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J. Biol. Chem., Vol. 277, Issue 51, 49186-49199, December 20, 2002

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An ACTH- and ATP-regulated Background K⁺ Channel in Adrenocortical Cells Is TREK-1^*

John J. Enyeart, Lin Xu, Sanjay Danthi^§, and Judith A. Enyeart

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

Received for publication, July 18, 2002, and in revised form, September 10, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Bovine adrenal zona fasciculata (AZF) cells express a background K⁺ channel (I_AC) that sets the resting potential and acts pivotally in ACTH-stimulated cortisol secretion. We have cloned a bTREK-1 (KCNK2) tandem-pore K⁺ channel cDNA from AZF cells with properties that identify it as the native I_AC. The bTREK-1 cDNA is expressed robustly in AZF cells and includes transcripts of 4.9, 3.6, and 2.8 kb. In patch clamp recordings made from transiently transfected cells, bTREK-1 displayed distinctive properties of I_AC in AZF cells. Specifically, bTREK-1 currents were outwardly rectifying with a large instantaneous and smaller time-dependent component. Similar to I_AC, bTREK-1 increased spontaneously in amplitude over many minutes of whole cell recording and was blocked potently by Ca²⁺ antagonists including penfluridol and mibefradil and by 8-(4-chlorophenylthio)-cAMP. Unitary TREK-1 and I_AC currents were nearly identical in amplitude. The native I_AC current, in turn, displayed properties that together are specific to TREK-1 K⁺ channels. These include activation by intracellular acidification, enhancement by the neuroprotective agent riluzole, and outward rectification. bTREK-1 current differed from native K⁺ current only in its lack of ATP dependence. In contrast to I_AC, the current density of bTREK-1 in human embryonic kidney-293 cells was not increased by raising pipette ATP from 0.1 to 5 mM. Further, the enhancement of I_AC current in AZF cells by low pH and riluzole was facilitated by, and dependent on, ATP at millimolar concentrations in the pipette solution. Overall, these results establish the identity of I_AC K⁺ channels, demonstrate the expression of bTREK-1 in a specific endocrine cell, identify potent new TREK-1 antagonists, and assign a pivotal role for these tandem-pore channels in the physiology of cortisol secretion. The activation of I_AC by ATP indicates that native bTREK-1 channels may function as sensors that couple the metabolic state of the cell to membrane potential, perhaps through an associated ATP-binding protein.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Bovine AZF¹ cells express a novel K⁺-selective channel (I_AC) that functions pivotally in the regulation of cortisol secretion (1-3). I_AC displays a number of properties typical of background or leak-type channels that function in setting the resting membrane potential of various cells. However, native I_AC channels also exhibit a combination of properties not observed previously in a single type of K⁺ channel.

In whole cell patch clamp recordings, I_AC appears as a non-inactivating, outwardly rectifying K⁺ current that displays weak voltage dependence and grows spontaneously over many minutes (1, 3, 4). At the single channel level, the conductance and rectifying properties of these channels depend strongly on the presence of divalent cations (5). In symmetrical K⁺, with physiological concentrations of Ca²⁺ and Mg²⁺, the channels are outwardly rectifying, whereas they become nearly ohmic in the absence of divalent cations (4, 5).

In whole cell patch clamp recordings, I_AC is inhibited potently by ACTH and angiotensin II, the two peptide hormones that regulate cortisol secretion physiologically (1, 6). I_AC channels are also inhibited by paracrine factors, including ATP and adenosine, which act through specific G protein-coupled nucleotide receptors (7, 8).

Second messengers synthesized or released upon activation of these receptors, including Ca²⁺ and cAMP, inhibit I_AC channels. However, inhibition appears to involve atypical signaling pathways, because responses to peptides and second messengers are insensitive to inhibition of cAMP and Ca²⁺-activated protein kinases (3, 5, 6, 9).

Among background K⁺ channels, I_AC channels are unique in their activation by hydrolyzable and non-hydrolyzable forms of ATP, applied intracellularly at physiological concentrations through a recording pipette (4). These background channels are also activated by UTP, GTP, and by inorganic polytriphosphate (PPP_i) (4, 5).

I_AC channels also display a unique pharmacological profile. Specifically, these channels are relatively insensitive to many standard K⁺ channel antagonists, including 4-AP and TEA (respective IC₅₀ values of 2.8 and 24.3 mM), whereas they are blocked by others such as quinidine (IC₅₀ = 24.1 µM) (10). Surprisingly, I_AC K⁺ channels are blocked potently by the Ca²⁺ antagonists penfluridol and mibefradil at far lower concentrations (respective IC₅₀ values of 0.20 and 0.50 µM) (10, 11).

Although I_AC K⁺ channels function as background or leak K⁺ channels that set the resting potential of AZF cells, their aggregate properties do not match the profile of any K⁺ channel yet described. Regardless, these channels appear to function critically in the physiology of cortisol secretion. In this capacity, they act as sensors that integrate hormonal and metabolic signals and couple these to depolarization-dependent Ca²⁺ entry.

Because of their distinctive properties as a metabolic sensor and role in cortisol secretion, it will be important to determine the molecular identity of I_AC channels. In this regard, K⁺-selective ion channels in mammals can be divided into several families, based on structure and membrane topology. Voltage-gated K⁺ channels include a major subunit that consists of one pore domain and six transmembrane segments (1P/6TMS) (12). A second family that consists of channel subunits containing a single pore domain and two membrane-spanning segments (1P/2TMS) includes inward rectifiers many of which contribute to the resting potential and some of which are inhibited directly by intracellular ATP (12-14).

K⁺-selective leak or background channels comprise the third and most recently described family of K⁺ channels. In mammals, the largest group of leak K⁺ channels possesses distinctive subunits that consist of two pore domains and four transmembrane segments. These 2P/4TMS channels are typically non-inactivating K⁺ channels that set the resting potential in a range of cells (15-17). Since 1995, more that a dozen members of the 2P/4TMS family have been identified and characterized by molecular cloning and patch clamp techniques (15-17).

Although similar in structure, 2P/4TMS K⁺ channels can be distinguished based on a variety of functional properties including unitary conductance, rectification, sensitivity to mechanical stimulation, pH and temperature, modulation by cAMP, fatty acids, membrane phospholipids, general anesthetics, and the neuroprotective agent riluzole (15-19). These leak-type channels also show variable sensitivity to standard K⁺ channel blockers (15-17). At least one member of this family is reported to acquire voltage-dependent gating upon phosphorylation (20).

Although I_AC functions in AZF cells as a classic leak-type K⁺ channel, its distinctive properties are not duplicated in any of the 2P/4TMS K⁺ channels yet described. In particular, direct modulation of K⁺ channels by ATP has only been demonstrated in K_ATP channels of the 1P/2TMS type, which are inhibited rather than activated by this nucleotide (13, 14).

We have cloned a 2P/4TMS channel TREK-1 (KCNK2) K⁺ channel cDNA from AZF cells that appears to code for I_AC K⁺ channels. When expressed in cell lines, bTREK-1 currents share several distinctive features with native I_AC currents. Evidence is presented that indicates that, in AZF cells, bTREK-1 channels acquire additional properties including ATP sensitivity.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials

Tissue culture media, antibiotics, fibronectin, and fetal bovine sera were obtained from Invitrogen. Culture dishes were purchased from Corning Glass (Corning, NY). Coverslips were from Bellco (Vineland, NJ). Phosphate-buffered saline, enzymes, ACTH (1-24), AMP-PNP (lithium salt), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, riluzole, forskolin, and 8-pcpt-cAMP, glibenclamide, and LaCl₃ were from Sigma. Penfluridol was obtained from Janssen Pharmaceuticals (Beerse, Belgium). p3-CD8 clone was kindly provided by Dr. Brian Seed, Department of Genetics, Massachusetts General Hospital, Boston, MA. Rat TREK-2 cDNA was kindly provided by Dr. D. Kim, Dept. of Physiology and Biophysics, Finch University of Health Sciences, The Chicago Medical School, Chicago, IL. HEK-293 and CHO-K1 cells were obtained from ATCC (Manassas, VA).

Methods

Isolation and Culture of AZF Cells-- Bovine adrenal glands were obtained from steers (age range 1 to 3 years) within 1 h of slaughter at a local slaughterhouse. Fatty tissue was removed immediately, and the glands were transported to the laboratory in ice-cold phosphate-buffered saline containing 0.2% dextrose. Isolated AZF cells were prepared as described previously (4). After isolation, cells were either resuspended in Dulbecco's modified Eagle's medium/F12 (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/F12+) and plated for immediate use or resuspended in fetal bovine serum/5% Me₂SO, divided into 1-ml aliquots each containing about 2 × 10⁶ cells, and stored in liquid nitrogen for future use. Cells were plated in either 60- or 35-mm dishes containing 9-mm² glass coverslips. Dishes or 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 plated in Dulbecco's modified Eagle's medium/F12+ and were maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO₂.

Cloning of the b-TREK-1 K⁺ Channel cDNA-- RNeasy columns (Qiagen, Valencia, CA) that had been treated with RNase-free DNase (Qiagen) to remove genomic contamination were used to extract total RNA from AZF cells that had been cultured overnight. poly(A)⁺ mRNA was extracted from total RNA using a Poly(A) Pure kit (Ambion, Austin, TX). Reverse transcription and cDNA amplification were performed using a Marathon cDNA amplification kit (Clontech, Palo Alto, CA). The resulting AZF double-stranded cDNA was used as the template for all PCR and RACE PCR reactions. RACE and PCR reactions were run as per Clontech instructions on a Thermolyne Temptronic thermal cycler.

Noting the sequence similarities among double-pore channels, particularly at the transmembrane and pore regions, degenerate primers were designed using human TREK-1 (AF171068), and human TRAAK (AF247042) pore (P1) and transmembrane (M2) sequences as templates. For an initial 3'RACE reaction, a degenerate primer designed from P1 5'-CTG GGA CCG TCA TCA CVA CCA TMG G-3') was used as the forward primer, and the Clontech adaptor primer 1 was used as the reverse primer. Using the product of this initial reaction, nested 3'RACE was performed using a degenerate primer from the M2 region (5'-TGG GGA TTC CGC TGT TTG GKW TYY T-3') as the forward primer, and the Clontech adaptor primer 2 was used as the reverse primer. The 3' nested RACE product was separated on a 1.2% agarose gel and visualized with ethidium bromide/UV transillumination. A 750-bp 3' RACE product was excised from the gel and cloned into pCR-TOPO vector (Invitrogen). Clones were sequenced using an Applied Systems sequencer, model 3700, version 3.6.1 software. Using this initial 750-bp product to design specific nested primers for 5' RACE PCR, the 5' end of bTREK-1 was obtained. For transfection, the full-length bTREK-1 clone was produced by PCR using a low error rate DNA polymerase (Pfu DNA polymerase; Invitrogen), bTREK-1-specific primers 5'-/5phos/GCG ATT CCG TGT CTT CTC-3' as the forward primer and 5'-AAG GTC TAA TTG CTA TGC CTG AG-3' as the reverse primer, and AZF double-stranded cDNA as the template. The PCR product obtained was subcloned into the pCR3.1-Uni expression vector (Invitrogen) for transfection into HEK-293 cells or CHO-K1.

Transient Transfection and Visual Identification of HEK-293 Cells Expressing bTREK-1-- For patch clamp recording of bTREK-1 currents, HEK-293 cells were co-transfected with a mixture of pCR^®3.1-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 (21).

Measurement of bTREK-1 mRNA-- 7 µg of poly(A)⁺ mRNA, isolated as described above, was separated on a denaturing 8% formaldehyde, 1.0% agarose gel, and transferred to a nylon membrane (Gene Screen Plus; PerkinElmer Life Sciences). The RNA was fixed to the membrane by UV-cross-linking using a Stratalinker (Stratagene, La Jolla, CA). Northern blots were prehybridized in heat-sealable plastic bags for 2 h at 42 °C in ULTRAhyb (Ambion, Austin, TX) for probing using either a 1415-bp fragment that contained the entire coding region of bTREK-1 or a 206-bp BamHI fragment primarily from the bTREK-1 5'-untranslated end. The blots were hybridized with a random-primed ³²P--dCTP radiolabeled probe (Prime-it-II; Stratagene, La Jolla, CA) overnight in minimal volume of hybridization solution. After 18-24 h the blots were washed twice at room temperature in 2× saline/sodium phosphate/EDTA for 15 min, twice at 40 °C in 2× saline/sodium phosphate/EDTA, 1% SDS for 30 min with a final wash at 65 °C with 0.1 × saline/sodium phosphate/EDTA, 1% SDS for 20 min. Autoradiograms were obtained by exposing the blots for 2 to 5 h to Eastman Kodak Co. X-Omat AR film at 70 °C.

Patch Clamp Experiments-- Whole cell patch clamp recordings of K⁺ currents were made from AZF cells or bTREK-1-transfected HEK-293 cells. The standard pipette solution was 120 mM KCl, 2 mM MgCl₂, 1 mM CaCl₂, 20 mM HEPES, 11 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, 5 mM MgATP, and 200 µM GTP, with pH buffered to 7.2 using KOH. Deviations from these solutions with respect to nucleotide and pH are described in the text. The external solution consisted of 140 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, 10 mM HEPES, and 5 mM glucose, pH 7.35, using NaOH. All solutions were filtered through 0.22-µm cellulose acetate filters.

AZF cells were used for patch clamp experiments 2-12 h after plating. Typically, cells with diameters of <15 µm and capacitances of 8-15 picofarads were selected. Coverslips were transferred from 35-mm culture dishes to the recording chamber (volume, 1.5 ml), which was perfused continuously 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 2.0-5.0 megohms were fabricated from Corning 0010 glass (WPI, Sarasota, FL). K⁺ currents were recorded at room temperature (20-23 °C) following the procedure of Hamill et al. (22), using an Axopatch 1D patch clamp amplifier (Axon Instruments, Inc., Union City, CA) or List EPC 7 patch clamp amplifier.

HEK-293 or CHO-K1 cells were used for patch clamping 24-72 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 bTREK-1 K⁺ currents in transfected cells were recorded as described for AZF cells. Smaller cells with capacitances of 10-15 pF were selected for recording.

Pulse generation and data acquisition were done using a personal computer and PCLAMP software with a TL-1 interface (Axon Instruments, Inc., Union City, CA). Currents were digitized using an 8-pole Bessel filter (Frequency Devices, Haverhill, MA) Linear leak and capacity currents were subtracted from current records using hyperpolarizing steps of 1/3 to 1/4 amplitude. Data were analyzed and plotted using PCLAMP 5.5 and 6.04 (Clampan and Clampfit) and SigmaPlot (version 5.0).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

bTREK-1 in AZF Cells-- Previous studies using whole cell and single channel patch clamp recording showed that I_AC channels resembled 2P/4TMS channels of the TREK-1 and TRAAK with regard to single channel conductance and rectification (5, 16). Degenerate primers were designed using the P1 and M2 regions of hTREK-1 and hTRAAK as templates, and 3'RACE PCR was performed using AZF cell double-stranded cDNA generated from poly(A)⁺ mRNA. A 750-bp product was obtained that showed 92% nucleotide sequence identity to human TREK-1. No PCR products were obtained that were homologous to hTRAAK. Specific primers were designed from this TREK-1-like product for extension in the 5' direction using nested 5' RACE PCR. Combining the 3' and 5' RACE products yielded a 1534-nt cDNA (GenBank^TM accession number AY148474). This coding sequence consists of an open reading frame of 411 amino acids. The predicted amino acid sequence for bTREK-1 is given in Fig. 1, with the four transmembrane and two pore sequences noted. The bovine sequence AAN37591-1 shows 99% protein sequence identity to human TREK-1 (protein ID, NP_055032.1), 96% identity to rat TREK-1 (protein ID, AAL95708), and 96.4% to mouse TREK-1 (Protein ID, XP_123605).

View larger version (48K): [in this window] [in a new window]   Fig. 1.   Predicted amino acid sequence and expression of bovine adrenal TREK-1 cDNA. A, the amino acid sequence for bovine adrenal TREK-1 cDNA. Amino acids are represented by their single letter abbreviations. Transmembrane sections are underlined, and pore regions are in italics. B, bTREK-1 expression in the bovine adrenal cortex. Either full-length bTREK-1 cDNA (left) or a 206-bp BamHI fragment from the 5'untranslated end of bTREK-1 (right) labeled with [32P]-dCTP was hybridized to size-fractionated poly(A)+ mRNA (7 µg) extracted from AZF cells. Northern blot analysis was carried out as described under "Methods." Bands correspond to transcripts of ~4.9, 3.6, and 2.8 kb.

Northern blot analysis of poly(A)⁺ mRNA showed that bTREK-1 mRNA is expressed highly in the bovine adrenal cortex. A 1415-bp DNA probe that included the full-length coding region of bTREK-1 hybridized to separate ~4.9-, 3.6-, and 2.8-kb mRNA transcripts isolated from AZF cells (Fig. 1B, left lane). To eliminate the possibility that these transcripts could be the result of the full-length TREK-1 probe hybridizing to mRNA coding for homologous channels present in the adrenal cortex, a TREK-1-specific probe consisting of a 206-bp BamHI fragment derived primarily from the 5'untranslated end of bTREK-1 was used to probe a separate poly(A)⁺ blot. The 206-bp fragment also hybridized to the same three transcripts (Fig. 1B, right lane), with the strongest hybridization signal originating from the 3.6- and 2.8-kb transcripts.

Properties of bTREK-1 Currents-- Properties of bTREK-1 currents were studied in transiently transfected HEK-293 cells. Successfully transfected cells were identified based on decoration with anti-CD-8 antibody-coated beads, as described under "Methods." In whole cell patch clamp recording, bTREK-1 was remarkably similar in several respects to the native I_AC current in bovine AZF cells. Specifically, in physiological saline bTREK-1 appeared as a non-inactivating current that increased spontaneously in amplitude to a maximum value over many minutes of whole cell recording (Fig. 2A).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Properties of bTREK-1 K+ current in HEK-293 cells. Whole cell K+ currents were recorded from HEK-293 cells that had been transiently co-transfected with pCR®3.1-Uni-bTREK-1 and P3-CD8 cDNAs. Cells decorated with anti-CD8 antibody-coated beads were selected for patch clamping (see "Methods"). Mock-transfected cells were transfected with the expression plasmid pCR®3.1-Uni (no insert) and P3-CD8 cell surface antigen. A, time-dependent increase in bTREK-1 currents. K+ currents were activated by voltage steps to +20 mV applied at 30-s intervals from a holding potential of 80 mV. Traces and associated graph illustrate currents recorded from a single cell at indicated times after initiating whole cell recording. B, bTREK-1 current-voltage relationship. Whole cell K+ currents were recorded from bTREK-1-transfected HEK-293 and control cells in response to voltage steps applied from 80 mV in 10-mV increments to test potentials between 70 and +60 mV. Current traces from bTREK-1-transfected and control cells and associated I-V plots are shown.

In the experiment illustrated in Fig. 2A, during 17 min of recording, bTREK-1 amplitude doubled from its initial value of ~1250 pA. This spontaneous time-dependent increase in the amplitude of bTREK-1 current was observed in nearly all of the more than 75 cells tested. It occurred at pipette solution pHs ranging from 6.4 to 7.3 and was independent of ATP concentrations between 0.1 and 5.0 mM. Further, this phenomenon was not specific to HEK-293 cells. The growth of bTREK-1 current with time was also observed in transfected CHO-K1 cells (data not shown). Although the time-dependent growth of I_AC in whole cell recordings has been established as a hallmark of this current in AZF cells (1, 3, 4), this phenomenon has not been reported previously for other native or cloned TREK-1 channels.

Similar to I_AC, when bTREK-1 is activated from a holding potential of 80 mV to various test potentials, this current consists of a large instantaneous component and a smaller time-dependent component that could be fit with a single exponential (Fig. 2, A (left) and B). In physiological saline, bTREK-1 is outwardly rectifying (Fig. 2B).

Comparative Pharmacology of I_AC and Cloned bTREK-1-- If native I_AC and cloned bTREK-1 K⁺ channels are identical then they should display a similar pharmacological profile. The pharmacology of 2P/4TMS K⁺ channels, including TREK-1, is currently being investigated. Murine and human TREK-1 channels are relatively insensitive to standard organic K⁺ channel antagonists, including TEA and 4-AP (23, 24). High concentrations of these two drugs are also required to inhibit native I_AC channels (10). However, I_AC channels are inhibited potently by several organic Ca²⁺ antagonists, including diphenylbutylpiperidines (DPBPs) and mibefradil (10, 11).

In the present study, we found that the DPBP penfluridol and mibefradil also potently inhibit cloned bTREK-1 channels expressed in mammalian cell lines. DPBPs such as penfluridol inhibit I_AC K⁺ channels with IC₅₀ values of ~0.2-0.4 µM (10). Penfluridol inhibited bTREK-1 expressed in HEK-293 cells with an IC₅₀ of 0.35 µM (Fig. 3A and C). Inhibition of bTREK-1 by penfluridol was independent of voltage at test potentials of 40 to +40 mV (Fig. 3B). bTREK-1 was also inhibited by a second DPBP, pimozide (data not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Penfluridol blocks bTREK-1 in transfected cells. K+ currents were recorded from bTREK-1-transfected HEK-293 cells in response to voltage steps to +20 mV applied at 30-s intervals from a holding potential of 80 mV. When bTREK-1 reached a maximum value, cells were superfused with penfluridol at concentrations between 0.1 and 2.5 µM. A, current traces and associated plot of bTREK-1 current amplitudes for a cell superfused with 2.5 µM penfluridol. Numbers on traces correspond to those on graph at right. B, penfluridol on current-voltage relationship. bTREK-1 K+ currents were recorded from transiently transfected HEK-293 cells in response to voltage steps applied from 80 mV to test potentials between 50 and +40 mV, before and 6 min after superfusing the cell with 2.5 µM penfluridol. Current amplitudes are plotted against test potential. C, inhibition curve for penfluridol constructed from experiments as in A. Fraction of unblocked bTREK-1 current is plotted against penfluridol concentration. Data were fit with an equation of the form, I/Imax = 1/[1 + B/IC50)]X, where B is the penfluridol concentration, IC50 is the concentration that produced half-maximal inhibition, and X is the Hill slope. The IC50 estimated from the fit is 3.5 × 107 M. Data are normalized mean values ± S.E. of the indicated number of measurements.

Mibefradil inhibits I_AC K⁺ channels with an IC₅₀ of 0.5 µM (11). This drug also inhibited cloned bTREK-1 channels with similar potency (Fig. 4A). At concentrations of 0.5 and 2.5 µM, mibefradil inhibited bTREK-1 by 52 ± 15% (n = 4) and 81 ± 8.3% (n = 9), respectively. Other agents including La³⁺ and glibenclamide inhibited bTREK-1 less potently but at concentrations similar to those that inhibited native I_AC (Fig. 4, B, C, and E).

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Pharmacology of bTREK-1 K+ channels. K+ currents were recorded from bTREK-1-transfected HEK-293 or CHO-K1 cells in response to voltage steps to +20 mV, applied at 30-s intervals from a holding potential of 80 mV. When bTREK-1 reached a maximum value, cells were superfused with mibefradil, La3+, glibenclamide, or 8-pcpt-cAMP as indicated. A, mibefradil, current traces and associated plot of bTREK-1 current amplitudes for a HEK-293 cell superfused with 0.5 and 2.5 µM mibefradil. Numbers on traces correspond to those on graph at right. B, La3+, plot of bTREK-1 amplitudes for CHO-K1 cell superfused with 100 and 250 µM La3+. C, glibenclamide, plot of bTREK-1 amplitudes for CHO-K1 cell superfused with 100 and 200 µM glibenclamide. D, 8-pcpt-cAMP, plot of bTREK-1 amplitude for CHO-K1 cell superfused with 500 µM 8-pcpt-cAMP. E, comparative inhibition of bTREK-1 and native IAC current by 250 µM La3+, 100 µM glibenclamide, and 500 µM 8-pcpt-cAMP is illustrated. Values are mean ± S.E. of from three to 23 determinations.

Cloned TREK-1 channels and bovine I_AC channels are inhibited by cAMP (3, 23). The membrane-permeable cAMP analog 8-pcpt-cAMP inhibited native I_AC and cloned bTREK-1 channels with equal effectiveness (Fig. 4, D and E).

bTREK-1 and ATP-- Despite these similarities, bTREK-1 expressed in HEK-293 cells differed from native I_AC in that bTREK-1 activity was independent of intracellular ATP. Specifically, in whole cell recordings with patch pipettes containing 0.1 mM MgATP, bTREK-1 reached a maximum current density of 116.7 ± 28.7 pA/pF (n = 8), compared with 111.3 ± 25.2 pA/pF (n = 9) when pipettes containing 5 mM MgATP.

Unitary bTREK-1 and I_AC Currents-- Unitary bTREK-1 currents were recorded from transfected CHO-K1 cell membrane patches in the inside-out configuration and compared with unitary I_AC currents recorded from AZF cells under the same conditions. Amplitude analysis of single channel bTREK-1 and native I_AC currents showed that these were nearly identical in size (Fig. 5, A and B). In the experiment illustrated unitary bTREK-1 and I_AC currents measured at +30 mV had amplitudes of 3.57 and 3.60 pA, respectively. Solutions in contact with the cytoplasmic membrane face in these recordings contained 0.1 mM ATP. In excised inside-out patches activity of I_AC and bTREK-1 channels were both independent of ATP.

View larger version (32K): [in this window] [in a new window]   Fig. 5.   Comparison of unitary native IAC and cloned bTREK-1 currents. Unitary IAC and bTREK-1 currents were recorded in excised inside-out patches from AZF cells and bTREK-1-transfected CHO-K1 cells, respectively. Patches were excised into standard internal solutions used in whole cell recording, supplemented with 0.1 mM MgATP (see "Methods"). Voltage steps of 300-ms duration to +30 mV were applied at 10-s intervals from a holding potential of 40 mV. Amplitude histograms were constructed from idealized unitary currents that were distributed into bins 0.180 pA in width. A and B, unitary currents (top) and corresponding amplitude histograms (bottom) from AZF cells (A) and bTREK-1-transfected CHO-K1 cells (B). Currents were filtered at a cutoff frequency of 2 kHz and sampled at a frequency of 5 kHz. Continuous lines in the histograms are fits of Gaussian distributions to the data.

Activation of I_AC Current in AZF Cells by Intracellular Acidification-- When expressed in HEK-293 cells, bTREK-1 current displayed several distinctive properties of the native I_AC of AZF cells. Experiments were done to determine whether I_AC, in turn, possessed well established properties of TREK-1 channels cloned previously. These include activation by acidification and the neuroprotective agent riluzole (18, 25).

Extensive patch clamp and molecular cloning studies have identified only two types of K⁺ channels in bovine AZF cells, a rapidly inactivating, voltage-gated Kv1.4-type current and the non-inactivating I_AC current (1, 4, 26, 27). Although present at low density upon initiating whole cell recordings, I_AC often grows dramatically over a period of minutes provided that ATP is present at millimolar concentrations in the pipette solution (1, 4, 5).

The absence of time- and voltage-dependent inactivation of the I_AC current allows it to be isolated and measured 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, I_AC can be measured near the end of a voltage step when the transient current has inactivated (Fig. 6, left traces). Alternatively, I_AC can be activated selectively after a 10-s prepulse to 20 mV has fully inactivated the Kv1.4-type current (Fig. 6A, right traces).

View larger version (14K): [in this window] [in a new window]   Fig. 6.   pH-dependent activation of IAC K+ current. Whole cell K+ currents were recorded at 30-s intervals in response to voltage steps to +20 mV from a holding potential of 80 mV using patch pipettes containing standard solutions at pH levels between 6.2 and 7.5. A, time-dependent expression of IAC current at intracellular pH levels of 6.2 and 7.4. Current traces recorded with (right) and without (left) depolarizing prepulses immediately after initiating whole cell recording (1) and after IAC had reached a stable maximum amplitude (2). IAC amplitude with () and without () depolarizing prepulses are plotted against time at right. B, maximum average IAC current densities (expressed as pA/pF) were determined from experiments as in A at seven different pH levels and plotted against pH. Values are mean ± S.E. of indicated number of determinations.

Of the 2P/4TMS K⁺ channels described thus far, TREK-1 and TREK-2 channels (KCNK2 and KCNK10) are unique in the pronounced increase in activity observed in response to lowering intracellular pH (25, 28). To determine whether the activity of the ATP-dependent I_AC current was enhanced by cytoplasmic acidification, K⁺ currents were recorded with pipette solution that was supplemented with 5 mM MgATP at pH levels between 6.2 and 7.5.

As shown in Fig. 6, the time-dependent development of the non-inactivating K⁺ current was enhanced markedly and selectively at successively lower pH levels. Overall, I_AC current density varied from a minimum of 14.6 ± 1.7 pA/pF (n = 15) at pH 7.4 to 118.3 ± 9.2 pA/pF (n = 8) at pH 6.2 (Fig. 6, A and B).

Cooperative Activation of I_AC by pH and ATP-- The magnitude of I_AC K⁺ current recorded in the presence of 5 mM ATP was enhanced markedly at low pH levels. Conversely, the enhancement of I_AC by acidification was found to depend on the concentration of ATP in the pipette solutions.

The cooperative actions of ATP and pH on I_AC expression are illustrated in Fig. 7. When I_AC K⁺ currents were recorded at pH 6.5 (Fig. 7A) or pH 7.0 (Fig. 7B), the time-dependent increase in I_AC was ~4-fold greater in the presence of 5.0 mM ATP compared with 0.5 mM ATP. Overall, with 0.5 ATP in the pipette lowering pH from 7.5 to 7.0 and 6.5 increased maximum I_AC current density from a value of 11.7 ± 3.4 pA/pF (n = 5) to 13.1 ± 3.0 pA/pF (n = 4) and 40.4 pA/pF (n = 5), respectively. By comparison, with pipettes containing 5.0 mM ATP, reducing pH from 7.5 to 7.0 and 6.5 increased maximum I_AC current density from 15.0 ± 3.1 (n = 7) to 63.5 ± 8.4 pA/pF (n = 5) and 114 ± 9.8 pA/pF (n = 6), respectively (Fig. 7C).

View larger version (17K): [in this window] [in a new window]   Fig. 7.   ATP facilitates pH-dependent expression of IAC. Whole cell K+ currents were recorded from AZF cells using the voltage protocols described in the legend of Fig. 4 with pipettes containing standard solution supplemented with either 0.5 or 5 mM MgATP and with pH levels adjusted to 6.5, 7.0, or 7.5. A and B, K+ currents recorded at pH 6.5 (A) or pH 7.5 (B) with pipettes containing 0.5 or 5 mM MgATP. Numbered traces recorded with (left) or without (right) depolarizing prepulses were recorded at times indicated on corresponding graph. IAC amplitudes are plotted against time at right. Open and closed symbols indicated currents recorded with and without depolarizing prepulses, respectively. C, maximum IAC current densities expressed as pA/pF were determined from experiments such as those shown in A and B and plotted against pH at two ATP concentrations. Values are mean ± S.E. for the indicated number of determinations.

Effects of Penfluridol, ACTH, and Riluzole on pH-activated K⁺ Current-- The cooperative enhancement of the background K⁺ current of AZF cells by acidity and ATP suggest that both agents act on the same channels. They suggest further that I_AC is a TREK-1 type K⁺ channel that acquires ATP sensitivity when expressed in its native cell type.

If the pH-activated K⁺ current and I_AC are one and the same, selective I_AC antagonists such as penfluridol should inhibit the pH-activated current with similar potency. Accordingly, penfluridol (2.5 µM) inhibited the non-inactivating K⁺ current recorded at pH 6.4 by 85 ± 5% (n = 5) (Fig. 8A).

View larger version (22K): [in this window] [in a new window]   Fig. 8.   Modulation of pH-activated K+ current in AZF cells by penfluridol, ACTH, and riluzole. Whole cell K+ currents were recorded at 30-s intervals in response to voltage steps from a holding potential of 80 mV to a test potential of +20 mV with (right traces) or without (left traces) as illustrated. Pipettes contained standard solution at pH 6.4 supplemented with 0.1 mM ATP. When IAC reached a stable maximum amplitude cells were superfused with penfluridol with 2.5 µM penfluridol (A), 200 pM ACTH (B), or 100 µM riluzole (C). Current amplitudes are plotted at right recorded with (open circles) and without (closed circles) depolarizing prepulses. Numbers on traces correspond to those on graph at right.

ACTH inhibits I_AC current in AZF cells at subnanomolar concentrations (1). This peptide hormone also inhibited the acid-stimulated K⁺ current potently in these cells. In four experiments, ACTH (200 pM) completely inhibited the non-inactivating K⁺ current activated at pH 6.4 with pipette solutions containing ATP at either 0.5 or 5.0 mM (Fig. 8B).

In addition to low pH, cloned human and rat TREK-1 K⁺ channels are activated by the neuroprotective agent riluzole (2-amino-6-(trifluoromethoxy) benzothrazole) (18). However, the riluzole-induced increase in K⁺ current through cloned TREK-1 channels was reported to be transient and followed by inhibition, in response to a secondary rise in intracellular cAMP (18).

If the pH-activated K⁺ current of AZF cells is because of TREK-1 channels, then riluzole should also modulate this current. In the experiment illustrated in Fig. 8C, whole cell currents were recorded at pH 6.4 with 0.5 mM ATP in the pipette. After the pH-dependent current had reached maximum amplitude, AZF cells were superfused with saline containing 100 µM riluzole, whereas continuing to monitor currents at 30-s intervals.

Riluzole produced a pronounced increase in the non-inactivating K⁺ current, which reached a maximum within several minutes and then declined toward the control value. In a total of eight cells, riluzole increased the non-inactivating current to 331 ± 59% of its control value. In six of these cells, the non-inactivating K⁺ current declined to 71.9 ± 6.6% of its maximum value after 10 min in the continued presence of riluzole. In the remaining two cells, the riluzole-induced increase was sustained, with no transient component. The riluzole-induced increase in non-inactivating K⁺ current is consistent with the notion that the drug acts on native TREK-1-type K⁺ channels. The variation in the kinetics of the response likely reflects differences in the extent of dilution of intracellular components (see below). The inhibition of the acid-stimulated current by penfluridol and ACTH, and its enhancement by riluzole are consistent with the hypothesis that I_AC is a pH and ATP-dependent bTREK-1 current expressed in AZF cells.

Rectifying Properties of Native and Transfected bTREK-1 Channels-- If the ATP- and pH-dependent background K⁺ currents of AZF cells are because of K⁺ flux through bTREK-1 channels, then each of these currents should display similar voltage-dependent rectification. The rectifying properties of non-inactivating K⁺ currents in AZF cells, activated by acidification of pipette solution to pH 6.4 or by 5 mM ATP were compared with those of bTREK-1 currents in transfected HEK-293 cells using ramp voltage protocols. Fig. 9A shows non-inactivating K⁺ currents recorded in response to 2-s linear ramp voltages, applied between +60 and 140 mV. Each of the three currents displayed strong outward rectification, crossing the voltage axis near the Nerst equilibrium potential for K⁺. Scaling of the three currents using factors derived from the maximum current amplitudes measured at +60 mV resulted in three nearly superimposable traces (Fig. 9B). This result suggests that these currents result from K⁺ flow through identical channels.

View larger version (13K): [in this window] [in a new window]   Fig. 9.   Comparison of rectifying properties of bTREK-1 with ATP- and pH-activated background currents on AZF cells. Linear voltage ramps of 100 mV/s were applied from a holding potential of 0 mV to potentials between +60 and 140 mV in whole cell recordings from HEK-293 transiently transfected with bTREK-1 and bovine AZF cells. Pipette solution for bTREK-1-transfected cells contained 100 µM MgATP with pH 6.4. Pipette solutions for AZF cells contained either 100 µM MgATP with pH 6.4 or 5 mM MgATP with pH 7.2 as indicated. A, current traces recorded in response to ramp voltage protocols from bTREK-1-transfected HEK-293 cells or AZF cells wit pipette solutions as indicated. B, current traces in A were scaled by multiplying each of the currents by an appropriate scaling factor determined from the maximum value of each current measured at +60 mV.

Enhancement of ATP-dependent I_AC by Riluzole-- If the ATP-dependent I_AC K⁺ channels are of the TREK-1 type, then riluzole should increase the amplitude of I_AC in AZF cells. In the experiments illustrated in Fig. 10A, whole cell recordings were made with pipettes containing 5 mM ATP at pH 7.1. When I_AC reached a stable maximum amplitude, cells were superfused with riluzole at concentrations between 1 and 100 µM.

View larger version (16K): [in this window] [in a new window]   Fig. 10.   Time- and concentration-dependent enhancement of IAC K+ current by riluzole. A, time-dependent effects of riluzole. Whole cell 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 inactivating 10-s prepulse to 20 mV. After IAC reached a maximum, cells were superfused with 100 µM riluzole. IAC amplitudes recorded with () and without () depolarizing prepulses are plotted against time for three separate cells. B, concentration dependence. IAC current amplitudes obtained from experiments as in A at riluzole concentrations from 1 to 100 µM are expressed as percent of maximum value recorded in control saline. Values are mean ± S.E. of the indicated number of determinations. C, rectifying properties of riluzole-activated current. Left traces, linear voltage ramps of 100 mV/s were applied from a holding potential of 0 mV to potentials between +60 and 140 mV after IAC reached a maximum in control saline (control), after a maximum increase was reached in response to riluzole (Riluzole (max)), and after a steady increase in riluzole was achieved (Riluzole (SS)). Right traces, traces at left were scaled against the riluzole (max) trace, using the maximum value of each trace measured at +60 mV to determine the appropriate scaling factors.

Riluzole produced a concentration-dependent, reversible increase in non-inactivating component of K⁺ current that was indistinguishable from I_AC (Fig. 10). At maximally effective concentrations of 75 and 100 µM, riluzole increased this current to 418 ± 19% (n = 6) and 410 ± 39% (n = 18) of its control value (Fig. 10B).

Although riluzole (50-100 µM) induced rapid 2- to 8-fold increases in the amplitude of the non-inactivating K⁺ current in each of 33 cells tested, the temporal pattern of the responses varied widely (Fig. 10A). In the majority of cells (21/33) the riluzole-induced increase was characterized by distinct transient and sustained components (Fig. 10A, middle panel). In four cells, the transient increase in current was followed by near complete inhibition of the non-inactivating current (Fig. 10A, left panel). In these cells, a second application of riluzole, after washing with control saline, failed to produce any increase.

When riluzole was applied to the remaining 12 cells, it induced a sustained increase in current with no detectable decrement during at least 10 min of exposure (Fig. 10A, right panel). In these cells, a second application of riluzole was again effective at enhancing the non-inactivating K⁺ current.

The varying temporal pattern of the riluzole-induced responses was correlated with the measured series resistance of the recording pipette. In the cells where riluzole-induced increase in current was sustained, series resistance averaged 3.6 ± 0.3 megohms (n = 12). By comparison, in experiments where the riluzole response displayed a distinct transient component, averaged series resistance was more than twice as great (8.6 ± 0.7 megohms) (n = 21). These results suggest that the inhibitory component of the riluzole response is dependent on the continued presence of cytoplasmic contents that are lost more rapidly when currents are recorded with low resistance pipettes.

The varying transient and sustained components of the riluzole-induced increases in non-inactivating current could suggest that this drug enhances the activity of two different K⁺ channels in AZF cells. Several lines of evidence indicate that this is not the case and that riluzole enhances current flow only through pre-activated I_AC channels.

Ramp voltage protocols applied before and after exposing AZF cells to riluzole showed that the transient and sustained components of non-inactivating K⁺ current induced by this drug were indistinguishable from I_AC. In Fig. 10C (left), ramp voltage protocols were applied after the K⁺ current reached a maximum value in control saline (control), immediately after the current reached a maximum value in riluzole (Riluzole max) and after a steady state value was reached (Riluzole SS).

Scaling of the three currents using factors derived from the maximum current amplitudes measured at +60 mV and plotting these on a separate set of axis yielded three current traces that were nearly superimposable (Fig. 10C, right traces). Thus, the transient and sustained components of the riluzole-induced current had rectifying properties identical to I_AC.

Cooperative Activation of I_AC by Riluzole and ATP-- The riluzole-stimulated increases in non-inactivating K⁺ current were observed to be proportional to the amplitude of the pre-existing I_AC current present in individual cells. This suggested that riluzole-enhanced current flow through I_AC channels that had been pre-activated by ATP. To test this hypothesis, we compared the effects of riluzole in experiments with pipettes containing ATP at several concentrations.

In whole cell recordings made with pipette solutions containing ATP at concentrations of 1 mM or less, I_AC was small and failed to grow significantly with time. Further, under these conditions, riluzole (100 µM) was much less effective at increasing the amplitude of the non-inactivating K⁺ current (Fig. 11, A (left traces) and B).

View larger version (18K): [in this window] [in a new window]   Fig. 11.   ATP- and PPPi-dependent activation of IAC by riluzole. Whole cell K+ currents were recorded from AZF cells at 30-s intervals in response to voltage steps to +20 mV with or without depolarizing prepulses, using patch pipettes containing MgATP (0.1, 0.5, or 5.0 mM), AMP-PNP (1 mM), or 5 mM PPPi plus 1 mM MgATP. When IAC reached a maximum amplitude, the cell was superfused with 100 µM riluzole. A, K+ current records made with pipettes containing MgATP or AMP-PNP at the indicated concentrations. Numbers on traces recorded with (bottom) and without (top) depolarizing prepulses correspond to currents recorded immediately after initiating whole cell recording (1), after IAC reached a maximum value in control saline (2), and after the maximum current in the presence of riluzole (3). B, the effect of riluzole in combination with ATP, AMP-PNP, and PPPi on IAC current density was determined from experiments as in A above. IAC current density, expressed as pA/pF, was determined by dividing the maximum IAC current amplitude for each condition by the cell capacitance. Values are mean ± S.E. of the indicated number of determinations.

By comparison, when recordings were made with pipettes containing 5 mM ATP, I_AC currents reached much larger maximum values, whereas riluzole produced far greater absolute increases in a non-inactivating component of K⁺ current that was not distinguishable from I_AC (Fig. 11, A (middle traces) and B).

The non-hydrolyzable ATP analog AMP-PNP is more potent than ATP as an activator of I_AC K⁺ channels (5) (4). Accordingly, with 1 mM AMP-PNP in the recording pipette, I_AC reached current densities severalfold greater than those observed with 1 mM ATP. Further, in these experiments, riluzole again produced much larger increases in non-inactivating K⁺ current in the presence of 1 mM AMP-PNP, compared with 1 mM ATP (Fig. 11, A (right panel) and B).

In addition to nucleotide triphosphates, PPP_i activates I_AC effectively in whole cell recordings (5). With pipettes containing 1 mM ATP and 5 mM PPPi, I_AC current density was severalfold larger than that observed with ATP alone. Accordingly, riluzole produced 6-fold larger absolute increases in non-inactivating K⁺ current in the presence of PPPi and ATP, compared with ATP alone (Fig. 11B). Overall, these results are consistent with the hypothesis that riluzole enhances only the activity of I_AC K⁺ channels that have been pre-activated by ATP and PPP_i.

Inhibition of Riluzole-activated K⁺ Current by ACTH and cAMP-- Results demonstrating cooperative effects of ATP, PPP_i, and riluzole on expression of background K⁺ current in AZF cells suggest that these agents all enhance only the activity of I_AC channels and that these are bTREK-1 channels. However, the possibility that multiple K⁺ channels are involved has not been excluded. For example, TRAAK-type K⁺ channels exhibit some properties of I_AC, including activation by riluzole (18, 29). In this regard, unlike native I_AC and cloned TREK-1 channels, TRAAK channels are not inhibited by cAMP (29). If riluzole activates TRAAK in AZF cells, this current would be resistant to inhibition by ACTH and cAMP.

ACTH was found to inhibit all non-inactivating K⁺ currents completely in AZF cells, including that induced by riluzole. In the experiment illustrated in Fig. 12A, I_AC reached a stable maximum value before the cell was superfused with 100 µM riluzole, which produced a 3-fold enhancement of the current. Superfusion of the cell with ACTH (200 pM) in the continued presence of riluzole produced complete inhibition of the non-inactivating current. Nearly identical results were obtained in each of four experiments.

View larger version (15K): [in this window] [in a new window]   Fig. 12.   Inhibition of Riluzole-activated K+ Current by ACTH and cAMP. K+ currents were recorded from AZF cells at 30-s intervals in response to voltage steps of increasing size, applied from a holding potential of 80 mV with () or without () depolarizing prepulses to 20 mV. IAC amplitudes are plotted against time. A and B, inhibition of riluzole-activated current by ACTH. A, after IAC reached a maximum in control saline, cell was superfused sequentially with saline containing 100 µM riluzole followed by 100 µM riluzole plus 200 pM ACTH. B, after IAC reached a maximum amplitude in control saline, cell was superfused sequentially with saline containing 200 pM ACTH and 200 pM ACTH plus 100 µM riluzole. C and D, inhibition of riluzole-activated K+ current by cAMP. C, after IAC reached a maximum amplitude, cell was superfused sequentially with saline containing 500 µM 8-pcpt-cAMP followed by 500 µM 8-pcpt-cAMP and 100 µM riluzole. D, K+ currents were recorded with a patch pipette containing 100 µM cAMP. After 7 min, cell was superfused with saline containing 100 µM riluzole as indicated.

In other experiments, I_AC was allowed to grow to a stable maximum amplitude before sequentially perfusing 200 pM ACTH and 100 µM riluzole, respectively. After complete inhibition of I_AC by ACTH, riluzole was totally ineffective in activating any current (Fig. 12B). Similar findings were obtained in three other experiments.

cAMP was equally effective as ACTH in suppressing riluzole-induced K⁺ currents. In the experiment illustrated in Fig. 12C, I_AC was permitted to reach a maximum amplitude before sequentially superfusing a membrane-permeable cAMP analog (8-pcpt-cAMP; 500 µM), followed by riluzole (100 µM). After complete inhibition of I_AC current by 8-pcpt-cAMP, riluzole failed to produce any increase in K⁺ current.

When 8-pcpt-cAMP was applied intracellularly through the recording pipette, I_AC was suppressed, and riluzole was again ineffective (Fig. 12D). In a total of six similar experiments, riluzole increased I_AC from a control value of 53.6 ± 7.6 pA (n = 6) to a final value of only 68.8 ± 6.1 pA. These experiments demonstrate that the riluzole activated K⁺ channel is identical to I_AC with respect to sensitivity to inhibition by cAMP and ACTH.

Effect of Penfluridol on Riluzole-activated Current-- Penfluridol is a potent antagonist of both native I_AC and cloned bTREK-1 currents. If the riluzole-activated current in AZF cells is composed only of I_AC, then it should be inhibited by this drug with similar potency. In the experiment shown in Fig. 13A, I_AC was allowed to approach a maximum value (trace 2) before superfusing with saline containing riluzole (100 µM), which produced transient (trace 3) and sustained (trace 4) increases in the non-inactivating current. Subsequent superfusion of saline containing riluzole and penfluridol (2.5 µM) resulted in near complete inhibition of the remaining non-inactivating K⁺ current (trace 5). Overall, in four similar experiments, penfluridol inhibited the sustained K⁺ current by 92.6 ± 5.8%.

View larger version (16K): [in this window] [in a new window]   Fig. 13.   Inhibition of riluzole-activated K+ current by penfluridol. K+ currents were recorded from AZF cells at 30-s intervals in response to voltage steps to +20 mV from a holding potential of 80 mV with () and without () depolarizing prepulses to 20 mV. Cells were superfused with 100 µM riluzole and/or 2.5 µM penfluridol as indicated. A, after IAC reached a maximum amplitude in control saline, cell was superfused sequentially with saline containing 100 µM riluzole followed by one containing 100 µM riluzole and 2.5 µM penfluridol. Numbers on traces at left correspond to those on plot of IAC amplitude against time at right. B, after IAC reached a maximum amplitude in control saline, cell was superfused with saline containing 2.5 µM penfluridol followed by one containing 2.5 µM penfluridol and 100 µM riluzole. Numbers on traces at left correspond to those on plot of IAC amplitude against time at right.

Experiments such as those illustrated in Fig. 13A did not eliminate the possibility that the transiently activated component of non-inactivating current induced by riluzole was a separate penfluridol-insensitive K⁺ current. This possibility was excluded by experiments like that illustrated in Fig. 13B in which I_AC was permitted to reach a maximum amplitude (trace 2) before subsequently superfusing the cell with saline containing penfluridol (2.5 µM), followed by one containing both penfluridol (2.5 µM) and riluzole (100 µM). Penfluridol inhibited the I_AC current completely (trace 3) and nearly eliminated any riluzole-induced increase (trace 4). In this experiment, the previously reported spontaneous time-dependent decrease in Kv1.4 current that accompanies the growth of I_AC is evident after treatment with penfluridol (trace 3) (30). At this concentration, penfluridol blocks I_AC selectively. Similar results were obtained in each of three experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this study, a b-TREK-1 K⁺ channel cDNA was cloned and shown to be expressed robustly in bovine AZF cells. When bTREK-1 cDNA was transiently expressed in HEK-293 or CHO-K1 cells, the corresponding membrane current displayed prominent characteristics of the native I_AC background K⁺ current of AZF cells. Conversely, I_AC displayed distinctive properties of cloned bTREK-1 channels (Table I). Overall, these results provide convincing evidence that I_AC is a bTREK-1 channel expressed in AZF cells. Apparently, when expressed in AZF cells, these native channels retain properties, such as ATP sensitivity, that are lost when the same channels are expressed in cell lines.

Comparison of bTREK-1 to Other TREK-1 Channels-- bTREK-1 codes for a 411-amino acid protein that is 96 and 99% homologous in amino acid sequence to mouse and human TREK-1 channels of the same length (24, 31). However, Northern blot analysis of TREK-1 transcripts from several mouse tissues and human brain identified a single 3.8-kb transcript (24, 31). In contrast, Northern blot analysis of AZF cell mRNA identified three bTREK-1 transcripts of 4.9, 3.6, and 2.8 kb. This result indicates that several splice variants of bTREK-1 channels may be expressed in bovine AZF cells. Multiple variants of a number of K⁺ channel subtypes are expressed in various tissues (32). Kv1.4 transcripts of 3.4 and 4.4 kb are expressed by bovine AZF cells (27).

Comparison of bTREK-1 Currents to I_AC-- The time-dependent increase in bTREK-1 current observed in transiently transfected HEK-293 and COS-1 cells had not been observed previously in whole cell recordings of cloned TREK-1 channels but is typical of I_AC in AZF cells (1, 3-5). The molecular mechanism underlying this phenomenon is unknown, but dilution of an endogenous cytoplasmic inhibiting factor common to all three cell types appears likely.

Whole cell bTREK-1 current waveforms resemble I_AC currents with respect to kinetics of activation. Both consist of an instantaneous component and a smaller time-dependent fraction that activates with first order kinetics (3). This two-component current has also been described for cloned mouse TREK-1 expressed in a mammalian cell line (31).

The time-dependent component of bTREK-1 and I_AC currents observed in response to membrane depolarization are indicative of voltage-dependent gating. In a previous study, we demonstrated a weak voltage-dependent activation of I_AC in which open probability increased by 0.3 between test voltages of 40 and +40 mV (4). Recently, others have shown a voltage dependence activation of native and cloned mouse and rat TREK-1 channels that may or may not depend on the phosphorylation state of the channel (20, 33). Regardless, bTREK-1, I_AC, and TREK-1 currents from several species display similar kinetics and voltage-dependent gating.

In physiological solutions, cloned bTREK-1 current displayed identical rectification with both the ATP- and pH-activated background K⁺ current recorded from AZF cells. This rectification is typical of that observed for mouse and human TREK-1 channels (24, 31).

bTREK-1 channels displayed a pharmacological profile similar to that described previously for I_AC in AZF cells (10, 11). In particular, the potent inhibition of bTREK-1 and I_AC currents by penfluridol and mibefradil establishes an interesting pharmacological similarity between the two channels. It also identifies these two Ca²⁺ channel blockers as the most potent antagonists of TREK-1 channels yet described. In this respect, penfluridol and mibefradil are at least 1000- to 10,000-fold more potent than K⁺ channel antagonists such as TEA and 4-AP (16, 23, 31). It will be interesting to determine whether these Ca²⁺ channel antagonists will also inhibit other tandem-pore K⁺ channels.

In whole cell recordings from transfected HEK-293 cells, bTREK-1 currents were expressed independently of the concentration of ATP in the pipette solution. This is the single significant property distinguishing these cloned channels from native I_AC channels. Direct activation by nucleotides or phosphates has not been reported for any cloned TREK-1 channel. It is possible that cloned TREK-1 channels lose their ATP dependence and sensitivity, because an ATP-binding protein similar to the K_ATP channel associated sulfonylurea receptor is missing in the host cell (34). If ATP binding to a TREK-associated inhibitory subunit frees this channel for activation, then absence of this protein in the host cell could explain the ATP-independent activity of bTREK-1 channels expressed in HEK-293. In this regard, we have been unable to demonstrate ATP-dependent activation of I_AC channels in excised inside-out patches from AZF cells. In fact, on patch excision, channel activity typically increases spontaneously with time, regardless of the ATP concentration at the cytoplasmic membrane surface (5). This result indicates that ATP-mediated modulation of I_AC activity requires AZF cell components that are lost after patch excision.

Relatedly, cloned TREK-1 channels are temperature-sensitive and activated dramatically by heat. The thermal activation of TREK-1 is lost on patch excision (35). It appears that the thermal sensitivity of TREK-1 channels also depends on factors present in an intact cell.

Cooperative Activation of I_AC by H⁺ and ATP-- ATP and pH acted cooperatively in promoting the activity of I_AC channels. Expression of the native I_AC current in the presence of 5 mM ATP was enhanced strongly at acidic pHs and nearly eliminated by raising the intracellular pH to 7.5 or above. These results suggest that I_AC is a 2P/4TMS channel of the TREK variety. Of the tandem pore K⁺ channels cloned thus far, only murine and human TREK-1 and TREK-2 channels are activated by acidification of the cytoplasm (17, 25, 28).

In this regard, it is unlikely that I_AC is TREK-2, because TREK-2 is strongly inwardly rectifying in symmetrical K⁺ solutions and insensitive to 100 µM quinidine (28). By comparison, in symmetrical K⁺ solutions, I_AC channels are nearly ohmic whereas they are blocked by quinidine with an IC₅₀ of 26 µM (5, 10). Further, Northern blot analysis of AZF cell mRNA using a rat TREK-2 cDNA as a probe indicated that TREK-2 mRNA is not expressed in these cells.²

The cooperative activation of I_AC by pH and ATP was also evident in the ATP-dependent enhancement of I_AC current by acidification. With pipettes containing ATP at a concentration of 0.5 mM, successive reductions in pH were far less effective at enhancing I_AC than in the presence of 5 mM ATP.

There seems to be little doubt that the non-inactivating K⁺ current activated by ATP and/or acidification is due to a single type of K⁺ channel. In addition to the cooperative actions of ATP and pH described above, currents activated by either agent were inhibited by ACTH and penfluridol equivalently. Further, riluzole enhanced both pH- and ATP-activated K⁺ current in AZF cells. Finally, pH- and ATP-activated currents displayed identical rectification at potentials between +60 and 140 mV.

Enhancement of I_AC by Riluzole-- Several lines of evidence indicated that the riluzole-activated K⁺ current in AZF cells was I_AC and not a second non-inactivating K⁺ current. Specifically, the riluzole-activated current was indistinguishable from I_AC with regard to current waveform, voltage-dependent rectification, and inhibition by penfluridol, ACTH, and cAMP.

Further, the activation of non-inactivating K⁺ current by riluzole varied directly with the concentration of ATP in the pipette solution. At low ATP concentrations (1 mM), I_AC was poorly expressed, and riluzole induced only very small absolute increases in I_AC current. These results are consistent with the hypothesis that riluzole enhances current through I_AC channels that have been pre-activated by ATP. The facilitory effects of AMP-PNP and PPP_i on riluzole-mediated increases in K⁺ current are also consistent with this model.

The identification of I_AC as the riluzole-activated K⁺ current in AZF cells provides further evidence that this current flows through bTREK-1 channels. Of the 2P/4TMS K⁺ channels riluzole has been reported to increase only the activity of TRAAK and TREK-1 (18). Of these two, only TREK-1 type channels are inhibited by cAMP (18, 31). Because cAMP and ACTH inhibited riluzole-induced increases completely in the non-inactivating current, it is unlikely that this current was TRAAK and consistent with the hypothesis that it is TREK-1. In addition, TRAAK has been shown to be a neuron-specific channel (15, 29).

The riluzole-induced increase in I_AC current was similar to that reported for activation of cloned rat TREK-1 channels with respect to concentration dependence but differed with respect to kinetics. The inhibition that followed transient activation of cloned TREK-1 channels has been attributed to a riluzole-induced increase in cAMP, subsequent to inhibition of phosphodiesterase (18). The temporal pattern of this response, and the inhibition in particular, would depend on the presence of required cytoplasmic components. Within this framework, the absence of the inhibitory component of the riluzole response that we observed with low resistance electrodes would occur through more rapid dilution of cytoplasmic contents by the pipette solution. We believe that the varying temporal pattern of the riluzole response in AZF cells is consistent with the activation of only I_AC (bTREK-1)-type channels.

Identity of I_AC and bTREK-1 Channels-- In summary, we have cloned a bTREK-1 K⁺ channel cDNA that is expressed robustly in bovine AZF cells. When expressed in cell lines, bTREK-1 current displays many of the properties of the endogenous I_AC current of AZF cells. These include time-dependent growth in whole cell recordings, instantaneous and time-dependent components in response to depolarization, potent inhibition by penfluridol and mibefradil, similar rectification, and unitary current amplitude.

Native I_AC in turn displays essential properties of bTREK-1, as well as those of human, rat, and mouse TREK-1 channels expressed in cell lines (20, 24, 29). In addition to similarities mentioned previously in single channel conductance, rectification, and inhibition by cAMP, I_AC channels have now been shown to be activated by riluzole and intracellular acidification. In other experiments, we have observed that additional agents that activate TREK-1 channels, including unsaturated fatty acids, lysophospholipids, and the volatile anesthetic chloroform also activate I_AC current in AZF cells.³ Overall, convincing evidence indicates that the cloned bTREK-1 channel is the primary subunit of AZF cell I_AC channels.

Significance-- Earlier studies have shown that TREK-1 channels are expressed primarily in the CNS and in primary sensory neurons (31, 35). TREK-1 channels may also be present in heart, kidney, lungs, ovary, and stomach (16, 24, 31, 36). This is the first study demonstrating that TREK-1 channels are expressed by a specific endocrine cell.

Further, when combined with previous studies, the present work assigns a pivotal role to bTREK-1 in the physiology of cortisol secretion. Apparently, these channels set the resting potential of bovine AZF cells and couple ACTH and angiotensin II receptor activation to depolarization-dependent Ca²⁺ entry through T-type channels (1-3, 6). Because I_AC channels are also expressed in bovine glomerulosa cells where they are inhibited by ACTH and angiotensin II, it is also likely that bTREK-1 channels have a similar function in regulating aldosterone secretion (6). It will be interesting to determine whether TREK-1 is expressed in other steroid hormone-secreting cells.

Previous studies showed that TREK-1 K⁺ channels set the resting membrane potential and conferred sensitivity of the cell to diverse stimuli, including mechanical force, temperature, pH, fatty acids, phospholipids, and transmitters (25, 31, 35-37). The activation of I_AC by ATP and inorganic phosphate expands the role of TREK-1 channels to that of a metabolic sensor that may couple the metabolic state of the cell to membrane potential and electrical activity. In AZF cells, bTREK-1 channels may integrate both hormonal and metabolic signals linking these to depolarization-dependent Ca²⁺ entry and cortisol secretion.

	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. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY148474.

To whom correspondence should be addressed: Dept. of Neuroscience, The Ohio State University, College of Medicine, 5190 Graves Hall, 333 W. 10th Ave., Columbus, OH 43210-1239. Tel.: 614-292-3511; Fax: 614-688-8742; E-mail: enyeart.1@osu.edu.

^§ Supported in part by the National Science Foundation under Agreement 0112050.

Published, JBC Papers in Press, October 3, 2002, DOI 10.1074/jbc.M207233200

² J. J. Enyeart, L. Xu, S. Danthi, and J. A. Enyeart, unpublished observations.

³ S. Danthi and J. J. Enyeart, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: AZF, adrenal zona fasciculata; ACTH, adrenocorticotropic hormone; PPPI, inorganic polytriphosphate; 4-AP, aminopyridine; TEA, tetraethyl ammonium; P, pore; TM, transmembrane; TMS, TM segments; AMP-PNP, adenosine 5'-(,-imino)triphosphate; 8-pcpt-cAMP, 8-(4-chlorophenylthio)-cAMP; HEK, human embryonic kidney; CHO, Chinese hamster ovary; RACE, rapid amplification of cDNA ends; pF, picofarad; pA, picoampere; DPBP, diphenylbutylpiperidine.

	REFERENCES

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