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RECEPTORS AND SIGNAL TRANSDUCTION

Mechanism of inhibition of TREK-2 (K_2P10.1) by the G_q-coupled M₃ muscarinic receptor

¹Department of Physiology and Biophysics, Rosalind Franklin University of Medicine and Science, The Chicago Medical School, North Chicago, Illinois; and ²Medical Research Center for Neural Dysfunction and Department of Physiology, Gyeongsang National University College of Medicine, Jinju, Korea

Submitted 1 February 2006 ; accepted in final form 19 April 2006

	ABSTRACT

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TREK-2 is a member of the two-pore domain K⁺ channel family and provides part of the background K⁺ current in many types of cells. Neurotransmitters that act on receptors coupled to G_q strongly inhibit TREK-2 and thus enhance cell excitability. The molecular basis for the inhibition of TREK-2 was studied. In COS-7 cells expressing TREK-2 and M₃ receptor, acetylcholine (ACh) applied to the bath solution strongly inhibited the whole cell current, and this was markedly reduced in the presence of U-73122, an inhibitor of PLC. The inhibition was also observed in cell-attached patches when ACh was applied to the bath solution. In inside-out patches, direct application of guanosine 5'-O-(3-thiotriphosphate) (10 µM), Ca²⁺ (5 µM), or diacylglycerol (DAG; 10 µM) produced no inhibition of TREK-2 in >75% of patches tested. Phosphatidic acid, a product of DAG kinase, had no effect on TREK-2. Pretreatment of cells with 20 µM wortmannin, an inhibitor of phosphatidylinositol kinases, did not affect the inhibition or the recovery from inhibition of TREK-2, suggesting that phosphatidylinositol 4,5-bisphosphate depletion did not mediate the inhibition. Pretreatment of cells with a protein kinase C inhibitor (bisindolylmaleimide, 10 µM) markedly inhibited ACh-induced inhibition of TREK-2. Mutation of two putative PKC sites (S326A, S359C) abolished inhibition by ACh. Mutation of these amino acids to aspartate to mimic the phosphorylated state resulted in diminished TREK-2 current and no inhibition by ACh. These results suggest that the agonist-induced inhibition of TREK-2 via M₃ receptor occurs primarily via PKC-mediated phosphorylation.

two-pore domain potassium channel; G_q protein; background potassium conductance; protein kinase C; phosphatidylinositol 4,5-bisphosphate

Recent studies show that neurotransmitters that act on receptors coupled to G_q inhibit TREK-2 in expression systems as well as in neurons (4, 28). Therefore, TREK-2 is an important target of receptor agonists that modulate cell excitability. In fact, inhibition of a background K⁺ current such as TREK-2 may be one of the central mechanisms that regulate action potential firing in excitable cells. The molecular basis of agonist-induced inhibition of TREK-2 via receptor coupled to G_q is still poorly understood. Because G_q is known to stimulate phospholipase C (PLC), signaling molecules generated by PLC have been tested. phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), inhibited TREK-2, suggesting that PKC might mediate the agonist-induced inhibition (14). Another study reported that agonist-induced inhibition of TREK-2 was due to a direct action of diacylglycerol (DAG) and phosphatidic acid that are generated via PLC (4). Other studies have reported that receptor agonists inhibit TREK-1, a K_2P channel member that is closely related to TREK-2 in function and amino acid identity (63%), via depletion of phosphatidylinositol 4,5-bisphosphate (PIP₂) levels (30), ATP-dependent pathways (10), directly by elevated intracellular Ca²⁺ levels (10), or phosphorylation by PKC (33). These findings suggest the possibility that TREKs may be modulated not by a single mechanism but by several signaling pathways and that different receptors modulate TREKs via distinct pathways.

In this study, we specifically examined the signaling pathway by which binding of ACh to muscarinic receptor coupled to G_q modulates TREK-2 expressed in COS-7 cells. All relevant signaling molecules generated by PLC pathway were tested using wild-type and mutant TREK-2. Both cell-attached and whole cell recording modes were employed to assess the nature of the signaling molecules. Our results provide evidence that the primary pathway by which agonists inhibit TREK-2 via G_q is through activation of PKC that phosphorylates two serine residues at the COOH terminus of TREK-2.

	METHODS

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Transfection in COS-7 cells. Full-length rat TREK-2 (GenBank accession no. NM 207261) and TASK-3 (AF192366) were cloned previously in this laboratory (1, 24). pcDNA3.1 containing the coding region of human M₃ receptor was obtained from the Guthrie cDNA resource center. The coding region of TREK-2 and TASK-3 were subcloned into pcDNA3.1 vector (Invitrogen, Carlsbad, CA). COS-7 cells were seeded at a density of 2 x 10⁵ cells per 35-mm dish 24 h before transfection in 10% bovine serum in Dulbecco's modified Eagle's medium (DMEM). COS-7 cells were cotransfected with DNA fragments encoding a K_2P channel, M₃ receptor, G protein-coupled inward-rectifying K⁺ channel (GIRK)1, GIRK4, and green fluorescent protein (GFP) in pcDNA3.1 as desired using Lipofectamine and Opti-MEM I reduced serum medium (Life Technologies). Green fluorescence from cells expressing GFP was detected with the aid of a Nikon microscope equipped with a mercury lamp light source. Cells were used 1–3 days after transfection.

TREK-2 mutants. Single and double point mutations were done using a QuikChange site-directed mutagenesis kit (Stratagene) to generate serine-to-alanine and serine-to-asparate mutations in pcDNA3.1. Both strands of mutated TREK-2 DNA fragments were sequenced for confirmation.

Electrophysiological studies. Electrophysiological recording was performed using a patch-clamp amplifier (Axopatch 200; Axon Instruments, Union City, CA). Single-channel currents were digitized with a digital data recorder (VR10; Instrutech, Great Neck, NY), and stored on videotape. The recorded signal was filtered at 3 kHz using an eight-pole Bessel filter (–3 dB; Frequency Devices, Haverhill, MA) and transferred to a computer (Dell) using the Digidata 1320 interface (Axon Instruments) at a sampling rate of 20 kHz. Whole cell currents were recorded after canceling the capacitive transients. Whole cell and single-channel currents were analyzed with the pCLAMP program (version 8). For single-channel analysis, the filter dead time was 100 µs (0.3/cutoff frequency) such that events shorter than 50 µs in duration would be missed. Data were analyzed to obtain channel activity (NP_o, where N is the number of channels in the patch and P_o is the probability of a channel being open). NP_o was determined from 1 min of current recording. The single-channel current tracings shown were filtered at 2 kHz. In experiments using cell-attached and excised patches, pipette and bath solutions contained (in mM) 150 KCl, 1 MgCl₂, 5 EGTA, and 10 HEPES (pH 7.3). In cell-attached and whole cell recordings to study the effect of ACh on TREK-2, bath solution contained (in mM) 135 NaCl, 5 KCl, 1 CaCl₂, 1 MgCl₂, 5 glucose, and 10 HEPES (pH 7.3). For whole cell current recording, the pipette solution contained 150 mM KCl, 1 mM MgCl₂, 5 mM EGTA, 1 mM ATP, 100 µM GTP, and 10 mM HEPES (pH 7.3). Whole cell configuration was formed by rupturing the membrane using suction. An outside-out patch was formed from the whole cell configuration by gently lifting up the pipette. Free Ca²⁺ concentration in the bath solution was adjusted to desired levels using the Maxchelator program (www.stanford.edu/cpatton/maxc.html). Purified bovine brain -subunit was purchased from Calbiochem. Wortmannin was purchased from Biomol. All other chemicals and enzymes were purchased from Sigma Chemical. For statistics, Student's t-test was used with P < 0.05 as the criterion for significance. Data are represented as means ± SE unless specified otherwise.

	RESULTS

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Inhibition of TREK-2 by ACh via M₃ receptor. Inhibition of TREK-2 by ACh was first tested using COS-7 cells expressing TREK-2 and G_q-coupled M₃ receptor. Whole cell configuration was formed, and the cell membrane potential was held at –80 mV in physiological bath solution containing 5 mM KCl. Ramp voltage pulses from –120 to +60 mV (duration, 865 ms) were applied once every 5 s to record whole cell current. Application of 100 µM ACh to the perfusion solution produced a 66 ± 5% decrease in the whole cell current within 10 s (Fig. 1A). Whole cell current recovered fully in 3 min to the control level after washout of ACh (data not shown).

View larger version (36K): [in this window] [in a new window]   Fig. 1. Inhibition of TREK-2 by ACh in whole cell and cell-attached patches. A: whole cell currents were recorded from COS-7 cells expressing TREK-2 and M3 receptor before and after application of ACh and after washout. Cell membrane potential was held at –80 mV, and ramp pulses were applied from –120 to +60 mV once every 5 s. B: a schematic diagram showing the experimental setup and ionic conditions when cell-attached patches were used. Pipette solution contained 150 mM KCl, and bath solution contained 5 mM KCl and 135 mM NaCl. Pipette potential was held at 0 mV to record inward currents. C: cell-attached patches were formed, and ACh was applied briefly to the bath solution as shown. Channel openings at a higher time resolution are shown at appropriate times (a–c). Bar graph shows the effect of ACh and its washout on TREK-2 channel activity. Each bar represents the mean ± SE of 10 experiments. *P < 0.05, significant difference from the control value.

If a soluble messenger mediates the inhibition, the agonist may be able to inhibit TREK-2 in the cell-attached patch when the agonist is applied to the bath solution. To test this idea, we formed cell-attached patches with pipette solution containing 150 mM KCl and bath solution containing 5 mM KCl, as shown schematically in Fig. 1B. Under these experimental conditions, channel openings in the inward current direction are recorded when the pipette potential is set at 0 mV. When ACh was applied to the bath perfusion solution, a strong inhibition of TREK-2 channel activity was observed (Fig. 1C). The decrease in TREK-2 channel activity was largely due to reduced frequency of opening. A small decrease in single-channel amplitude was also observed, probably due to the resulting depolarization of the cell (Fig. 1C). Washout of ACh resulted in slow recovery of channel activity, similar to the time course of recovery observed at the whole cell level. These results show that the inhibition of TREK-2 by a receptor agonist via G_q involves a signaling molecule that is easily mobile within the cytoplasm (soluble) or the lipid bilayer.

Because M₃ receptor is coupled to G_q that stimulates PLC, we tested whether an inhibitor of PLC reduces ACh-mediated inhibition. Therefore, cells were preincubated for 5 min with 5 µM U-73122, a PLC inhibitor (16), and ACh was applied to the bath solution while the whole cell current was recorded. ACh produced only a small inhibition (17 ± 6%, n = 4) of TREK-2 proteins in the presence of U-73122 (Fig. 2B) compared with 57 ± 4% inhibition in its absence (Fig. 2A). These results are summarized in Fig. 2C and show that the inhibitory signaling pathway is most likely via PLC.

View larger version (15K): [in this window] [in a new window]   Fig. 2. U-73122 reduces inhibition of TREK-2 by ACh. A: ACh-induced inhibition of TREK-2 in COS-7 cells. B: COS-7 cells expressing TREK-2 and M3 receptor were preincubated with 5 µM U-73122 for 5 min. In the presence of U-73122, ACh was applied to the perfusion solution. C: bar graph shows %inhibition of TREK-2 produced by ACh in the presence and absence of U-73122 measured at +60 mV. Each bar presents means ± SE of 4 experiments. *P < 0.05, significant difference from the control value.

View larger version (45K): [in this window] [in a new window]   Fig. 3. Effect of wortmannin on the inhibition of TREK-2 and TASK-3 and recovery from inhibition by ACh. A: cell-attached patches were formed on COS-7 cells expressing TREK-2 and M3 receptor. ACh was applied to the bath perfusion solution and then washed off (top tracing). In other patches, wortmannin was applied 10 min before application of ACh and remained present throughout the experiment (bottom tracing). Pipette potential was 0 mV. B: same protocol as in A except that COS-7 cells expressed TASK-3 and M3 receptor. C: bar graph shows the inhibitory effects of ACh on TREK-2 and TASK-3 in the presence and absence of wortmannin (20 µM). Each bar represents means ± SE of 5 determinations. *P < 0.05, significant difference from the control value.

Given that IP₃ causes a rise in cytosolic free Ca²⁺ concentration by releasing Ca²⁺ from endoplasmic reticulum, it is possible that the rise in intracellular Ca²⁺ concentration causes the inhibition. To test this directly, we formed inside-out patches containing TREK-2 and raised the Ca²⁺ concentration in the bath solution to 5 µM from the basal level of 2 nM (calculated based on Maxchelator program). Ca²⁺ (5 µM) showed no effect on TREK-2 in 18 patches tested but, interestingly, produced a significant inhibition (47 ± 10%) in 3 patches. This suggested the possibility that Ca²⁺ also may be interacting with a Ca²⁺-sensitive modulator that is very loosely bound to the inner surface of the plasma membrane. Such a modulator may be present in a few patches, whereas it is washed off from the membrane in most patches. Because TREK-2 in the majority of patches does not respond to DAG or Ca²⁺, it is most likely that these two molecules do not directly inhibit TREK-2. Nevertheless, the inconsistent effects of DAG and Ca²⁺ were further examined using TREK-2 mutants.

Bisindolylmaleimide (PKC inhibitor) blocks ACh-induced inhibition of TREK-2. DAG is well known to activate PKC in many cell types. To test whether phosphorylation of TREK-2 by PKC mediates ACh-induced inhibition, we preincubated cells for 5 min with 10 µM bisindolylmaleimide I, a potent inhibitor of PKC. In the continuous presence of bisindolylmaleimide, whole cell currents were recorded before and after application of ACh. In control cells in the absence of bisindolylmaleimide, ACh inhibited TREK-2 by 68 ± 4%, as before (Fig. 4A). In the presence of bisindolylmaleimide, ACh produced a much smaller inhibition of TREK-2 (Fig. 4A), suggesting that phosphorylation of TREK-2 by PKC may underlie the inhibition of TREK-2 produced by ACh via PLC-generated DAG. In keeping with the role of PKC, direct application of PMA (10 µM), a potent activator of PKC, also caused inhibition of TREK-2, and this was nearly abolished by pretreatment with bisindolylmaleimide (Fig. 4B). In cell-attached patches, application of ACh to the bath solution produced 74% inhibition of TREK-2 channel activity, as before (Fig. 4C). When bisindolylmaleimide was present 5 min before the application of ACh, no significant inhibition of TREK-2 was observed. The results from whole cell recordings are summarized in Fig. 4D.

View larger version (24K): [in this window] [in a new window]   Fig. 4. Bisindolylmaleimide (Bisindol) blocks ACh-induced inhibition of TREK-2. A: whole cell currents were recorded from COS-7 cells expressing TREK-2 and M3 receptor using the ramp pulse method. ACh was applied to the bath solution in the presence or absence of Bisindol (10 µM). Bisindol was present 5 min before addition of ACh. B: same protocol as in A except that PMA (10 µM) was applied to the bath solution in the presence or absence of Bisindol (10 µM). Bisindol was present 5 min before addition of PMA. C: cell-attached patches were formed on COS-7 cells. ACh was applied briefly to the bath solution in the absence (left) or presence (right) of Bisindol. Pipette potential was 0 mV. D: bar graph shows the effect of ACh and PMA on TREK-2 whole cell current measured at +60 mV. Each bar represents means ± SE of 4 determinations. *P < 0.05, significant difference from the corresponding control value.

ACh caused marked inhibition (50–70%) of all of the mutants except S326A and S359C. S326A and S359C mutants were insensitive to ACh (Fig. 5A), suggesting that phosphorylation at these amino acid residues may underlie the inhibition of TREK-2 by ACh. Therefore, these residues were mutated to aspartate, which mimics the phosphorylated state, and these mutants (S326D, S359D) were expressed in COS-7 cells together with M₃ receptor. TREK-2 currents generated by these mutants were significantly smaller compared with that obtained with TREK-2 wild type (Fig. 5B), consistent with the hypothesis that phosphorylation of these residues leads to channel inhibition. Single channels of S326D and S359D mutants could be recorded from cell-attached patches, and application of arachidonic acid (20 µM) or negative pressure (–100 mmHg) to inside-out patches produced an increase in channel activity, as well as the number of open levels. Therefore, S326D and S359D mutants were functionally expressed in the membrane. These results provide additional evidence that phosphorylation by PKC underlies the inhibition of TREK-2 by ACh.

View larger version (32K): [in this window] [in a new window]   Fig. 5. TREK-2 mutants and response to ACh. A: potential PKC sites were mutated to prevent phosphorylation and expressed in COS-7 cells together with M3 receptor. Whole cell currents were recorded using the ramp pulse protocol before and after application of ACh. B: bar graphs show the current expression level of each mutant (top) and %inhibition of TREK-2 produced by ACh for each single amino acid mutation at +60 mV (bottom). Each bar represents means ± SE of 5–8 determinations. *P < 0.05, significant difference from the value observed with wild-type TREK-2. C: bar graphs show current expression levels of TREK-2 double mutants (top) and %inhibition produced by ACh measured at +60 mV (bottom). Each bar represents means ± SE of 7–11 determinations (indicated on each bar). *P < 0.05, significant difference from the value observed with S326A/S359A mutant. The last 3 bars were significantly different from each other (P < 0.05).

Lack of effect of DAG and Ca²⁺ on S326A:S359A mutant. Because DAG and Ca²⁺ were able to inhibit TREK-2 in some inside-out patches (see Is inhibition of TREK-2 due to PLC-generated molecules?), their effects were also tested on S326A/S359A mutant that cannot be phosphorylated at these sites. The idea behind this experiment was that perhaps DAG or Ca²⁺ somehow caused partial activation of PKC that may be present near the membrane in some patches (perhaps due to different shape of the patch membrane or incomplete washout) and caused inhibition of TREK-2. Such inhibition should not occur with the S326A/S359A mutant. Inside-out patches were formed from COS-7 cells expressing TREK-2/M3 receptor, and 10 µM DAG or 5 µM Ca²⁺ was applied to the bath solution. In all patches tested, both DAG (n = 9) and Ca²⁺ (n = 11) failed to inhibit the S326A/S359A mutant. These results therefore provide additional evidence that DAG and Ca²⁺ are not direct inhibitors of TREK-2. It seems likely that in some patches, DAG or Ca²⁺ caused small inhibition of wild-type TREK-2 via partial activation of PKC remaining with the patch membrane.

	DISCUSSION

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Inhibition of K⁺ channels by receptor agonists as a mechanism for increasing cell excitability is a well-recognized phenomenon. The molecular basis of K⁺ channel inhibition by an agonist that act on G_q-coupled receptor is reasonably well defined for some K⁺ channels (such as M and GIRK channels) but not at all clear for other types of K⁺ channels. In this study, we investigated the signaling pathway by which a receptor agonist coupled to G_q inhibits TREK-2, a member of the two-pore domain K⁺ channel family. TREK-2 is a background (leak) K⁺ channel and therefore should help to set and stabilize the resting membrane potential in those cells that express it. Because TREK-2 is active at rest, its inhibition by receptor agonists should lead to reduced leak K⁺ current and increased cell excitability. Agonists that act on receptors coupled to G_q are well known to stimulate PLC, leading to generation of a number of second messenger molecules that in turn produces or activates other effectors. Study of the effects of these molecules generated by PLC on TREK-2 and its mutants suggests that signaling via PKC may be the primary mechanism for agonist (ACh)-induced suppression of TREK-2 via the muscarinic (M₃) receptor.

Mechanisms for agonist-induced inhibition of K⁺ channels via G_q. One of the most extensively studied signaling pathway by which receptor agonists inhibit a K⁺ channel is the muscarinic receptor-induced inhibition of the M current that is a heteromeric channel made up of KCNQ2 and KCNQ3 subunits (38). Carbachol produces a strong inhibition of the M current that recovers easily upon washout of the agonist. The recovery that occurs upon washout of the agonist is inhibited when wortmannin is present. This was the first evidence showing that inhibition of the M current by carbachol was via depletion of PIP₂, because wortmannin at high concentrations (10–20 µM) blocks the action of PI4-kinase that regenerates PIP₂. More recently, fluorescence measurements showed that carbachol caused translocation of EGFP-PLC-PH (that binds PIP₂) from the membrane to the cytosol, and this was closely associated with the decrease in M current (16). Studies using overexpression and inhibition of PI4-kinase also showed that PIP₂ was the major signaling molecule that determined the activity of the M channels (40). Thus there is now a general agreement that PLC-induced depletion of membrane PIP₂ is the major process that underlies the inhibition of the M current by carbachol.

In addition to M current, other inwardly rectifying K⁺ channels such as GIRK and IRK also are modulated similarly by PIP₂ (9, 17, 26). Is this signaling pathway in which PIP₂ concentration determines the K⁺ current a common theme among G_q-modulated K⁺ channels? Based on the evidence from recent studies as well as from the present study, the answer is clearly no. For example, M₁ and bradykinin receptor agonists that are both coupled to G_q do not share exactly the same mechanism for inhibition of M current in superior cervical ganglion neurons (40). A significant part of the bradykinin-induced inhibition may involve Ca²⁺/calmodulin action on the channel (40). Even with the same agonist (ACh) acting on the same receptor (M₃) and the same cell (COS-7), our results show that different K⁺ channels (TASK-3 and TREK-2) are inhibited via distinct pathways. Like M and GIRK channels, neuronal Ca²⁺ channels also are inhibited by G_q-dependent hydrolysis of PIP₂ (2, 7, 11). In a recent study, activation of G_q in cardiac myocytes was found to inhibit L-type Ca²⁺ channels through PI3-kinase, and PLC was not required (31). In sympathetic neurons, M₁ agonist produced a strong inhibition of Ca²⁺ channels, whereas bradykinin that also acts on G_q-coupled receptor had little or no effect (3). These studies show that ion channels modulated by G_q pathway can be achieved by signaling other than PIP₂ hydrolysis. Therefore, the individual specificity of the signaling pathway for agonist-induced inhibition must be considered for each agonist/receptor and each ion channel for each cell type, as also suggested by earlier studies (5, 8).

Mechanism of inhibition of TREK-2 by ACh via M₃ receptor. Several earlier studies have examined the mechanism by which a receptor agonist inhibit TREK-1, a close relative of TREK-2 that shares 63% amino acid identity with TREK-1 (1). Although the proximal regions of the COOH termini of TREK-1 and TREK-2 are similar, TREK-2 COOH terminus is 101 residues longer than that of TREK-1. Because the COOH terminus regulates the gating of these K⁺ channels (25, 36), the signaling pathway for inhibition of TREK-1 may not be the same as that for TREK-2 and must be tested directly. Moreover, the results obtained from different laboratories do not agree and allow no general conclusions to be reached as to the mechanism of TREK-1 inhibition by receptor agonists. For example, the first study reported that DAG and phosphatidic acid (generated by DAG kinase), but not PIP₂, inhibit TREK-1 (4). With bovine TREK-1, separate Ca²⁺- and ATP-dependent pathways were found to mediate the inhibition by angiotensin II via G_q (10). In that study, the inhibitory effect of Ca²⁺ was observed in inside-out patches. Similar to M and TASK channels, PIP₂ hydrolysis was reported to underlie the agonist-induced inhibition of TREK-1 expressed in oocytes (30). Finally, agonists failed to inhibit TREK-1 when cells were treated with PKC inhibitor (bisindolylmaleimide), and mutation of PKC sites abolished inhibition by agonists (33). Thus many of the molecules generated or reduced by PLC have been implicated in TREK-1 inhibition. These results obtained with TREK-1 could perhaps be because the signaling mechanisms might depend critically on the cell and receptor types used in each study.

Our results with TREK-2 expressed in COS-7 cells agree well with only one of the mechanisms proposed for TREK-1, i.e., phosphorylation via PKC, and are not consistent with the other reported mechanisms including the involvement of PIP₂. Simultaneous experiments with TASK-3 as positive control was important in providing evidence that TREK-2 inhibition by ACh via M₃ receptor was not due to PIP₂ depletion. Thus wortmannin nearly completely abolished the recovery of TASK-3 after inhibition by ACh, whereas it had no effect on the recovery of TREK-2. Our studies using PKC inhibitor (bisindolylmaleimide) suggest that TREK-2 inhibition by ACh occurs via PKC-mediated phosphorylation. Additional evidence comes from the use of TREK-2 mutants, where putative PKC sites were mutated to prevent phosphorylation. Two TREK-2 mutants (S326A and S359C) were found to be insensitive to ACh, providing additional molecular evidence that phosphorylation at these sites is crucial for inhibition of TREK-2. In the earlier study by Murbartian et al. (33) using HEK-293 cells stably expressing thyrotropin releasing hormone receptor or orexin receptor, the critical role of PKC in agonist-induced inhibition of TREK-1 was also reported. Although we have not directly measured the cell surface expression of TREK-2 mutants, TREK-1 mutations at the homologous phosphorylation positions (S300 and S333) did not affect cell surface expression (33). Thus these results are most consistent with the interpretation that agonists that act on G_q-coupled receptors inhibit TREK-1 and TREK-2 via PKC-mediated phosphorylation of two serine residues at the COOH terminus of the channel that presumably interact with the channel gate to modulate the opening frequency.

The inconsistent inhibition of TREK-2 by DAG and Ca²⁺ that we have observed is interesting but rather difficult to comprehend. An earlier study also reported that Ca²⁺ inhibits bovine TREK-1 in about one-half of the inside-out patches tested (10). It was suggested that Ca²⁺ acts via binding to calmodulin that may already be bound to some but not all TREK-1 protein. For TREK-2, we were unable to observe any additional effect of calmodulin on Ca²⁺-induced inhibition (data not shown). Because the S326A/S359A double mutant of TREK-2 did not respond to DAG or Ca²⁺, we speculate that somehow DAG or Ca²⁺ induces partial phosphorylation of TREK-2 in some inside-out patches, possibly due to the presence of low amounts of cytoplasmic proteins that has not been washed off. In any case, our results support the view that these two molecules do not directly act on TREK-2 to cause inhibition. In our experiments, we have observed that the inhibition of the whole cell TREK-2 current by ACh does not diminish even after several minutes, suggesting that the inhibitory signaling molecule does not easily dialyze out of the cell during recording. Molecules such as DAG and PKC are closely associated with the plasma membrane after activation and may not easily diffuse out of the cell, whereas small molecules such as Ca²⁺ are expected to leave the cell faster. Therefore, Ca²⁺ is clearly not the direct inhibitor of TREK-2.

In summary, the molecular basis of agonist-induced inhibition of TREK-2 was examined using COS-7 cells expressing TREK-2 and M₃ receptor. Because the inhibition is observed in cell-attached patches when the agonist is applied to the bath solution, the signaling most likely involves a messenger that is easily diffusible. Our studies suggest that this signaling pathway involves activation of PKC. The results are most consistent with the mechanism in which two serine residues at the COOH terminus of TREK-2 are phosphorylated by PKC activated via agonist-mediated stimulation of G_q-dependent PLC. There was no need to invoke an involvement of PIP₂ in this inhibitory process.

	GRANTS

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This work was supported by National Heart, Lung, and Blood Institute Grant HL-55363 (to D. Kim) and in part by Basic Research Program of the Korea Science and Engineering Foundation Grant R13-2005-012-01002-0 (to D. Kang and J. Han).

	FOOTNOTES

 

Address for reprint requests and other correspondence: D. Kim, Dept. of Physiology and Biophysics, Chicago Medical School, Rosalind Franklin Univ. of Medicine and Science, 3333 Green Bay Road, North Chicago, IL 60064 (e-mail: donghee.kim{at}rosalindfranklin.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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