Modulation of the Two-pore Domain Acid-sensitive K+ Channel TASK-2 (KCNK5) by Changes in Cell Volume
2001; Elsevier BV; Volume: 276; Issue: 46 Linguagem: Inglês
10.1074/jbc.m107192200
ISSN1083-351X
AutoresMarı́a Isabel Niemeyer, L. Pablo Cid, L. Felipe Barros, Francisco V. Sepúlveda,
Tópico(s)Electrochemical Analysis and Applications
ResumoThe molecular identity of K+channels involved in Ehrlich cell volume regulation is unknown. A background K+ conductance is activated by cell swelling and is also modulated by extracellular pH. These characteristics are most similar to those of newly emerging TASK (TWIK-related acid-sensitive K+ channels)-type of two pore-domain K+ channels. mTASK-2, but not TASK-1 or -3, is present in Ehrlich cells and mouse kidney tissue from where the full coding sequences were obtained. Heterologous expression of mTASK-2 cDNA in HEK-293 cells generated K+ currents in the absence intracellular Ca2+. Exposure to hypotonicity enhanced mTASK-2 currents and osmotic cell shrinkage led to inhibition. This occurred without altering voltage dependence and with only slight decrease in pK a in hypotonicity but no change in hypertonicity. Replacement with other cations yields a permselectivity sequence for mTASK-2 of K+ > Rb+ ≫ Cs+ > NH 4+ > Na+ ≅ Li+, similar to that for the native conductance (IK, vol). Clofilium, a quaternary ammonium blocker of IK, vol, blocked the mTASK-2-mediated K+ current with an IC50 of 25 μm. The presence of mTASK-2 in Ehrlich cells, its functional similarities with IK, vol, and its modulation by changes in cell volume suggest that this two-pore domain K+ channel participates in the regulatory volume decrease phenomenon. The molecular identity of K+channels involved in Ehrlich cell volume regulation is unknown. A background K+ conductance is activated by cell swelling and is also modulated by extracellular pH. These characteristics are most similar to those of newly emerging TASK (TWIK-related acid-sensitive K+ channels)-type of two pore-domain K+ channels. mTASK-2, but not TASK-1 or -3, is present in Ehrlich cells and mouse kidney tissue from where the full coding sequences were obtained. Heterologous expression of mTASK-2 cDNA in HEK-293 cells generated K+ currents in the absence intracellular Ca2+. Exposure to hypotonicity enhanced mTASK-2 currents and osmotic cell shrinkage led to inhibition. This occurred without altering voltage dependence and with only slight decrease in pK a in hypotonicity but no change in hypertonicity. Replacement with other cations yields a permselectivity sequence for mTASK-2 of K+ > Rb+ ≫ Cs+ > NH 4+ > Na+ ≅ Li+, similar to that for the native conductance (IK, vol). Clofilium, a quaternary ammonium blocker of IK, vol, blocked the mTASK-2-mediated K+ current with an IC50 of 25 μm. The presence of mTASK-2 in Ehrlich cells, its functional similarities with IK, vol, and its modulation by changes in cell volume suggest that this two-pore domain K+ channel participates in the regulatory volume decrease phenomenon. family of K+ channels with two P regions in tandem and four putative transmembrane helices Goldman-Hodgkin-Katz regulatory volume decrease tandem of P domains in weak inward rectifier K+ channel TWIK-related acid-sensitive K+channel TWIK-relatedalkali-activated K+channel reverse transcription-polymerase chain reaction base pair(s) human expressed sequence tag 3-(cyclohexylamino)propanesulfonic acid 4-morpholineethanesulfonic acid kilobase(s) Potassium channels are multimeric membrane proteins capable of allowing the passage of K+ ions across the membrane down their electrochemical potential gradient. Their functions range from the propagation of the action potential and the control of excitability to transepithelial transport and the homeostasis of cell volume. There are many varieties of K+ channels distinguishable by their functional properties and pharmacological sensitivities. From the molecular point of view, three major families have been distinguished (1North R.A. Trends Neurosci. 2000; 23: 234-235Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar): voltage-gated KV channels, Kir inward rectifiers and SKCa/IKCaCa2+-dependent K+ channels. These previously described K+ channels have only one pore domain (P) and form tetramers with each monomer contributing one P domain to the selectivity filter. A novel family of K+ channels which, exceptionally, have two P regions in tandem and four putative transmembrane helices (2P-4TM1) has recently emerged, with 13 mammalian homologues described at the time of this writing. In heterologous systems they give rise to K+-selective conductances open at all voltages and, generally, showing little rectification besides that expected from the Goldman-Hodgkin-Katz (GHK) prediction (2Lesage F. Lazdunski M. Am. J. Physiol. 2000; 279: F793-F801Crossref PubMed Google Scholar, 3Goldstein S.A. Bockenhauer D. O'Kelly I. Zilberberg N. Nat. Rev. Neurosci. 2001; 2: 175-184Crossref PubMed Scopus (561) Google Scholar). A diagnostic feature of these channels is their insensitivity (or low sensitivity) to a range of conventional K+ channel blockers, including various toxins, Ba2+, tetraethylammonium, and 4-aminopyridine. 2P-4TM channels are thought to underlie the leak or background conductances. These conductances maintain the passive properties of the cell. They have also been implicated in the regulation of excitability by neurotransmitters, second messengers, O2, or volatile anesthetics. The discovery of the 2P-4TM channel family provides molecular counterparts for these relatively ill-understood conductances and allows the study of their modulation (1North R.A. Trends Neurosci. 2000; 23: 234-235Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 2Lesage F. Lazdunski M. Am. J. Physiol. 2000; 279: F793-F801Crossref PubMed Google Scholar, 3Goldstein S.A. Bockenhauer D. O'Kelly I. Zilberberg N. Nat. Rev. Neurosci. 2001; 2: 175-184Crossref PubMed Scopus (561) Google Scholar). The best studied is TASK-1 (KCNK3), which is thought to be the background K+ conductance closed by neurotransmitters to enhance excitability in the central nervous system (4Millar 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 (237) Google Scholar, 5Talley E.M. Lei Q. Sirois J.E. Bayliss D.A. Neuron. 2000; 25: 399-410Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar). An increase in cell volume is followed in most cells by regulatory volume decrease (RVD) mediated by efflux of K+, Cl−, organic osmolytes and osmotically obliged water leading to volume recovery. The pathway through which K+exits Ehrlich cells during RVD is not known at the molecular level but has been characterized recently through electrophysiology and flux measurements (6Riquelme G. Sepúlveda F.V. Jørgensen F. Pedersen S. Hoffmann E.K. Biochim. Biophys. Acta. 1998; 1371: 101-106Crossref PubMed Scopus (32) Google Scholar, 7Niemeyer M.I. Hougaard C. Hoffmann E.K. Jørgensen F. Stutzin A. Sepúlveda F.V. J. Physiol. (London). 2000; 524: 757-767Crossref Scopus (39) Google Scholar, 8Hougaard C. Niemeyer M.I. Hoffmann E.K. Sepúlveda F.V. Pflügers Arch. 2000; 440: 283-294PubMed Google Scholar, 9Jørgensen N.K. Christensen S. Harbak H. Brown A.M. Lambert I.H. Hoffmann E.K. Simonsen L.O. J. Membr. Biol. 1997; 157: 281-299Crossref PubMed Scopus (67) Google Scholar, 10Hougaard C. Jørgensen F. Hoffmann E.K. Pfluegers Arch. 2001; 442: 622-633Crossref PubMed Scopus (30) Google Scholar). This current (IK, vol) is independent of intracellular Ca2+, has a current-voltage relation that obeys the GHK formalism, suggesting the channels involved lack intrinsic voltage dependence, and is selective to K+and Rb+, with PK > PRb.IK, vol is rather insensitive to a number of conventional K+ channel inhibitors but is efficiently blocked by the quaternary ammonium derivative clofilium.IK, vol in Ehrlich cells is markedly dependent upon extracellular pH, being strongly inhibited at pH 6.4 and enhanced at pH 8.4, compared with the control at pH 7.4. The properties of IK, vol in Ehrlich cells, particularly the lack of voltage dependence and insensitivity to many conventional blockers, is reminiscent of the characteristics of the 2P-4TM K+ channels. In addition, its strong dependence on extracellular pH approaches them to a group within the 2P-4TM family that can be distinguished by their sensitivity to extracellular pH. These have been termed TASK, for TWIK (Tandem of P domains in Weak Inward rectifier K+ channels)-related acid-sensitive K+ channels, and more recently TALK, for alkali-activated K+ channels. 2An alternative naming plan, i.e. the KCNK nomenclature, has been proposed in which the allusion to a possible functional description that might not yet be firmly established is avoided (3Goldstein S.A. Bockenhauer D. O'Kelly I. Zilberberg N. Nat. Rev. Neurosci. 2001; 2: 175-184Crossref PubMed Scopus (561) Google Scholar). In this, TASK-1, -2, and 3 become KCNK-3, -5, and -9, respectively. 2An alternative naming plan, i.e. the KCNK nomenclature, has been proposed in which the allusion to a possible functional description that might not yet be firmly established is avoided (3Goldstein S.A. Bockenhauer D. O'Kelly I. Zilberberg N. Nat. Rev. Neurosci. 2001; 2: 175-184Crossref PubMed Scopus (561) Google Scholar). In this, TASK-1, -2, and 3 become KCNK-3, -5, and -9, respectively.TASK channels, but not TALK-1 and -2 (=TASK-4), which are active only at alkaline pH (11Decher 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, 12Girard 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 (146) Google Scholar), provide good candidates forIK, vol. The aim of this work was to investigate whether a member of the TASK group of the 2P-4TM channel family could be responsible for K+ efflux during RVD. It is demonstrated here that, of the three murine TASK K+channels known this far, only mTASK-2 transcript is present in Ehrlich cells. mTASK-2, studied by heterologous expression in HEK-293 cells, is shown to share pharmacological blockade and ion selectivity with the native conductance activated by osmotic swelling of Ehrlich cells. Importantly, osmotic cell swelling can enhance the activity of TASK-2 whereas shrinkage decreases its activity. We therefore propose it to be the molecular counterpart of IK, vol. Total RNA was prepared from adult mouse tissue immediately after euthanasia by cervical dislocation or from Ehrlich cells grown and collected as described before (7Niemeyer M.I. Hougaard C. Hoffmann E.K. Jørgensen F. Stutzin A. Sepúlveda F.V. J. Physiol. (London). 2000; 524: 757-767Crossref Scopus (39) Google Scholar). RNA was isolated using the RNeasy kit (Qiagen) according to the manufacturer's instructions, and cDNA was synthesized from 2–3 μg of RNA using SuperScript (Life Technologies, Inc.), oligo(dT), and random primers in the presence of RNase inhibitors (RNasin, Promega). All animal manipulations were approved by the local ethics committee. The PCR amplification procedures were carried out as described before (13Cid L.P. Niemeyer M.I. Ramı́rez A. Sepúlveda F.V. Am. J. Physiol. 2000; 279: C1198-C1210Crossref PubMed Google Scholar). For mTASK-1 (14Kim Y. Bang H. Kim D. Am. J. Physiol. 1999; 277: H1669-H1678PubMed Google Scholar) the primers used were: sense 5′-CGTCGTGCTGCGCCTCAA-3′ and antisense 5′-AGCCTGGCCGTTGTGCGT-3′, corresponding to nucleotides coding for Arg44-Ala50 and Arg245-Ala251 in the Mus musculus TBAK-1 (GenBankTM accession numberAB008537). The expected product is 624 bp long. Conditions were: denaturation at 94 °C for 2 min, followed by 30 cycles at 94 °C for 15 s, annealing at 58° for 30 s, extension at 72 °C for 1 min, and a final extension at 72 °C for 5 min. mTASK-3 primers, based on the rat homologue (15Kim Y. Bang H. Kim D. J. Biol. Chem. 2000; 275: 9340-9347Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar), were: sense 5′-ATGCGCGAIGAGGAGAAACT-3′ and antisense 5′-TCTTGATICGCTTCAGCAGG-3′. They correspond to peptide segments Met35-Leu41and Thr121-Ser127 of rTASK-3 (GenBankTM access number AF192366). The expected product is 338 bp long. PCR conditions were as for mTASK-1. For mTASK-2, the amino acid sequence for (human) hTASK-2 (16Reyes 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 (323) Google Scholar) was used to search for murine expressed sequence tags (ESTs) with the NCBI tBLASTn program in the National Library of Medicine data bases. ESTs AW31846 and AW31955 were used to design primers 5′-AGTGATTAGTGAACCCGG-3′ (sense) and 5′-CCAGTGGCTTCCTCTCACG-3′, which should correspond to 5′- and 3′-untranslated segments. The expected size of the amplicon is around 1623 bp. PCR conditions were: initial denaturation at 94 °C for 2 min, followed by 5 cycles at 94 °C for 15 s, annealing at 68 °C for 45 s, extension at 72 °C for 1 min, 25 cycles at 94 °C for 15 s, annealing at 54 °C for 45 s, extension at 72 °C for 1 min, and a final extension at 72 °C for 5 min. PCR products resolved by agarose gel electrophoresis were excised and extracted for DNA, which was cloned into pGEM-T vector (Promega). Sequencing was performed automatically. For Northern analysis, total RNA was run in a denaturing agarose gel. After transferring to a nylon membrane, it was probed with digoxigenin-labeled antisense riboprobe (50 ng ml−1) synthesized by in vitro transcription using mTASK-2 cDNA as a template. Hybridization was conducted at 68 °C. Detection was with an anti- digoxigenin-alkaline phosphatase conjugate (Roche Molecular Biochemicals, Mannheim, Germany), which was used for colorimetric visualization. Western blot was done with crude membrane fractions (17Gallardo P. Cid L.P. Vio C.P. Sepúlveda F.V. Am. J. Physiol. 2001; 281: G856-G863PubMed Google Scholar). Ehrlich cells, grown as described elsewhere (7Niemeyer M.I. Hougaard C. Hoffmann E.K. Jørgensen F. Stutzin A. Sepúlveda F.V. J. Physiol. (London). 2000; 524: 757-767Crossref Scopus (39) Google Scholar), were homogenized in a buffer containing 250 mm sucrose and 10 mm triethanolamine. Homogenates were centrifuged at 2000 × g for 10 min at 4 °C. The supernatants were spun down at 100,000 × g for 1 h at 4 °C, and pellets were resuspended. Protein concentration was determined by the Bradford method. SDS-polyacrylamide gel electrophoresis was done using Laemmli buffers on 10% polyacrylamide minigels. Enhanced chemiluminescence was used to reveal antigen-antibody reaction. The anti-TASK-2 antibody (APC-07, Alomone Laboratories, Israel) was used at 1:200 dilution. Preadsorption was done by preincubating the antibody with the antigen peptide for 1 h at a 1/1 μg ratio. The mTASK-2 plasmid used in the electrophysiology studies was subcloned in the expression vector pCR3.1 (Invitrogen) and transfected into HEK-293 cells as described previously (13Cid L.P. Niemeyer M.I. Ramı́rez A. Sepúlveda F.V. Am. J. Physiol. 2000; 279: C1198-C1210Crossref PubMed Google Scholar). CD8 cotransfection was used to identify effectively transfected cells. The CD8 antigen was revealed with microspheres (Dynabeads) coated with an anti-CD8 antigen. In cation selectivity studies, the bath solution contained 135 mm XCl, 5 mm KCl, 2 mmCaCl2, 1 mm MgCl2, 30 mm sucrose, 10 mm, Hepes/Tris, pH 7.4.X stands for either Rb, Cs, Li, Na, K, or NH4, as indicated. The pipette solution contained 140 mmKCl, 1 mm MgCl2, 10 mm EGTA, 1 mm Na3ATP, 0.1 mm GTP, 10 mm Hepes, pH 7.4. Alternatively, in experiments at low Cl− concentration this anion was replaced by gluconate to give a final Cl− concentration of 10 mmsolution either in the pipette or bath. In experiments to measure the pH dependence of the currents HEPES (used for pH 7.0, 7.5, and 8.0) in the bathing medium was replaced with CAPS (pH 10 and 11), Tris (pH 8.5 and 9), or MES (pH 6.0). Standard whole-cell patch-clamp recordings were performed as described elsewhere (13Cid L.P. Niemeyer M.I. Ramı́rez A. Sepúlveda F.V. Am. J. Physiol. 2000; 279: C1198-C1210Crossref PubMed Google Scholar, 18Dı́az M. Sepúlveda F.V. Pfluegers Arch. 1995; 430: 168-180Crossref PubMed Scopus (39) Google Scholar). All chemicals were from Sigma Chemical Co. (St. Louis, MO). When necessary, calculated correction for changes in junction potential were made (19Barry P.H. J. Neurosci. Meth. 1994; 51: 107-116Crossref PubMed Scopus (545) Google Scholar). Changes in cell water volume were assessed in single cells by measuring changes in concentration of an intracellularly trapped fluorescent dye (20Alvarez-Leefmans F.J. Altamirano J. Crowe W.E. Methods Neurosci. 1995; 27: 361-391Crossref Scopus (54) Google Scholar) exactly as described previously (21Stutzin A. Torres R. Oporto M. Pacheco P. Eguiguren A.L. Cid L.P. Sepúlveda F.V. Am. J. Physiol. 1999; 277: C392-C402Crossref PubMed Google Scholar). HEK-293 transfected with mTASK-2 cDNA were plated on 25-mm No. 1 round coverslips, loaded with calcein-AM (5 μm, for 5 min) and then superfused with iso-osmotic solution for 30 min before starting the experiment. The experiments were performed using a confocal laser imaging system (LSM5 Pascal, Carl Zeiss, Germany). Excitation light was 488 nm, and emitted light was measured at wavelengths longer than 515 nm. Pictures were obtained at 30-s intervals, and fluorescence of a selected area inside the cell was measured. Under the conditions of the experiment there was no apparent dye photobleaching. The data are presented asF0/Ft, whereF0 = fluorescence in iso-osmotic solution, att = 0, and Ft = fluorescence at time = t. The ratioF0/Ft is proportional to cell volume. Transfected cells were identified by the presence of microbeads as described above. The possible presence of TASK transcripts in Ehrlich cells was assayed in RT-PCR experiments. In Fig. 1 Aspecific primers for mTASK-1 were used. When using RNA from mouse heart as a positive control, a product of a size compatible with the 624 nucleotides of the expected amplicon was seen in the electrophoresis run. However, there was no detectable amplification of mTASK-1 using RNA from Ehrlich cells or from mouse liver, a negative control known to lack mTASK-1 mRNA (2Lesage F. Lazdunski M. Am. J. Physiol. 2000; 279: F793-F801Crossref PubMed Google Scholar, 22Kim D. Fujita A. Horio Y. Kurachi Y. Circ. Res. 1998; 82: 513-518Crossref PubMed Scopus (113) 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 (142) Google Scholar). A similar result was obtained for mTASK-3, as seen in Fig. 1 B. There was clear amplification of a product of a size compatible with the expected 338 nucleotides amplicon in brain, the site of most abundance of this transcript (15Kim Y. Bang H. Kim D. J. Biol. Chem. 2000; 275: 9340-9347Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar,24Rajan S. Wischmeyer E. Liu G.X. Preisig-Muller R. Daut J. Karschin A. Derst C. J. Biol. Chem. 2000; 275: 16650-16657Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). No detectable amplification was seen in either Ehrlich cells or small intestine, a negative control (15Kim Y. Bang H. Kim D. J. Biol. Chem. 2000; 275: 9340-9347Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, 24Rajan S. Wischmeyer E. Liu G.X. Preisig-Muller R. Daut J. Karschin A. Derst C. J. Biol. Chem. 2000; 275: 16650-16657Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). To search for the presence of mTASK-2 in Ehrlich cells and mouse kidney by RT-PCR, the protein sequence for (human) hTASK-2 (16Reyes 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 (323) Google Scholar) was used to search for murine expressed sequence tags (ESTs). Two ESTs (AW31846 and AW31955) were identified that contained putative start and stop codons for an mTASK-2 (mouse). Primers were designed to flank these, and RT-PCR with mouse kidney and Ehrlich cell RNA gave products of around 1600 nucleotides as shown in Fig. 1 C, suggesting that mTASK-2 was present in Ehrlich cells. This was confirmed by the Northern blot shown in Fig.1 D that revealed a ∼3.3-kb transcript. The amplicons were subcloned and sequenced confirming the presence of an open reading frame that on translation gave a 502-amino acid sequence containing the two P regions in tandem as well as four putative trans-membrane helices. This predicted polypeptide was 88.8% homologous to hTASK-2. The mTASK-2 sequence information has been deposited in the GenBankTM under accession number AF319542. These data suggest that Ehrlich cells express TASK-2 but not TASK-1 or -3. 3Western analysis with anti-hTASK-1 antibody has suggested the presence of this protein in mouse Ehrlich cells (10Hougaard C. Jørgensen F. Hoffmann E.K. Pfluegers Arch. 2001; 442: 622-633Crossref PubMed Scopus (30) Google Scholar). We do not understand the origin of this discrepancy with the present data, but as discussed below, the cation selectivity and lack of voltage dependence of the pH effect on IK, vol separate the channel in Ehrlich cells from TASK-1. mTASK-2 expression was also checked by Western blot of membranes from Ehrlich cells, as shown in Fig. 1 E. The analysis revealed a major band of about 70 kDa (lane 1). This, as well as other minor bands, could be abolished by previous incubation of the antibody with the antigenic peptide (lane 2). The mass of mTASK-2 simply derived from the predicted amino acid sequence is 55 kDa. Glycosylation, for which a consensus site is present, could account for this discrepancy. No K+ currents were seen in mock- or untransfected HEK-293 cells (not shown). After transfection with mTASK-2 cDNA, sizeable currents occurred that had voltage and pH dependence characteristics identical to those reported for the human orthologue (16Reyes 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 (323) Google Scholar). These currents showed GHK behavior at all pH values tested (not shown). The selectivity of mTASK-2 was analyzed by cation replacement. Fig. 2 shows currents evoked by the pulse protocol in Fig. 2 A. The pipette solution was high in K+ (140 mm), and the external solution contained 140 (Fig. 2 E) or 5 mm K+and 135 mm of either Na+, Rb+, or Cs+ (Fig. 2, B–D). In Na+-rich medium, instant rectification and a moderate activation/deactivation were observed. With K+-rich medium the currents appeared Ohmic with decreased time dependence. In Cs+ the rectification was more marked, and there was little evidence of time dependence. In Rb+-rich medium, on the other hand, the instantaneous current was large at all potentials and relaxed slightly to a lower absolute value toward the end of the pulse at negative potentials. The current-voltage relations for these experiments as measured at the end of the pulse are shown in Fig. 2 (F andG). In symmetrical K+ solutions, it was linear with a reversal potential at 0 mV. Replacement of all but 5 mm K+ by Na+ shiftedErev to more negative than −70 mV. The current-voltage relation was described by the GHK formalism albeit with a lower PK value than for symmetrical K+ condition (2.4 × 10−11 and 2.8 × 10−11 cm3 s−1, respectively). In low K+ solution and at very depolarized voltages, the fit deviated from the experimental points, which are lower than expected. Fig.2 G compares the current-voltage relations measured in Rb+-, Cs+-, and Li+-rich solutions. The curve in the Li+-replaced solution was indistinguishable from that in Na+. In Cs+, although the reversal was as in Na+, current was depressed in the entire voltage range examined. The reversal in Rb+-rich solution was shifted in the depolarizing direction compared with that in Na+. The permeability ratios for different cations were calculated from the shifts in reversal potential. They are given in Fig. 2 H and compared with those reported for mTASK-1 (KCNK3) (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 (142) Google Scholar). The main difference between TASK-1 and -2 is that the former presents aPRb > PK permeability sequence. Clofilium is an inhibitor ofIK, vol in Ehrlich cells (7Niemeyer M.I. Hougaard C. Hoffmann E.K. Jørgensen F. Stutzin A. Sepúlveda F.V. J. Physiol. (London). 2000; 524: 757-767Crossref Scopus (39) Google Scholar). The effect of this compound on mTASK-2 currents is shown in Fig. 3. In theinset to the graph in Fig. 3 A it is seen that 30 μm clofilium inhibited mTASK-2 current evoked by an 80-mV pulse without affecting the kinetics of its development. Current-voltage relations are shown under control conditions (squares) and after superfusing the cells with 20 and 50 μm clofilium. There was a graded inhibition of the current at all potentials explored without affecting the reversal potential. In Fig. 3 B a summary of results for clofilium inhibition of mTASK-2 mediated currents is shown. The measurements were taken at 0 mV (ECl) and are expressed as percent inhibition caused by the drug. The result obtained in mTASK-2 assays (circles) is compared with that obtained previously forIK, vol (7Niemeyer M.I. Hougaard C. Hoffmann E.K. Jørgensen F. Stutzin A. Sepúlveda F.V. J. Physiol. (London). 2000; 524: 757-767Crossref Scopus (39) Google Scholar). The solid linerepresents the best fit to the Hill equation to the mTASK-2 data with an IC50 of 25 μm andnH of 2. The data presented above make TASK-2 a likely candidate to be the molecular counterpart of IK, vol. The sensitivity of mTASK-2 current to changes in tonicity was, therefore, tested. Swelling untransfected HEK-293 cells, by exposure to hypotonic solution, resulted in the activation of Cl−current of a type present in other cells (25Dı́az M. Valverde M.A. Higgins C.F. Rucareanu C. Sepúlveda F.V. Pfluegers Arch. 1993; 422: 347-353Crossref PubMed Scopus (121) Google Scholar, 26Valverde M.A. Hardy S.P. Sepúlveda F.V. FASEB J. 1995; 9: 509-515Crossref PubMed Scopus (48) Google Scholar), but no K+ current developed (not shown). In mTASK-2-expressing cells, hypotonic exposure elicited an increase in K+current as measured at ECl (upper trace in Fig. 4 A). As expected, this was accompanied by activation of a small Cl− current measured at EK (lower panel in Fig. 4 A). The current-voltage relations measured in isotonicity under physiological (open circles) or symmetrical (triangles) K+ concentrations (Fig.4 B) showed the expected rectification properties and reversal potentials. The current-voltage relationship measured in physiological K+ concentration under hypotonicity is shown in Fig. 4 C. There was a general increase in current magnitude and a shift in Erev fromEK to a more depolarized value, consistent with concomitant activation of a Cl− conductance. The current at ECl retained pH sensitivity as shown by theblack triangle (pH 8.5) and circle (pH 6.0). In symmetrical K+, Erev changed to 0 mV as expected. As seen in Fig. 4 A, the effect of cell swelling was slowly reversible upon return to isotonicity. Notice the slower recovery time for K+ current, which has been seen before for the native currents (8Hougaard C. Niemeyer M.I. Hoffmann E.K. Sepúlveda F.V. Pflügers Arch. 2000; 440: 283-294PubMed Google Scholar). Cells shrunk in hypertonic medium showed a significant reduction in mTASK2-mediated current. Fig.5 A shows an experiment where a cell was superfused with a solution made 100 mosmhypertonic, through the addition of mannitol without change in ion composition. Hypertonic exposure led to a rapid decrease in mTASK-2-mediated current. This decrease was reversible and was not accompanied by alterations in the current at EK. Fig. 5 B shows current-voltage plots taken before (circles) or after (downward triangles) shrinking the cell by changing the tonicity of the extracellular fluid from 300 to 400 mosm. The current depressed by this maneuver had the same reversal potential, and the effect was similar in the entire voltage range as shown by experiments with symmetrical K+(not shown). Fig. 6 A shows a summary of experiments demonstrating the osmosensitivity of mTASK-2. The average relative changes in mTASK-2 K+ current at 0 mV (ECl) are shown. Statistically significant increases or decreases in current were seen in hypo- and hypertonicity, respectively. The increase in current in hypotonicity occurred with a small but significant change in pH sensitivity. As shown in Fig.6 B the pH dependence of K+ current at 0 mV could be described by a Hill equation with pK a of 8.30 ± 0.07 and nH of 0.81 ± 0.08 (n = 13). In hypotonicity the respective values were 7.99 ± 0.07 and 0.81 ± 0.07 (n = 8), with pK a being significantly reduced with respect to the control (p = 0.0077). This small shift in pK a only accounts for ∼60% of the hypotonicity effect. The pH dependence of the current in hypertonicity was not altered significantly, with a pK a andnH values of 8.24 ± 0.13 and 0.87 ± 0.05 (n = 4), respectively. The effect of mTASK-2 expression on the ability of cells to undergo regulatory volume decrease was tested. As in previous experiments, co-expression of the CD-8 antigen revealed with an antibody conjugated to microbeads was used to identify transfected cells. Fig.7 A shows a group of such HEK-293 cells loaded with calcein to measure changes in their volume (20Alvarez-Leefmans F.J. Altamirano J. Crowe W.E. Methods Neurosci. 1995; 27: 361-391Crossref Scopus (54) Google Scholar, 21Stutzin A. Torres R. Oporto M. Pacheco P. Eguiguren A.L. Cid L.P. Sepúlveda F.V. Am. J. Physiol. 1999; 277: C392-C402Crossref PubMed Google Scholar). Two of the cells in the group (labeled 3 and4) are decorated with beads showing that they are expressing the foreign DNA. Two non-ex
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