1Department of Physiology and Neuroscience, 2Department of Pediatric Cardiology, and 3Department of Biochemistry, New York University School of Medicine, New York, New York 10016
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ABSTRACT |
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Vega-Saenz de Miera, Eleazar, David H. P. Lau, Maria Zhadina, David Pountney, William A. Coetzee, and Bernardo Rudy. KT3.2 and KT3.3, Two Novel Human Two-Pore K+ Channels Closely Related to TASK-1. J. Neurophysiol. 86: 130-142, 2001. We report the cloning of human KT3.2 and KT3.3 new members of the two-pore K+ channel (KT) family. Based on amino acid sequence and phylogenetic analysis, KT3.2, KT3.3, and TASK-1 constitute a subfamily within the KT channel mammalian family. When Xenopus oocytes were injected with KT3.2 cRNA, the resting membrane potential was brought close to the potassium equilibrium potential. At low extracellular K+ concentrations, two-electrode voltage-clamp recordings revealed the expression of predominantly outward currents. With high extracellular K+ (98 mM), the current-voltage relationship exhibited weak outward rectification. Measurement of reversal potentials at different [K+]o revealed a slope of 48 mV per 10-fold change in K+ concentration as expected for a K+-selective channel. Unlike TASK-1, which is highly sensitive to changes of pH in the physiological range, KT3.2 currents were relatively insensitive to changes in intracellular or extracellular pH within this range due to a shift in the pH dependency of KT3.2 of 1 pH unit in the acidic direction. On the other hand, the phorbol ester phorbol 12-myristate 13-acetate (PMA), which does not affect TASK-1, produces strong inhibition of KT3.2 currents. Human KT3.2 mRNA expression was most prevalent in the cerebellum. In rat, KT3.2 is exclusively expressed in the brain, but it has a wide distribution within this organ. High levels of expression were found in the cerebellum, medulla, and thalamic nuclei. The hippocampus has a nonhomogeneous distribution, expressing at highest levels in the lateral posterior and inferior portions. Medium expression levels were found in neocortex. The KT3.2 gene is located at chromosome 8q24 1-3, and the KT3.3 gene maps to chromosome 20q13.1.
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INTRODUCTION |
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Potassium channels are
ubiquitously expressed and play critical roles in both excitable and
nonexcitable cells. They help to determine the resting potential as
well as the excitability and firing properties of excitable cells
(Hille 1992; Llinas 1988
; Rudy
1988
). A large number of mammalian potassium-channel subunits have been described (Chandy and Gutman 1993
;
Coetzee et al. 1999
; Isomoto et al. 1997
;
Jan and Jan 1997
). The latest addition to this expanding
set of mammalian K+-channel subunits is a group
of subunits containing two separate pore regions within a single
subunit. The first prototypes of this group YOK1/TOK1 was discovered in
yeast (Ketchum et al. 1995
; Lesage et al.
1996a
) and consist of subunits containing eight transmembrane
domains (TMDs) and two pore regions. K+-channel
subunits having two pore regions within a single polypeptide (KT) have
also been described in mammals. However, all of these subunits
described to date contain only four TMDs (termed M1-M4) with the two
pore regions (P1 and P2), respectively, located between TMDs 1 and 2 and between TMDs 3 and 4. K+-channel subunits
having this last structural organization have been identified in
invertebrates (Goldstein et al. 1996
; Wei et al.
1996
), mammals (Chavez et al. 1999
;
Duprat et al. 1997
; Fink et al. 1996
,
1998
; Lesage et al. 1996b
; Pountney et
al. 1999
; Reyes et al. 1998
; Salinas et
al. 1999
), and plants (Czempinski et al. 1997
),
suggesting a common ancestral origin before the evolutionary
diversification of plants and animals. Seven genes of the KT family
have been identified in mammals. In the order of their discovery, they
are: TWIK1 (Lesage et al. 1996b
), TREK (Fink et
al. 1996
), TASK-1 (Duprat et al. 1997
;
Leonoudakis et al. 1998
), TRAAK/KT4.1 (Fink et
al. 1998
; A. Ozaita and E. Vega-Saenz de Miera,
unpublished data), TASK-2 (Reyes et al. 1998
),
TWIK-2/TOSS (Chavez et al. 1999
; Pountney et al.
1999
), and KCNK6/KCNK7 (Salinas et al. 1999
).
Here, we describe the cloning of two new members of this emerging group of mammalian K+-channel subunits, human KT3.2 and KT3.3, having a sequence similarity closest to TASK-1. KT3.2 subunits express channels that differ from TASK-1 channels in pH sensitivity, modulation by PMA, and tissue distribution.
A portion of these results was presented at the 44th annual meeting of the Biophysical Society.
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METHODS |
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Bioinformational analysis
Initial identification of the sequences was obtained by using
the Blast algorithm (http://www.ncbi.nlm.nih.gov/blast/blast.cgi). Exon
identification was performed by using Blast of two sequences (http://www.ncbi.nlm.nih.gov/gorf/bl2.html), followed by sequence translation (http://www.expasy.ch/tools/dna.html) and identification of
sequences by comparison to the amino acid sequence of human TASK-1 and
finding the consensus sequence for intron/exon boundaries (Stephens and Schneider 1992). Hydropathy analysis was
performed using the Kyte and Doolittle algorithm (Kyte and
Doolittle 1982
) implemented in ProtScale web page
(http://www.expasy.ch/cgi-bin/protscale.pl). The prediction of
phosphorylation sites and posttranslational modifications analysis was
performed using ProfileScan
(http://www.isrec.isb-sib.ch/software/PFSCAN_form.html).
Cloning of human KT3.2
Forward and reverse primers to amplify the full coding sequence
of KT3.2 by polymerase chain reaction, using human brain cDNA (Clontech) as template, were designed by comparison of a human genomic
sequence (Accession No. AC007869) and the sequence of human TASK-1
(Duprat et al. 1997). The sequence for the forward primer was (TTG CTG GCG GCC ATG AAG AG) and for the reverse primer was
(CTA AAC GGA CTT CCG GCG TTT C). The PCR conditions were: 35 cycles,
54°C annealing, 72°C extension, and 94°C denaturation, each step
for 1 min. The band was subcloned into pCR2.1 using TA cloning
(Invitrogen), and both strands were sequenced.
To obtain additional 5' UTR sequences, rapid amplification of cDNA ends (RACE) experiments were performed using nested primers. The first round of amplification was performed using a specific oligonucleotide (AAC CTA TGG TGG TGA TGA CCG TGA TCG) complementary to the sequence that encodes for the amino acid sequence ITVITTIG in KT3.2, and the forward primer AP1 form Clontech Marathon-Ready human brain cDNA Kit. A second nested amplification was performed using the oligonucleotide complementary to the area that encodes for the amino acid sequence LIVCTFTYL in the human KT3.2 cDNA (AGC AGG TAG GTG AAG GTG CAG ACG ATG AGG) and the internal forward primer AP2 form the Clontech Marathon-Ready human brain cDNA kit.
Cloning of rat KT3.2
Two degenerated oligonucleotides were designed against areas
with significant sequence conservation in human mouse and rat TASK-1,
human KT3.2, and Caenorhabditis elegans n2p38
(Vega-Saenz de Miera and Lin 1992). The forward primer
(ATG AAG AGG CAR AAY RTN MGN AC) was designed using the sequences
around the starting methionine (MKRQNV/IRT). The reverse primer (AAG
GCA CCG ATN ACN GTN ARN CC) was designed to be complementary to the
amino acid sequence (GLTVIGA) located in the fourth TMD of these
channels. The latter sequence is also conserved in the human KT3.3
sequence. These primers were used for PCR amplification from cDNAs
prepared from mRNA isolated from thalamus and cortex (Vega-Saenz
de Miera and Lin 1992
). The PCR conditions were identical to
those used for the cloning of the human KT3.2 except that the annealing
temperature was 48°C.
Cloning of human KT3.3
PCR was performed using the primers GCC ATG CGG AGG CCG AGC GT (forward) and CTA TGG TGG TGA TGA CCG TGA T (reverse), under the following PCR conditions: 35 cycles of 58°C for 1 min and 97°C for 1 min, using human genomic DNA as well human brain cDNAs as templates. The PCR products were separated in 2% agarose gels, and the amplified bands were subcloned as in the case of human KT3.2. The final sequence of KT3.3 was assembled by combining the sequences obtained from the expressed sequence tag (EST) clones (see RESULTS) and from the PCR band using an internal NotI site. The integrity of this construct was confirmed by double-strand sequencing.
mRNA isolation
Total RNA was prepared using the guanidinium-thiocyanate method
(Chomczyznski and Sacchi 1987) from tissues obtained
from adult (150-175 g) Sprague-Dawley rats. Poly(A) RNA was isolated with oligo(dT) cellulose columns as previously described (Rudy et al. 1988
).
Northern and dot blot analysis
Human multi-tissue master dot blot (Clontech) was hybridized using Stratagene QuickHyb solution. The complete coding sequence of KT3.2 was labeled by random priming and used as a probe. The blot was hybridized for 1 h at 68°C with two washes at RT in 2× SSC 0.1% SDS and a final wash at 60°C in 0.1 SSC, 0.1% SDS for 30 min. The blot was placed at RT in a phosphoimager cartridge for 48 h. The values are reported as the values of the average signal measured by a circle that contains all the pixels in the dot minus the background, which was estimated by measuring an equal area free of signal but in the vicinity of the dot. The value was finally corrected for mRNA concentration applied to the blot and is reported as arbitrary digital counts per microgram of mRNA. Measurements of signal intensity were performed using Scion Imager.
The mRNAs isolated from rat tissues were electrophoresed in
formaldehyde-denaturing gels and transferred to Duralon membranes (Stratagene). The membranes containing the bound mRNAs as well as a
Clontech multi tissue rat Northern blot were hybridized with the first
719 nucleotides of the coding sequence of rat KT3.2. The probe was
labeled with [32P]dCTP using the Boehringer
random primers kit. The blots were hybridized in QuikHyb (Stratagene)
containing 1× 106 cpm/ml (0.5-1.0 ng of DNA/ml)
for 1 h at 68°C. They were then washed twice in 2× SSC, 0.1%
SDS at room temperature for 15 min each, a final wash in 0.1× SSC,
0.3% SDS at 60°C for 30 min. The blots were exposed to X-ray film
(X-OMAT AR Kodak) at 70°C for 1-2 days.
In situ hybridization
The in situ hybridization experiments were performed as
described (Vega-Saenz de Miera et al. 1997), with the
exception that a cDNA fragment made of the first 719 nucleotides of the
coding sequence of rat KT3.2 was used as template for probe synthesis. Blast search using this sequence shows areas with
81% identity (105 bp) and 74% (474 bp) to rat Task-1 and 87% (41 bp), 81% (198 bp),
and 76% (205 bp) to KT3.3 but not significant identities to other
cDNAS. Specificity was further checked by hybridization of this probe
against a blot containing synthetic RNA of rTASK-1 (Leonoudakis
et al. 1998
), rat KT3.2, and human KT3.3. A strong signal was
with rat KT3.2, but no signal with rTASK or KT3.3 was obtained after
24 h (data not shown). Another evidence of specificity was
obtained by the results of the Northern blot experiments in which the
distribution as well as the size of the mRNA band do not mach to the
ones described for rTASK (Leonoudakis et al. 1998
).
Two-electrode voltage clamp of Xenopus laevis oocytes
The recombinant pCR2.1 plasmid containing KT3.2 was linearized
by digestion with ScaI, and cRNA was transcribed in vitro
using T7 RNA polymerase (Stratagene). Stage IV-V Xenopus
oocytes were harvested, treated with collagenase (1 mg/ml), and
micro-injected with 50 nl cRNA (~20 ng) (Iverson and Rudy
1990). All electrophysiology experiments were carried out at
room temperature (22°C) 1-2 days following injection. Recordings
were made in solutions containing ND96 (in mM): 1.8 CaCl2, 1 MgCl2, 96 NaCl, 2 KCl, and 5 HEPES, pH 7.5, or KD98, which is similar in composition to
ND96 except that NaCl was replaced with KCl. Intermediate
concentrations of potassium were obtained by mixing ND96 and KD98.
Square voltage-clamp pulses were applied from a holding potential of
90 mV (ND96),
40 mV (KD14),
30 mV (KD26)
20 mV (KD50), or 0 mV
(KD98) to test potentials ranging from
150 to +50 mV in 10-mV
increments with a 3-s inter-pulse interval. Acquisition of data and
subsequent analysis was performed using pClamp 6.0 (Axon Instruments)
and Microsoft Excel.
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RESULTS |
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Human KT3.2
Screening of the GeneBank EST (expressed sequence tags) database
using the Blast algorithm (Altschul et al. 1997) with
the sequence of TASK-1 cDNA (Accession No. AF006823) as a probe yielded
two EST sequences, AI690321 and AI739096, distinctly different from but
related to TASK-1. The clones were incomplete at the 5' end and are
partial sequences of KT3.3 (vide infra). Using the partial sequences of
KT3.3 found in the EST database, a search in the high throughput
genomic sequences database (HTGS) identified two human genomic
sequences containing two new KT genes. One of the genes encodes a KT
channel that had 58% identity and 72% similarity to the published
human TASK-1 sequence (Accession No. AC007869) These values increase to
75% identity, 86% similarity if only the core region is used for
comparison. We refer to this gene as KT3.2.
Analysis of the KT3.2 genomic sequence identified two exons that cover
the full coding sequence of this channel. The exons are separated by an
intron that is larger than 80 kb (Fig.
1A). Using the predicted
coding sequence as a query in a search against the EST database
revealed a sequence that contains the carboxy terminus of a KT3.2
transcript (AA349574) and served to confirm the position of the stop
codon. A third exon, encoding the 3' UTR, was also identified. The
first splice junction occurs at the first glycine of the GYG sequence
in the first pore region. Oligonucleotide primers designed against
sequences surrounding the predicted starting methionine and the stop
codon of KT3.2 were used for PCR amplification with human brain cDNA
(Clontech) as template. A band of 1,137 bp was observed with gel
electrophoresis, and the cDNA was purified and subcloned. Three
independent clones were identical in sequence and differed only at
nucleotide 636 of the coding sequence from the putative sequence
predicted by the analysis of the genomic sequence contained in AC007869 (a thymidine-to-cytosine substitution). This substitution does not
alter the amino acid sequence and may represent a single nucleotide polymorphism (Fig. 1C). To confirm that the assigned
starting methionine was the real starting methionine, five different
RACE experiments using different primers were attempted. In each case, but in the last one, we obtain sequence related to KT3.2 that did not
reach the starting methionine. In the last case, using the primers for
the RACE experiments described in METHODS, we isolated 75 clones, 50 of which contained additional sequence related to KT3.2.
From this experiment, we obtained 74 extra nucleotides without a stop
codon in frame. The coding sequence found in the identified sequence
predicts a protein of 374 aa with a molecular weight of 42,264 Da.
Hydrophobicity analysis (Kyte and Doolittle 1982)
predicts the presence of four transmembrane domains (Fig. 1B). The sequence contains two regions between the first and
second and between the third and fourth TMDs having the canonical
GYG/GFG sequence that is part of the pore of potassium channels
(Heginbotham et al. 1994
) (Fig. 1C). A
potential N-glycosylation site (Fig. 1C,
)
(Miletich and Broze 1990
) is found in the linker
between M1 and P1. Interestingly, as in TASK-1, this new protein does not have a cysteine in the extracellular M1-P1 that is found in all
other mammalian KT channels and that has been suggested to be critical
for channel assembly (Lesage et al. 1996c
). The carboxy terminal sequence of the predicted protein contains three consensus sequences for phosphorylation by protein kinase C (Fig. 1C,
) (Woodgett et al. 1986
) and one for PKA (Fig.
1C,
) (Feramisco et al. 1980
; Glass
et al. 1986
).
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Human KT3.3
A Blast search using the partial EST sequences of KT3.3 described in the preceding text identified a new EST sequence, AI968607, which contains 140 bp in the 5' that do not match the previous ESTs. Translation of this extra sequence in one of the three reverse frames produces a 25 amino acid sequence with high degree of similarity to the sequences in the amino terminal of TASK-1 and KT3.2. We speculated that this could represent an inversion of the 5' sequence of the EST produced during library construction. PCR experiments using human genomic DNA or human brain cDNA with primers designed against the starting methionine predicted by the inverted 5' sequence and an internal primer based on the previous KT3.3 partial sequences produced a 283-bp product that was cloned and sequenced. The sequence of this product confirmed that the antisense sequence of AI968607 corresponds to the 5' sequence of KT3.3. Combination of the sequence obtained from the PCR experiment with the one obtained from EST AI690321 produced a 1,286-bp-long sequence with a 41-bp polyadenylation track. The open reading frame of this cDNA predicts a protein of 330 amino acids with a molecular weight of 36,130 Da (Fig. 1F).
Hydrophobicity analysis (Kyte and Doolittle 1982)
predicts a protein containing four TMDs. The protein also includes two
loops containing the canonical GYG/GFG signature of the
K+ channel pore (Heginbotham et al.
1994
), located between the first and second TMDs and the third
and fourth TMDs (Fig. 1E). Two putative sites for protein
kinase C (Fig. 1F,
) and one for protein kinase A (Fig.
1F,
) are also observed.
A Blast search of the HTGS database of GeneBank, using KT3.3 sequence as a probe identified a genomic sequence containing the KT3.3 gene (Accession No. AL118522). Analysis of KT3.3 sequence and the genomic clone AL118522 using Blast 2 sequences shows that the cDNA sequence is made by the participation of two different exons, separated by a 3,936-bp intron (Fig. 1D).
Expression of KT3.2 in Xenopus oocytes
Xenopus oocytes were injected with KT3.2 cRNA and
studied after 18-48 h in a bath solution of ND96. These oocytes had a
resting potential of 89 ± 2 mV (n = 15;
P < 0.05), which was ~45 mV more negative than that
of uninjected oocytes (
44 ± 2 mV; n = 7). This
membrane potential was close to the K+
equilibrium potential (calculated to be
90 mV under our experimental conditions), suggesting that the expressed channels are selectively permeable to K+ and can play an important role in
the generation of the resting potential.
Using two-electrode voltage-clamping techniques (in ND96
solution), mainly outward currents were observed. The currents
activated almost instantaneously (Fig.
2A). These currents were
absent in control water-injected oocytes (Fig.
3C). At high extracellular K+ concentrations (KD98), large inward currents
were detected (Fig. 2B) although a weak outward
rectification can be observed; (Fig. 2E). In this respect,
KT3.2 differs from the closely related TASK-1, which shows a linear
current-voltage relationship in high extracellular K+ concentrations (Duprat et al.
1997; Leonoudakis et al. 1998
). The reversal
potentials obtained from Fig. 2E, plotted as a function of
the K+ concentration, yielded a linear
relationship with a slope of 48 ± 1.3 mV per 10-fold change in
K+ concentration (n = 7; Fig.
2F), a value that is close to the theoretical value of 58 mV, as predicted by the Nernst equation for a
K+-selective channel. We were unable to detect
any current (recorded in ND96 with a pH that ranges from pH 6.5 to 8.5)
or shift in the resting potential in oocytes injected with KT3.3 cRNA.
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Modulation of KT3.2 currents by pharmacological agents and by pH
Currents produced by KT3.2 were relatively insensitive to
extracellular application of TEA or 4-aminopyridine (4-AP; ~20% reduction in outward current was observed when all
Na+ was replaced by TEA or 4-AP; data not shown).
Moderate block was observed by extracellular quinidine (~20% block
occurred with 100 µM and 80% block with 1 mM). Similar results were
obtained using quinine (data not shown). Ba2+ (3 mM) added to the perfusion solution blocked the current. This blockade
was released at potentials positive to the reversal potential in a
time-dependent manner (Fig. 3B). After reaching steady
state, inward currents are blocked proportionally more than outward
currents (~80% block occurred at 150 mV, whereas only ~20%
block occurred at +50 mV; Fig. 3C). This effect of
Ba2+ is not due to changes in membrane surface
charge because increasing Ca2+ to a final
concentration of 4.8 mM or Mg2+ to a final
concentration of 4 mM had no effect on KT3.2 currents (112 ± 6%,
n = 3, for Ca2+ and 106 ± 6%, n = 3, for Mg2+).
KT3.2 currents were inhibited by the phorbol ester PMA, which is a
potent PKC activator. A 50% inhibition was observed within 20 min of
application of 10 nM PMA. An increase in PMA concentration to 100 nM
produced similar results (Fig. 4). At
1 h after application, an 80% inhibition was observed (Fig. 4).
The currents were not affected by incubation with the inactive phorbol
ester 4 4alpha-phorbol 12,13-didecanoate (PDD) (Fukushima et
al. 1996
) or in the absence of phorbol ester application (Fig.
4), suggesting that specific activation of PKC was responsible for the
decrease in current produced by PMA. In contrast TASK-1 has been
described to be insensitive to 100 nM PMA (Duprat et al.
1997
).
|
KT3.2 and TASK-1 channels also differ in pH sensitivity. A
characteristic of TASK-1 channels, from which its name was derived (TASK: two pore acid
sensitive K+ channel), is
their high sensitivity to pH changes around the physiological pH range,
(current magnitude depends on pH with a pK of 7.29 ± 0.03 and is
nearly completely blocked at pH 6.5) (Duprat et al.
1997; Leonoudakis et al. 1998
). KT3.2 currents were also pH sensitive (Fig. 5), but the
pH dependence is shifted toward more acid pH's by over 1 pH
unit, such that KT3.2 currents were relatively insensitive to changes
in the pH 7.0 to 8.0 range (Fig. 5). Furthermore, KT3.2 currents were
not affected by application of dinitrophenol (1 mM for
20 min, data
not shown), a treatment that presumably acidifies the cytosol and
produces 50% reduction of TASK-1 currents after 6-min incubation
(Leonoudakis et al. 1998
).
|
Tissue distribution of human KT3.2
The pattern of KT3.2 tissue expression was studied by using
multiple human tissues mRNA dot blots (Clontech). We found expression to occur mainly in neuronal tissue, particularly in cerebellum, although hybridization signals were also detected in samples of adrenal
gland, kidney and lung mRNA (Fig.
6C). This expression pattern
of KT3.2 is different from that of human TASK-1, which expresses more
predominantly in placenta and pancreas followed by brain, lung,
prostate, and heart (Duprat et al. 1997).
|
Distribution of KT3.2 in rat tissues
To study the expression of the KT3.2 gene in rat, we
generated species-specific probes. We used RT-PCR with degenerate
oligonucleotides (see METHODS) with cortex and thalamus rat
cDNAs as template. Two different sequences were isolated from
these experiments. One was identical to the rat TASK-1 sequence
(Leounoudakis et al. 1998), whereas the other encoded a
sequence of 237 aa with 95% identity (99.6% similarity) to human
KT3.2 (Fig. 7). This rat ortholog of
KT3.2 was used as a probe for in-situ hybridization and Northern blot
experiments.
|
Northern blot analysis revealed that the KT3.2 gene is expressed in the
CNS but not in heart, lung, liver, kidney, intestine, or skeletal
muscle. This pattern of expression is different from that of TASK-1,
which in rat and mouse expresses mainly in heart and lung
(Duprat et al. 1997; Leonoudakis et al.
1998
) (Fig. 8). In situ
hybridization experiments revealed a wide expression pattern in rat
brain (Fig. 10, Table 1). The highest
expression levels were found in the olfactory nuclei, piriform cortex,
cerebellum, anterodorsal thalamic nucleus, pontine nucleus, dorsal
raphe, and several nuclei in the medulla (Fig.
9). An intriguing expression pattern was
found in hippocampus, with low levels of expression in the medial,
anterior, and superior areas and higher levels in the lateral and
ventral areas (Fig. 9, A, B, F, G vs. H and B). Diffuse expression was also observed throughout the
cortex (Fig. 9, A-H and Table 1). In spinal cord, the
channel is expressed in the anterior and posterior horns and more
intensely in layer 9 of Rexed (Fig. 9M and Table 1).
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Chromosomal localization of KT3.2 and KT3.3
THE KT3.2 GENE IS LOCALIZED TO 8q24.1-3. A database search using the genomic sequence containing KT3.2 identified three sequence-tagged sites (STS), having Accession Nos. Z53064, Z53078, and G23634. These STSs correspond to markers D8S1741 and D8S1743, which are, respectively, localized in the Geneton frame at 161.51 Mbp (536.87 cR3000 in GeneMap'99 (http://www.ncbi.nlm.nih.gov/genemap/loc.cgi?ID=19131) and WI-14922, located at 698 cR from the top of chromosome 8 in the MIT-Whitehead frame (http://carbon.wi.mit.edu:8000/cgi-bin/contig/sts_info?sts=EST275360&database=release), and 536.87 cR3000 in GeneMap'99 (http://www.ncbi.nlm.nih.gov/genemap/loc.cgi?ID=19130).The fact that the three markers are located in the same general area strongly supports the localization of this gene to this area. Although these frames are not anchored to the cytogenetic map, it is very probable that, based on the data obtained from GeneMap'99, this gene is located at 8q24.1-3.
THE KT3.3 GENE IS LOCATED TO CHROMOSOME 20q13. Searching the STS database using the genomic sequence found in AL118522, we obtained four human STS Accession Nos., G18091, G18129, G07367, and AL031792. All of these sequences map to human chromosome 20. The sequence G07367 corresponds to the human STS marker WI-9624. The MIT map (http://carbon.wi.mit.edu:8000/cgi-bin/contig/sts_info/406) localized this marker to the chromosome 20 reference map at 286.01 cR from top of the Chr20 linkage group. In GeneMap 98 (http://www.ncbi.nlm.nih.gov/genemap98/map.cgi?MAP=GB4&BIN=581&MARK=WI-9624) it is localized at 56.6 cM (235.78 cR3000) from the top of chromosome 20. Although there is no strict correlation between these frames and the cytogenetic maps, it is very probable, based on the data contained in GeneMap98, that this gene is located at 20q12-13.1.
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DISCUSSION |
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We report here the identification of two new members of the
emerging four TMDs or two-pore (KT) family of K+
channel pore forming subunits: KT3.2 and KT3.3. The KT family constitutes the largest family of K+-channel
proteins in the C. elegans genome, with close to 40 KT genes
identified (Wang et al. 1999). Sequence analysis suggest that about half of these genes can be grouped into subfamilies containing at least two members, while the remainder appear to be
unique genes. Counting KT3.2 and KT3.3, a total of nine different KT
genes have been identified in mammals. The identities in the core
region (TMD1-TMD4) of mammalian KT proteins range from 25 to 75%
(Table 2). As the number of identified
genes grows, it becomes possible to identify groups within the same
species having particularly large similarities that can be considered
separate subfamilies (Fig. 10).
|
|
With the cloning of the human KT3.2 and KT3.3 genes, a clear new
subfamily with more than one member in the same species can be defined
(Fig. 10). Human KT3.2 is 58% identical (72% similarity) to human
TASK-1. In the core region (TMD1-TMD4), these values increase to 75%
identity and 86% similarity, while KT3.3 shows 68% identity (81%
similarity) to TASK-1. Thus TASK-1, KT3.2, and KT3.3 can be considered
members of a subfamily that has at least three members in the same
species (human). At least two members of this subfamily also exist in
rat (rat KT3.2, this paper; rat TASK-1: Leonoudakis et al.
1998). Interestingly, the members of this subfamily are the
only mammalian KT genes for which a clear ortholog can be found in the
C. elegans genome, the n2p38 gene (Wang et al.
1999
) (Table 2; Figs. 10 and
11).
|
HTWIK (Lesage et al. 1996b) together with TWIK-2/TOSS
(Chavez et al. 1999
; Pountney et al.
1999
) and TRAAK/KT4.1 (Fink et al. 1998
; A. Ozaita and E. Vega-Saenz de Miera, unpublished results) together with
TREK (Patel et al. 1999
) may also define two additional KT subfamilies (Fig. 10), although the similarities within these subfamilies are less than in the TASK group (48.6 and 51.2% identity, respectively, in the core region; Table 2). Interestingly, another gene
related to TREK, human KT2.2, obtained by analysis of genomic sequences
(Accession No. AL122021), has 70% identities in the core region
(Vega-Saenz de Miera, personal observation).
The emergence of several KT subfamilies led us to implement a
nomenclature system following the model developed by Chandy et
al. (1991) for the pore forming subunits of voltage-gated (Kv) K+ channels. A similar nomenclature has been
applied to the inward rectifier family (Isomoto et al.
1997
). Following these models, each independent KT gene was
denominated KTx.y where the K stands for potassium and T stands for
"two pore." The "x" refers to the subfamily number and the
"y" to the member's number in that subfamily. Subfamilies and
members within a subfamily are numbered according to their order of
publication. Accordingly TASK-1 would be called KT3.1 because it was
the first identified member of the third identified subfamily of KT
genes. Hence, the newly discovered genes published in this manuscript
were named KT3.2 and KT3.3. Although these genes could also be called
TASK-2 and TASK-3, we should point out that the name TASK-2 has already
been used in the literature for a gene that also expresses pH-sensitive
K+ channels (TASK: two
pore acid-sensitive
K+ channel) but is evolutionary more
distantly related (Fig. 10, Table 2).
In the case of the Kv family of
K+-channel-forming subunits, the classification
into subfamilies turned out to be quite useful since the subfamilies,
in addition to reflecting closely related homologs, also appear to
constitute functional groups. Kv subunits can form homomultimeric
channels that are believed to be tetrameric (Coetzee et al.
1999). Subunits of the same subfamily can also interact and
form functional heteromultimeric channels (reviewed in Coetzee
et al. 1999
). In the KT family, it is believed that the
channels are formed by dimers of KT subunits (Lesage et al. 1996c
). Whether KT subunits also form heteromeric channels and whether this is or not restricted to members of the same subfamilies remains to be investigated.
FUNCTIONAL ROLES OF KT CHANNELS.
Several of the cloned KT subunits express K+
currents in heterologous expression systems, presumably by forming
homodimers (Lesage et al. 1996). A few others, including
TOSS (Pountney et al. 1999
), KCNK6, KCNK7
(Salinas et al. 1999
), and KT3.3 (this paper), do not
express currents when introduced into heterologous expression systems.
CHROMOSOMAL MAPPING OF KT3.2 AND KT3.3.
The KT3.2 gene was mapped to human chromosome 8q24.1-3. Several
diseases have been mapped to this area, including benign neonatal febrile convulsion/KCNQ3 (Charlier et al. 1998;
Lewis et al. 1993
), childhood absence epilepsy
(Fong et al. 1998
), idiopathic general epilepsy
(Zara et al. 1995
), benign adult familial myoclonic
epilepsy (Mikami et al. 1999
), Lom syndrome, an
autosomal recessive peripheral neuropathy with deafness
(Kalaydjieva et al. 1996
), and Meleda disease, a cardiac
disease with cardiomegaly, electrocardiographic abnormalities, and
episodes of ventricular tachycardia (Fischer et al.
1998
). On the other hand, the KT3.3 gene was mapped to 20q13.1,
an area in which BFNC, a rare kind of epilepsy, has been mapped
(Singh et al. 1999
). More interestingly, several tumors (Bossolasco et al. 1999
; De Angelis et al.
1999
; Palmedo et al. 1999
; Tanner et al.
1995
) show a duplication of this chromosomal area with multiple
copies being present in the same cell, which may explain why all the
KT3.3 ESTs found in the GenBank database are from tumor cells
(AI690321, moderately differentiated endometrial adenocarcinoma;
AW167075, uterus serous papillary carcinoma; AI739096, colon tumor;
AI968607, pooled germ cell tumors; and AW073155 and AI097455,
glioblastoma). A possible linkage of KT3.2 or KT3.3 to any of these
diseases is worth further investigation.
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ACKNOWLEDGMENTS |
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This research was supported by National Institute of Neurological Disorders and Stroke Grants NS-30989 and NS-35215 to B. Rudy and by American Heart Association Grant-in-Aid 9951070T to E. Vega-Saenz de Miera.
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FOOTNOTES |
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Address for reprint requests: E. Vega-Saenz de Miera, Dept. of Physiology and Neuroscience, New York University School of Medicine, 550 First Ave., New York, NY 10016 (E-mail: vegae01{at}endeavor.med.nyu.edu).
Received 27 April 2000; accepted in final form 8 March 2001.
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NOTE ADDED IN PROOF |
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While this paper was under review, three papers reporting the sequence and functional properties of KT3.2 (TASK-3) in rat, guinea pig, and human were published (J Biol Chem 275: 8340-9347; J Biol Chem 275: 16650-16657; Mol Brain Res 82: 74-83).
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REFERENCES |
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