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J Biol Chem, Vol. 273, Issue 47, 30863-30869, November 20, 1998
From the A complementary DNA encoding a novel
K+ channel, called TASK-2, was isolated from human
kidney and its gene was mapped to chromosome 6p21. TASK-2 has a low
sequence similarity to other two pore domain K+ channels,
such as TWIK-1, TREK-1, TASK-1, and TRAAK (18-22% of amino acid
identity), but a similar topology consisting of four potential
membrane-spanning domains. In transfected cells, TASK-2 produces
noninactivating, outwardly rectifying K+ currents with
activation potential thresholds that closely follow the K+
equilibrium potential. As for the related TASK-1 and TRAAK channels, the outward rectification is lost at high external K+
concentration. The conductance of TASK-2 was estimated to be 14.5 picosiemens in physiological conditions and 59.9 picosiemens in
symmetrical conditions with 155 mM K+. TASK-2
currents are blocked by quinine (IC50 = 22 µM) and quinidine (65% of inhibition at 100 µM) but not by the other classical K+ channel
blockers tetraethylammonium, 4-aminopyridine, and Cs+. They
are only slightly sensitive to Ba2+, with less than 17% of
inhibition at 1 mM. As TASK-1, TASK-2 is highly sensitive
to external pH in the physiological range. 10% of the maximum current
was recorded at pH 6.5 and 90% at pH 8.8. Unlike all other cloned
channels with two pore-forming domains, TASK-2 is essentially absent in
the brain. In human and mouse, TASK-2 is mainly expressed
in the kidney, where in situ hybridization shows that it is
localized in cortical distal tubules and collecting ducts. This
localization, as well as its functional properties, suggest that TASK-2
could play an important role in renal K+ transport.
Potassium channels are present in virtually all living cells. They
conduct the flux of potassium ions through the membrane, and in doing
so, they are involved in the control of numerous cellular functions,
such as neuronal firing, muscle contraction, volume regulation, and
hormone secretion (1, 2). In the kidney, they are more particularly
involved in the K+ secretion that is fundamental for
K+ homeostasis (3).
Recently, K+ channels with unusual structures have been
identified. The yeast channel subunit TOK/YKC/YORK/DUK contains two pore-forming (P) domains and eight
TMSs1 in a single subunit
(4-7), whereas TWIK-1, the founding member of a new mammalian class,
has two P domains and four TMSs (8, 9). The TWIK-related channels
TREK-1, TASK (also called cTBAK1), and TRAAK exhibit the same overall
structure despite their low similarity in amino acid sequence (10-14).
Structurally related channels have also been identified in
Drosophila, Caenorhabditis elegans, and plants
(15-17).
In mammals, these 2P domain channels are extraordinarily diverse in
terms of both distribution and functional properties. TWIK-1,
TREK-1, and TASK are expressed in many tissues with
specific patterns, whereas TRAAK is only expressed in
neuronal cells (18). From a functional point of view, TWIK-1 expressed
baseline weakly inward-rectifying currents that are stimulated by
protein kinase C and inhibited by internal acidification (8). TREK-1
produces arachidonic acid-activated mechano-sensitive outwardly
rectifying currents that are inhibited by both protein kinase A and
protein kinase C (10, 19). On the other hand, TASK and TRAAK currents behave like K+-selective "holes," with no rectification
other than that predicted from the constant-field assumptions for an
open channel (11-14). Despite this common property, TASK and TRAAK
exhibit very different modulations of their activity. TASK is regulated
by external pH variations and is inhibited by a small drop of the
external pH near the physiological range (11, 13, 14). TRAAK is
stimulated by arachidonic acid, as well as other unsaturated fatty
acids (12). Despite their different functional properties, all of these
2P domain K+ channels express quasi-instantaneous and
noninactivating currents that do not display
voltage-dependent activation thresholds. They are open at
the resting potential, and their expression is associated with a strong
membrane polarization. All of these properties suggest that the 2P
domain K+ channels are involved in the generation and the
modulation of the resting potential of many cell types (18). This paper
describes the cloning, the gene localization, the tissue distribution,
and the functional characterization of a novel member of this emerging family.
Cloning of TASK-2--
The sequences of mammalian two P domain
K+ channels were used to search homologs in public DNA data
bases by using the tBLASTn alignment program (20) and led to the
identification of an expressed sequence tag (GenBank accession number
H01932). The 5' end of this expressed sequence tag encoded a domain
similar to the M1P1 extracellular loop of TWIK-1, but its 3'-extremity
did not encode the P1 and M2 domains of a two P domain channels as
expected. We postulated that this expressed sequence tag was a tandem
cDNA or was issued from an unspliced mRNA, and we used only the
5' part of the sequence to design oligonucleotides. These
oligonucleotides were further used to carry out 3'-rapid amplification
of cDNA ends polymerase chain reaction (PCR) experiments on human
brain cDNA by standard methods. A DNA fragment was obtained that
extended the region homologous to TWIK-1 and was previously
identified in H01932. Two novel oligonucleotides were deduced from the homologous part of this DNA fragment: sense strand,
5'-CACAGAAGCTGCATCTGCTCA-3'; antisense strand,
5'-CCCTCAGTCTCCATGAATAGGA-3'. They were used to amplify a 430-base pair
fragment that was 32P-labeled and used to screen a human
kidney cDNA library as described previously (8). From 3 × 105 phages screened, 37 positive clones were obtained.
Seven clones were excised from Analysis of TASK-2 mRNA Distribution--
For Northern blot
analysis, human multiple tissue Northern blots were purchased from
CLONTECH and hybridized at 65 °C in ExpressHyb solution with 0.6- and 1.2-kilobase SmaI
32P-labeled fragments from pBS-TASK-2 following the
manufacturer's protocol. For RT-PCR experiments, total RNAs were
extracted from adult mouse tissues and from mouse embryos with the SNAP
total RNA isolation kit (Invitrogen). After a DNase treatment, 15 µg of total RNA were reverse-transcribed according to the manufacturer's instructions (Life Technologies, Inc.).
In situ hybridization was performed as described previously
(21) on 7-µm paraffin sections of human kidneys fixed in 4% paraformaldehyde. A specific antisense cRNA probe was generated with T7
RNA polymerase (Promega) by in vitro transcription using (35S) Human Chromosomal Mapping--
The Genebridge 4 RH DNA panel
(Research Genetics) was screened by PCR using primers deduced from the
3'-untranslated part of TASK-2 cDNA (sense primer, base
positions 1971-1991, 5'-CTTCCTAACCTTCCATCATCC-3', and antisense
primer, positions 2455-2564, 5'-CTTGACCTGAGACAGGGAAC-3'). PCR
conditions were 40 cycles of 30 s at 94 °C, 30 s at
50 °C, and 30 s at 72 °C. PCR products were separated by
electrophoresis on agarose then transferred onto charged nylon
membranes. Blots were probed at high stringency with a
32P-labeled DNA probe spanning the amplified region of the
TASK-2 gene. The results were analyzed by using RHMAPPER
program at the Whitehead Institute
(http://www-genome.wi.mit.edu).
Electrophysiological Measurements in Xenopus Oocytes--
The
sequence coding for TASK-2 was amplified by PCR using a low error rate
polymerase (PWO pol, Boehringer Mannheim) and subcloned into the pEXO
vector (22) to give pEXO-TASK. Capped-cRNAs were synthesized in
vitro from the linearized plasmid by using the T7 RNA polymerase
(Stratagene). Xenopus laevis oocytes were purchased from
CRBM (Montpellier, France). Preparation and cRNA injection of oocytes
has been described elsewhere (23). Oocytes were used for
electrophysiological studies 2-4 days following injection (20 ng/oocyte). In a 0.3-ml perfusion chamber, a single oocyte was impaled
with two standard microelectrodes (1-2.5 M Patch-Clamp Recordings in Transfected COS Cells--
A
NotI/EcoRI fragment of 3.2 kilobases was excised
from pBS-TASK-2 and subcloned into the pIRES-CD8 vector to give
pIREScd8-TASK-2. The pIRES-CD8 vector was obtained by replacing the
neor gene in the original vector pIRESneo
(CLONTECH) by the coding sequence of the surface
marker CD8 of the T type lymphocyte. COS cells were seeded at a density
of 20,000 cells per 35-mm dish, 24 h prior transfection. Cells
were then transiently transfected by the classical DEAE-dextran method
with 1 µg of pIREScd8-TASK-2 plasmid per 35-mm dish. Transiently
transfected cells were visualized 48 h after transfection using
the anti-CD8 antibody-coated beads method (24). For whole cell
recordings, the internal solution contained 150 mM KCl, 3 mM MgCl2, 5 mM EGTA, and 10 mM HEPES at pH 7.2 with KOH, and the external solution
contained 150 mM NaCl, 5 mM KCl, 3 mM MgCl2, 1 mM CaCl2,
10 mM HEPES at pH 7.4 with NaOH.
Cloning and Primary Structure of TASK-2--
An expressed sequence
tag was identified by homology screening with the two P domain
K+ channels using the tBlastn alignment algorithm (20).
This sequence was amplified by PCR and used to screen a human kidney
cDNA library. A full-length cDNA of 3.5 kilobases was isolated.
It contains an extended open reading frame that codes for a polypeptide
of 499 residues with a calculated molecular mass of 55.1 kDa. The predicted product displays all the hallmarks of the 2P domain K+ channels (Fig.
1A). Analysis of its
hydropathy profile indicates the presence of four TMSs, designated M1
to M4; the M1 and M2 segments flank the first P domain (P1), and the M3
and M4 flank a second P domain (P2). An extended M1P1 interdomain that
is characteristic of this channel family is also found that is expected
to be extracellular, as for TWIK-1. This region contains a potential
N-linked glycosylation site and a cysteine residue (position
51) that are conserved in TWIK-1, TREK-1, and TRAAK. In TWIK-1, this
cysteine residue has been shown to be implicated in the formation of an
interchain disulfide bond (25). Despite this overall structural
conservation, the novel subunit is only distantly related to the other
cloned 2P domain K+ channels (between 18 and 22% of amino
acid identity). The dendrogram shown in Fig. 1B also
suggests than the novel subunit is not more related to TASK than to
TWIK-1, TREK-1, or TRAAK. However, it was called TASK-2 to emphasize
the fact that it produces K+ currents that are
acid-sensitive, like TASK-1, as shown below. For this reason, TASK is
now called TASK-1.
Tissue Distribution of TASK-2--
The tissue distribution of
TASK-2 in adult human was analyzed by Northern blot (Fig.
2A). A 4-kilobase transcript
is abundantly expressed in the kidney and is present to a lesser extent
in the pancreas, the liver, the placenta, and the small intestine. The expression of TASK-2 was also analyzed by RT-PCR from mouse
tissues (Fig. 2B). The mouse TASK-2 message was
found in the kidney, the liver, and the small intestine, in the same
relative abundance as in human. As expected, the RT-PCR method is more
sensitive than the Northern blot technique, and faint positive signals
were also obtained in mouse brain, heart, skeletal muscle and colon. Surprisingly, TASK-2 expression levels in uterus, lung and
pancreas are different between human and mouse.
Fig. 2B compares the distribution of TASK-2 to
those of the other cloned 2P domain K+ channels,
TWIK-1, TREK-1, and TASK-1. As shown previously
by Northern blot analyses, the TWIK-1, TREK-1, and
TASK-1 channels are widely distributed but with unique
patterns. The expression pattern of TASK-2 is also unique.
TASK-2 is preferentially expressed in the kidney and the
liver and is quasi-absent in the nervous system, unlike TWIK-1,
TREK-1, or TASK-1 (Fig. 2B).
TRAAK is exclusively expressed in neuronal cells (12).
Both Northern blot and RT-PCR analyses indicate that the tissue that
expresses the highest levels of TASK-2 is the kidney. The
distribution of TASK-2 in the human kidney cortex was
observed at higher resolution after in situ hybridization.
Fig. 3 shows that the expression of
TASK-2 is restricted to the distal tubules and the
collecting ducts. No specific signal was observed in the proximal
tubules or over the glomeruli.
Chromosomal Mapping of TASK-2--
The chromosomal assignment of
human TASK-2 was carried out by radiation hybrid panel
analysis. As shown in Fig. 4, the gene encoding TASK-2 lies on chromosome 6p and is 5.45 cR centromeric to the
framework marker WI-4142 (LOD score of 21). Although radiation hybrid
maps are not anchored to the cytogenic maps, the most likely localization of the TASK-2 gene is 6p21.31-p21.33.
TWIK-1 has been previously mapped to chromosome 1q42-1q43
(26), and TREK and TASK have been mapped to
chromosomes 1q41 and 2p23, respectively (27).
Biophysical and Pharmacological Properties of
TASK-2--
TASK-2-transfected COS cells display
noninactivating currents (Fig.
5A) that are not present in
control cells (not shown). The activation kinetics of TASK-2 currents
are rapid. They are fitted with a single exponential characterized by
time constants of 60.9 ± 6 ms at +50 mV and 62.6 ± 6 ms at
0 mV (n = 8). The current-voltage (I-V) relationship is
outwardly rectifying, and almost no inward currents were recorded in an
external medium containing 5 mM K+ (Fig.
5B). When cells were perfused with a K+-rich
solution (155 mM), the TASK-2 currents presented an almost linear I-V relationship, and the inward currents recorded at very negative potentials were noisy (Fig. 5, A and B).
The relationship between the reversal potential and
[K+]o is close to the predicted Nernst value
(58.9 ± 4.5 mV/decade, n = 4), as expected for a
highly selective K+ channel (Fig. 5C). TASK-2
was also expressed in Xenopus oocytes, where it shows
similar properties (Fig. 5D). Activation kinetics of the
TASK currents in oocytes are slightly slower, with time constants of
112.6 ± 16.8 ms at +50 mV and 102.9 ± 7.3 ms at 0 mV
(n = 6). As previously shown with TWIK-1, TREK-1,
TRAAK, and TASK-1 (8, 10-12), the membrane potential of oocytes
expressing TASK-2 is strongly polarized (
Single channel TASK-2 currents were recorded in outside-out patches
from transfected COS cells. They are very flickery and show substates
(Fig. 5E). In 5 mM external K+, the
single channel I-V relationship is almost linear between -80 and +40
mV and presents a saturation at potentials more positive than +40 mV
(Fig. 5F). The slope conductance measured between -60 mV
and +20 mV is 14.5 ± 1.4 pS (n = 22). In 155 mM external K+, the slope conductance is
greatly increased (59.9 ± 3.1 pS, n = 19) and
saturates both at very negative and positive potentials.
The effects of various pharmacological agents on currents elicited by
voltage pulses to +50 mV have been studied in TASK-2-expressing COS
cells. The "classical" K+ channels blockers
tetraethylammonium (1 mM), 4-aminopyridine (100 µM), and Cs+ (1 mM) were inactive
on the recorded currents. Ba2+ only slightly diminished the
current at +50 mV (16.9 ± 1.6% at 1 mM,
n = 3). Quinine induced a dose-dependent
inhibition of TASK-2 currents characterized by an IC50 of
22.4 ± 1.8 µM (n = 9) (not shown),
whereas 100 µM quinidine induced a 65 ± 3.8%
inhibition of the current (n = 4). A strong effect was
observed with lidocaine (1 mM) and bupivacaine (1 mM) with inhibitions of 60.4 ± 1.5% and 80.9 ± 4.5%, respectively (n = 4). Zinc (100 µM) was also tested and induced a slight decrease of the
current (15.3 ± 2.2%, n = 5). Unlike TRAAK (12),
TASK-2 is not sensitive to arachidonic acid (10 µM).
Regulation of TASK-2 Activity by External pH--
TASK-2 currents
were insensitive to the activation of adenyl cyclase obtained by
increasing intracellular cAMP with a mixture of isobutylmethylxanthine
(1 mM) and forskolin (10 µM) or by perfusion of the permeant 8-chloro-cAMP (500 µM), or to the
activation of protein kinase C obtained by an application of the
phorbol ester phorbol 12-myristate 13-acetate (70 nM).
Interestingly, TASK-2 currents are highly sensitive to external pH,
like TASK-1 (11). The I-V relationships recorded at pH 6.0, 7.4, and
8.6 are presented in Fig. 6A.
For an external pH of 6.0, a drastic block was observed at all
potentials, whereas an activation was recorded at pH 8.6, also at all
potentials. The inhibition and activation produced no modification of
current kinetics (Fig. 6B). The pH dependence of the TASK-2
channel is shown in Fig. 6C. For currents recorded at A New Member in the TWIK Family of K+
Channels--
TASK-2 is a novel member of the emerging family of 2P
domain K+ channels. Its cloning extends to five the number
of these channels identified to date in mammals. Despite an overall
structure conservation, TASK-2 does not share more than 18-22% of
amino acid identity with the four other cloned channels and does not
seem to be more related to any one of them from a phylogenetic point of view.
Both Shaker and inwardly rectifying K+ channel
families of K+ channel subunits comprise numerous members
corresponding to different genes. Within each of these two
superfamilies, different subclasses can be distinguished according to
their sequence similarities. Moreover, sequence conservations within
each family are associated with similar functional properties. For
instance, within the inwardly rectifying K+ channel family,
the Kir3.x subunits share 55-60% of amino acid identity (28). All
these Kir3.x proteins form G-protein-activated K+ channels.
Kir subunits belonging to the other subgroups are more distant from the
point of view of sequences and form inward rectifier channels that are
not activated by G proteins (29-31). On the other hand, the
Shaker-related voltage-dependent Kv1.x
K+ channels (32-34) or the
Ca2+-dependent SK channels (35) form subsets of
proteins sharing 70-85% of amino acid identity. However, despite a
similar structure with six TMSs and one P domain, Kv1.x and SK channels
have less than 20% of overall amino acid identity. The question then
arises of whether equivalent structural and functional subfamilies can be distinguished in the 2P domain K+ channel family. With a
low sequence conservation between the different channels cloned up till
now (18-38% of identity), the usual criteria of sequence similarity
cannot be used. Nevertheless, our work leads us to propose a functional
classification. TWIK-1 forms a first functional group because it is the
only one to express weakly inward rectifying currents. TREK-1 and TRAAK
form a second group of channels that produce outwardly rectifying
currents stimulated by arachidonic acid and polyunsaturated fatty acids
(10, 12, 19). The fact that both channels share 38% of amino acid
identity instead of the 18-22% of identity usually found between the
2P domain K+ channels could signify that they have evolved
from a common ancestral gene. However, TREK-1 and TRAAK probably have
different physiological significance, because they have quite different
tissue distributions, as well as different electrophysiological and
regulation properties. The quasi-ubiquitous TREK-1 channel is inhibited
by cAMP, but the neuronal TRAAK channel is not. TRAAK loses its outward
rectification in high external [K+], but TREK-1 does not.
The last functional group of 2P domain K+ channels is
composed of TASK-1 and TASK-2. Both channels produce open rectifier
K+ currents that are inhibited by a drop of external pH in
the physiological range (11, 13). Their pharmacological behaviors are
also similar. TASK-2, like TASK-1, is relatively insensitive to
classical K+ channel blockers such as Ba2+,
Cs+, tetraethylammonium, and 4-aminopyridine, and both
TASK-1 and TASK-2 are blocked by the local anesthetics lidocaine and
bupivacaine. Their sequences are only distantly related, and for this
reason, it is extremely difficult to know whether they have evolved
from a common gene coding for an ancestral pH-sensitive K+
channel. TASK-1 and TASK-2 have different tissue distributions. TASK-1 is widely expressed in excitable as well as
nonexcitable tissues (11), whereas TASK-2 seems to be
preferentially present in epithelia. They also show significant
differences in terms of electrophysiological and regulation properties.
Unlike TASK-1 activity, TASK-2 activity is not inhibited by variations
of intracellular cAMP (13), and TASK-2 is the sole 2P domain
K+ channel cloned to date that displays relatively slow
activation kinetics (11, 13, 14).
TASK-2, a Novel Renal K+ Channel--
As discussed
previously, TASK-2 has a unique tissue distribution. The
TASK-2 message is poorly expressed or absent in the nervous
and muscular systems but is present in epithelial tissues, such as
lung, colon, intestine, stomach, liver, and particularly in the kidney.
In this organ, TASK-2 is more precisely located in the
cortical distal tubules and collecting ducts. In these structures,
K+-selective currents are postulated to play a major role
in the volume regulation and in the control of the negative potential of tubule cells, in the K+ recycling across the basolateral
membranes in conjunction with the Na-K-ATPase, and in the
K+ secretion into the tubular lumen in concert with
Na+ influx through amiloride-sensitive Na+
channels (for reviews, see Refs. 3 and 36). Principal cells of the
collecting ducts express at least two types of apical K+
currents sharing common properties, such as inhibition by
Ba2+ and ATP, as well as by internal acidification (3). The
first one is a K+ channel with a large conductance and a
low probability of opening that is activated by membrane depolarization
and internal Ca2+ and that is inhibited by
tetraethylammonium (37, 38). The second one is a small conductance (25 pS) K+ channel with a high Po and inward rectification and
that is insensitive to tetraethylammonium (39, 40). A cloned
K+ channel that has the same properties is ROMK2 (41). On
the other hand, three other K+ currents have been described
at the basolateral membrane of the collecting duct cells: a small
conductance K+ channel (28 pS) up-regulated by protein
kinase C, nitric oxide, and cGMP (36, 42, 43); an intermediate
conductance (85 pS) K+ channel activated by protein kinase
A and hyperpolarization (44); and a large conductance (147 pS)
K+ channel (42). The principal biophysical and
pharmacological properties of TASK-2 do not fit those of these native
K+ channels. A possibility would be that TASK-2 channels
are present in kidney cells but have not yet been recorded, which would
not be surprising because of the lack of a specific pharmacology. Another possibility would be that TASK-2 associates with yet
unidentified pore-forming subunits or regulatory proteins to produce an
active channel in native cells with properties different from those of the cloned channel, as has been observed for some other K+
channels (45-47). The inhibition of K+ channels by
acidification is consistent with the effect of metabolic acidosis,
which decreases secretion in distal tubules (36).
New insights into the mechanism of K+ secretion have been
recently provided by the cloning of several renal K+
channels and by fine studies of their distribution and cellular localization. Work presented in this paper opens the possibility of
progress in four different directions: (i) the search for a native
renal K+ channel with the properties of TASK-2, (ii) the
search for a potent pharmacology specific of the TASK-2 channel, (iii)
the determination of the localization of this channel type, and (iv) the knockout of the TASK-2 gene in mice. On the other hand,
genetic diseases associated with channelopathies are now discovered
with an increasing frequency (48), and it might turn out that there are
human diseases associated with kidney, pancreas, and/or liver dysfunctions corresponding to mutations in the TASK-2 gene.
We thank M. Fay, M. Jodar, and N. Leroudier
for expert technical assistance and V. Briet for secretarial assistance.
*
This work was supported by the CNRS and the Association
Française contre les Myopathies.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF084830.
§
The first three authors contributed equally to this work.
The abbreviations used are:
TMS, transmembrane
segments; 2P, two pore-forming; PCR, polymerase chain reaction; RT-PCR, reverse transcription-PCR; pBS, pBluescriptII SK
Cloning and Expression of a Novel pH-sensitive Two Pore Domain
K+ Channel from Human Kidney*
§,§,§,,,
Institut de Pharmacologie Moléculaire
et Cellulaire, CNRS-UPR 411, 660 route des Lucioles, Sophia Antipolis,
06560 Valbonne, France and the ¶ Faculté de Médecine
Xavier Bichat, INSERM U478, BP 416, 16 rue Henri Huchard, 75870 Paris
Cédex 18, France
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
ZAPII XR vector into pBluescriptII
SK
(pBS) and analyzed by restriction analysis and by sequencing of
their extremities. The longer cDNA insert (pBS-TASK-2) was
completely sequenced on both strands by using the dideoxy nucleotide
termination method using an automatic sequencer (Applied Biosystems,
model 373A).
µl of each sample
was used as template for PCR amplification (Taq DNA
polymerase, Life Technologies, Inc.) by using TASK-2 (base positions 358-381 (5'-CTGCTCACCTCGGCCATCATCTTC-3') and 901-924 (5'-GTAGAGGCCCTCGATGTAGTTCCA-3')) and GAPDH
(CLONTECH) primers. PCR conditions were 30 cycles
of 30 s at 94 °C, 30 s at 60 °C, and 30 s at
72 °C. TASK-2-amplified fragments were transferred onto
nylon membranes and then probed at high stringency with a 32P-labeled SmaI DNA fragment of pBS-TASK-2
(nucleotides 116-1305).
-UTP from a EcoRI-linearized plasmid
containing a 337-base pair NcoI/ClaI fragment of
the 5'-untranslated sequence of TASK-2 cDNA inserted
into pBS. The same plasmid was linearized by XhoI, and T3
RNA polymerase was used for the synthesis of a control sense probe. The
probes were hybridized, and then slides were covered with NTB2 emulsion
(Kodak) and exposed for 32 days at 20 °C. After development,
slides were stained with toluidine blue and photographed. The fragments
of human kidney were obtained from surgical ablation of renal cancers
(pieces of tissues surrounding the tumor).
resistance) filled with
3 M KCl and maintained under voltage clamp by using a Dagan
TEV 200 amplifier, in standard ND96 solution (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 2 mM MgCl2, 5 mM HEPES, pH 7.4, with
NaOH). Stimulation of the preparation, data acquisition, and analysis were performed using pClamp software (Axon Instruments). Drugs were
applied externally by addition to the superfusate (flow rate, 3 ml/min). All experiments were performed at room temperature (21-22 °C).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (91K):
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Fig. 1.
Sequences of TASK-2 and comparison to cloned
TWIK-related K+ channels. A,
nucleotide and deduced amino acid sequences of TASK-2. The four
potential transmembrane segments are boxed, and the two P
domains are underlined. A consensus site for
N-linked glycosylation (*) is indicated. B,
dendrogram of the five 2P domain K+ channels cloned in
mammals.
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Fig. 2.
Tissue distribution of TASK-2 in human and
mouse. A, multiple tissue Northern blots from
CLONTECH were probed at high stringency with a
TASK-2 cDNA probe and reprobed with a 
-actin probe as a control.
sk. muscle, skeletal muscle; sm. intestine, small
intestine; PBL, peripheral blood leukocytes. B,
RT-PCR analysis from mouse tissues. The amplified products were
analyzed by Southern blot using a specific TASK-2 probe. To check the
integrity of cDNAs, a GAPDH fragment was amplified and separated by
electrophoresis before staining by ethidium bromide.
View larger version (40K):
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Fig. 3.
Mapping of the TASK-2 gene.
Ideogram of human G-banded chromosome 6p and localization of the
TASK-2 gene relative to markers mapped in the Genebridge 4 Radiation Hybrid DNA panel from Research Genetics.
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Fig. 4.
TASK-2 mRNA distribution in human adult
kidney. In situ hybridization was performed with
antisense (A) and sense (B) probes. Specific
signal (A) was observed over distal tubules and collecting
ducts (indicated by stars in the tubular lumen), whereas a
low nonspecific labeling was apparent over the glomerulus
(G) and proximal tubule (PT). Sense probe
(B) gave a uniform signal. Magnification, × 520.
78.6 ± 2.7 mV,
n = 9) compared with control oocytes (42.2 ± 3.1 mV, n = 6).
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Fig. 5.
Expression of TASK-2 in COS cells and
Xenopus oocytes. A, TASK-2 whole cell
currents recorded in transfected COS cells in 5 or 155 mM
external K+, during voltage pulses ranging from 150 to
+50 in 50 mV steps. The holding potential was 80 mV; the dotted lines
indicate the zero current level. B, current-voltage
relationship recorded as in A, with voltage ramps ranging
from 150 to +50 mV, 500 ms in duration. C, relationship
between the reversal potential measured in COS cells and the external
K+ concentration; data (mean ± S.E.,
n = 4) are shown with the linear regression
(line). D, current-voltage relationship recorded
in a TASK-2-expressing oocyte in 2 mM external
K+, with voltage ramps ranging from 150 to +50 mV, 500 ms
in duration. Inset, currents recorded in 2 mM
external K+, during voltage pulses ranging from 150 to
+50 in 50 mV steps. The holding potential was 80 mV. E,
single channel currents recorded in transfected COS cells in
outside-out patch at various potentials ranging from 80mV to +80 mV,
in 40-mV steps. The dotted lines indicate the zero current
level. F, single channel current-potential relationships
recorded as in E in 5 mM (n = 22) and 155 mM (n = 19) external
K+.
50
mV, 0 mV, and +50 mV, the inhibition by acidic pH levels was
characterized by pHm values (for 50% of inhibition) of 8.6 ± 0.1, 8.3 ± 0.1, and 7.8 ± 0.1 units, respectively
(n = 6). The Hill coefficient at +50 mV is 0.69 ± 0.09 (n = 6). Fig. 6D shows the pH
sensitivity of TASK-2 single channel currents in the outside-out
configuration. A large inhibition of the current was recorded at pH 6.5 and an increase at pH 9.1. Fig. 6E shows that the pH effects
are due to a variation in N.Po and not in the single channel
conductance (Fig. 6F). 10% of the maximum current was
obtained at pH 6.5 ± 0.1 (n = 6) and 90% at pH
8.8 ± 0.1 (n = 6). These results indicate that
TASK-2 is very sensitive to extracellular pH in the physiological range.
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Fig. 6.
Sensitivity of TASK-2 currents to external
pH. A, current-voltage relationships deduced from
currents elicited by voltage pulses ranging from 150 to +50 mV, in
50-mV steps, measured at three different external pH levels (6.0, 7.4, and 8.6). Steps lasted 500 ms and started from an holding potential of
80 mV. B, whole cell currents elicited as in A.
C, relationship between the current measured at 50, 0, and
+50 mV and the external pH. Data (mean ± S.E.) were fitted with a
Boltzman relation (pHm = 7.8 ± 0.1, n = 17, at +50 mV). D, effect of pH 6.5, 7.3, and 9.1 on single
channel current recorded at 0 mV in the outside-out patch
configuration. E, effect of pH 6.5, 7.3, and 9.1 on
N.Po calculated from mean single channel currents recorded
in outside-out patches at 0 mV during 30 s and from slope
conductance between -20 and +20 mV (n = 5).
F, effect of pH 6.5, 7.3, and 9.1 on single channel currents
recorded at 0 mV (n = 13).
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed. Tel.:
33-4-93-95-77-02 or 33-4-93-95-77-03; Fax: 33-4-93-95-77-04; E-mail.ipmc{at}ipmc.cnrs.fr.
; pS, picosiemens.
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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