-dependent inhibition of AKAP150-associated KCNQ2/3 K+ channels (27). In contrast, calphostin C did not affect the potency of ANG II as an inhibitor of bTREK-1. Thus, PKC does not contribute to TREK-1 inhibition by ANG II regardless of concentration.
Other kinases. Antagonists of other kinases activated by ANG II in bovine AZF cells were also ineffective at suppressing the inhibition of bTREK-1 by ANG II. Although each of these antagonists was used at concentrations many fold higher than their reported IC50s, it is possible that the targeted enzymes were not completely inhibited under the conditions of our experiments. However, at the concentrations utilized, each of the inhibitors has been shown to effectively inhibit the specified kinase in intact cells (9, 11, 19, 57, 58).
The failure of individual antagonists to alter bTREK-1 inhibition by ANG II suggested the possibility that this response was mediated through the activation of multiple kinases, each of which was sufficient to produce inhibition. However, the ineffectiveness of U0126, BIM, and wortmannin applied together through the pipette in suppressing bTREK-1 inhibition by ANG II argues against this possibility. At the very least, this result indicates that simultaneous inhibition of the three major kinases activated by ANG II in these cells does not blunt the ANG II response.
In a previous study, we found that antagonists of other ANG II-activated kinases, including tyrosine kinases and JAK2 kinases, were ineffective at suppressing bTREK-1 inhibition by ANG II (60). These results may indicate that ANG II inhibits bTREK-1 by an, as yet, unidentified kinase. Alternatively, the possibility that the ATP hydrolysis-dependent inhibition of bTREK-1 is mediated by an ATPase cannot be excluded. The remarkable activation of bTREK-1 by 6-PhEt-ATP and by nucleotide triphosphates and inorganic phosphates resembles that of CFTR, wherein the gating of the Cl– channel is regulated through a cycle of ATP binding and hydrolysis (1, 20, 29, 60, 62).
Modulation of TREK-1 channel gating by PIP2 and ATP. ATP and PIP2 have been reported to comodulate the gating of many ion channels. In addition to the CFTR Cl– channel, members of each of the major classes of K+ channels, including voltage-gated channels, inward rectifiers, and 4TMS/2P background channels, are reportedly modulated by this nucleotide and phosphoinositide (12, 23, 31, 32, 48, 52). In some cases, these modulators compete for identical or related sites on the channel (32). Interestingly, for all of these channels, gating is controlled by the synthesis and hydrolysis of these phosphate-containing molecules.
In the present study, DiC8PI(4,5)P2 (40 µM) was a relatively weak activator of bTREK-1 when included in the patch electrode with 1 mM NaATP. By comparison, under the same conditions, 6-PhEt-ATP (50 µM) produced a fourfold greater increase in bTREK-1 activity. The activation of bTREK-1 by 6-PhEt-ATP resembles its activation of CFTR Cl– channels, wherein it binds with more than 50-fold higher affinity than ATP (62). The ATP binding site associated with bTREK-1 channels has not been identified.
In contrast to our results, PIP2 was reported to robustly activate native neuronal or transfected TREK-1 channels by interacting with a cluster of positively charged amino acids on the carboxy terminal domain (7, 31). These seemingly conflicting results could be due to differing experimental conditions. In our experiments, bTREK-1 was measured in whole cell recordings with 1 mM NaATP in the pipette, while in the above-mentioned studies, bTREK-1 was recorded from excised patches in the absence of ATP. The relative ineffectiveness of DiC8PI(4,5)P2 as an activator of bTREK-1 suggests that, under physiological conditions where ATP is present at millimolar concentrations, fluctuations in PIP2 may be less important in regulating bTREK-1 activity in AZF cells.
bTREK-1 inhibition and PIP2 hydrolysis. Overall, our results do not support a role for PLC-dependent PIP2 hydrolysis and depletion in any of the separate pathways by which ANG II inhibits bTREK-1 channels. First, the ATP-dependent inhibition of bTREK-1 can occur under conditions where PLC activation has been suppressed by strong Ca2+ buffering and the PLC antagonists U73122 [GenBank] and edelfosine. Further, PLC-coupled PIP2 hydrolysis does not require the hydrolysis of ATP. Therefore, ANG II-stimulated PIP2 depletion would not be prevented by substituting UTP in the patch pipette. However, the ATP-dependent inhibition of bTREK-1 by ANG II is eliminated in the absence of hydrolyzable ATP. Finally, the ATP-dependent inhibition of bTREK-1 is not affected by adding DiC8PI(4,5)P2 to the recording pipette.
Although the Ca2+-dependent inhibition of bTREK-1 by ANG II proceeds through a PLC-dependent mechanism, this pathway was also unaffected by including DiC8PIP2 in the pipette. Ca2+-dependent inhibition of bTREK-1 appears to involve IP3-stimulated Ca2+ release, rather than PIP2 hydrolysis and depletion (14).
Comparison to modulation of other TREK-1 channels. The combined results of this and a previous study have shown that ANG II can produce near-complete inhibition of bTREK-1 in AZF cells by two pathways that are completely independent of PKC, DAG, and PIP2 hydrolysis (14). In this regard, considerable controversy exists regarding the modulation of other TREK-1 channels by these three signals. Murbartian et al. (38) reported that Gq-coupled receptors inhibit TREK-1 in transfected HEK293 cells through activation of PKC and phosphorylation of a specific amino acid. They also found that PDBu (1 µM) robustly inhibited TREK-1. However, at this concentration, a large fraction of bTREK-1 inhibition by PDBu may be unrelated to PKC activation.
In contrast to these findings, Chemin et al. (6) report that TREK-1 inhibition through PLC-coupled receptors is not mediated through DAG activation of PKC, but through direct inhibition of the channel by DAG (50 µM). However, lipid soluble diacylglycerol analogs nonspecifically affect the function of many ion channels when used at micromolar concentrations (24).
Finally, Lopes et al. (31) report that inhibition of cloned bTREK-1 channels by ACh through PLC-coupled muscarinic receptors is not mediated through PKC or DAG, but through PIP2 hydrolysis and depletion. In this study, it was reported that PIP2 converts TREK-1 from a voltage-gated outwardly rectifying channel to an open leak channel by inducing a large negative shift in the voltage dependence of activation. Conversely, receptor-mediated PIP2 hydrolysis inhibited bTREK-1 through a marked depolarizing shift in activation voltage (31). Under the condition of our experiments on native bTREK-1 channels, PIP2 did not change the voltage-dependent rectifying properties of bTREK-1. Further, inhibition of bTREK-1 by ANG II was voltage independent, a result consistent with our finding that this response is not mediated through PIP2 hydrolysis.
Overall, studies exploring the modulation of native and cloned TREK-1 channels through PLC-coupled receptors demonstrate that inhibition of these channels occurs through activation of multiple signaling pathways, involving one or more kinases, Ca2+, phospholipids, and perhaps, ATPases. Given the rich and complex network of modulators, it is not surprising that studies on native TREK-1, as well as cloned channels expressed in oocytes and cell lines, have produced varied and conflicting results.
In recent years, multiple studies have demonstrated that G protein-coupled receptors modulate ion channels through cell type- and species-specific signaling complexes that include scaffolding proteins, enzymes, guanine nucleotide exchange factors, and other nucleotides (26, 27). It is highly unlikely that the native signaling complexes are present in the cell lines where modulation of transfected channels is often studied. The emerging picture highlights the importance of studying receptor-mediated modulation of a channel in its native cell.
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) or without (
) prepulses are plotted against time. Numbers on traces correspond to those on the plot. Pipette solution: 5 mM NaUTP (A) or 5 mM MgATP (B, C). D: Summary of experiments as in (A–C). Bars indicate the percentage of bTREK-1 blocked by ANG II under control conditions, with U73122 (3 µM) or edelfosine (10 µM) in the pipette solution with edelfosine in both external and pipette solutions. Values are means ± SE for the indicated number of determinations.
) or DiC8PI(
), as indicated. Right: Summary of bTREK-1 current densities (expressed in pA/pF). Values are means ± SE of indicated number of determinations. B: effect of DiC8PI(
