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TASK, a human background K+ channel to sense external pH variations near physiological pH

TASK, a human background K+ channel to sense external pH variations near physiological pH The EMBO Journal Vol.16 No.17 pp.5464–5471, 1997 TASK, a human background K channel to sense external pH variations near physiological pH and four structural classes of auxiliary subunits (Kvβ, Fabrice Duprat, Florian Lesage, Michel Fink, Kcaβ, SUR and IsK) (Takumi et al., 1988; Knaus et al., Roberto Reyes, Catherine Heurteaux and 1 1994; Pongs, 1995; Inagaki et al., 1996). All Shaker-type Michel Lazdunski subunits have a conserved hydrophobic core containing six Institut de Pharmacologie Mole´culaire et Cellulaire, CNRS, 660 route transmembrane segments (TMS). Associations of Shaker- des Lucioles, Sophia Antipolis, 06560 Valbonne, France type subunits with accessory subunits such as Kvβ, Kcaβ or IsK give rise to voltage-dependent K channels (Pongs, Corresponding author e-mail: [email protected] 1995; Barhanin et al., 1996; Fink et al., 1996a; Sanguinetti et al., 1996) and Ca -dependent K channels (MacCobb F.Duprat, F.Lesage and M.Fink contributed equally to this work et al., 1995; MacManus et al., 1995). Subunits of inward rectifier K channels (IRK) have only two TMS (Doupnik TASK is a new member of the recently recognized et al., 1995; Lesage et al., 1995; Fakler and Ruppersberg, TWIK K channel family. This 395 amino acid poly- 1996). Some IRKs give rise to ATP-sensitive K channels peptide has four transmembrane segments and two P when they are associated with sulfonylurea receptor (SUR) domains. In adult human, TASK transcripts are found subunits (Inagaki et al., 1996). Despite a very low overall in pancreas<placenta<brain<lung, prostate<heart, sequence similarity, Shaker and IRK pore-forming subunits kidney<uterus, small intestine and colon. Electro- share a conserved domain called the P domain. This physiological properties of TASK were determined peculiar motif is an essential element of the K -selective after expression in Xenopus oocytes and COS cells. filter of the aqueous pore and is considered as the TASK currents are K -selective, instantaneous and signature of K channel-forming proteins (Heginbotham non-inactivating. They show an outward rectification et al., 1994). when external [K ] is low ([K ]  2 mM) which is out We recently have described a new family of mammalian not observed for high [K ] (98 mM). The rectification out K channel subunits. Despite a low sequence similarity can be approximated by the Goldman–Hodgkin–Katz between them (28% amino acid identity), both cloned current equation that predicts a curvature of the members of this family (TWIK-1 and TREK-1) possess current–voltage plot in asymmetric K conditions. the same overall structure, with four TMS and two P This strongly suggests that TASK lacks intrinsic voltage domains (Fink et al., 1996b; Lesage et al., 1996a, 1997). sensitivity. The absence of activation and inactivation The conservation of this structure is not associated with kinetics as well as voltage independence are character- a conservation of functional properties: TWIK-1 gives istic of conductances referred to as leak or background rise to weakly inward rectifier K currents (Lesage et al., conductances. For this reason, TASK is designated as 1996a) while TREK-1 produces outward rectifier K a background K channel. TASK is very sensitive to currents (Fink et al., 1996b). However, both channels are variations of extracellular pH in a narrow physiological open at the resting potential and are able to drive the range; as much as 90% of the maximum current is resting membrane potential near the K equilibrium poten- recorded at pH 7.7 and only 10% at pH 6.7. This tial. This common property suggests that these channels property is probably essential for its physiological control the resting membrane potential in a large set of function, and suggests that small pH variations may cell types. Here, we describe the cloning, tissue distribution serve a communication role in the nervous system. and expression of a novel human member of this new Keywords: in situ hybridization/leak/membrane potential/ structural family. To our knowledge, this channel, called potassium channels/rectification TASK for TWIK-related acid-sensitive K channel, is the first cloned mammalian channel that produces K currents that possess all the characteristics of background conduct- ances. They are instantaneous with voltage changes, and Introduction their current–voltage relationships fit the curves predicted from the constant field theory for simple electrodiffusion Potassium channels are ubiquitous membrane proteins that through an open K -selective pore, indicating that TASK form the largest family of ion channels in terms of both currents are voltage insensitive. The activity of this back- function and structure. By determining and modulating ground channel is strongly dependent on the external pH the membrane potential, they play a major role in neuronal in the physiological range, suggesting that this particular integration, muscular excitability and hormone secretion channel is a sensor of external pH variations. (Rudy, 1988; Hille, 1992). More than 40 genes encoding K channel subunits are now identified in mammals. Results These subunits fall into two structural classes of pore- forming subunits [Shaker and inward rectifier K channel Cloning and primary structure of TASK (IRK)] (Pongs, 1992; Jan and Jan, 1994; Doupnik et al., TWIK-1 and TREK-1 sequences were used to search 1995; Fakler and Ruppersberg, 1996; Kohler et al., 1996) related sequences in the GenBank database by using 5464 © Oxford University Press TASK, a TWIK-related acid-sensitive K channel Fig. 1. Nucleotide and deduced amino acid sequences of human TASK and partial amino acid sequence of mouse TASK. Consensus sites for N-linked glycosylation (*) and phosphorylation by protein kinase C (j), protein kinase A (m) and tyrosine kinase (d) in human TASK. These sites have been identified by using the prosite server (European Bioinformatics Institute) with the ppsearch software (EMBL Data library) based on the MacPattern program. The sequences of human and mouse TASK have been deposited in the GenBank/EMBL database under the accession numbers AF006823 and AF006824, respectively. the Blast alignment program. We identified two mouse kinase C (residues 358 and 383), tyrosine kinase (residue expressed sequence tags (ESTs, accession numbers 323) and protein kinase A (residues 392 and 393). All W36852 and W36914) that overlap and give a contig these phosphorylation sites are located in the C-terminal fragment of 560 bp whose deduced amino acid sequence part of the protein. Except for a 19 residue cluster (amino presents significant similarity to TWIK-1 and TREK-1. A acids 276–294 in the human sequence), mouse and human corresponding DNA fragment was amplified by RT–PCR proteins share a high overall sequence conservation (85% and used to screen a mouse brain cDNA library. Eight of identity), indicating that they are probably products of independent clones were isolated. The 1.8 kb cDNA insert ortholog genes (Figure 1). Sequence alignments presented of the longer clone bears in its 5 part an open reading in Figure 2 clearly show that the cloned protein is a new frame (ORF) coding for a 405 amino acid polypeptide member of the TWIK-related K channel family. Like (Figure 1). This ORF does not begin with an initiating TWIK-1 and TREK-1, TASK has four putative transmem- methionine codon, suggesting that the brain cDNA clones brane segments (M1–M4) and two P domains (P1 and P2) were partial. Ten additional positive clones were isolated (Figure 2A and B). TASK is 58 amino acids longer than from a mouse heart cDNA library. Analysis of their 5 TWIK-1 and 24 amino acids longer than TREK-1 because sequences showed that none these clones were longer than its C-terminus is more extended. the clones previously isolated from brain. The 5 sequence has a very high GC content and is probably associated with Distribution of TASK secondary structures that could have promoted premature The expression of TASK in adult human and mouse tissues stops of RNA reverse transcription during the construction was examined by Northern blot analysis. Three different of both mouse cDNA libraries. To overcome this problem, transcripts were detected in the human tissues, with we cloned the complete cDNA in another species. The estimated sizes of 6.8, 4.2 and 2.6 kb (Figure 3), the DNA probe was used to screen a cDNA library from shorter one having the same size as the cloned cDNAs. human kidney, a tissue that express both TWIK-1 and The other two transcripts (4.2 and 6.8 kb) may result from TREK-1 channels. Two hybridizing clones were character- alternative polyadenylation signals in the 3-non-coding ized. Both contain an ORF of 1185 nucleotides encoding sequence and/or correspond to alternatively spliced or a 394 amino acid polypeptide (Figure 1). The human immature forms of the transcript. TASK is expressed in protein sequence contains consensus sites for N-linked many different tissues but particularly in pancreas and glycosylation (residue 53), and phosphorylation by protein placenta. Lower levels of expression were found in 5465 F.Duprat et al. Fig. 3. Northern blot analysis of TASK distribution in adult human tissues. Human multiple tissue Northern blots from Clontech were probed at high stringency with a TASK cDNA probe. Each lane contains 2 μg of poly(A) RNA. Autoradiograms were exposed for 48 h at –70°C. The blots were re-probed with a β-actin cDNA probe for control. sk. muscle, skeletal muscle; sm. intestine, small intestine; PBL, peripheral blood leukocytes. the cerebral cortex, in the CA1–CA4 pyramidal cell layer, in the granule cells of the dentate gyrus, in the habenula, in the paraventricular thalamic nuclei, in the amyloid nuclei, in the substantia nigra and in the Purkinje and granular cells of the cerebellum. In the heart, a high level of TASK expression was found in the atria (Figure 4D), while ventricular cells did not express this channel. Biophysical properties of TASK currents For functional studies, TASK cRNAs were injected into Xenopus oocytes. A non-inactivating current, not present in uninjected oocytes (not shown), was measured by two-electrode voltage-clamp (Figure 5A). The activation kinetics of the TASK current are almost instantaneous (10 ms). The current–voltage (I–V) relationship is out- wardly rectifying and almost no inward currents were recorded in the ND96 external medium containing 2 mM K (Figure 5B). However, inward currents were revealed when the external K concentration ([K ] ) was Fig. 2. Sequence comparison and membrane topology of out TWIK-related channels. (A) Alignment of human TWIK-1, mouse increased gradually to 98 mM K (Figure 5A and B). TREK-1 and human TASK sequences. Identical and conserved Figure 5B shows the I–V relationships of the current in K - residues are shown in black and gray, respectively. Dashes indicate rich solutions ranging from 2 to 98 mM. The relationship gaps introduced for a better alignment. The relative positions of between the reversal potential and [K ] was close to out putative transmembrane segments (M1–M4) and P domains (P1 and the predicted Nernst value (52.1 mV/decade, n  4), as P2) of human TASK are also indicated. The M1–M4 domains were deduced from a hydropathy profile computed with a window size of expected for a highly selective K channel (Figure 5C, 11 amino acids according to the method of Kyte and Doolittle (1982). upper panel). On the other hand, external K enhanced (B) Putative membrane topology of TWIK-1, TREK-1 and TASK the outward currents in a concentration-dependent manner channels. as illustrated in Figure 5C (lower panel). The half- maximum activation by K was observed at a K of 2.06 0.5 brainlung, prostateheart, kidneyuterus, small mM. The theoretical I–V relationships in various [K ] out intestine and colon. As shown in Figure 4A, the TASK calculated according to the Goldman–Hodgkin–Katz cur- probe detected a single transcript in the mouse with rent equation are shown in Figure 5D. These I–V relation- an estimated size of 4.2 kb. TASK is expressed in ships are very close to the I–V relationships corresponding heartlungbrain and kidney. No expression was seen to recorded TASK currents (Figure 5B). This strongly in liver and skeletal muscle. The TASK distribution was suggests that TASK currents show no rectification other studied further in adult mouse brain and heart by in than that predicted from the constant field assumptions, situ hybridization. A wide and heterogeneous pattern of and that TASK lacks intrinsic voltage sensitivity. The expression was obtained in the brain (Figure 4B and C). slight deviations between experimental and theoretical TASK mRNA was detected throughout the cell layers of points are probably due to small endogenous chloride TASK, a TWIK-related acid-sensitive K channel Fig. 4. Distribution of TASK mRNA in adult mouse. (A) Northern blot analysis. Each lane contains 2 μg of poly(A) RNA. Autoradiograms were exposed for 72 h at –70°C. The blots were re-probed with a β-actin cDNA probe for control. (B–D) In situ hybridization analysis from a coronal section at the level of the forebrain (B), the cerebellum (C) and the heart (D). Warmer colors represent higher levels of expression. CA1–CA3, fields CA1–3 of Ammon’s horn; Cx, cerebral cortex; DG, dentate gyrus; Gl, granular layer; Hb, habenula; SN, substantia nigra; PLCo, postero lateral cortical amygdaloid nuclei; PVP, paraventricular thalamic nucleus; A, atrium; V, ventricule. conductance and/or a K loading of the oocytes. We have the same Goldman–Hodgkin–Katz type outward rectific- shown previously that oocytes expressing TWIK-1 or ation as in oocytes. TREK-1 are more polarized that control oocytes, the resting membrane potential (E ) reaching a value close Regulation of the TASK channel to the K equilibrium potential (E ). In oocytes expressing TASK currents were insensitive to internal Ca changes TASK, the E was –85  0.8 mV (n  23, in standard obtained by injection of inositol triphosphate (IP3, 1 mM) ND96) instead of –44  2.6 mV (n  9) in non-injected or EGTA (100 mM), to the activation of adenyl cyclase oocytes. This result demonstrates that TASK, like other by perfusion of IBMX (1 mM) and forskolin (10 μM), or TWIK or TREK channels, is able to drive E close to to the activation of protein kinase C (PKC) by application E . The effect of various pharmacological agents on of phorbol 12-myristate 13-acetate (PMA; 40 nM). TASK currents elicited by voltage pulses to 50 mV has been currents were insensitive to the internal acidification or studied in TASK-expressing oocytes. Less than 20% of alkalization obtained by injection of solutions at pH 2 or TASK currents were inhibited in the presence of quinine 8.7 respectively (n  3). However, their very interesting (100 μM), quinacrine (100 μM) or quinidine (100 μM). property is that they are highly sensitive to external pH. The ‘classical’ K channel blockers tetraethylammonium The current–potential relationships recorded from a TASK- (TEA, 1 mM) and 4-aminopyridine (4AP, 1 mM) were expressing oocyte at pH 6.5, 7.4 and 8.4 are presented in also inactive. Cs (100 μM) induced a voltage-dependent Figure 6A. For an external pH of 6.5, a drastic block was block of 31  2% (n  4) of the inward current, recorded observed at all potentials, while an activation was recorded at –150 mV, in 50 mM external K . In the same conditions, at pH 8.4, also at all potentials. The inhibition and Ba (100 μM) was ineffective, with a variation of 6  activation produced no modification of current kinetics 1% (n  4) of the inward current. (Figure 6A, inset). The pH dependence of the TASK The biophysical properties of TASK were then verified channel is shown in Figure 6B. For currents recorded at in transfected COS cells. Untransfected cells did not 50 mV, the inhibition by acidic pHs was characterized express this K channel activity (not shown). Figure 5E by an apparent pK of 7.34  0.04 units (n  3) and a shows whole-cell currents recorded in mammalian COS Hill coefficient of 1.54  0.08 (n  3). For currents cells transiently transfected with TASK, in external solu- recorded at 0 and –50 mV, the pKs were 7.32  0.02 and tions containing 5 and 155 mM K . The currents were 7.30 0.01 respectively (n 3), showing that the blocking instantaneous and non-inactivating, as in Xenopus oocytes. effect of external protons is not voltage dependent. The Figure 5F presents the I–V relationships of the current in resting membrane potential of TASK-expressing oocytes various external K concentrations. The currents show was –84  1mV(n  6) at pH 7.4 and shifted to –47 5467 F.Duprat et al. 6mV(n  6) at pH 6.4 (not shown). Finally, Figure 6C structural similarity to TWIK-1 and TREK-1 channels and D shows that the strong pH sensitivity of TASK that suggests a common ancestral origin. Despite this currents was also observed in transfected COS cells. A similar structural organization, the amino acid identity large inhibition or activation of the current was recorded, between TASK and the two other related mammalian at all potentials, when the pH was changed from 7.4 to channels is very low (25–28%). Sequence homologies are 6.1 or 8.4 respectively (Figure 6C). The kinetics of the no higher between TASK and a recently cloned Drosophila current were unmodified at both pH values (Figure 6C, channel that also belongs to the structural TWIK channel inset). Figure 6D shows that the pH effects were also class (Goldstein et al., 1996). The highest degree of non-voltage dependent in COS cells. The external pH sequence conservation is in the two P domains and the dependence of TASK, at 50 mV, indicates a pK value M2 segment. In these regions, the amino acid identity of 7.29  0.03 (n  5) and a Hill coefficient of 1.57 reaches ~50%. Like other TWIK-related channels, TASK 0.07 (n  5). Currents recorded at 0 and –50 mV presented contains an extended M1P1 interdomain. This peculiar pKs of 7.29  0.04 (n  5) and 7.32  0.05 (n  4) domain has been shown to be extracellular in the case of respectively. Ten percent of the maximum current was TWIK-1 and to be important for the self-association of obtained at pH 6.68  0.08 (n  4) and 90% at pH 7.66 two TWIK-1 subunits. The TWIK-1 homodimers are 0.05 (n  4). These results confirm that TASK is extremely covalent because of the presence of an interchain disulfide sensitive to extracellular pH in the physiological range. bridge between cysteines 69 located in the M1P1 inter- domain (Lesage et al., 1996b). This particular cysteine Discussion residue is conserved in TREK-1 (residue 93) but not in TASK, strongly suggesting that TASK probably does not Here we report the isolation and the characterization of a form covalent dimers as observed for TWIK1 (Lesage novel human K channel. This channel has an overall et al., 1996b) and TREK-1 (unpublished data). The biophysical and regulation properties of TASK are unique. TWIK-1 has a mild inward rectification that involves an internal block by Mg (Lesage et al., 1996a). TREK-1 expresses an outward rectification which seems to result from a voltage sensitivity intrinsic to the channel protein (Fink et al., 1996b). In the case of TASK, the outward rectification observed at physiological external Fig. 5. Biophysical properties of TASK in Xenopus oocytes and COS cells. (A) TASK currents recorded from a Xenopus oocyte injected with TASK cRNA and elicited by voltage pulses from –150 to 50 mV in 40 mV steps, 500 ms in duration, from a holding potential of –80 mV in low (2 mM K ) or high K solutions (98 mM K ). The zero current level is indicated by an arrow. (B) Current–voltage relationships. Mean currents were measured over the last 50 ms at the end of voltage pulses from –150 to 50 mV in 10 mV steps as in (A). Modified ND96 solutions containing 2 mM K and 96 mM TMA were used, TMA was then substituted by K to obtain solutions ranging from 2 to 98 mM K . TASK currents are not sensitive to external TMA, no changes were observed upon substitution of NaCl by TMA (data not shown). (C) Upper panel: reversal potentials of TASK currents as a function of external K concentration (mean SEM, n  3). Lower panel: slope conductance measured between 10 and 50 mV on current–voltage relationships as in (B), plotted as a function of the external K concentration (mean  SEM, n  3). The mean values were fitted with a hyperbolic function. (D) Theoretical current–voltage relationship under the same conditions as in (B), calculated according to the following modified Goldman–Hodgkin– Katz (GHK) current relationship: 2   –V F/RT [K ] V F [K ] –[K ] ·e out m in out I P · · · K  K ()() –V F/RT K  [K ] RT 1–e 0.5 out where I  is the potassium current, P  is the apparent permeability K K for K , K the half maximum activation by K ,[K ] and [K ] 0.5 out in are the external and internal K concentrations, V the membrane potential, F, R and T have their usual meanings. The classical GHK relationship has been modified with [K ] /K  [K ] to take into out 0.5 out account the sensitivity of the conductance to external K .(E) TASK currents recorded from a transfected COS cell and elicited by voltage pulses from –150 to 50 mV in 40 mV steps, 500 ms in duration, from a holding potential of –80 mV, in low (5 mM K ) or high K solutions (155 mM K ). The zero current level is indicated by an arrow. (F) Current–voltage relationships. Mean currents were measured over the last 50 ms at the end of voltage pulses ranging from –150 to 50 mV in 10 mV steps as in (E). Solutions containing 5 mM K and 150 mM TMA were used, TMA was then substituted by K to obtain solutions ranging from 5 to 155 mM K . TASK, a TWIK-related acid-sensitive K channel tivities, and their absence of specific pharmacology has delayed their extensive electrophysiological and physio- logical characterization. Cloning of TASK, the first ‘true’ background mammalian K channel, should help to characterize further this peculiar functional family of K channels at the molecular level and identify specific and high affinity pharmacological agents that would block these channels and facilitate analysis of their physio- logical roles. TASK behaves as a K -selective ‘hole’, but this does not mean that its activity cannot be modulated. Unlike TWIK-1 and TREK-1 channels, its activity is not changed by activation of protein kinase A or C (Fink et al., 1996b; Lesage et al., 1996a). The probably very important property of TASK is that it is extremely sensitive to extracellular pH in the physiological range, i.e. between 6.5 and 7.8. The Hill coefficient of ~1.6 found for the H concentration dependence of the TASK current is consistent with the idea that the channel is formed by the assembly of two subunits, as previously demonstrated for TWIK1. These two subunits would be in strong cooperative interactions with regard to H . The modulation by external protons probably has important implications for the physiological function of the TASK channel. Stimulus-elicited pH shifts have been Fig. 6. pH regulation of TASK in Xenopus oocytes and COS cells. characterized in a wide variety of neural tissues by using (A) Current–voltage relationships recorded from a TASK-expressing extracellular pH-sensitive electrodes (reviewed in Chesler, oocyte with a ramp ranging from –150 to 50 mV, 500 ms in 1990; Chesler and Kaila, 1992). They can be observed in duration, from a holding potential of –80 mV, in ND96 solution at physiopathological situations such as epileptiform activity pH 6.5, 7.4 or 8.4. Inset: currents elicited by voltage pulses to 50 mV, 500 ms in duration, under the same conditions as above. The and spreading depression in which acid shifts are usually zero current level is indicated by an arrow. (B) pH dependence of preceded by alkaline transients (Siesjo¨ et al., 1985; TASK activity in a Xenopus oocyte recorded at –50, 0 and 50 mV Nedergaard et al., 1991). They can be observed of course (mean  SEM, n  3) as in (A). Data were fitted with a Boltzman in ischemia where large acidifications of the extracellular relationship. (C) Current–voltage relationship recorded from a TASK-expressing COS cell with a ramp ranging from –150 to medium have been recorded (Kraig et al., 1983; Mutch 50 mV, 500 ms in duration, from a holding potential of –80 mV, in and Hansen, 1984). However, they can also be observed 5mMK solution at pH 6.1, 7.4 and 8.4. Inset: currents elicited by in physiological conditions such as electrical stimulation voltage pulses to 50 mV, 500 ms in duration, under the same of Schaeffer collateral fibers in the hippocampal slice conditions as above. The zero current level is indicated by an arrow. (D) pH dependence of TASK activity recorded in a COS cell at –50, 0 (Krishtal et al., 1987), or light stimulation of the retina and 50 mV (mean  SEM, n  3) as in (C). Data were fitted with a (Borgula et al., 1989; Yamamoto et al., 1992), or parallel Boltzman relationship. fibers in cerebellum (Kraig et al., 1983). All these pH shifts correspond to bursts of H or OH creating small K concentrations can be approximated to the rectification pH variations from the external physiological pH value predicted by the Goldman–Hodgkin–Katz current equa- of 7.4 in the alkaline or acidic direction (up to 0.3 pH tion, suggesting that this rectification simply results from units) and are rapid, in the 1–30 s range. They might the asymmetric concentrations of K on both sides of the actually be larger in range or shorter in time course in the membrane. In other words, this would mean that TASK vicinity of the synaptic cleft. A particularly interesting lacks intrinsic voltage sensitivity and behaves like a K - issue of course is whether these relatively small activity- selective ‘hole’. This behavior is, to our knowledge, dependent pH changes have significant modulatory effects. unique among cloned mammalian K channels. Voltage In other words, does H serve a transmitter role in the and time independences are classical criteria to describe nervous system? The discovery of this new TASK channel the so-called leak or background K channels. Some of that can fully open or close within a range of only 0.5 pH these channels have been described in invertebrates, the unit around the physiological pH (7.4) will certainly best characterized of which are the S channels in Aplysia strengthen the idea that pH could be a natural modulator sensory neurons (Siegelbaum et al., 1982), and in verte- of neuronal activity (Chesler and Kaila, 1992). brates, for example in bullfrog sympathetic ganglia (Koyano et al., 1992), guinea-pig submucosal neurons Materials and methods (Shen et al., 1992), rat carotid bodies (Buckler, 1997), and guinea-pig ventricular myocytes (Backx and Marban, Cloning of TASK and RNA analysis TWIK-1 and TREK-1 were used to search homologs in gene databases 1993). These channels are open at all membrane potentials by using the tBlastn sequence alignment program (Altschul et al., 1990). and probably play a pivotal role in the control of the Translation of two overlapping EST sequences (GenBank accession Nos resting membrane potential and in the modulation of W36852 and W36914) in one frame presented significant sequence electrical activity of both neurons and cardiac cells. similarities with TWIK-1 and TREK-1. A 560 bp DNA fragment was However, their lack of kinetics, voltage and time sensi- amplified by PCR from mouse brain poly(A) cDNAs and subcloned 5469 F.Duprat et al. into pBluescript (Stratagene) to give pBS-852/914. This fragment was the internal solution contained 150 mM KCl, 3 mM MgCl ,5mM P-labeled and used to screen mouse brain and heart cDNA libraries. EGTA, and 10 mM HEPES at pH 7.2 with KOH, and the external Filters were hybridized and washed as previously described (Fink et al., solution 150 mM NaCl, 5 mM KCl, 3 mM MgCl , 10 mM HEPES at 1996b). Eight positive clones from brain and 10 from heart were pH 7.4 with NaOH. obtained. cDNA inserts were characterized by restriction analysis and by partial or complete sequencing on both strands by the dideoxynucleo- Acknowledgements tide chain termination method using an automatic sequencer (Applied Biosystems). All the clones were shown to only contain a partial ORF. We thank Drs Jacques Barhanin and G.Romey for very helpful discus- The cDNA insert of the longer mouse clone (designated pBS-mTASK) sions, M.Jodar, N.Leroudier and G.Jarretou for technical assistance and was P-labeled and used to screen a human kidney cDNA library. Two D.Doume for secretarial assistance. This study was supported by the independent hybridizing clones were isolated and sequenced. Both clones Centre National de la Recherche Scientifique (CNRS), the Ministe`re de (2.5 kb long) were shown to contain the full-length ORF. The longer l’Enseignement Supe´rieur et de la Recherche (Contract MESR ACC one was designated pBS-hTASK. SV9 No. 9509113) and Bristol-Myers Squibb (unrestricted award). For Northern blot analysis, poly(A) RNAs were isolated from adult mouse tissues and blotted onto nylon membranes as previously described (Lesage et al., 1992). The blot was probed with the P-labeled insert References of pBS-mTASK in 50% formamide, 5 SSPE [0.9 M sodium chloride, 50 mM sodium phosphate (pH 7.4), 5 mM EDTA], 0.1% SDS, Altschul,S.F., Gich,W., Miller,W., Myers,E.W. and Lipman,D.J. (1990) 5 Denhardt’s solution, 20 mM potassium phosphate (pH 6.5) and Basic local alignment search tool. J. Mol. Biol., 215, 403–410. 250 μg of denatured salmon sperm DNA at 50°C for 18 h and washed Backx,P.H. and Marban,E. (1993) Background potassium current active stepwise at 55°C to a final stringency of 0.2 SSC, 0.3% SDS. For during the plateau of the action potential in guinea-pig ventricular hybridization of human multiple tissue Northern blots from Clontech, myocytes. Circ. Res., 72, 890–900. the procedure was identical except that the probe was derived from pBS- Barhanin,J., Lesage,F., Guillemare,E., Fink,M., Lazdunski,M. and hTASK. The cDNA insert of pBS-hTASK contains different repeat Romey,G. (1996) KvLQT1 and IsK (minK) proteins associate to form sequences [AluJb, MIR and (CGG) ] in the untranslated regions (UTR), the IKs cardiac potassium current. Nature, 384, 78–80. and a SmaI–ApaI restriction fragment of 1390 bp spanning the coding Borgula,G.A., Karwoski,C.J. and Steinberg,R.H. (1989) Light-evoked sequence was chosen as a probe that does not contain these repeats. changes in extracellular pH in frog retina. Vision Res., 29, 1069–1077. In situ hybridization experiments were performed on adult Balb/c Buckler,K.J. (1997) A novel oxygen-sensitive potassium current in rat mice by using standard procedures (Fink et al., 1996b). An antisense carotid body type I cells. J. Physiol. (Lond.), 498, 649–662. oligonucleotide (48mer, 5-CACCAGCAGGTAGGTGAAGGTGCAC- Chesler,M. (1990) The regulation and modulation of pH in the nervous ACGATGAGAGCCAACGTGCGCAC-3) complementary to the mouse system. Prog. Neurobiol., 34, 401–427. cDNA sequence of TASK (from nucleotides 7 to 54) was used to detect Chesler,M. and Kaila,K. (1992) Modulation of pH by neuronal activity. the expression of TASK transcripts in frozen fixed brain sections (10 μm). Trends Neurosci., 15, 386–402. The probe was 3-end-labeled with [α- P]dATP. Sections were digested Doupnik,C.A., Davidson,N. and Lester,H.A. (1995) The inward rectifier with 5 μg/ml of proteinase K for 15 min at 37°C and acetyled for 10 potassium channel family. Curr. Opin. Neurobiol., 5, 268–277. min in 0.25% acetic anhydride in 0.1 M triethanolamine. Hybridization Fakler,B. and Ruppersberg,J.P. (1996) Functional and molecular diversity was carried out overnight at 37°C in 2 SSC, 50% formamide, 10% classifies the family of inward-rectifier K channels. Cell. Physiol. dextran sulfate, 1 Denhardt’s solution, 5% sarcosyl, 500 μg of denatured Biochem., 6, 195–209. salmon sperm DNA, 250 mg/ml yeast tRNA, 20 mM dithiothreitol and Fink,M., Duprat,F., Lesage,F., Heurteaux,C., Romey,G., Barhanin,J. and 20 mM NaPO with 0.2 ng/ml of radiolabeled probe (sp. act.  810 Lazdunski,M. (1996a) A new K channel β subunit to specifically d.p.m./μg). Slides were then washed in 1 SSC before dehydratation, enhance Kv2.2 (CDRK) expression. J. Biol. Chem., 271, 26341–26348. drying and apposition to hyperfilm-βmax (Amersham) for 6 days. The Fink,M., Duprat,F., Lesage,F., Reyes,R., Romey,G., Heurteaux,C. and specificity of labeling was verified by in situ hybridization using cold Lazdunski,M. (1996b) Cloning, functional expression and brain displacement of radioactive probe with a 500-fold excess of unlabeled localization of a novel unconventional outward rectifier K channel. oligonucleotide. EMBO J., 15, 6854–6862. Goldstein,S.A.N., Price,L.A., Rosenthal,D.N. and Pausch,M.H. (1996) Electrophysiological measurements in Xenopus oocytes ORK1, a potassium-selective leak channel with two pore domains A 2480 bp SmaI–XhoI fragment from pBS-hTASK containing 14 bp of cloned from Drosophila melanogaster by expression in Saccharomyces 5 UTR, the coding sequence and the entire 3 UTR was subcloned into cerevisiae. Proc. Natl Acad. Sci. USA, 93, 13256–13261. the pEXO vector (Lingueglia et al., 1993) to give pEXO-TASK. Capped Guillemare,E., Honore,E., Pradier,L., Lesage,F., Schweitz,H., Attali,B., cRNAs were synthesized in vitro from the linearized plasmid by using Barhanin,J. and Lazdunski,M. (1992) Effects of the level of messenger the T7 RNA polymerase (Stratagene). Xenopus laevis were purchased RNA expression on biophysical properties, sensitivity to neurotoxins, from CRBM (Montpellier, France). Preparation and cRNA injection of and regulation of the brain delayed-rectifier K channel Kv1.2. oocytes have been described elsewhere (Guillemare et al., 1992). Oocytes Biochemistry, 31, 12463–12468. were used for electrophysiological studies 2–4 days following injection Heginbotham,L., Lu,Z., Abramson,T. and MacKinnon,R. (1994) (20 ng/oocyte). In a 0.3 ml perfusion chamber, a single oocyte was Mutations in the K channel signature sequence. Biophys. 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(1994) Potassium channels and their evolving superfusate (flow rate: 3 ml/min) or intracellularly injected by using a gates. Nature, 371, 119–122. pressure microinjector (InjectMatic, Switzerland). All experiments Jurman,M.E., Boland,L.M. and Yellen,G. (1994) Visual identification of were performed at room temperature (21–22°C). individual transfected cells for electrophysiology using antibody- coated beads. BioTechniques, 17, 876–881. Patch–clamp recording in transfected COS cells Knaus,H.G., Folander,K., Garciacalvo,M., Garcia,M.L., Kaczorowski, The 2480 bp SmaI–XhoI fragment of pBS-TASK was subcloned into G.J., Smith,M. and Swanson,R. (1994) Primary sequence and the pCi plasmid (Promega) under the control of the cytomegalovirus immunological characterization of beta-subunit of high conductance promoter to give pCi-TASK. COS cells were seeded at a density of Ca -activated K channel from smooth muscle. J. Biol. Chem., 269, 70 000 cells per 35 mm dish 24 h prior transfection. Cells were then 17274–17278. transfected by the classical calcium phosphate precipitation method with Ko¨hler,M., Hirschberg,B., Bond,C.T., Kinzie,J.M., Marrion,N.V., 2 μg of pCI-TASK and 1 μg of CD8 plasmids. Transfected cells were Maylie,J. and Adelman,J.P. (1996) Small-conductance, calcium- visualized 48 h after transfection using the anti-CD8 antiboby-coated activated potassium channels from mammalian brain. Science, 273, beads method (Jurman et al., 1994). For electrophysiological recordings, 1709–1714. TASK, a TWIK-related acid-sensitive K channel Koyano,K., Tanaka,K. and Kuba,K. (1992) A patch–clamp study on the muscarine-sensitive potassium channel in bullfrog sympathetic ganglion cells. J. Physiol. (Lond.), 454, 231–246. Kraig,R.P., Ferreira-Filho,C.R. and Nicholson,C. (1983) Alkaline and acid transients in cerebellar microenvironment. J. Neurophysiol., 49, 831–851. Krishtal,O.A., Osipchuk,Y.V., Shelest,T.N. and Smirnoff,S.V. (1987) Rapid extracellular pH transients related to synaptic transmission in rat hippocampal slices. Brain Res., 436, 352–356. Kyte,J. and Doolittle,R. (1982) A simple model for displaying the hydropathic character of a protein. J. Mol. Biol., 157, 105–106. Lesage,F., Attali,B., Lazdunski,M. and Barhanin,J. (1992) Developmental expression of voltage-sensitive K channels in mouse skeletal muscle and C2C12 cells. FEBS Lett., 310, 162–166. Lesage,F., Guillemare,E., Fink,M., Duprat,F., Heurteaux,C., Fosset,M., Romey,G., Barhanin,J. and Lazdunski,M. (1995) Molecular properties of neuronal G-protein-activated inwardly rectifying K channels. J. Biol. Chem., 270, 28660–28667. Lesage,F., Guillemare,E., Fink,M., Duprat,F., Lazdunski,M., Romey,G. and Barhanin,J. (1996a) TWIK-1, a ubiquitous human weakly inward rectifying K channel with a novel structure. EMBO J., 15, 1004–1011. Lesage,F., Lauritzen,I., Duprat,F., Reyes,R., Fink,M., Heurteaux,C. and Lazdunski,M. (1997) The structure, function and distribution of the mouse TWIK-1 K channels. FEBS Lett., 402, 28–32. Lesage,F., Reyes,R., Fink,M., Duprat,F., Guillemare,E. and Lazdunski,M. (1996b) Dimerization of TWIK-1 K channel subunits via a disulfide bridge. EMBO J., 15, 6400–6407. Lingueglia,E., Voilley,N., Waldmann,R., Lazdunski,M. and Barbry,P. (1993) Expression cloning of an epithelial amiloride-sensitive Na channel. A new channel type with homologies to Caenorhabditis elegans degenerins. FEBS Lett., 318, 95–99. MacCobb,D.P., Fowler,N.L., Featherstone,T., Lingle,C.J., Saito,M., Krause,J.E. and Salkoff,L. (1995) A human calcium-activated potassium channel gene expressed in vascular smooth muscle. Am. J. Physiol.-Heart Circ. Physiol., 38, H767–H777. MacManus,O.B., Helms,L.M.H., Pallanck,L., Ganetzki,B., Swanson,R. and Leonard,R.J. (1995) Functional role of the beta subunit of high conductance calcium-activated potassium channels. Neuron, 14, 645–650. Mutch,W.A.C. and Hansen,A.J. (1984) Extracellular pH changes during depression and cerebral ischemia: mechanisms of brain pH regulation. J. Cereb. Blood Flow Metab., 4, 17–27. Nedergaard,M., Kraig,R.P., Tanabe,J. and Pulsinelli,W.A. (1991) Dynamics of interstitial and intracellular pH in evolving brain infarct. Am. J. Physiol., 260, R581–R588. Pongs,O. (1992) Molecular biology of voltage-dependent potassium channels. Physiol. Rev., 72, S69–S88. Pongs,O. (1995) Regulation of the activity of voltage-gated potassium channels by beta subunits. Semin. Neurosci., 7, 137–146. Rudy,B. (1988) Diversity and ubiquity of K channels. Neuroscience, 25, 729–749. Sanguinetti,M.C., Curran,M.E., Zou,A., Shen,J., Spector,P.S., Atkinson,D.L. and Keating,M.T. (1996) Coassembly of KvLQT1 and MinK (IsK) proteins to form cardiac I potassium channel. Nature, KS 384, 80–83. Shen,K.Z., North,R.A. and Surprenant,A. (1992) Potassium channels opened by noradrenaline and other transmitters in excised membrane patches of guinea-pig submucosal neurones. J. Physiol. (Lond.), 445, 581–599. Siegelbaum,S.A., Camardo,J.S. and Kandel,E. (1982) Serotonin and cyclic AMP close single K channels in Aplysia sensory neurones. Nature, 229, 413–417. Siesjo¨,B.K., von Hanwehr,R., Nerglius,G., Nevander,G. and Ingvar,M. (1985) Extra- and intracellular pH in the brain during seizures and in the recovery period following the arrest of seizure activity. J. Cereb. Blood Flow Metab., 5, 47–57. Takumi,T., Ohkubo,H. and Nakanishi,S. (1988) Cloning of a membrane protein that induces a slow voltage-gated potassium current. Science, 242, 1042–1045. Yamamoto,F., Borgula,G.A. and Steinberg,R.H. (1992) Effects of light and darkness on pH outside rod photoreceptors in the cat retina. Exp. Eye Res., 54, 685–697. Received on May 22, 1997; revised on June 10, 1997 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

TASK, a human background K+ channel to sense external pH variations near physiological pH

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Copyright © European Molecular Biology Organization 1997
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Abstract

The EMBO Journal Vol.16 No.17 pp.5464–5471, 1997 TASK, a human background K channel to sense external pH variations near physiological pH and four structural classes of auxiliary subunits (Kvβ, Fabrice Duprat, Florian Lesage, Michel Fink, Kcaβ, SUR and IsK) (Takumi et al., 1988; Knaus et al., Roberto Reyes, Catherine Heurteaux and 1 1994; Pongs, 1995; Inagaki et al., 1996). All Shaker-type Michel Lazdunski subunits have a conserved hydrophobic core containing six Institut de Pharmacologie Mole´culaire et Cellulaire, CNRS, 660 route transmembrane segments (TMS). Associations of Shaker- des Lucioles, Sophia Antipolis, 06560 Valbonne, France type subunits with accessory subunits such as Kvβ, Kcaβ or IsK give rise to voltage-dependent K channels (Pongs, Corresponding author e-mail: [email protected] 1995; Barhanin et al., 1996; Fink et al., 1996a; Sanguinetti et al., 1996) and Ca -dependent K channels (MacCobb F.Duprat, F.Lesage and M.Fink contributed equally to this work et al., 1995; MacManus et al., 1995). Subunits of inward rectifier K channels (IRK) have only two TMS (Doupnik TASK is a new member of the recently recognized et al., 1995; Lesage et al., 1995; Fakler and Ruppersberg, TWIK K channel family. This 395 amino acid poly- 1996). Some IRKs give rise to ATP-sensitive K channels peptide has four transmembrane segments and two P when they are associated with sulfonylurea receptor (SUR) domains. In adult human, TASK transcripts are found subunits (Inagaki et al., 1996). Despite a very low overall in pancreas<placenta<brain<lung, prostate<heart, sequence similarity, Shaker and IRK pore-forming subunits kidney<uterus, small intestine and colon. Electro- share a conserved domain called the P domain. This physiological properties of TASK were determined peculiar motif is an essential element of the K -selective after expression in Xenopus oocytes and COS cells. filter of the aqueous pore and is considered as the TASK currents are K -selective, instantaneous and signature of K channel-forming proteins (Heginbotham non-inactivating. They show an outward rectification et al., 1994). when external [K ] is low ([K ]  2 mM) which is out We recently have described a new family of mammalian not observed for high [K ] (98 mM). The rectification out K channel subunits. Despite a low sequence similarity can be approximated by the Goldman–Hodgkin–Katz between them (28% amino acid identity), both cloned current equation that predicts a curvature of the members of this family (TWIK-1 and TREK-1) possess current–voltage plot in asymmetric K conditions. the same overall structure, with four TMS and two P This strongly suggests that TASK lacks intrinsic voltage domains (Fink et al., 1996b; Lesage et al., 1996a, 1997). sensitivity. The absence of activation and inactivation The conservation of this structure is not associated with kinetics as well as voltage independence are character- a conservation of functional properties: TWIK-1 gives istic of conductances referred to as leak or background rise to weakly inward rectifier K currents (Lesage et al., conductances. For this reason, TASK is designated as 1996a) while TREK-1 produces outward rectifier K a background K channel. TASK is very sensitive to currents (Fink et al., 1996b). However, both channels are variations of extracellular pH in a narrow physiological open at the resting potential and are able to drive the range; as much as 90% of the maximum current is resting membrane potential near the K equilibrium poten- recorded at pH 7.7 and only 10% at pH 6.7. This tial. This common property suggests that these channels property is probably essential for its physiological control the resting membrane potential in a large set of function, and suggests that small pH variations may cell types. Here, we describe the cloning, tissue distribution serve a communication role in the nervous system. and expression of a novel human member of this new Keywords: in situ hybridization/leak/membrane potential/ structural family. To our knowledge, this channel, called potassium channels/rectification TASK for TWIK-related acid-sensitive K channel, is the first cloned mammalian channel that produces K currents that possess all the characteristics of background conduct- ances. They are instantaneous with voltage changes, and Introduction their current–voltage relationships fit the curves predicted from the constant field theory for simple electrodiffusion Potassium channels are ubiquitous membrane proteins that through an open K -selective pore, indicating that TASK form the largest family of ion channels in terms of both currents are voltage insensitive. The activity of this back- function and structure. By determining and modulating ground channel is strongly dependent on the external pH the membrane potential, they play a major role in neuronal in the physiological range, suggesting that this particular integration, muscular excitability and hormone secretion channel is a sensor of external pH variations. (Rudy, 1988; Hille, 1992). More than 40 genes encoding K channel subunits are now identified in mammals. Results These subunits fall into two structural classes of pore- forming subunits [Shaker and inward rectifier K channel Cloning and primary structure of TASK (IRK)] (Pongs, 1992; Jan and Jan, 1994; Doupnik et al., TWIK-1 and TREK-1 sequences were used to search 1995; Fakler and Ruppersberg, 1996; Kohler et al., 1996) related sequences in the GenBank database by using 5464 © Oxford University Press TASK, a TWIK-related acid-sensitive K channel Fig. 1. Nucleotide and deduced amino acid sequences of human TASK and partial amino acid sequence of mouse TASK. Consensus sites for N-linked glycosylation (*) and phosphorylation by protein kinase C (j), protein kinase A (m) and tyrosine kinase (d) in human TASK. These sites have been identified by using the prosite server (European Bioinformatics Institute) with the ppsearch software (EMBL Data library) based on the MacPattern program. The sequences of human and mouse TASK have been deposited in the GenBank/EMBL database under the accession numbers AF006823 and AF006824, respectively. the Blast alignment program. We identified two mouse kinase C (residues 358 and 383), tyrosine kinase (residue expressed sequence tags (ESTs, accession numbers 323) and protein kinase A (residues 392 and 393). All W36852 and W36914) that overlap and give a contig these phosphorylation sites are located in the C-terminal fragment of 560 bp whose deduced amino acid sequence part of the protein. Except for a 19 residue cluster (amino presents significant similarity to TWIK-1 and TREK-1. A acids 276–294 in the human sequence), mouse and human corresponding DNA fragment was amplified by RT–PCR proteins share a high overall sequence conservation (85% and used to screen a mouse brain cDNA library. Eight of identity), indicating that they are probably products of independent clones were isolated. The 1.8 kb cDNA insert ortholog genes (Figure 1). Sequence alignments presented of the longer clone bears in its 5 part an open reading in Figure 2 clearly show that the cloned protein is a new frame (ORF) coding for a 405 amino acid polypeptide member of the TWIK-related K channel family. Like (Figure 1). This ORF does not begin with an initiating TWIK-1 and TREK-1, TASK has four putative transmem- methionine codon, suggesting that the brain cDNA clones brane segments (M1–M4) and two P domains (P1 and P2) were partial. Ten additional positive clones were isolated (Figure 2A and B). TASK is 58 amino acids longer than from a mouse heart cDNA library. Analysis of their 5 TWIK-1 and 24 amino acids longer than TREK-1 because sequences showed that none these clones were longer than its C-terminus is more extended. the clones previously isolated from brain. The 5 sequence has a very high GC content and is probably associated with Distribution of TASK secondary structures that could have promoted premature The expression of TASK in adult human and mouse tissues stops of RNA reverse transcription during the construction was examined by Northern blot analysis. Three different of both mouse cDNA libraries. To overcome this problem, transcripts were detected in the human tissues, with we cloned the complete cDNA in another species. The estimated sizes of 6.8, 4.2 and 2.6 kb (Figure 3), the DNA probe was used to screen a cDNA library from shorter one having the same size as the cloned cDNAs. human kidney, a tissue that express both TWIK-1 and The other two transcripts (4.2 and 6.8 kb) may result from TREK-1 channels. Two hybridizing clones were character- alternative polyadenylation signals in the 3-non-coding ized. Both contain an ORF of 1185 nucleotides encoding sequence and/or correspond to alternatively spliced or a 394 amino acid polypeptide (Figure 1). The human immature forms of the transcript. TASK is expressed in protein sequence contains consensus sites for N-linked many different tissues but particularly in pancreas and glycosylation (residue 53), and phosphorylation by protein placenta. Lower levels of expression were found in 5465 F.Duprat et al. Fig. 3. Northern blot analysis of TASK distribution in adult human tissues. Human multiple tissue Northern blots from Clontech were probed at high stringency with a TASK cDNA probe. Each lane contains 2 μg of poly(A) RNA. Autoradiograms were exposed for 48 h at –70°C. The blots were re-probed with a β-actin cDNA probe for control. sk. muscle, skeletal muscle; sm. intestine, small intestine; PBL, peripheral blood leukocytes. the cerebral cortex, in the CA1–CA4 pyramidal cell layer, in the granule cells of the dentate gyrus, in the habenula, in the paraventricular thalamic nuclei, in the amyloid nuclei, in the substantia nigra and in the Purkinje and granular cells of the cerebellum. In the heart, a high level of TASK expression was found in the atria (Figure 4D), while ventricular cells did not express this channel. Biophysical properties of TASK currents For functional studies, TASK cRNAs were injected into Xenopus oocytes. A non-inactivating current, not present in uninjected oocytes (not shown), was measured by two-electrode voltage-clamp (Figure 5A). The activation kinetics of the TASK current are almost instantaneous (10 ms). The current–voltage (I–V) relationship is out- wardly rectifying and almost no inward currents were recorded in the ND96 external medium containing 2 mM K (Figure 5B). However, inward currents were revealed when the external K concentration ([K ] ) was Fig. 2. Sequence comparison and membrane topology of out TWIK-related channels. (A) Alignment of human TWIK-1, mouse increased gradually to 98 mM K (Figure 5A and B). TREK-1 and human TASK sequences. Identical and conserved Figure 5B shows the I–V relationships of the current in K - residues are shown in black and gray, respectively. Dashes indicate rich solutions ranging from 2 to 98 mM. The relationship gaps introduced for a better alignment. The relative positions of between the reversal potential and [K ] was close to out putative transmembrane segments (M1–M4) and P domains (P1 and the predicted Nernst value (52.1 mV/decade, n  4), as P2) of human TASK are also indicated. The M1–M4 domains were deduced from a hydropathy profile computed with a window size of expected for a highly selective K channel (Figure 5C, 11 amino acids according to the method of Kyte and Doolittle (1982). upper panel). On the other hand, external K enhanced (B) Putative membrane topology of TWIK-1, TREK-1 and TASK the outward currents in a concentration-dependent manner channels. as illustrated in Figure 5C (lower panel). The half- maximum activation by K was observed at a K of 2.06 0.5 brainlung, prostateheart, kidneyuterus, small mM. The theoretical I–V relationships in various [K ] out intestine and colon. As shown in Figure 4A, the TASK calculated according to the Goldman–Hodgkin–Katz cur- probe detected a single transcript in the mouse with rent equation are shown in Figure 5D. These I–V relation- an estimated size of 4.2 kb. TASK is expressed in ships are very close to the I–V relationships corresponding heartlungbrain and kidney. No expression was seen to recorded TASK currents (Figure 5B). This strongly in liver and skeletal muscle. The TASK distribution was suggests that TASK currents show no rectification other studied further in adult mouse brain and heart by in than that predicted from the constant field assumptions, situ hybridization. A wide and heterogeneous pattern of and that TASK lacks intrinsic voltage sensitivity. The expression was obtained in the brain (Figure 4B and C). slight deviations between experimental and theoretical TASK mRNA was detected throughout the cell layers of points are probably due to small endogenous chloride TASK, a TWIK-related acid-sensitive K channel Fig. 4. Distribution of TASK mRNA in adult mouse. (A) Northern blot analysis. Each lane contains 2 μg of poly(A) RNA. Autoradiograms were exposed for 72 h at –70°C. The blots were re-probed with a β-actin cDNA probe for control. (B–D) In situ hybridization analysis from a coronal section at the level of the forebrain (B), the cerebellum (C) and the heart (D). Warmer colors represent higher levels of expression. CA1–CA3, fields CA1–3 of Ammon’s horn; Cx, cerebral cortex; DG, dentate gyrus; Gl, granular layer; Hb, habenula; SN, substantia nigra; PLCo, postero lateral cortical amygdaloid nuclei; PVP, paraventricular thalamic nucleus; A, atrium; V, ventricule. conductance and/or a K loading of the oocytes. We have the same Goldman–Hodgkin–Katz type outward rectific- shown previously that oocytes expressing TWIK-1 or ation as in oocytes. TREK-1 are more polarized that control oocytes, the resting membrane potential (E ) reaching a value close Regulation of the TASK channel to the K equilibrium potential (E ). In oocytes expressing TASK currents were insensitive to internal Ca changes TASK, the E was –85  0.8 mV (n  23, in standard obtained by injection of inositol triphosphate (IP3, 1 mM) ND96) instead of –44  2.6 mV (n  9) in non-injected or EGTA (100 mM), to the activation of adenyl cyclase oocytes. This result demonstrates that TASK, like other by perfusion of IBMX (1 mM) and forskolin (10 μM), or TWIK or TREK channels, is able to drive E close to to the activation of protein kinase C (PKC) by application E . The effect of various pharmacological agents on of phorbol 12-myristate 13-acetate (PMA; 40 nM). TASK currents elicited by voltage pulses to 50 mV has been currents were insensitive to the internal acidification or studied in TASK-expressing oocytes. Less than 20% of alkalization obtained by injection of solutions at pH 2 or TASK currents were inhibited in the presence of quinine 8.7 respectively (n  3). However, their very interesting (100 μM), quinacrine (100 μM) or quinidine (100 μM). property is that they are highly sensitive to external pH. The ‘classical’ K channel blockers tetraethylammonium The current–potential relationships recorded from a TASK- (TEA, 1 mM) and 4-aminopyridine (4AP, 1 mM) were expressing oocyte at pH 6.5, 7.4 and 8.4 are presented in also inactive. Cs (100 μM) induced a voltage-dependent Figure 6A. For an external pH of 6.5, a drastic block was block of 31  2% (n  4) of the inward current, recorded observed at all potentials, while an activation was recorded at –150 mV, in 50 mM external K . In the same conditions, at pH 8.4, also at all potentials. The inhibition and Ba (100 μM) was ineffective, with a variation of 6  activation produced no modification of current kinetics 1% (n  4) of the inward current. (Figure 6A, inset). The pH dependence of the TASK The biophysical properties of TASK were then verified channel is shown in Figure 6B. For currents recorded at in transfected COS cells. Untransfected cells did not 50 mV, the inhibition by acidic pHs was characterized express this K channel activity (not shown). Figure 5E by an apparent pK of 7.34  0.04 units (n  3) and a shows whole-cell currents recorded in mammalian COS Hill coefficient of 1.54  0.08 (n  3). For currents cells transiently transfected with TASK, in external solu- recorded at 0 and –50 mV, the pKs were 7.32  0.02 and tions containing 5 and 155 mM K . The currents were 7.30 0.01 respectively (n 3), showing that the blocking instantaneous and non-inactivating, as in Xenopus oocytes. effect of external protons is not voltage dependent. The Figure 5F presents the I–V relationships of the current in resting membrane potential of TASK-expressing oocytes various external K concentrations. The currents show was –84  1mV(n  6) at pH 7.4 and shifted to –47 5467 F.Duprat et al. 6mV(n  6) at pH 6.4 (not shown). Finally, Figure 6C structural similarity to TWIK-1 and TREK-1 channels and D shows that the strong pH sensitivity of TASK that suggests a common ancestral origin. Despite this currents was also observed in transfected COS cells. A similar structural organization, the amino acid identity large inhibition or activation of the current was recorded, between TASK and the two other related mammalian at all potentials, when the pH was changed from 7.4 to channels is very low (25–28%). Sequence homologies are 6.1 or 8.4 respectively (Figure 6C). The kinetics of the no higher between TASK and a recently cloned Drosophila current were unmodified at both pH values (Figure 6C, channel that also belongs to the structural TWIK channel inset). Figure 6D shows that the pH effects were also class (Goldstein et al., 1996). The highest degree of non-voltage dependent in COS cells. The external pH sequence conservation is in the two P domains and the dependence of TASK, at 50 mV, indicates a pK value M2 segment. In these regions, the amino acid identity of 7.29  0.03 (n  5) and a Hill coefficient of 1.57 reaches ~50%. Like other TWIK-related channels, TASK 0.07 (n  5). Currents recorded at 0 and –50 mV presented contains an extended M1P1 interdomain. This peculiar pKs of 7.29  0.04 (n  5) and 7.32  0.05 (n  4) domain has been shown to be extracellular in the case of respectively. Ten percent of the maximum current was TWIK-1 and to be important for the self-association of obtained at pH 6.68  0.08 (n  4) and 90% at pH 7.66 two TWIK-1 subunits. The TWIK-1 homodimers are 0.05 (n  4). These results confirm that TASK is extremely covalent because of the presence of an interchain disulfide sensitive to extracellular pH in the physiological range. bridge between cysteines 69 located in the M1P1 inter- domain (Lesage et al., 1996b). This particular cysteine Discussion residue is conserved in TREK-1 (residue 93) but not in TASK, strongly suggesting that TASK probably does not Here we report the isolation and the characterization of a form covalent dimers as observed for TWIK1 (Lesage novel human K channel. This channel has an overall et al., 1996b) and TREK-1 (unpublished data). The biophysical and regulation properties of TASK are unique. TWIK-1 has a mild inward rectification that involves an internal block by Mg (Lesage et al., 1996a). TREK-1 expresses an outward rectification which seems to result from a voltage sensitivity intrinsic to the channel protein (Fink et al., 1996b). In the case of TASK, the outward rectification observed at physiological external Fig. 5. Biophysical properties of TASK in Xenopus oocytes and COS cells. (A) TASK currents recorded from a Xenopus oocyte injected with TASK cRNA and elicited by voltage pulses from –150 to 50 mV in 40 mV steps, 500 ms in duration, from a holding potential of –80 mV in low (2 mM K ) or high K solutions (98 mM K ). The zero current level is indicated by an arrow. (B) Current–voltage relationships. Mean currents were measured over the last 50 ms at the end of voltage pulses from –150 to 50 mV in 10 mV steps as in (A). Modified ND96 solutions containing 2 mM K and 96 mM TMA were used, TMA was then substituted by K to obtain solutions ranging from 2 to 98 mM K . TASK currents are not sensitive to external TMA, no changes were observed upon substitution of NaCl by TMA (data not shown). (C) Upper panel: reversal potentials of TASK currents as a function of external K concentration (mean SEM, n  3). Lower panel: slope conductance measured between 10 and 50 mV on current–voltage relationships as in (B), plotted as a function of the external K concentration (mean  SEM, n  3). The mean values were fitted with a hyperbolic function. (D) Theoretical current–voltage relationship under the same conditions as in (B), calculated according to the following modified Goldman–Hodgkin– Katz (GHK) current relationship: 2   –V F/RT [K ] V F [K ] –[K ] ·e out m in out I P · · · K  K ()() –V F/RT K  [K ] RT 1–e 0.5 out where I  is the potassium current, P  is the apparent permeability K K for K , K the half maximum activation by K ,[K ] and [K ] 0.5 out in are the external and internal K concentrations, V the membrane potential, F, R and T have their usual meanings. The classical GHK relationship has been modified with [K ] /K  [K ] to take into out 0.5 out account the sensitivity of the conductance to external K .(E) TASK currents recorded from a transfected COS cell and elicited by voltage pulses from –150 to 50 mV in 40 mV steps, 500 ms in duration, from a holding potential of –80 mV, in low (5 mM K ) or high K solutions (155 mM K ). The zero current level is indicated by an arrow. (F) Current–voltage relationships. Mean currents were measured over the last 50 ms at the end of voltage pulses ranging from –150 to 50 mV in 10 mV steps as in (E). Solutions containing 5 mM K and 150 mM TMA were used, TMA was then substituted by K to obtain solutions ranging from 5 to 155 mM K . TASK, a TWIK-related acid-sensitive K channel tivities, and their absence of specific pharmacology has delayed their extensive electrophysiological and physio- logical characterization. Cloning of TASK, the first ‘true’ background mammalian K channel, should help to characterize further this peculiar functional family of K channels at the molecular level and identify specific and high affinity pharmacological agents that would block these channels and facilitate analysis of their physio- logical roles. TASK behaves as a K -selective ‘hole’, but this does not mean that its activity cannot be modulated. Unlike TWIK-1 and TREK-1 channels, its activity is not changed by activation of protein kinase A or C (Fink et al., 1996b; Lesage et al., 1996a). The probably very important property of TASK is that it is extremely sensitive to extracellular pH in the physiological range, i.e. between 6.5 and 7.8. The Hill coefficient of ~1.6 found for the H concentration dependence of the TASK current is consistent with the idea that the channel is formed by the assembly of two subunits, as previously demonstrated for TWIK1. These two subunits would be in strong cooperative interactions with regard to H . The modulation by external protons probably has important implications for the physiological function of the TASK channel. Stimulus-elicited pH shifts have been Fig. 6. pH regulation of TASK in Xenopus oocytes and COS cells. characterized in a wide variety of neural tissues by using (A) Current–voltage relationships recorded from a TASK-expressing extracellular pH-sensitive electrodes (reviewed in Chesler, oocyte with a ramp ranging from –150 to 50 mV, 500 ms in 1990; Chesler and Kaila, 1992). They can be observed in duration, from a holding potential of –80 mV, in ND96 solution at physiopathological situations such as epileptiform activity pH 6.5, 7.4 or 8.4. Inset: currents elicited by voltage pulses to 50 mV, 500 ms in duration, under the same conditions as above. The and spreading depression in which acid shifts are usually zero current level is indicated by an arrow. (B) pH dependence of preceded by alkaline transients (Siesjo¨ et al., 1985; TASK activity in a Xenopus oocyte recorded at –50, 0 and 50 mV Nedergaard et al., 1991). They can be observed of course (mean  SEM, n  3) as in (A). Data were fitted with a Boltzman in ischemia where large acidifications of the extracellular relationship. (C) Current–voltage relationship recorded from a TASK-expressing COS cell with a ramp ranging from –150 to medium have been recorded (Kraig et al., 1983; Mutch 50 mV, 500 ms in duration, from a holding potential of –80 mV, in and Hansen, 1984). However, they can also be observed 5mMK solution at pH 6.1, 7.4 and 8.4. Inset: currents elicited by in physiological conditions such as electrical stimulation voltage pulses to 50 mV, 500 ms in duration, under the same of Schaeffer collateral fibers in the hippocampal slice conditions as above. The zero current level is indicated by an arrow. (D) pH dependence of TASK activity recorded in a COS cell at –50, 0 (Krishtal et al., 1987), or light stimulation of the retina and 50 mV (mean  SEM, n  3) as in (C). Data were fitted with a (Borgula et al., 1989; Yamamoto et al., 1992), or parallel Boltzman relationship. fibers in cerebellum (Kraig et al., 1983). All these pH shifts correspond to bursts of H or OH creating small K concentrations can be approximated to the rectification pH variations from the external physiological pH value predicted by the Goldman–Hodgkin–Katz current equa- of 7.4 in the alkaline or acidic direction (up to 0.3 pH tion, suggesting that this rectification simply results from units) and are rapid, in the 1–30 s range. They might the asymmetric concentrations of K on both sides of the actually be larger in range or shorter in time course in the membrane. In other words, this would mean that TASK vicinity of the synaptic cleft. A particularly interesting lacks intrinsic voltage sensitivity and behaves like a K - issue of course is whether these relatively small activity- selective ‘hole’. This behavior is, to our knowledge, dependent pH changes have significant modulatory effects. unique among cloned mammalian K channels. Voltage In other words, does H serve a transmitter role in the and time independences are classical criteria to describe nervous system? The discovery of this new TASK channel the so-called leak or background K channels. Some of that can fully open or close within a range of only 0.5 pH these channels have been described in invertebrates, the unit around the physiological pH (7.4) will certainly best characterized of which are the S channels in Aplysia strengthen the idea that pH could be a natural modulator sensory neurons (Siegelbaum et al., 1982), and in verte- of neuronal activity (Chesler and Kaila, 1992). brates, for example in bullfrog sympathetic ganglia (Koyano et al., 1992), guinea-pig submucosal neurons Materials and methods (Shen et al., 1992), rat carotid bodies (Buckler, 1997), and guinea-pig ventricular myocytes (Backx and Marban, Cloning of TASK and RNA analysis TWIK-1 and TREK-1 were used to search homologs in gene databases 1993). These channels are open at all membrane potentials by using the tBlastn sequence alignment program (Altschul et al., 1990). and probably play a pivotal role in the control of the Translation of two overlapping EST sequences (GenBank accession Nos resting membrane potential and in the modulation of W36852 and W36914) in one frame presented significant sequence electrical activity of both neurons and cardiac cells. similarities with TWIK-1 and TREK-1. A 560 bp DNA fragment was However, their lack of kinetics, voltage and time sensi- amplified by PCR from mouse brain poly(A) cDNAs and subcloned 5469 F.Duprat et al. into pBluescript (Stratagene) to give pBS-852/914. This fragment was the internal solution contained 150 mM KCl, 3 mM MgCl ,5mM P-labeled and used to screen mouse brain and heart cDNA libraries. EGTA, and 10 mM HEPES at pH 7.2 with KOH, and the external Filters were hybridized and washed as previously described (Fink et al., solution 150 mM NaCl, 5 mM KCl, 3 mM MgCl , 10 mM HEPES at 1996b). Eight positive clones from brain and 10 from heart were pH 7.4 with NaOH. obtained. cDNA inserts were characterized by restriction analysis and by partial or complete sequencing on both strands by the dideoxynucleo- Acknowledgements tide chain termination method using an automatic sequencer (Applied Biosystems). All the clones were shown to only contain a partial ORF. We thank Drs Jacques Barhanin and G.Romey for very helpful discus- The cDNA insert of the longer mouse clone (designated pBS-mTASK) sions, M.Jodar, N.Leroudier and G.Jarretou for technical assistance and was P-labeled and used to screen a human kidney cDNA library. Two D.Doume for secretarial assistance. This study was supported by the independent hybridizing clones were isolated and sequenced. Both clones Centre National de la Recherche Scientifique (CNRS), the Ministe`re de (2.5 kb long) were shown to contain the full-length ORF. The longer l’Enseignement Supe´rieur et de la Recherche (Contract MESR ACC one was designated pBS-hTASK. SV9 No. 9509113) and Bristol-Myers Squibb (unrestricted award). For Northern blot analysis, poly(A) RNAs were isolated from adult mouse tissues and blotted onto nylon membranes as previously described (Lesage et al., 1992). The blot was probed with the P-labeled insert References of pBS-mTASK in 50% formamide, 5 SSPE [0.9 M sodium chloride, 50 mM sodium phosphate (pH 7.4), 5 mM EDTA], 0.1% SDS, Altschul,S.F., Gich,W., Miller,W., Myers,E.W. and Lipman,D.J. (1990) 5 Denhardt’s solution, 20 mM potassium phosphate (pH 6.5) and Basic local alignment search tool. J. Mol. Biol., 215, 403–410. 250 μg of denatured salmon sperm DNA at 50°C for 18 h and washed Backx,P.H. and Marban,E. (1993) Background potassium current active stepwise at 55°C to a final stringency of 0.2 SSC, 0.3% SDS. For during the plateau of the action potential in guinea-pig ventricular hybridization of human multiple tissue Northern blots from Clontech, myocytes. Circ. Res., 72, 890–900. the procedure was identical except that the probe was derived from pBS- Barhanin,J., Lesage,F., Guillemare,E., Fink,M., Lazdunski,M. and hTASK. The cDNA insert of pBS-hTASK contains different repeat Romey,G. (1996) KvLQT1 and IsK (minK) proteins associate to form sequences [AluJb, MIR and (CGG) ] in the untranslated regions (UTR), the IKs cardiac potassium current. Nature, 384, 78–80. and a SmaI–ApaI restriction fragment of 1390 bp spanning the coding Borgula,G.A., Karwoski,C.J. and Steinberg,R.H. (1989) Light-evoked sequence was chosen as a probe that does not contain these repeats. changes in extracellular pH in frog retina. Vision Res., 29, 1069–1077. In situ hybridization experiments were performed on adult Balb/c Buckler,K.J. (1997) A novel oxygen-sensitive potassium current in rat mice by using standard procedures (Fink et al., 1996b). An antisense carotid body type I cells. J. Physiol. (Lond.), 498, 649–662. oligonucleotide (48mer, 5-CACCAGCAGGTAGGTGAAGGTGCAC- Chesler,M. (1990) The regulation and modulation of pH in the nervous ACGATGAGAGCCAACGTGCGCAC-3) complementary to the mouse system. Prog. Neurobiol., 34, 401–427. cDNA sequence of TASK (from nucleotides 7 to 54) was used to detect Chesler,M. and Kaila,K. (1992) Modulation of pH by neuronal activity. the expression of TASK transcripts in frozen fixed brain sections (10 μm). Trends Neurosci., 15, 386–402. The probe was 3-end-labeled with [α- P]dATP. Sections were digested Doupnik,C.A., Davidson,N. and Lester,H.A. (1995) The inward rectifier with 5 μg/ml of proteinase K for 15 min at 37°C and acetyled for 10 potassium channel family. Curr. Opin. Neurobiol., 5, 268–277. min in 0.25% acetic anhydride in 0.1 M triethanolamine. Hybridization Fakler,B. and Ruppersberg,J.P. (1996) Functional and molecular diversity was carried out overnight at 37°C in 2 SSC, 50% formamide, 10% classifies the family of inward-rectifier K channels. Cell. Physiol. dextran sulfate, 1 Denhardt’s solution, 5% sarcosyl, 500 μg of denatured Biochem., 6, 195–209. salmon sperm DNA, 250 mg/ml yeast tRNA, 20 mM dithiothreitol and Fink,M., Duprat,F., Lesage,F., Heurteaux,C., Romey,G., Barhanin,J. and 20 mM NaPO with 0.2 ng/ml of radiolabeled probe (sp. act.  810 Lazdunski,M. (1996a) A new K channel β subunit to specifically d.p.m./μg). Slides were then washed in 1 SSC before dehydratation, enhance Kv2.2 (CDRK) expression. J. Biol. Chem., 271, 26341–26348. drying and apposition to hyperfilm-βmax (Amersham) for 6 days. The Fink,M., Duprat,F., Lesage,F., Reyes,R., Romey,G., Heurteaux,C. and specificity of labeling was verified by in situ hybridization using cold Lazdunski,M. (1996b) Cloning, functional expression and brain displacement of radioactive probe with a 500-fold excess of unlabeled localization of a novel unconventional outward rectifier K channel. oligonucleotide. EMBO J., 15, 6854–6862. Goldstein,S.A.N., Price,L.A., Rosenthal,D.N. and Pausch,M.H. (1996) Electrophysiological measurements in Xenopus oocytes ORK1, a potassium-selective leak channel with two pore domains A 2480 bp SmaI–XhoI fragment from pBS-hTASK containing 14 bp of cloned from Drosophila melanogaster by expression in Saccharomyces 5 UTR, the coding sequence and the entire 3 UTR was subcloned into cerevisiae. Proc. Natl Acad. Sci. USA, 93, 13256–13261. the pEXO vector (Lingueglia et al., 1993) to give pEXO-TASK. 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Journal

The EMBO Journal – Springer Journals

Published: Sep 1, 1997

Keywords: in situ hybridization; leak; membrane potential; potassium channels; rectification

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