From the Institut für Normale und Pathologische
Physiologie, ¶ Institut für Humangenetik, Marburg
University, and § Max Planck Institute for Biophysical
Chemistry, 37070 Göttingen, Germany
Received for publication, October 2, 2000, and in revised form, November 1, 2000
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ABSTRACT |
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Two cDNAs encoding novel K+
channels, THIK-1 and THIK-2 (tandem pore domain
halothane inhibited K+
channel), were isolated from rat brain. The proteins of 405 and 430 amino acids were 58% identical to each other. Homology analysis showed
that the novel channels form a separate subfamily among tandem pore
domain K+ channels. The genes of the human orthologs were
identified as human genomic data base entries. They possess one intron
each and were assigned to chromosomal region 14q24.1-14q24.3 (human (h) THIK-1) and 2p22-2p21 (hTHIK-2). In rat (r), THIK-1 (rTHIK-1) is
expressed ubiquitously; rTHIK-2 expression was found in several tissues
including brain and kidney. In situ hybridization of brain slices showed that rTHIK-2 is strongly expressed in most brain regions,
whereas rTHIK-1 expression is more restricted. Heterologous expression
of rTHIK-1 in Xenopus oocytes revealed a K+
channel displaying weak inward rectification in symmetrical
K+ solution. The current was enhanced by arachidonic acid
and inhibited by halothane. rTHIK-2 did not functionally express.
Confocal microscopy of oocytes injected with green fluorescent
protein-tagged rTHIK-1 or rTHIK-2 showed that both channel subunits are
targeted to the outer membrane. However, coinjection of rTHIK-2 did not
affect the currents induced by rTHIK-1, indicating that the two channel subunits do not form heteromers.
The family of tandem pore domain potassium (2P
K+)1 channels can
be divided into several subfamilies: (i) the acid-sensitive 2P
K+ channels TASK-1 to -3 (1-6), (ii) the mechanosensitive
2P K+ channels TREK-1/-2 and TRAAK (7-11) and (iii)
the weakly inwardly rectifying 2P K+ channels TWIK-1/-2,
and the structurally related but nonfunctional KCNK7
(12-15). All 2P K+ channels have four transmembrane
regions (M1-M4) and two typical pore-forming regions (P1 and P2)
including the K+ selectivity filter consensus sequence
TxG[YF]G (12) and a large extracellular loop between M1 and P1. The
extracellular loop is thought to participate in dimerization of
subunits, which in some 2P K+ channels may involve
disulfide bond formation (16). The N terminus of the 2P K+
channels is usually very short, whereas the C-terminal domain is much
larger and determines many functional properties.
Relatively little is known about the function of 2P K+
channels in vivo. Recently, endogenous currents with
properties similar to those of TASK channels have been found in the
heart (17), in arterial chemoreceptor cells (18), in zona
glomerulosa cells of the adrenal cortex (19), in cerebellar
granular cells (20), and in motoneurons (21). TASK-1 currents have been
shown to be coupled to the activation of thyrotropin-releasing hormone receptor 1 (TRH-R1; Ref. 21) and angiotensin II receptors (AT1a; Ref.
19); thus, TASK-1 channels are regulated also by mechanisms other than
extracellular pH. Furthermore, another member of this subfamily,
TASK-3, is activated by depolarization and modulated by extracellular
divalent cations (5).
With TREK-1, TREK-2, and TRAAK, three mechanosensitive channels have
been cloned (10, 22). Other factors that have been reported to modulate
the activity of these mechanosensitive channels include heat (TREK-1;
Ref. 23), lysophospholipids (TREK-1, TRAAK; Ref. 24), arachidonic
acid (TRAAK; Ref. 7), and intracellular pH (TREK-1; Ref. 25). The
third subfamily of 2P K+ channels so far comprises TWIK-1
and TWIK-2, both of which are expressed in multiple tissues (12, 13,
15). TWIK-1 shows weak inward rectification with symmetrical
K+ concentrations
(12).2 The current carried by
TWIK-2 is heat-sensitive and shows rapid time-dependent
inactivation at 37 °C (15). Both channels may contribute to setting
the resting membrane potential, but their specific function in various
tissues is not yet clear.
In this paper we describe the cloning of the first two members of a
novel subfamily of 2P K+ channels. One of these channels,
rTHIK-1, was found to be expressed in all tissues tested. Heterologous
expression of rTHIK-1 in Xenopus oocytes induced a current
that could be activated by arachidonic acid and inhibited by the
volatile anesthetic halothane. The second novel 2P K+
channel, THIK-2, is closely related to THIK-1 (58% identity at the
amino acid level) but could not be functionally expressed. THIK-2 was
strongly expressed in several tissues including stomach, liver, and
kidney and was particularly abundant in the brain.
Data Base Analysis--
BLAST searches of the expressed sequence
tag data base (dbEST), the genomic survey sequence data base (dbGSS),
and the human genomic data base (htgs) identified several human and rat
EST clones, one human GSS clone, and three human genomic data base entries of novel 2P K+ channel subunits. One rat EST clone
(GenBankTM accession number AI070460) and one human GSS
clone (GenBankTM accession number AQ898820) were purchased
from Research Genetics (Huntsville, AL) and completely sequenced.
Isolation of Rat THIK-1 and THIK-2 cDNAs--
About 1 × 106 clones of a Sequence Analysis--
For sequence analysis, the GCG program
package implemented at the Heidelberg Unix Sequence Analysis Resources
(HUSAR) was used. The program PROSITE was used for identification of
sequence consensus sites, the program GAP for pairwise sequence
alignments, the program CLUSTAL W for creation of multiple alignments,
and the program CLUSTREE for computation of the phylogenetic tree. Standard default parameters were used for the calculations.
Reverse Transcription-PCR Analysis of rTHIK-1 and rTHIK-2
Expression--
RNA from different rat tissues was prepared using the
TRIZOL method (Life Technologies, Inc.) and reverse-transcribed with Superscript II reverse transcriptase (Life Technologies, Inc.). PCR
with rTHIK-2-specific primers (5'-CCTTCCTCCGGCACTACGAG-3'; 5'-ATGAAGGCCAGCAGCGAGAT-3') amplifying a 250-bp intron-spanning DNA
fragment or rTHIK-1-specific primers (5'-CGTGGGCACAGTGGTAACTA-3'; 5'-GCTCCACAGGAGATGGCTAC-3') amplifying a 326-bp intron-spanning DNA
fragment was performed with AmpliTaq Gold DNA polymerase (Applied Biosystems). PCR conditions were as follows: denaturation for 30 s
at 94 °C, annealing for 30 s at 52 °C, and elongation for 1 min at 72 °C for 35 cycles each (with initial enzyme activation for
6 min at 94 °C and final additional elongation for 5 min at 72 °C). PCR fragments were analyzed on a 4% Nusieve-agarose
gel (Biozym, Hessisch-Oldendorf, Germany).
Chromosomal Assignment of Human THIK Genes--
Physical map
positions were determined by segregation analysis of gene markers using
gene-specific amplification of clone DNAs from the GeneBridge 4 human
radiation hybrid panel (26). The following primers were used for
amplification: hTHIK-2/KCNK12 (5'-TTCACAAGCTCATCCACAGC-3'; 5'-ATGGCTTCTTTTGGGTTCCT-3'),
hTHIK-1/KCNK13 (5'-CAGGGTTTGGGATGACAACT-3';
5'-CGTAGTACACGGAGGGCTTC-3'), TREK-2/KCNK10 (5'-GAGGGTCCATGTCTGCATCT-3'; 5'-CACATACCTGGTGGCCTCTT-3'). PCR conditions were as follows: denaturation for 30 s at 94 °C,
annealing for 30 s at 55 °C, elongation for 1 min at 72 °C,
each for 30 cycles. Data vectors (Table I) based on two
independent PCR analyses of the entire panel, with data arranged in the
order specified for the Whitehead/Mit on-line Radiation Hybrid Mapper
Program, were submitted to two-point maximum-likelihood analysis.
Electrophysiological Analysis--
cDNAs encoding rTHIK-2,
rTHIK-1, and chimeras between the two were cloned into the expression
vector pSGEM (a gift of Dr. M. Hollmann) for expression in
Xenopus laevis oocytes. Capped run-off poly(A+)
cRNA transcripts were synthesized and injected individually or in
combination into defolliculated oocytes at constant amounts (~3 ng
each). Oocytes were incubated at 19 °C in ND96 solution (96 mM NaCl, 2 mM KCl, 1 mM
MgCl2, 1 mM CaCl2, 5 mM
HEPES, pH 7.4 -7.5), supplemented with 100 µg/ml gentamicin and 2.5 mM sodium pyruvate, and assayed 48 h after injection.
Two-electrode voltage-clamp measurements were performed with a Turbo
Tec-10 C amplifier (npi, Tamm, Germany), which has a settling
time of 100 µs for a 100-mV voltage pulse. Data were recorded via an
EPC9 (Heka Electronics, Lambrecht, Germany) interface at sampling rates
up to 20 kHz using Pulse/Pulsefit software (Heka). The Oocytes were
placed in a small-volume perfusion chamber and bathed with ND96 or
"high K+ " solution (96 mM KCl, 2 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM HEPES, pH
7.4-7.5).
In Situ Hybridization--
Wistar rats were decapitated under
ether anesthesia, and their brains were removed and frozen on powdered
dry ice. Tissue was stored at
Synthetic oligonucleotides were chosen from the open reading frame with
least homology to other known sequences to minimize cross-hybridization. Antisense oligonucleotides designed for a minimal
tendency of forming hairpins and self-dimers were as follows (base
position on coding strand indicated). rTHIK-2: (i) (834) 5'-TATGGTCGACACCACGGTGCCCACGAAGTA GAAGGCTCCAGGGAAG3' and (ii) (1618-1662) 5'-CTCGGAAAGCTGCTTCTGCAGCAGTGCCAGCGACACCTTGTTGGA-3'. rTHIK-1: (i) (1074-1117)
5'-GTCGCCCATCAGTGTCACTTTCCATCACCCCGTCCGTCTCTATG-3', and (ii)
(1280- 1328)
5'-GCTACTTCTGGTCCCTACCTGTCCCCACTGGTCTCTGCCAACCTGTTAT-3'. Oligonucleotides were 3' end-labeled with 35S-dATP or
[33P]dATP (PerkinElmer Life Sciences; 1200/1000
Ci/mmol) by terminal deoxynucleotidyltransferase (Roche Molecular
Biochemicals) and used for hybridization at concentrations of 2-10
pg/µl (4 × 105 cpm/100 µl of hybridization
buffer/slide). Slides were air-dried and hybridized for 20-24 h at
43 °C in 100 µl of buffer containing 50% formamide, 10% dextran
sulfate, 50 mM DTT, 0.3 M NaCl, 30 mM Tris-HCl, 4 mM EDTA, 1× Denhardt's
solution, 0.5 mg/ml denatured salmon sperm DNA, 0.5 mg/ml polyadenylic
acid, and the labeled probe. After hybridization, slides were washed
2 × 30 min in 1× SSC (0.15 M NaCl and 0.015 M sodium citrate) plus 50 mM
Cloning of Two Novel 2P K+ Channel cDNAs--
Two
cDNA populations coding for novel 2P K+ channels were
isolated from two rat cDNA libraries. The first channel was
isolated from a rat brain and a rat heart library and was named THIK-1 for tandem pore domain halothane
inhibited K+ channel. The second,
closely related channel was found only in the rat brain library and was
named THIK-2 despite the fact that it was not functional when expressed
in Xenopus oocytes. Sequence analysis of the isolated
cDNA clones revealed complete open reading frames (Fig.
1) for rTHIK-1 (2032 bp) and rTHIK-2
(1900 bp). The nucleotide sequences predict proteins of 405 (rTHIK-1)
and 430 (rTHIK-2) amino acids, respectively, which show the typical
features of 2P K+ channels: four transmembrane regions, two
pore-forming regions, a large extracellular linker between M1 and P1, a
short N terminus and a larger C terminus. As indicated in Fig. 1,
A and B, several putative intracellular
phosphorylation sites for protein kinase A, protein kinase C, and
casein kinase were found in both sequences. In the M1-P1 linker region,
rTHIK-1 harbors two putative glycosylation sites
(N-{P}-[S/T]-{P}), whereas rTHIK-1 possesses only one.
The rTHIK-2 protein is significantly longer at the N terminus,
including three putative protein kinase C phosphorylation sites and
several repetitive amino acid elements.
Sequence comparison identified the human orthologs of THIK-2 in data
base entry AC009600 and of THIK-1 in two overlapping data base entries
(AL355074, AL137128). In accordance with the human nomenclature
committee (HUGO), the two novel channel genes were named
KCNK12 (THIK-2) and KCNK13 (THIK-1),
respectively. The human channel subunits are 98% (hTHIK-2) and 84%
(hTHIK-1), identical to their rat orthologs. hTHIK-1 and hTHIK-2 are
61.8% identical and 67.5% similar to each other, whereas only about 25-35% identity was found to other 2P K+ channels (Fig.
2B). Interestingly, the
intracellular M2-M3 linker was 16-20 amino acids longer than that of
other cloned 2P K+ channels. The cytosolic C-terminal
domains of the THIK channels are 57% identical to each other (see
supplemental material), whereas no significant homology was found to
other C-terminal domains of 2P K+ channel. The pore regions
of both THIK channels are nearly identical (no mismatch in a stretch of
13 amino acids in the first pore region and only one mismatch in the
second pore), whereas there is considerable difference to other 2P
K+ channels (Fig. 2C). From the homology scores,
the unique structural features of THIK channels and the phylogenetic
clustering (Fig. 2A), it is obvious that THIK 1 (KCNK13) and THIK-2 (KCNK12) form a novel
subfamily among the 2P K+ channels. Supplemental material
(multiple sequence alignment excluding the unrelated C-terminal domains
and pairwise alignment of THIK-1 and THIK-2 C-terminal domains) is
available in the on-line version.
Analysis of the entries of the human genome databases showed that both
genes described here have a similar structure, with a single large
intron of 48.1 kilobases (KCNK12) and 121.5 kilobases (KCNK13), respectively, splitting the coding region at the
first pore GYG motif. This intron is conserved in all mammalian 2P
K+ channel genes (KCNK1-10) cloned so
far. Chromosomal localization assigned KCNK12 to the human
chromosomal region 2p22-2p21 between markers WI-10633 and SGC34238.
KCNK13 was assigned to chromosomal region 14q24.1-14q24.3
between markers IB3608 and SGC30527, which is very close to the human
TREK-2 (KCNK10) gene. The results of the chromosomal
assignments are summarized in Table I.
The tissue distribution of the rTHIK-1 and rTHIK-2 was obtained by
reverse transcription-PCR analysis. Intron-spanning primers were used to avoid false positive results due to genomic contamination. rTHIK-1-specific products were amplified from all tissues tested. In
contrast, rTHIK-2 expression was found in brain, lung, kidney, liver,
stomach, and spleen but not in skeletal muscle, heart, and testis (Fig.
3).
In Situ Hybridization of Rat Brain--
The mRNA distribution
of rTHIK-1 and rTHIK-2 in the adult rat brain as detected by in
situ hybridization is highly differential, with little overlap
(summarized in Table II). Two different
antisense probes were used for either subunit. The resulting labeling
profiles were identical for each subunit, indicating that the probes
were specific. rTHIK-2 mRNA was found to be widely expressed in
most brain regions, with highest levels in the cerebellar granule cell layer, the mitral and granule cell layers of the olfactory bulb (Fig.
4E), and in the anterodorsal
and anteroventral nuclei of the thalamus (Fig. 4F). Strong
signals were also found in the thalamic ventral posterior nuclei (Fig.
4G), cortex, hippocampus, the pontine nucleus, the red
nucleus (Fig. 4E), the oculomotor nucleus, and some nuclei
of the amygdala. In the brainstem, elevated levels were found in all
trigeminal sensory nuclei, in the tegmental and reticular nuclei, and
in the ventral cochlear nuclei. rTHIK-2 mRNA was also found in some
non-neuronal cells such as the ependymal lining of the ventricles.
rTHIK-2 was found to be absent from the substantia nigra, gracile
nuclei, inferior olive, and from most parts of the caudate putamen and
septal nuclei as well as all white matter pathways.
In contrast, rTHIK-1 mRNA expression was found to be rather weak
and restricted to only a few brain regions and nuclei (Fig. 4,
A-D). Substantial expression levels were detected only in
the granule cell layer of the olfactory bulb, in the olfactory
tubercle, the lateral septum (Fig. 4A), and in distinct
hypothalamic and thalamic nuclei (ventromedial hypothalamic nucleus,
lateral mamillary nucleus, reticular nucleus, reunions nuclei (Fig. 4,
B and C)). Other rTHIK-1-positive structures were
only partially labeled. The cortex, for example, was positive only in
layer II, the striatum was labeled only in the caudal part, and the
dentate gyrus granule cell layer of the dentate gyrus exhibited
particularly high mRNA levels in the caudal-ventral part (Fig.
4D). Similarly, within a nuclear group, only specific
subnuclei were positive, e.g. the dorsal subnucleus of the
lateral septum (Fig. 4A), the magnocellular part of the red
nucleus (Fig. 4D), a subnucleus of the lateral habenula
(Fig. 4, A and C), one lateral parabrachial
subnucleus, and the ventral nucleus of the cochlear nuclei.
Heterologous Expression in Xenopus Oocytes--
At day 2 after
injection of rTHIK-1 cRNA into Xenopus oocytes, large
currents were recorded using the two-electrode voltage clamp (Fig.
5A), which were not seen under
control conditions. The steady-state amplitude of the currents measured
at +60 mV was 21.85 ± 4.45 µA (n = 7) in the
presence of 2 mM external K+. Current
activation in response to depolarizing voltage steps was not
instantaneous but, when fitted to a single exponential, showed an
activation time constant of 0.97 ± 0.25 ms at +60 mV. During a
500-ms voltage pulse, currents did not inactivate. The steady-state
current-voltage relationship of rTHIK-1 showed only weak voltage
dependence. With low external K+, moderate outward
rectification was observed, whereas with nearly symmetrical
K+, weak inward rectification was found (Fig.
5B). When external K+ was altered to examine the
ion selectivity of rTHIK-1, the measured reversal potentials were very
close to the K+ equilibrium potential (calculated on the
assumption of an intracellular K+ concentration of 100 mM). The linear regression fit to the data gave a slope of
In contrast, the currents measured in Xenopus oocytes after
injection of rTHIK-2 (Fig. 5D) were not significantly
different from those of noninjected or water-injected oocytes
(n = 10). To test whether both channel proteins were
translated and targeted to the surface membrane, rTHIK-1 and rTHIK-2
were tagged with EGFP at the N terminus. 48 h after injection of
EGFP-rTHIK-2, confocal microscopy showed strong membrane fluorescence
that was very similar to that found after injection of EGFP-rTHIK-1
(Fig. 6A). Thus rTHIK-2
subunits appear to be targeted to the outer membrane. The reason for
the lack of functional expression of rTHIK-2 was further studied by
constructing chimeric subunits in which the first part of the
rTHIK-2-coding region (M1-P1-M2) was fused to the second half of
rTHIK-1 (M3-P2-M4) and vice versa. Injection of the chimeric
cRNAs did not induce any current in Xenopus oocytes.
Some voltage-activated (Kv) or inwardly rectifying (Kir) K+
channel subunits that do not functionally express as homomers can modify the activity of other subunits by heteromerization. Therefore we
investigated the possibility that rTHIK-1 and rTHIK-2 subunits might
coassemble to form heterodimeric channels. When rTHIK-1 and rTHIK-2
cRNAs were injected at equimolar amounts (n = 5), all
macroscopic current properties were virtually indistinguishable from
rTHIK-1 currents, and current amplitudes were not significantly different (Fig. 6, B and C). Furthermore, the
currents induced by injection of rTHIK-1 were unaffected by injection
of larger amounts of rTHIK-2 cRNAs (ratio 1:5). The apparent absence of heteromerization of rTHIK-2 and rTHIK-1 subunits suggests that rTHIK-2 is not a regulatory subunit of the rTHIK-1 conductance.
Regulation of rTHIK-1 Channels--
To test for functional
similarities to other members of the two pore domain K+
channel family, we studied the modulation of rTHIK-1 channel activity
by various experimental interventions such as extracellular acidification, application of the polyunsaturated fatty acid
arachidonic acid, or application of the volatile anesthetic halothane
(Fig. 7A). The outward current
carried by rTHIK-1 was only weakly inhibited by extracellular
acidification to pH 6, which is in marked contrast to the pronounced pH
sensitivity of TASK-1 (KCNK3; half-maximal block at pH
7.38), as illustrated in Fig. 7B. Lowering the pH to 4.5 inhibited the outward current carried by rTHIK-1 by 34 ± 8%
(n = 5). Similar to TASK-3, proton block of rTHIK-1
occurred with a fast time course (Fig. 7A) and was
independent of the membrane potential (Fig. 7B). The
sensitivity of rTHIK-1 to intracellular pH was tested by the "rebound
acidification" technique. 20 mM NH4Cl was
applied for 5 min and then removed, which should decrease the
intracellular pH by about 1 unit. This intervention produced no
measurable change in the current carried by rTHIK-1 (n = 3), indicating that rTHIK-1 is not modulated by intracellular pH.
Since TREK-1 has been reported to be heat-sensitive (23), we also
studied the temperature dependence of rTHIK-1. Raising the temperature
from 22 to 37 °C increased the current amplitude by a factor of 1.6 (n = 3), in agreement with van't Hoff's rule, whereas
TREK-1 is augmented by a factor of 10 under the same conditions (23).
These findings suggest that, unlike TREK-1, rTHIK-1 is not a
heat-sensitive channel. Lysophosphatidylcholine (3 µM)
has also been shown to activate TREK-1 and TRAAK (24). Application of
10 µM lysophosphatidylcholine to Xenopus
oocytes expressing rTHIK-1 induced only a very minor (up to 20%)
increase in current amplitude, which may be attributable to a
nonspecific effect of the lysophospholipid.
Application of arachidonic acid to the bath solution induced a rapid
increase in the outward current carried by rTHIK-1 channels (Fig.
7A). This effect could be washed out within 5 min. In the presence of 5 µM arachidonic acid, the current was
increased by 85 ± 24% (n = 5) at +30 mV. As can
be seen from Fig. 7C, the current activated by arachidonic
acid was outwardly rectifying. It reversed at the calculated potassium
equilibrium potential when the external K+ concentration
was altered. The concentration dependence of the effect of arachidonic
acid on rTHIK-1 could be described by a Kd of 0.98 µM and a Hill coefficient of 1.97 (Fig. 7C). The effects of arachidonic acid reported here are similar to the effects found in TREK-1 (KCNK2) and TRAAK (KCNK4)
(7, 9, 24).
Two of the known 2P K+ channels, TREK-1 and TASK-1, are
activated by the volatile anesthetic halothane (27). Surprisingly, our
whole-cell recordings in Xenopus oocytes showed that the
current carried by rTHIK-1 was rapidly and reversibly inhibited by
halothane (Fig. 7, A and D). When the membrane
potential was held at +30 mV, application of 5 mM halothane
reduced rTHIK-1 currents by 56 ± 5% (n = 5). The
fit of the concentration-effect curve gave a Kd of
2.83 mM and a Hill coefficient of 1.06. Under the same
experimental conditions, application of chloroform (1 mM)
had no effect (n = 3; data not shown).
THIK-1 and THIK-2 are the first two members of a novel 2P
K+ channel subfamily. Although they show the typical
features of other 2P K+ channels, such as four
transmembrane regions, two pore-forming regions, and a large
extracellular M1-P1 linker region, the two novel channels are only
about 25-35% identical to the other known 2P K+ channels
but 61.8% identical to each other. One notable difference is the
larger cytosolic M2-M3 linker region, containing three (rTHIK-2) or one
(rTHIK-1) putative phosphorylation site(s). In addition, the THIK-2
protein has an unusual N terminus containing several repeats of
proline, arginine, and cysteine residues. The C-terminal domain of
THIK-1 and THIK-2 shows no significant homology to other mammalian 2P
K+ channels.
The reversal potential of the current induced by injection of rTHIK-1
cRNA in Xenopus oocytes followed the calculated
K+ equilibrium potential when external K+ was
changed. This suggests that the THIK channels are mainly permeable to
K+ ions. The whole-cell current induced by heterologous
expression of rTHIK-1 displayed outward rectification at physiological
external K+ and weak inward rectification with
approximately symmetrical K+ concentrations. It could be
activated by arachidonic acid (Kd, 0.98 µM; Hill coefficient, 1.97) and inhibited by halothane
(Kd, of 2.8 mM; Hill coefficient, 1.06).
Chloroform had no effect on rTHIK-1. Another 2P K+ channel,
TWIK-2, which is almost absent in the brain, has recently been found to
be inhibited by both halothane and chloroform (15). In contrast, both
TREK-1 and TASK-1 are activated by halothane and isoflurane, and TREK-1
is additionally activated by chloroform and diethyl ether (27). TREK-1,
TASK-1, and THIK-1 are all expressed in specific regions of the brain.
The findings reported here suggest that the effects of volatile
anesthetics on the brain may be more complex than hitherto assumed. We
are aware of the fact that the IC50 for the effects of
halothane on THIK-1 is higher than the EC50 for the
anesthetic effects in vivo ( Injection of rTHIK-2 cRNA in Xenopus oocytes did not produce
any measurable currents. The lack of functional expression of rTHIK-2
was apparently not due to inadequate targeting, because confocal
microscopy showed EGFP-tagged rTHIK-2 channels in the outer cell
membrane (Fig. 6). To localize possible structural constraints in
rTHIK-2 that prevent expression of functional channels, we constructed
rTHIK-1/rTHIK-2 and rTHIK-2/rTHIK-1 chimeras. However, since neither of
the two chimeras was functional, the reason for the nonfunctional state
of rTHIK-2 in Xenopus oocytes remains unclear. Another
possibility is that rTHIK-2 might be a regulator of rTHIK-1 conductance
by coassembling with rTHIK-1. This is unlikely, because whole cell
currents produced by injection of rTHIK-1 cRNA were unaffected by
coinjection even of 5-fold larger amounts rTHIK-2 cRNA. In
situ hybridization in the brain showed little overlap between
rTHIK-1 and rTHIK-2, which also argues against heteromerization. In
conclusion, the high expression of rTHIK-2 in cerebral cortex, hippocampus, and olfactory bulb and the specific expression in several
nuclei (Fig. 6) support the idea that rTHIK-2 is functionally important
in neurons, but the available experimental evidence suggests that
rTHIK-2 requires an accessory subunit or specific intracellular ligands
to form a conducting pore. The strong expression in lung, kidney, and
stomach suggests that rTHIK-2 may also play a role in epithelial cells.
Both human THIK genes described here have only one very large intron,
121.5 kilobases in the THIK-1 gene (KCNK13) and 48.1 kilobases in the THIK-2 gene (KCNK12), that splits the
coding region of the first pore (GYG motif). In this respect, the THIK genes are similar to the TASK-3 gene (KCNK9). The genes of
the TWIK and the TREK-/TRAAK families possess several introns in the coding region (10, 29, 30), but an intron splitting the GYG motif of
the first pore region is conserved in all mammalian 2P K+
channel genes cloned so far and, in addition, in most of the 2P
K+ channels of Caenorhabditis elegans (31) and
Drosophila melanogaster. Another interesting feature is the
colocalization of 2P K+ channel genes at the same
chromosomal region. We have shown that THIK-1 (KCNK13) maps
to the same region on the long arm of chromosome 14 as TREK-2
(KCNK10). The other 2P K+ channel gene pairs
known so far are TRAAK (KCNK5) and KCNK7 on chromosome 11q13 (29) and TWIK-1 (KCNK1) and TREK1
(KCNK2) on chromosome 11q41-43 (32). These data are
consistent with a common ancestral gene for all 2P K+
channels that was subject to several duplication events.
As illustrated in Fig. 2, mammalian 2P K+ channels can be
subdivided in five subfamilies: 1) TWIK/KCNK7, 2)
TREK/TRAAK, 3) TASK-1/TASK-3, 4) THIK, and 5) TASK-2. These channels
display a wide variety of electrophysiological and regulatory
characteristics that are usually not confined to one of the
subfamilies. The steady-state current voltage relation measured in the
whole-cell configuration at symmetrical K+ concentrations
was found to be inwardly rectifying (TWIK-1, TWIK-2, and THIK-1),
outwardly rectifying (TREK-1, TASK-3), or linear (TREK-1, TASK-1,
TRAAK). Some of the 2P K+ channels show a pronounced
sensitivity to intracellular (TREK-1) or extracellular pH (TASK-1,
TASK-2, TASK-3). The pharmacological profile of the 2P K+
channels is also very diverse and not related to subfamilies. Some 2P
K+ channels are activated by volatile anesthetics (TREK-1,
TASK-1); other channels are inhibited (TWIK-2, THIK-1). Some channels
are activated by fatty acids such as arachidonic acid (TREK-1, TRAAK, TWIK-2, THIK-1) or by phospholipids such as lysophosphatidylcholine (TREK and TRAAK). In addition, some of the 2P K+ channels
are mechanosensitive (TREK-1, TREK-2, and TRAAK) or heat-sensitive
(TREK-1). The most remarkable common characteristic of all 2P
K+ channels known so far is that their regulation by
physical and chemical stimuli is very complex. The difficulty in
identifying their function is probably related to this complex
regulation, which needs to be studied in the native cells in which the
channels are expressed.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-ZAPII rat brain or a
-ZAPII rat
heart cDNA library (Stratagene) plated with XL1-Blue MRF' cells were screened with Digoxigenin-labeled THIK-2 and
THIK-1 fragments derived from the EST or GSS clones,
respectively, and positive clones were detected using CSPD
(Roche Molecular Biochemicals) as chemoluminescence substrate. After
purification of isolated plaques by two further screenings, pBSK+
plasmids containing the cDNAs were excised from
-clones and sequenced.
20 °C until cutting. Sixteen-µm
sections were cut on a cryostat, thaw-mounted onto silane-coated
slides, and air-dried. After fixation for 10 min in 4%
paraformaldehyde dissolved in PBS, slides were washed in PBS,
dehydrated, and stored in ethanol until hybridization.
-mercaptoethanol, 1 h in 1× SSC at 60 °C, and 10 min in
0.1× SSC at room temperature. Specimens were then dehydrated,
air-dried, and exposed to Kodak BIOMAX x-ray film for 14-28 days. For
cellular resolution, selected slides were dipped in photographic
emulsion Kodak NTB2, incubated for 4-12 weeks, and then developed in
Kodak D-19 for 2.5 min. For identification and confirmation of brain
structures with bright- and dark-field optics, sections were
Nissl-counterstained with cresyl violet. Controls sections were
(a) digested with RNase A (50 ng/ml) for 30 min at 37 °C
before hybridization or (b) hybridized with a probe
containing a 20-50-fold excess of unlabeled oligonucleotides. These
control hybridizations resulted in a complete loss of specific hybridization signal.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (144K):
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Fig. 1.
cDNA and amino acid sequence of rTHIK-1
(A) and rTHIK-2 (B).
Transmembrane regions are shown in black, and pore regions
are marked gray. In addition, putative glycosylation sites
(h), protein kinase A phosphorylation sites ( ),protein kinase C
phosphorylation sites (
) and casein kinase phosphorylation sites
(
) are indicated below the amino acid sequence. Consensus
sites were identified using the program PROSITE.
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Fig. 2.
Sequence comparison of eleven 2P
K+ channels. A, phylogenetic tree. The
GenBankTM accession numbers for the different channels are:
hTHIK-1, AF287303; hTHIK-2, AF287302; hTASK-1, AF006823; hTASK-3,
AF212829; hTREK-1, AF129399; rTREK-2, AF196965; hTRAAK, AF247042;
hTWIK-1, U33632; hTWIK-2, AF117708; KCNK7, AF110522; and
mTASK-2, AF084830. The CLUSTAL/CLUSTREE algorithm (Heidelberg UNIX
Sequence Analysis Resources) was used for computation. B,
pairwise sequence identity scores of THIK channels. The program GAP was
used for calculation. C, multiple sequence alignment of the
two pore regions of the 2P K+ channels. Conserved residues
are shown in bold, and the only residue different in
THIK-1 and THIK-2 pore domains is labeled with an asterisk.
Additional information on sequence comparison (multiple alignment
excluding C-terminal domains of the above mentioned channels, pairwise
alignment of THIK-1 and THIK-2 C-terminal domains) is available on the
on-line version.
GeneBridge four-panel radiation hybrid mapping data
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Fig. 3.
Tissue distribution of rTHIK-1 and
rTHIK-2. cDNA from the tissues indicated was analyzed for the
presence of a 326-bp fragment of rTHIK-1 or a 250-bp fragment of
rTHIK-2, indicated as a white arrow. Note that both primer
pairs were intron-spanning to exclude possible genomic contamination. A
pBR322/HaeIII marker was used in the left-most
lane.
Distribution of rTHIK-1 and rTHIK-2 mRNA in the adult rat brain
View larger version (85K):
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Fig. 4.
In situ hybridization of rTHIK-1
and rTHIK-2 mRNA in rat brain. X-ray film images of adjacent
sagittal (A and E) and coronal (B-D,
F-H) sections through the adult rat brain show distribution
of rTHIK-1 (A-D) and rTHIK-2 (E-H) as detected
by in situ hybridization with 33P-labeled
oligonucleotide probes (exposure time, 26 days). Scale bars
represent 4 mm in A and E, 3 mm in all other
panels. AD and AV, anterodorsal and
anteroventral thalamic nuclei, respectively; CA1, CA1 field
of hippocampus; Cb, cerebellum; CPu, caudate
putamen; Cx, cortex; DEn, dorsal endopiriform
nucleus; DG, dentate gyrus of hippocampus; Hb,
habenula; LSD, lateral septum, dorsal nucleus;
MGD, medial geniculate nucleus; OB, olfactory
bulb; Pir, piriform cortex; Pn, pontine;
R, red nucleus; RMC, red nucleus magnocellular;
Re, reunions thalamic nucleus; Rt, reticular
thalamic nucleus; SCh, suprachiasmatic nucleus;
Sol, solitary nucleus; VMH, ventromedial
hypothalamic nucleus; VPM/L, ventral posteromedial/lateral
thalamic nucleus.
53 mV/decade (Fig. 5C), as would be expected for highly
selective K+ channels. THIK-1 could be partially blocked by
Ba2+ ions; application of 1 mM Ba2+
inhibited the instantaneous outward current measured at +60 mV (in the
presence of 2 mM external K+) by about 60%
(n = 4, not shown).
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Fig. 5.
Heterologous expression of rTHIK-1 and
rTHIK-2 in Xenopus oocytes. Whole-cell current
recordings from oocytes injected with cRNA of rTHIK-1 (A-C)
and rTHIK-2 (D), respectively. A, voltage steps
of 500-ms duration from a holding potential of 60 mV to potentials
between
140 to +60 mV. B, voltage ramps (105 mV
s
1) from
150 to +60 mV at external
potassium concentrations of 2, 5, 10, 25, and 96 mM
K+, respectively (the holding potential preceding the ramp
pulses was
60 mV). C, semi-logarithmic plot of the
measured zero-current (reversal) potentials versus
[K+]e. A linear regression line with a slope of
53 mV per decade was fitted to the data points. D, voltage
ramps in a Xenopus oocyte injected with rTHIK-2 cRNA. The
ramp protocol was identical to that shown for rTHIK-1 in
panel B.
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Fig. 6.
Targeting of rTHIK-1 and rTHIK-2 and
coexpression in Xenopus oocytes.
A, fluorescence signal of EGFP-labeled rTHIK-1 and rTHIK-2
in the oocyte membrane 48 h after cRNA injection. B,
whole-cell currents recorded from Xenopus oocytes after
expression of rTHIK-1 alone and after coexpression of rTHIK-1 together
with rTHIK-2 at RNA ratios of 1:1 and 1:5. Voltage ramps from 150 mV
to +60 mV were applied; the preceding holding potential was
60 mV.
C, bar graph of the currents measured at +60 mV.
No significant difference was found between oocytes injected with
rTHIK-1 alone and in combination with rTHIK-2.
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Fig. 7.
Regulation of rTHIK-1 by external pH,
arachidonic acid, and halothane. A, continuous current
recording at +30 mV from a rTHIK-1-injected oocyte superfused with
arachidonic acid (5 µM), halothane (5 mM), or
solution titrated to pH 4.5. The dotted line represents zero current.
B, the currents elicited by voltage ramps from 150 to +60
mV in extracellular solution titrated to pH 7.5 and 4.5, respectively
(left panel). On the right, the normalized current
inhibition for both rTHIK-1 and TASK-1 is plotted versus
extracellular pH. Data are least squares fits to the Hill equation (1/1 + ([A/Ki]n) with A
as a variable. The pH producing half-maximal block
(Ki) was 4.0, and the Hill coefficient
(n) was 0.74. C, the currents elicited by voltage
ramps in the presence and absence of 5 µM arachidonic
acid (left panel). On the right, the normalized current
increase is plotted against the concentration of arachidonic acid. The
fit of the data to the Hill equation (right panel) gave a
Kd of 0.99 µM and a Hill coefficient
of 1.98. The inset depicts the current augmentation by 10 µM arachidonic acid in an oocyte held at +30 mV.
D, the currents elicited by voltage ramps in the presence
and absence of the volatile anesthetic halothane (5 mM;
left panel). On the right, the normalized current inhibition
is plotted against concentration of halothane. A fit of the data as in
panel C gave a Ki of 2.83 mM
and a Hill coefficient of 1.06. The inset shows the
inhibition of the outward current (measured at a holding potential of
+30 mV) by 10 mM halothane.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
250 µM; Ref.
28). Nevertheless, we decided to name the new channels
tandem pore-domain halothane
inhibited K+ channels because this
discriminates them from some of the other 2P K+ channels.
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ACKNOWLEDGEMENTS |
---|
We thank Annette Hennighausen, Dirk Reuter, Hartmut Engel, and Antonio Mazzola for excellent technical assistance and Susanne Bamerny for invaluable secretarial help.
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FOOTNOTES |
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* This work was supported in part by Deutsche Forschungsgemeinschaft Grants Da177/7-3 and Ka1175/1-3, by the Ernst and Berta Grimmke Stiftung, and by the P. E. Kempkes Stiftung.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) AF287300 and AF287301 (for rat THIK-2 and THIK-1 cDNA) and AF287302 and AF287303 (for human THIK-2 and THIK-1 cDNA).
The on-line version of this article (available at
http://www.jbc.org) contains supplemental Fig. 8.
To whom correspondence should be addressed. Tel.:
49-551-2011665; Fax: 49-551-2011688; E-mail: akarsch@gwdg.de.
Published, JBC Papers in Press, November 1, 2000, DOI 10.1074/jbc.M008985200
2 E. Wischmeyer and A. Karschin, unpublished information.
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ABBREVIATIONS |
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The abbreviations used are: 2P K+ channels, tandem pore domain potassium channels; THIK, tandem pore domain halothane-inhibited K+ channel; rTHIK, rat THIK; hTHIK, human THIK; EST, expressed sequence tag; PCR, polymerase chain reaction; GSS, genomic survey sequence; EGFP, enhanced green fluorescent protein; bp, base pair(s).
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