Formation of Functional Heterodimers between the TASK-1 and TASK-3 Two-pore Domain Potassium Channel Subunits
2002; Elsevier BV; Volume: 277; Issue: 7 Linguagem: Inglês
10.1074/jbc.m107138200
ISSN1083-351X
Autores Tópico(s)Cardiac electrophysiology and arrhythmias
ResumoThe potassium channels in the two-pore domain family are widely expressed and regulate the excitability of neurons and other excitable cells. These channels have been shown to function as dimers, but heteromerization between the various channel subunits has not yet been reported. Here we demonstrate that two members of the TASK subfamily of potassium channels, TASK-1 and TASK-3, can form functional heterodimers when expressed in Xenopus laevis oocytes. To recognize the two TASK channel types, we took advantage of the higher sensitivity of TASK-1 over TASK-3 to physiological pH changes and the discriminating sensitivity of TASK-3 to the cationic dye ruthenium red. These features were clearly observed when the channels were expressed individually. However, when TASK-1 and TASK-3 were expressed together, the resulting current showed intermediate pH sensitivity and ruthenium red insensitivity (characteristic of TASK-1), indicating the formation of TASK-1/TASK-3 heterodimers. Expression of a tandem construct in which TASK-3 and TASK-1 were linked together yielded currents with features very similar to those observed when coexpressing the two channels. The tandem construct also responded to AT1a angiotensin II receptor stimulation with an inhibition that was weaker than the inhibition of homodimeric TASK-1 and greater than that shown by TASK-3. Expression of epitope-tagged channels in mammalian cells showed their primary presence in the plasma membrane consistent with their function in this location. Heteromerization of two-pore domain potassium channels may provide a greater functional diversity and additional means by which they can be regulated in their native tissues. The potassium channels in the two-pore domain family are widely expressed and regulate the excitability of neurons and other excitable cells. These channels have been shown to function as dimers, but heteromerization between the various channel subunits has not yet been reported. Here we demonstrate that two members of the TASK subfamily of potassium channels, TASK-1 and TASK-3, can form functional heterodimers when expressed in Xenopus laevis oocytes. To recognize the two TASK channel types, we took advantage of the higher sensitivity of TASK-1 over TASK-3 to physiological pH changes and the discriminating sensitivity of TASK-3 to the cationic dye ruthenium red. These features were clearly observed when the channels were expressed individually. However, when TASK-1 and TASK-3 were expressed together, the resulting current showed intermediate pH sensitivity and ruthenium red insensitivity (characteristic of TASK-1), indicating the formation of TASK-1/TASK-3 heterodimers. Expression of a tandem construct in which TASK-3 and TASK-1 were linked together yielded currents with features very similar to those observed when coexpressing the two channels. The tandem construct also responded to AT1a angiotensin II receptor stimulation with an inhibition that was weaker than the inhibition of homodimeric TASK-1 and greater than that shown by TASK-3. Expression of epitope-tagged channels in mammalian cells showed their primary presence in the plasma membrane consistent with their function in this location. Heteromerization of two-pore domain potassium channels may provide a greater functional diversity and additional means by which they can be regulated in their native tissues. Potassium channels play a pivotal role in adjusting the excitability of neurons and endocrine cells by controlling the resting membrane potential. Two large families of K+ channels have been identified so far that can drive the resting membrane potential (E m) toward hyperpolarization. Inwardly rectifying potassium channels conduct considerable currents around the K+ equilibrium potential, but their outward current diminishes upon depolarization. Therefore, these channels stabilize the resting E m without markedly influencing depolarizing membrane potential changes. In contrast, members of the two-pore domain (2P) 1The abbreviations used are:2Ptwo-pore domainECextracellularMES4-morpholineethanesulfonic acidHAhemagglutininFBSfetal bovine serumPBSphosphate-buffered salineRRruthenium red potassium channels provide substantial currents not only at the restingE m but also in the depolarized state. Moreover, the repolarizing current of 2P potassium channels is maintained at a given potential, because they activate instantaneously and, with the exception of TWIK-2 (1Chavez R.A. Gray A.T. Zhao B.B. Kindler C.H. Mazurek M.J. Mehta Y. Forsayeth J.R. Yost C.S. J. Biol. Chem. 1999; 274: 7887-7892Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), they do not inactivate. Therefore, in addition to controlling the resting E m, 2P potassium channels are also involved in the regulation of action potential duration and firing patterns in neurons (2Millar J.A. Barratt L. Southan A.P. Page K.M. Fyffe R.E. Robertson B. Mathie A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3614-3618Crossref PubMed Scopus (240) Google Scholar, 3Talley E.M. Lei Q. Sirois J.E. Bayliss D.A. Neuron. 2000; 25: 399-410Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar) as well as in the control of membrane potential changes in stimulated endocrine (4Czirják G. Fischer T. Spät A. Lesage F. Enyedi P. Mol. Endocrinol. 2000; 14: 863-874PubMed Google Scholar) and chemoreceptor (5Buckler K.J. Williams B.A. Honore E. J. Physiol. 2000; 525: 135-142Crossref PubMed Scopus (369) Google Scholar) cells (see Refs. 6Patel A.J. Honore E. Trends Neurosci. 2001; 24: 339-346Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar and 7Lesage F. Lazdunski M. Am. J. Physiol. 2000; 279: F793-F801Crossref PubMed Google Scholar for review). two-pore domain extracellular 4-morpholineethanesulfonic acid hemagglutinin fetal bovine serum phosphate-buffered saline ruthenium red Two-pore domain K+ channels are classified as a family based on their similar molecular architecture. Each subunit contains four transmembrane segments and two pore-forming domains: one located between the first and second transmembrane segments and another between the third and fourth transmembrane segments. This topology assumes that both the N and C termini of the subunits are intracellular. The selectivity filter of K+ channels being made up of four pore domains is the reason for the requirement to form dimers. In addition to these theoretical considerations, dimer formation of 2P channel subunits has been also demonstrated experimentally (8Lesage F. Lauritzen I. Duprat F. Reyes R. Fink M. Heurteaux C. Lazdunski M. FEBS Lett. 1997; 402: 28-32Crossref PubMed Scopus (106) Google Scholar, 9Lesage F. Reyes R. Fink M. Duprat F. Guillemare E. Lazdunski M. EMBO J. 1996; 15: 6400-6407Crossref PubMed Scopus (154) Google Scholar, 10Lopes C.M. Zilberberg N. Goldstein S.A. J. Biol. Chem. 2001; 276: 24449-24452Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). The cloned mammalian 2P channels have been classified into five subfamilies based on their primary sequences and electrophysiological and regulatory properties. The TWIK (tandem of pore domains in a weakly inwardly rectifyingK+ channel) subfamily contains TWIK-1 (11Lesage F. Guillemare E. Fink M. Duprat F. Lazdunski M. Romey G. Barhanin J. EMBO J. 1996; 15: 1004-1011Crossref PubMed Scopus (460) Google Scholar), TWIK-2 (1Chavez R.A. Gray A.T. Zhao B.B. Kindler C.H. Mazurek M.J. Mehta Y. Forsayeth J.R. Yost C.S. J. Biol. Chem. 1999; 274: 7887-7892Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), and KCNK7 (12Salinas M. Reyes R. Lesage F. Fosset M. Heurteaux C. Romey G. Lazdunski M. J. Biol. Chem. 1999; 274: 11751-11760Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) (although the latter channel could not be expressed functionally). TWIKs are regulated by protein kinase C and by changes in intracellular pH. Members of the TREK (TWIK-related K+channel) subfamily TREK-1 (13Fink M. Duprat F. Lesage F. Reyes R. Romey G. Heurteaux C. Lazdunski M. EMBO J. 1996; 15: 6854-6862Crossref PubMed Scopus (425) Google Scholar), TREK-2 (14Bang H. Kim Y. Kim D. J. Biol. Chem. 2000; 275: 17412-17419Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 15Lesage F. Terrenoire C. Romey G. Lazdunski M. J. Biol. Chem. 2000; 275: 28398-28405Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar), and TRAAK (16Fink M. Lesage F. Duprat F. Heurteaux C. Reyes R. Fosset M. Lazdunski M. EMBO J. 1998; 17: 3297-3308Crossref PubMed Scopus (398) Google Scholar) are mechanosensitive channels activated by unsaturated fatty acids. Of the THIK (tandem pore domainhalothane-inhibited K+channel) subfamily members, THIK-1 (17Rajan S. Wischmeyer E. Karschin C. Preisig-Muller R. Grzeschik K.H. Daut J. Karschin A. Derst C. J. Biol. Chem. 2001; 276: 7302-7311Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) can also be activated by arachidonic acid, whereas THIK-2 failed to display channel behavior upon heterologous expression, despite its targeting to the plasma membrane (17Rajan S. Wischmeyer E. Karschin C. Preisig-Muller R. Grzeschik K.H. Daut J. Karschin A. Derst C. J. Biol. Chem. 2001; 276: 7302-7311Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). The further subfamilies are interrelated by their strong pH sensitivity. TASK-2 (18Reyes R. Duprat F. Lesage F. Fink M. Salinas M. Farman N. Lazdunski M. J. Biol. Chem. 1998; 273: 30863-30869Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar), TALK-1 (19Girard C. Duprat F. Terrenoire C. Tinel N. Fosset M. Romey G. Lazdunski M. Lesage F. Biochem. Biophys. Res. Commun. 2001; 282: 249-256Crossref PubMed Scopus (148) Google Scholar), and TALK-2 (19Girard C. Duprat F. Terrenoire C. Tinel N. Fosset M. Romey G. Lazdunski M. Lesage F. Biochem. Biophys. Res. Commun. 2001; 282: 249-256Crossref PubMed Scopus (148) Google Scholar) (also termed as TASK-4 (20Decher N. Maier M. Dittrich W. Gassenhuber J. Bruggemann A. Busch A.E. Steinmeyer K. FEBS Lett. 2001; 492: 84-89Crossref PubMed Scopus (123) Google Scholar)) are activated by extracellular (EC) alkaline pH, whereas two other TASK (TWIK-relatedacid-sensitive K+) channels, TASK-1 (21Duprat F. Lesage F. Fink M. Reyes R. Heurteaux C. Lazdunski M. EMBO J. 1997; 16: 5464-5471Crossref PubMed Scopus (549) Google Scholar, 22Leonoudakis D. Gray A.T. Winegar B.D. Kindler C.H. Harada M. Taylor D.M.C.-R. Forsayeth J.R. Yost C.S. J. Neurosci. 1998; 18: 868-877Crossref PubMed Google Scholar, 23Lopes C.M. Gallagher P.G. Buck M.E. Butler M.H. Goldstein S.A. J. Biol. Chem. 2000; 275: 16969-16978Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar) and TASK-3 (24Kim Y. Bang H. Kim D. J. Biol. Chem. 2000; 275: 9340-9347Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar), are classified to another subfamily despite sharing the name with TASK-2 and TASK-4. Both TASK-1 and TASK-3 are inhibited by EC acidification, an effect mediated by histidine 98 of TASK-3 (25Rajan S. Wischmeyer E. Liu G.X. Müller R.P. Daut J. Karschin A. Derst C. J. Biol. Chem. 2000; 275: 16650-16657Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar), which is also conserved in TASK-1 (but not in TASK-2 or TASK-4). Recent studies indicated that stimulation of certain G protein-coupled receptors inhibits TASK-1 channels (2Millar J.A. Barratt L. Southan A.P. Page K.M. Fyffe R.E. Robertson B. Mathie A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3614-3618Crossref PubMed Scopus (240) Google Scholar, 3Talley E.M. Lei Q. Sirois J.E. Bayliss D.A. Neuron. 2000; 25: 399-410Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar, 4Czirják G. Fischer T. Spät A. Lesage F. Enyedi P. Mol. Endocrinol. 2000; 14: 863-874PubMed Google Scholar); this effect is mediated by phospholipase C activation (26Czirják G. Petheõ G.L. Spät A. Enyedi P. Am. J. Physiol. 2001; 281: C700-C708Crossref PubMed Google Scholar). In all of the above-mentioned studies, 2P channels were expressed as homodimers (for review see Ref. 27Goldstein S.A. Bockenhauer D. O'Kelly I. Zilberberg N. Nat. Rev. Neurosci. 2001; 2: 175-184Crossref PubMed Scopus (567) Google Scholar). Recently, it was also demonstrated that both the P1 and the P2 domains of KCNK3 (TASK-1) contribute to the pore within the homodimer (10Lopes C.M. Zilberberg N. Goldstein S.A. J. Biol. Chem. 2001; 276: 24449-24452Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). The possible heterodimer formation between THIK-1 and THIK-2 (17Rajan S. Wischmeyer E. Karschin C. Preisig-Muller R. Grzeschik K.H. Daut J. Karschin A. Derst C. J. Biol. Chem. 2001; 276: 7302-7311Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) and KCNK6 and KCNK7 (12Salinas M. Reyes R. Lesage F. Fosset M. Heurteaux C. Romey G. Lazdunski M. J. Biol. Chem. 1999; 274: 11751-11760Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) has been studied in detail. However, these studies concluded that neither THIKs nor these KCNKs can form heterodimers, because the THIK-1 current was not influenced by overexpression of the nonfunctional THIK-2, and coexpression of KCNK6 and KCNK7 did not yield a functional K+ channel (2Millar J.A. Barratt L. Southan A.P. Page K.M. Fyffe R.E. Robertson B. Mathie A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3614-3618Crossref PubMed Scopus (240) Google Scholar). In the present report we demonstrate for the first time that two members of the TASK subfamily, TASK-1 and TASK-3, are capable of constituting functional heterodimers when expressed in Xenopus laevis oocytes. The pharmacological and regulatory properties of the TASK-1/TASK-3 heterodimer is clearly different from those of each of the homodimers, adding to the diversity of potassium channels that affect the function of excitable cells. Enzymes and kits of molecular biology applications were purchased from Ambion (Austin, TX), Amersham Biosciences, Inc., Fermentas (Vilnius, Lithuania), New England Biolabs (Beverly, MA), and Promega (Madison, WI). All other chemicals of analytical grade were obtained from Fluka (Buchs, Switzerland), Promega, and Sigma. The coding region of TASK-3 was amplified from total RNA prepared from rat adrenal zona glomerulosa usingPfu turbo DNA polymerase after reverse transcription. PCR primers were designed on the basis of the published rat TASK-3 sequence. The forward primer was 5′-ggcatATGAAGCGGCAGAATGTGCG-3′ (T3-s), and the reverse primer (T3end-a) was 5′-cctctctagACTTAGATGGACTTGCGACG-3′. The additional bases at the 5′ ends (indicated by lowercase letters) introduced NdeI andXbaI restriction sites to the forward and reverse primers, respectively. The PCR product was digested with XbaI and cloned into the XbaI-EcoRV-digested Bluescript pKS phagemid (Stratagene). This construct was then subcloned into the pEXO vector containing the 5′- and 3′-untranslated regions of theXenopus globin gene. The sequence of the construct (pEXO-TASK-3) was determined from both directions by automated sequencing. Partially overlapping sense (T(3+1)LIGs: 5′-GTCCATCAAGCGGCAGAACGTGCGC-3′) and antisense (T(3+1)LIGa: 5′-CTGCCGCTTGATGGACTTGCGACGGATG-3′) oligonucleotides were designed for linking the coding sequences of TASK-3 and TASK-1. The sequence of the oligonucleotides covered the C-terminal end of the TASK-3 and the N-terminal beginning of the TASK-1 coding sequences in frame. PCR products were amplified (15 cycles) from pEXO-TASK-3 with the sense T3forw (corresponding to bases 268–290 of TASK-3) and the antisense T(3+1)LIGa primers. PCR was also performed from pEXO-TASK-1 with the sense T(3+1)LIGs and the antisense T1rev (corresponding to bases 611–631 of TASK-1). The two PCR products were then mixed, allowed to hybridize by their overlapping T(3+1)LIGs and T(3+1)LIGa primer regions, and elongated without primers. This was followed by further amplification (15 cycles) in the presence of T3forw and T1rev primers. The resulting T3forw-T1rev product was digested with Kpn2I (which cuts within the TASK-3 half of the sequence) and was ligated into the Kpn2I-SmaI digested pEXO-TASK-3. (SmaI creates a blunt end at the multiple cloning site of pEXO at the 3′ end of the TASK-3 insert). To add the missing piece of TASK-1 to this construct, the NcoI-BamHI fragment of the resulting clone was replaced with theNcoI-BamHI fragment of pEXO-TASK-1. (TheNcoI site is within the TASK-1 part of the T3forw-T1rev amplification product, and BamHI is in the pEXO cloning site downstream of the SmaI site.) The region of the two channels originating from the PCR amplification was verified by sequencing. The pEXO-TASK-(3+1) construct coded for a polypeptide chain, in which the last amino acid residue (Ile) of TASK-3 is followed by the second residue of TASK-1 (Lys). TASK-1, TASK-3, TASK-(3+1), TRAAK, TREK-1 and the angiotensin II (AT1a) receptor cRNA were synthesized in vitro according to the manufacturer's instructions (Ambion mMESSAGE mMACHINETM T7 in vitro transcription kit) using the XbaI linearized pEXO-TASK-1 (21Duprat F. Lesage F. Fink M. Reyes R. Heurteaux C. Lazdunski M. EMBO J. 1997; 16: 5464-5471Crossref PubMed Scopus (549) Google Scholar), pEXO-TASK-3, pEXO-TASK-(3+1), pEXO-TRAAK (28Maingret F. Fosset M. Lesage F. Lazdunski M. Honore E. J. Biol. Chem. 1999; 274: 1381-1387Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar), and pEXO-TREK-1 (13Fink M. Duprat F. Lesage F. Reyes R. Romey G. Heurteaux C. Lazdunski M. EMBO J. 1996; 15: 6854-6862Crossref PubMed Scopus (425) Google Scholar) constructs and the NotI linearized plasmid, comprising the coding sequence and 5′-untranslated region of rat AT1a angiotensin II receptor (kindly provided by Dr. K. E. Bernstein). Mature female X. laevis frogs were obtained from Amrep Reptielen (Breda, Netherlands). The frogs were anesthetized by immersion into benzocaine solution (0.03%). Ovarian lobes were removed, and the tissue was dissected and treated with collagenase (1.45 mg/ml, 148 units/mg, type I; Worthington Biochemical Corp., Freehold, NJ) and continuous mechanical agitation in Ca2+-free OR2 solution containing 82,5 mm NaCl, 2 mm KCl, 1 mm MgCl2, 5 mm HEPES, pH 7.5, for 1.5–2 h. Stage V and VI oocytes were defolliculated manually and kept at 18 °C in modified Barth's saline containing 88 mm NaCl, 1 mm KCl, 2.4 mm NaHCO3, 0.82 mmMgSO4, 0.33 mmCa(NO3)2, 0.41 mmCaCl2, 20 mm HEPES buffered to pH 7.5 with NaOH and supplemented with penicillin (100 units/ml), streptomycin (100 μg/ml), sodium pyruvate (4.5 mm), and theophyllin (0.5 mm). The oocytes were injected 1 day after defolliculation. Fifty nanoliters of the appropriate RNA solution was delivered with Nanoliter Injector (World Precision Instruments, Saratosa, FL). Electrophysiological experiments were performed 3 or 4 days after the injection. Membrane currents were recorded by two-electrode voltage clamp (OC-725-C; Warner Instrument Corp., Hamden, CT) using microelectrodes made of borosilicate glass (Clark Electromedical Instruments, Pangbourne, UK) with a resistance of 0.3–1 MΩ when filled with 3 m KCl. The currents were filtered at 1 kHz, digitally sampled at 1–2.5 kHz with a Digidata Interface (Axon Instruments, Foster City, CA), and stored on a PC/AT computer. Recording and data analysis were performed using pCLAMP software 6.0.4 (Axon Instruments). The experiments were carried out at room temperature, and the solutions were applied by a gravity-driven perfusion system. Low [K+] solution contained 95.4 mm NaCl, 2 mm KCl, 1.8 mmCaCl2, 5 mm HEPES. High [K+] solution contained 80 mm K+ (78 mmNa+ of the low [K+] solution was replaced with K+). Unless otherwise stated, the pH of every solution was adjusted to 7.5 with NaOH. Perifusing solutions with a pH of <6.5 were buffered by including 5 mm MES. Background K+ currents were measured in high EC [K+] at the end of 300-ms-long voltage steps to −100 mV applied in every 3 s. The holding potential was 0 mV. Where possible, the inward current in high [K+] was corrected for the small nonspecific leak measured in 2 mm EC [K+]. Oligonucleotide dimers coding for themyc (residues 410–419 of human c-myc, EQKLISEEDL) and HA (residues 99–107 of human influenza virus hemagglutinin, YPYDVPDYA) epitopes were ligated in the uniqueNruI and Kpn2I restriction enzyme sites of pEXO-TASK-1 and pEXO-TASK-3, respectively. Hybridization of the sensemyc oligonucleotide (5′-GAGGAGCAGAAGCTGATCTCAGAGGAGGACCTG-3′) with its reverse complement created blunt ends, whereas the sense (5′-CCGGGCTACCCTTACGACGTCCCTGACTACGCC-3′) and antisense (5′-CCGGGGCGTAGTCAGGGACGTCGTAAGGGTAGC-3′) HA oligonucleotides were designed to form overhanging Kpn2I-compatible ends in the appropriate reading frame. Correct insertion of the epitope coding regions was checked by sequencing. The epitope-tagged coding sequences were subcloned also into pcDNA3.1+ (Invitrogen) for expression in mammalian cells. For immunocytochemistry HEK cells were grown on glass coverslips in Dulbecco's modified Eagle's medium supplemented with 10% FBS and were transfected for 6 h with TASK-1-HA-pcDNA3 or TASK-3-HA-pcDNA3 constructs (1 μg) in 1.2 ml of Opti-MEM containing 2 μl of LipofectAMINE. Two days after the transfection the cells were fixed in 2% (w/v) paraformaldehyde in PBS for 10 min and washed three times with PBS containing 10% (v/v) fetal bovine serum (PBS-FBS). The cells were then incubated for 1 h with monoclonal anti-HA epitope antibody (Covance PRB 150C, 1:1000 dilution) in the presence of 0.2% saponin in PBS-FBS, followed by three 5-min washes before incubation for 1 h with Alexa Fluor-488 goat anti-mouse IgG for TASK-1 and with Alexa Fluor-594 goat anti-mouse IgG (Molecular Probes) for TASK-3 (each at 1:1000 dilution). For simultaneous detection of TASK-1 and TASK-3, the cells were cotransfected with TASK-1-myc-pcDNA3 and TASK-3-HA-pcDNA3 constructs (1 μg each, as above). During the immunostaining procedure and the incubation with the anti-HA epitope antibody and with the secondary Alexa Fluor 594 anti-mouse antibody (as above), the cells were washed and stained with a monoclonal fluorescein isothiocyanate-conjugated anti-mycantibody (Covance FITC-150L, 1:50 dilution). After three final washes (each for 5 min in PBS-FBS), the coverslips were mounted for confocal microscopy. The cells were examined by an inverted Zeiss 410 laser confocal microscope with a (100×) oil immersion lens. The data are expressed as the means ± S.E. Normalized dose-response curves were fitted (least squares method; Sigmaplot, Jandel Corporation) to the following Hill equation: y = 1/(1 + (c/K 12)n), where c is the concentration, K 12 is the concentration at which half-maximal inhibition occurs, and n is the Hill coefficient. Where the treatment failed to cause complete inhibition, a modified form of the equation was used: y = ϕ/(1 + (c/K 12)n) + (1 − ϕ), where ϕ is the fraction maximally inhibited by the treatment. In a recent study we found that TASK-1 and TASK-3 channels can be distinguished by their different sensitivity to the polycationic compound, ruthenium red (RR). 2G. Czirják and P. Enyedi, (2002)Mol. Endocrinol., in press With the aim of applying RR as a potential tool discriminating between different members of the 2P domain potassium channel family, we analyzed the inhibitory effect of RR on TASK-1, on TASK-3, and also on TRAAK and TREK-1 channels in more detail. The current was monitored at −100 mV in the presence of 80 mm extracellular [K+], a condition that gives a large signal of the expressed channels with only minor contamination by other endogenous currents of the oocytes (4Czirják G. Fischer T. Spät A. Lesage F. Enyedi P. Mol. Endocrinol. 2000; 14: 863-874PubMed Google Scholar). As shown in Fig. 1, TASK-3 and TRAAK currents were greatly reduced by RR with a half-maximal inhibitory concentration of 0.7 and 2 μm, respectively. Interestingly, the inhibition of TRAAK followed a much steeper dose-response relationship than the inhibition of TASK-3. This was also reflected in the Hill coefficients calculated for the inhibitory curves. The Hill coefficient for TASK-3 channels was found to be 1.0, indicating the binding of one RR molecule to one functional channel (dimer). In contrast, TRAAK inhibition by RR showed a Hill coefficient of 2.1, suggesting multiple RR-binding sites. The inhibitory effect of RR on both channels was found to be voltage-independent (Fig.2), indicating that the RR-binding sites on TASK-3 and TRAAK lay outside the transmembrane electrical field; accordingly a direct interference of the inhibitor with the pore of the channel is very unlikely. The rapid onset of the inhibition (not shown) suggests that RR acts from the extracellular side of the channels.Figure 2TASK-3 and TRAAK inhibition by ruthenium red is not voltage-dependent. Current-voltage relationships of an oocyte expressing TASK-3 (A) or TRAAK (B) were plotted in 2 mm EC [K+] (▪) and in 80 mm EC [K+] without (●) or with 3 μm ruthenium red (○). The currents were recorded at the end of 300-ms voltage steps from −120 mV to +20 mV in 10-mV increments from a holding potential of 0 mV.View Large Image Figure ViewerDownload (PPT) In contrast to the prominent inhibition of TASK-3 and TRAAK channels by RR, their close relatives, TASK-1 and TREK-1, respectively, were not inhibited by RR even at micromolar concentration (4.5 ± 1.2% inhibition of TASK-1 by 5 μm RR (n = 14) and less than 10% inhibition of TREK-1 by 20 μm(n = 5)). Based on these results, RR has been identified as an excellent tool to differentiate between the individual members within both the TASK and TREK family of potassium channels. As reported earlier, an additional feature discriminating between TASK-1 and TASK-3 channels is their sensitivity to extracellular acidification, TASK-1 (on the contrary to TASK-3) being highly sensitive to pH changes between 7.5 and 6.5 (21Duprat F. Lesage F. Fink M. Reyes R. Heurteaux C. Lazdunski M. EMBO J. 1997; 16: 5464-5471Crossref PubMed Scopus (549) Google Scholar, 22Leonoudakis D. Gray A.T. Winegar B.D. Kindler C.H. Harada M. Taylor D.M.C.-R. Forsayeth J.R. Yost C.S. J. Neurosci. 1998; 18: 868-877Crossref PubMed Google Scholar,24Kim Y. Bang H. Kim D. J. Biol. Chem. 2000; 275: 9340-9347Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar).2 First, we investigated the behavior of TASK-1 and TRAAK channels expressed simultaneously inXenopus oocytes in different ratios. Given the differential pH and ruthenium red sensitivities of the channels, their characteristics could be best evaluated when the degrees of inhibition by acidification (a pH step from 7.5 to 6.5) and RR (5 μm) were plotted one against the other (Fig.3). The positions of the two populations of homodimers (when only one form of the channels is expressed) is clearly separated in the extreme ends of the plot. TASK-1 homodimers that are insensitive to RR but highly sensitive to pH changes appear in the lower right, whereas TRAAK homodimers that are practically insensitive to acidification but are strongly inhibited by RR in the upper left area of the graph. When the characteristics of the coexpressed channels was plotted in the same graph, their data points all fell on the straight lineconnecting the clear TASK-1 and TRAAK populations (Fig. 3). Depending on the ratios of the two channels, the points fell closer or farther from the respective ends representing the homodimers. Such distribution of channel behavior is expected if various amounts of the two kind of homodimer channels operate independently, and therefore these data did not indicate that TASK-1 and TRAAK channels would form heterodimers. Clearly different results were obtained when TASK-1 and TASK-3 channels were coexpressed in the oocytes. TASK-1 or TASK-3 channels expressed alone are also presented with two, clearly distinguishable set of data points. The 70.4 ± 1.9 and 4.5 ± 1.2% (n = 14) inhibition of TASK-1 by acidification and RR, respectively, contrasted with the 19.3 ± 1.9 and 77.6 ± 2.3% (n = 14) inhibition by the same manipulations of TASK-3 channels. However, after coexpressions of the two channels, all the data points fell under the line connecting the TASK-1 and TASK-3 values (Fig.4 A). This result cannot be explained by formation of various proportions of simple homodimers and indicates the presence of significant amounts of heterodimer channels with new inhibitor sensitivity profiles. It is difficult to deduce the true characteristics of the heterodimer channels in terms of their pH and RR sensitivities in the above experiments because of the simultaneous presence of homodimers. To overcome this difficulty, we created a tandem version of TASK-3 and TASK-1 channels by joining the coding regions of the two channels to form one continuous protein. In this construct the C terminus of TASK-3 was fused to the N terminus of TASK-1. Such a tandem construct was expected to highly favor the assembly of the channels as heterodimers. Expressing the tandem channel in the oocytes induced large currents (13.3 ± 2.5 μA, n = 17), comparable in amplitude to the currents of TASK-1 and TASK-3. However, the pH and RR sensitivity values of the tandem channel was clearly distinct from both parent channels. It showed significant inhibition (36.4 ± 2.5%, n = 9) in response to a pH step form 7.5 to 6.5 (an intermediate pH sensitivity between TASK-1 and TASK-3 channels) and minimal inhibition (4.5 ± 1.5%,n = 9) by 5 μm RR (a feature of TASK-1 channels) (Fig. 4 B). Remarkably, the data set given by the tandem channel showed an overlapping distribution with those obtained in the coexpression experiments confirming the formation of heterodimers in the latter (Fig. 4 A). The pH sensitivity of the tandem channel was compared with that of TASK-1 and TASK-3 homo
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