From the Institut de Pharmacologie Moléculaire et Cellulaire, CNRS, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France
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ABSTRACT |
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Mouse KCNK6 is a new subunit belonging to the
TWIK channel family. This 335-amino acid polypeptide has four
transmembrane segments, two pore-forming domains, and a
Ca2+-binding EF-hand motif. Expression of KCNK6
transcripts is principally observed in eyes, lung, stomach and embryo.
In the eyes, immunohistochemistry reveals protein expression only in
some of the retina neurons. Although KCNK6 is able to dimerize as other
functional two-P domain K+ channels when it is expressed in
COS-7 cells, it remains in the endoplasmic reticulum and is unable to
generate ionic channel activity. Deletions, mutations, and chimera
constructions suggest that KCNK6 is not an intracellular channel but
rather a subunit that needs to associate with a partner, which remains
to be discovered, in order to reach the plasma membrane. A closely
related human KCNK7-A subunit has been cloned. KCNK7 displays an
intriguing GLE sequence in its filter region instead of the G(Y/F/L)G
sequence, which is considered to be the K+ channel
signature. This subunit is alternatively spliced and gives rise to the
shorter forms KCNK7-B and -C. None of the KCNK7 structures can generate
channel activity by itself. The KCNK7 gene is situated on
chromosome 11, in the q13 region, where several candidate diseases have
been identified.
Ion channels are present in excitable and nonexcitable eukaryotic
cells, where they control the electrical potential across the cell
membrane, secretion, and signal transduction (1). Electrophysiological
studies have allowed identification and characterization of a great
variety of ion channels, which are differentiated first by their
selectivity and then by their other biophysical properties, their
pharmacology, and their regulation (2). Recent cloning efforts and
analysis of structure-function relationships have now provided a
molecular basis for many of the biophysical properties. Thus, the
mechanism that determines ion channel selectivity is now fairly well
understood for K+ channels (3). K+ selectivity
is carried by a structural element called P domain (P for
pore-forming). This domain is highly conserved among the three main
structural classes of K+ channel subunits with
six-transmembrane, four-transmembrane, or two-transmembrane domains
(4).
The most recently described family of K+ channels has four
transmembrane domains (5-10). Members of this family have two
pore-forming domains (P1 and P2), an extended extracellular loop
between the M1 and P1 domains with a conserved cysteine residue (except
for TASK-1), and a short cytoplasmic N-terminal extremity. TWIK-1 has
been shown to form covalent homodimers (8, 11). Despite a relatively
low sequence similarity and different functional properties, these
two-P domain K+ channels all produce quasi-instantaneous
and noninactivating currents, although the TASK-2 currents displays
relatively slow activation kinetics. These new K+ channels
are presently classified into three distinct functional subfamilies.
TASK-1 and TASK-2 are background K+ channels sensitive to
small external pH variations near physiological pH (9, 10, 12). TREK-1
(13) and TRAAK (6, 14) are arachidonic acid-activated mechanosensitive
K+ channels; however, TREK-1 is inhibited by both protein
kinase A and protein kinase C (5), while TRAAK is not. TWIK-1 is a weakly inward rectifying K+ channel that is stimulated by
PKC and inhibited by internal acidification (7, 8). The different types
of regulations indicate that these K+ channels are probably
involved in a great diversity of physiological and pathophysiological roles.
More than 40 genes potentially encoding two-P domain K+
channel subunits have now been identified in the Caenorhabditis
elegans genome (15). This observation suggests that a large number
of two-P domain channels might also exist in mammals. The present paper
describes the cloning of two novel mammalian two-P domain channels
related to TWIK-1, one of mouse and the other of human origin.
Cloning of KCNK6 and KCNK7--
A BLAST search using the TWIK
K+ channel led to the identification of mouse and human
expressed sequence tags
(ESTs1; accession numbers
W18545 and AA777982). In order to characterize the corresponding
full-length cDNAs, called KCNK6 and KCNK7, 5'- and 3'-rapid
amplification of cDNA ends (RACE) were performed on adult mouse or
human brain cDNAs ligated with adapters as previously (16).
Two antisense primers for 5'-RACE
(5'-CTCCTCCAGGGCCTCAGGTGGCAA-3'/5'-GGCCATAAGCAGGAGCAGGTAT-3' specific
to KCNK6 and 5'-AGGCCTTTCCGCCTGGCGAT-3'/5'-GGAAGGTCCCAGGTCCTGCC-3' specific to KCNK7) and two sense primers for 3'-RACE
(5'-TGAGAGCACAGGCCCATGGAGTTT-3'/5'-GCATCCTCACCACCACCGGTTA- 3' specific
to KCNK6 and 5-CTGCGCTGCTGCAGGCAGTT-3'/5'-GCTGGTGCTGTGGGGCCTT-3' specific to KCNK7) were derived from ESTs W18545 and AA777982. Two
successive RACE reactions were performed with a mixture of Taq (Life Technologies, Inc.) and Pwo (Boehringer
Mannheim) DNA polymerases by using anchor primers
5'-TAGAATCGAGGTCGACGGTATC-3' and 5'-GATTTAGGTGACACTATAGAATCGA-3'. The
amplified products were subcloned into pBluescript (Stratagene), and
three clones of each products were sequenced on both strands (Applied
Biosystems model 373A). Primers flanking the KCNK6 and KCNK7-A coding
sequences (KCNK6, sense, 5'-CCGGAATTCGCCACCATGGGGAGTCTGAAACC-3', and
antisense, 5'-CGCGGATCCTCAAGGTCCCCCAACACGATC-3'; KCNK7-A, sense,
5'-CCGGAATTCGCCGCCATGGGGGGTCTAAGG-3', and antisense,
5'-CGCGGATCCTCAGCAAGCAGGGGCTTGT-3') and containing EcoRI or
BamHI restriction sites were used to amplify the coding sequences from mouse or human brain cDNA with a low error rate DNA
polymerase (Pwo DNA polymerase, Boehringer Mannheim). The PCR products were digested with EcoRI and BamHI
and subcloned into the pIRES-CD8 expression vector (6) and sequenced.
To exclude sequences errors due to the PCR, products issued from independent PCR experiments were subcloned and sequenced. In the same way, the coding sequences of both splicing forms KCNK7-B and
KCNK7-C were amplified with the KCNK7-A sense primer and with the
antisense primers: KCNK7-B, 5'-CGCGGATCCCTACCCCTCCCACGCCGT-3', and
KCNK7-C, 5'-CGCGGATCCTCCTAGTCCAGGCTCCTCTTC-3'.
Analysis of KCNK7 Exon Skipping--
Primers flanking the
variable region (primer F, 5'-CGTCTACTTCTGCTTCAGCTCG-3'; primer R,
5'-TCAGCAAGCAGGGGCTTGT-3') were used to amplify the different spliced
mRNA forms from human brain cDNA (10 ng) with the low error
rate DNA polymerase (Pwo). Fragments were separated by
agarose gel (2.5%) electrophoresis, and analysis was carried out by
Southern blot with two 32P-labeled oligonucleotides probes
(probe 1, 5'-CTACCCCTCCCACGCCGT-3'; probe 2, 5'-GTGCTGAGCGAGCTGAAG-3').
These fragments were subcloned for sequencing.
Analysis of KCNK6 and KCNK7 mRNA Distribution--
For
Northern blot analysis, mouse and human multiple-tissue Northern blots
(CLONTECH) were probed at 65 or 68 °C in
ExpressHyb solution with the 32P-labeled coding sequences
of KCNK6 or KCNK7 following the manufacturer's protocol. For reverse
transcription-PCR, total RNAs were extracted from mouse tissues with
the SNAP total RNA isolation kit (Invitrogen) and treated with DNase I. Fifteen µg of total RNA were reverse-transcribed following the
manufacturer's instructions (Life Technologies), and In Situ Hybridization--
Adult Swiss mice were killed by
a transcardial perfusion with 0.9% NaCl followed by ice-cold 4%
paraformaldehyde/phosphate-buffered saline (PBS, pH 7.4). The brain and
retina were postfixed in an ice-cold 4% (w/v) paraformaldehyde/PBS
solution for 3 h and then immersed overnight at 4 °C in a 20%
sucrose/PBS solution. Frozen sections (10 µm) were collected on
3-aminopropylethoxysilane-coated slides and stored at Production and Characterization of Anti-KCNK6
Antibodies--
DNA fragments coding for regions extending from
Gln29 to Asn89 and from Glu254 to
Pro335 were amplified by PCR and subcloned behind the
glutathione S-transferase (GST) coding sequence into the
pGEX3x plasmid. The resulting GST-KCNK6 fusion proteins were produced
in the BL21 (DE3 pLysS) Escherichia coli strain and purified
on glutathione-Sepharose according to the manufacturer's protocol
(Amersham Pharmacia Biotech). Antibodies were then raised in New
Zealand female rabbits immunized with a mixture containing 300 µg of
purified fusion protein in the presence of complete Freund's adjuvant
and then boosted every 4 weeks first with 150 µg of each protein in
incomplete Freund's adjuvant. For purification, the antisera was first
depleted of the anti-GST antibodies by absorption to nitrocellulose
strips saturated with the GST, followed by affinity purification of the anti-KCNK6 polyclonal antibodies against nitrocellulose strips saturated with the immunogen as described previously (18).
Western Blot--
For the preparation of microsomes, transfected
COS-7 cells (see below for the transfection) were washed, sonicated in
lysis buffer (140 mM NaCl, 20 mM Tris, pH 7.4, 1 mM phenylmethylsulfonyl fluoride, 10 mM
iodoacetamide, 2 mM EDTA), and the lysate was cleared by
centrifugation at 1000 × g for 10 min. The supernatant was then centrifuged at 100,000 × g for 20 min to
obtain microsomes. 0.5-1 µg were then loaded on a SDS-polyacrylamide
gel electrophoresis (9% gel) and transferred onto nitrocellulose
membrane (Hybond-C extra, Amersham Pharmacia Biotech). Blots were
saturated with 4% low fat dry milk in PBS and incubated 1 h with
affinity-purified anti-KCNK6 antibodies or M2 anti-tag monoclonal
antibodies (Eastman Kodak Co.) diluted 500-fold, followed by an
additional 1-h incubation with horseradish peroxidase-conjugated goat
anti-rabbit IgG or goat anti-mouse IgG (Jackson) with extensive washing
steps after antibody incubation, and finally revealed with Super Signal (Pierce).
Indirect Immunofluorescence on Transfected COS-7
Cells--
Transfected COS-7 cells were plated on glass coverslips
onto 15-mm Petri dishes. 24 h after plating, the cells were fixed for 15 min with a 4% (v/v) paraformaldehyde/PBS solution, rinsed with
PBS, and then permeabilized by incubation for 10 min with 0.1% Triton
X-100 (without Triton for nonpermeabilized conditions). Nonspecific
binding was eliminated by a 2-h incubation with 5% goat serum, 2%
bovine serum albumin in PBS at room temperature. The cells were then
incubated for 1 h with affinity-purified anti-KCNK6 antibody
(1:400) or anti-TRAAK antibody
(1:400)2 in 2% bovine serum
albumin/PBS, followed by washing with PBS and incubation for 1 h
with tetramethylrhodamine isothiocyanate-conjugated goat anti-rabbit Ig
(1:200; Sigma) or fluorescein isothiocyanate-conjugated goat
anti-rabbit Ig (1:200, Sigma). After washing in PBS and finally in 10 mM Tris-HCl, pH 7.5, cells were mounted in Vectashield
medium (Vector Laboratories, Inc.) and observed with a Leitz Aristoplan microscope (Wild Leitz) using an interference blue (fluorescein isothiocyanate) or green (rhodamine) filter and a × 40 lens.
Immunohistochemistry on Mouse Retina--
The eyes were
enucleated and immediately fixed with ice-cold 4% paraformaldehyde/PBS
for 4 h. The lens and the vitreous were removed, and the eye-cup
was cryoprotected in PBS containing 20% sucrose. Frozen sections (7 µm) were cut on a Leica cryostat and stored at Construction of N-terminally Tagged KCNK6 and KCNK6
Mutants--
The coding sequence of KCNK6 was subcloned into the
HpaI-NotI restriction sites of Flag-pRc/CMV
vector to create an N-terminally tagged KCNK6 protein as described
previously for the N-terminally tagged Kv2.2 protein (19). Deletion
channels and chimeras between KCNK6, mTREK1, and hTASK1 were
constructed by PCR as described (20). PCRs were performed on
Flag-pRc/CMVKCNK6 to keep the Flag peptide sequence at the amino
extremity of the constructions. Products were subcloned into
EcoRI-BamHI site of pIRES-CD8 vector and
verified by sequencing.
Cell Culture and Transfection--
COS-7 cells were grown in
Dulbecco's modified Eagle's medium (Life Technologies, Inc.)
supplemented with 10% fetal calf serum and antibiotics (60 mg/ml
penicillin, 50 mg/ml streptomycin). One day before transfection,
20 × 103 cells for electrophysiology experiment or
70 × 103 cells for immunofluorescence microscopy were
plated on cover glasses onto 35-mm Petri dishes. The cells were
transfected by the classical DEAE-dextran/chloroquine method using 1 or
2 µg of supercoiled DNA.
Human Chromosomal Mapping of KCNK7--
Gene mapping was
performed by PCR on the Genebridge 4 radiation mapping panel (Research
Genetics, Inc.) with the following primers: sense,
5'-CATGGCCCCACTATCGC-3'; antisense, 5'-CTGCAGTCGCCCTGAAGG-3'. The
results were analyzed by using the RHMAPPER program at the Whitehead
Institute with a logarithm of odds score of 21.
Amino Acid Sequence Alignments and Dendrogram--
The alignment
was generated with the ClustalW multiple sequence analysis program. The
dendrogram and the percentage of identity were deduced from the
conserved region extending from the M1 to the M4 transmembrane domains.
Accession numbers of the channel aligned are as follows: mTWIK,
AF033017; mTRAAK, AF056492; hTASK-1, AF006823; hTASK-2, AF084830;
mTREK1, U73488.
Cloning of KCNK6, a New Ion Channel Subunit--
The TWIK-1
sequence (7) was used to search related sequences in the
GenbankTM data base by using the Blast alignment program. A
mouse EST (accession number W18545) was identified encoding a portion
of a new Tissue Distribution of KCNK6--
In order to investigate KCNK6
mRNA expression, a Northern blot analysis was carried out using a
mouse multiple-tissue Northern blot from CLONTECH.
No KCNK6 transcripts were detected, indicating that KCNK6 is not
expressed or is only expressed at very low levels in the tested tissues
(heart, brain, spleen, lung, liver, skeletal muscle, kidney, and
testis). The expression of KCNK6 was further studied on a larger panel
of tissues with a more sensitive technique, reverse transcription-PCR
(Fig. 2A). KCNK6 mRNA was
detected in embryo (16-18 days), in eye, stomach, and lung. A weak
expression was found in colon and testis. In the other tissues, the
expression of KCNK6 mRNA was very low (atria, kidney, intestine,
bladder, uterus, ovary, salivary gland, thymus, and brain stem) or not detectable (brain, cerebellum, spinal cord, heart, ventricle, skeletal
muscle, liver, placenta, pancreas). In situ hybridization was performed on a vertical section of mouse retina. An antisense KCNK6
cRNA probe revealed an intense expression with a distinct stratification pattern. High labeling was apparent in the retinal ganglion cell layer (RGC) and in the inner nuclear layer
(INL) composed of nuclei of Müller cells and amacrine,
bipolar, and horizontal neurons (Fig. 2B). The distribution
of the hybridization signal suggests a neuronal localization of
transcripts. The control sense probe did not show significant
hybridization (data not shown). To confirm these results,
immunohistochemistry was performed on an equivalent section. The KCNK6
protein was immunodetected in the same neurons. A strong
immunoreactivity was observed in the retinal ganglion cell layer. In
addition, regular punctual staining was detected in a specific neuron
population of the distal area of the inner nuclear layer (Fig.
2C). To demonstrate the specificity of anti-KCNK6
antibodies, an equivalent section was incubated with anti-KCNK6
antiserum, which was previously absorbed with the GST-KCNK6 fusion
proteins. As expected, the staining was prevented (data not shown).
In situ hybridization and immunohistochemistry experiments
did not show significant detection in the brain (data not shown).
Heterologous Expression of KCNK6
Subunits--
Electrophysiological attempts to record KCNK6 channel
activity in various cell types like Xenopus oocytes, COS-7,
HEK293, and Sf9 cells were unsuccessful. In all of these
expression systems, attempts to activate channel activity with 1 µM Ca2+ at the intracellular side (because of
the presence of the EF-hand domain), with external pH variations from
pH 7.0 to 4.0, or with application of a phorbol ester (phorbol
12-myristate 13-acetate) (there is one potential protein kinase C site
(Fig. 1A)) were without any success. We also tried a
coexpression with the
Western blot analysis was then carried out to verify KCNK6 expression
in transfected cells. Under nonreducing conditions, anti-tag antibodies
(M2) detected a major band at a molecular mass of 83 kDa from COS-7
cells transfected with a tagged form of KCNK6. Under reducing
conditions (i.e. in the presence of Intracellular Retention of KCNK6--
One possible reason for the
lack of channel activity could of course be an intracellular
localization of the expressed KCNK6 protein. To test this point, the
KCNK6 subunit was localized by indirect immunofluorescence with the
anti-KCNK6 antibodies (Fig. 3C) on Triton-permeabilized
COS-7 cells. A strong fluorescence staining was detected at the
perinuclear region as well as in a fine reticular network extending
through the cytoplasm. This pattern was observed in all of the
KCNK6-expressing cells from multiple independent transfections. This
pattern is similar to that seen with known ER markers (27), suggesting
that KCNK6 is specifically sequestered in the ER and cannot reach the
plasma membrane. This pattern of labeling is very different from that obtained with the TRAAK channel. As shown in Fig. 3D,
TRAAK-expressing cells, which produce strong K+ currents
(6), show a typical surface labeling.
Why Is KCNK6 Unable to Reach the Plasma Membrane?--
The first
reason why KCNK6 cannot reach the surface membrane could reside in the
peptide sequence itself. Protein retention in intracellular
compartments is usually determined either by the presence of short
transmembrane domains (27-30) or by the presence of specific
cytoplasmic sequences. These are the ER retrieval signals (KDEL, KK, or
RR (31)), the peptide signals targeting to post-Golgi compartments (LL
or YXXO, where O is an amino acid with a bulky hydrophobic
group (32)), the peptide signals targeting to the trans-Golgi network
(SXYQRL (33), AYRV (34), or SDSEEDE (35, 36)), the signals
involved in endosomal trafficking (YKGL (35, 36)), or the endocytosis
signal (DAKTI or DAKSS (37)). The transmembrane domains of the KCNK6
protein present the requisite length to reach the plasma membrane as
for the other functional two-P domain K+ channels. Then a
possible retention signal was searched on the cytoplasmic regions of
the KCNK6 subunit. A particular sequence was found in the cytoplasmic
tail between Ser274 and Asp282. This sequence
SCKIIRTCKIIDEDQD is similar to a signal
required for trans-Golgi network localization,
SCKIIDSCKIIEEDE (CKII indicates amino acids
phosphorylated by casein kinase II) (35). This specific localization
requires a cluster of acidic amino acids and a pair of amino acids
phosphorylated by casein kinase II (38). Interestingly, this KCNK6
region overlaps with the EF-hand domain and in particular the residues
that are expected to be involved in the coordination of
Ca2+. One possibility would then be that the function of
the putative retention signal could be eliminated by Ca2+
fixation. To test this point, an ionomycin/Ca2+ (1 µM) treatment of 5, 10, or 15 min at 37 °C was
performed on transfected COS-7 cells, as described previously (39).
Unfortunately, indirect immunofluorescence microscopy with
anti-KCNK6 antibodies did not show any plasma membrane staining on
cells having undergone this treatment (not shown). In addition,
suppression of this potential retention signal in the KCNK6 Cloning of KCNK7, a Human Subunit Closely Related to
KCNK6--
Since KCNK6 was cloned from mice and did not produce a
functional channel, we then decided to clone a homologue of KCNK6 in humans in order to verify whether changing animal species would lead to
functional expression. A Blast search was performed on human EST data
bases with the KCNK6 peptide sequence. Two sequences (EST accession
numbers AA777982 and N39619) were identified corresponding to the
P1-M2-M3-P2 region of the two-P domain channel subunits and were very
close to KCNK6. The P2 domain showed a very unconventional sequence GLE
instead of GLG found in TWIK-1 and KCNK6 pore domains. As for KCNK6,
5'- and 3'-RACE reactions allowed the identification of a start codon
preceded by several stop codons. Interestingly, two 3'-RACE products
were found containing the poly(A) tail. Sequencing revealed that both
fragments are produced by splicing of a unique mRNA. Further
analyses, detailed below, revealed that exon skipping gives rise to
five spliced mRNAs. These five mRNAs code for three protein
forms, a subunit called KCNK7-A and two shorter variants called KCNK7-B
and KCNK7-C with truncated cytoplasmic carboxyl termini. PCR was used
to clone the complete coding sequences of KCNK7-A, KCNK7-B, and KCNK7-C from human brain cDNA. The independent cloning of these three forms
confirmed sequences, and especially the peculiar sequence GLE found in
the P2 domain. The KCNK7-A, KCNK7-B, and KCNK7-C cDNA have open
reading frames of 921, 756, and 771 base pairs and code for proteins of
307, 252, and 257 amino acids (Fig. 5) with a calculated molecular mass of 32, 26, and 27 kDa, respectively. KCNK7-A has the hallmarks of a functional subunit of two-P domain potassium channels. KCNK7-A shows 80 and 42% amino acid identity, and
94 and 64% homology with the core region of KCNK6 and TWIK-1, respectively (Fig. 1C). As expected, KCNK7-A is very close
to KCNK6 (Fig. 5). Several sites are conserved between both subunits such as the consensus sites for N-linked glycosylation
(residue 82) and the two sites for casein kinase II (residues 255 and
276). The short C-terminal tail of KCNK7-A also presents two potential Src homology 3 binding motifs. However, the EF-hand domain found in
KCNK6 is not conserved. A site for Ca2+ calmodulin kinase
(residue 8) at the cytoplasmic N-terminal tail could confer to KCNK7 an
indirect regulation by Ca2+. The spliced forms, KCNK7-B and
KCNK7-C, have shorter cytoplasmic carboxyl termini (Fig. 5). The M4
domain of KCNK7-B and KCNK7-C presents an abnormally short
As previously indicated, the analysis of the variable 3' region by
reverse transcription-PCR led us to the identification of five KCNK7
mRNA spliced forms (Fig.
6B). These different variants contain three alternative stop codons called stop-A, -B, and -C, as
depicted in Fig. 6A. Sequencing of the different splice
variants allowed us to identify the donor and acceptor splice sites
(Fig. 6D). The longest protein form (KCNK7-A) is produced as
the result of exclusion of an exon (exon skipping) containing an
in-frame stop codon. The quantification of the different forms was
performed by Southern blot and revealed that mRNAs coding for
KCNK7-A, KCNK7-B, and KCNK7-C subunits are in proportions of 46, 39, and 8%, respectively (Fig. 6C). We cannot distinguish
between KCNK7-B and KCNK7-C for the last 8% (Fig. 6, B and
C). KCNK7 is expressed in the brain.
As for KCNK6, neither human KCNK7 nor its spliced forms are able to
generate channel activity in transfected COS-7 cells. Attempts to
record currents in cells transfected with all possible combinations of
two of the KCNK6 and KCNK7-A, -B, and -C subunits also failed to
produce any ion current.
Gene assignment by radiation hybrid panel mapping localized the human
KCNK7 channel gene to chromosome 11q13 at 6.4 cRays from the
framework marker WI-1409 (logarithm of odds score >21) (Fig.
7).
This paper reports the cloning of KCNK6 (mouse) and KCNK7 (human)
subunits, which are structurally related to the growing family of
K+ channels with four TMDs and two P domains (5-10). They
are more particularly related to the TWIK-1 channel (40 and 42% amino
acid identity, respectively). The previously cloned channels of this family all produce instantaneous and noninactivating currents, except
TASK-2, which displays a relatively slow kinetic of activation. They
are open at resting potential and are able to drive the membrane potential near the K+ equilibrium potential. There are
three distinct functional subfamilies of two-P domain channels. TASK-1
and TASK-2 are background K+ channels sensitive to external
pH variations near physiological pH (9, 10). Both TREK-1 (13) and TRAAK
(6, 14) produce arachidonic acid-activated and mechanosensitive
K+ currents, and TREK-1 but not TRAAK is inhibited by both
protein kinase A and C (5). TWIK-1 is a weakly inward rectifying
K+ channel that is stimulated by protein kinase C
and inhibited by internal acidification (8). These
K+ channels probably contribute in a major way to
the regulation of the resting membrane potential and are
probably endowed with a diversity of physiological roles.
Voltage-sensitive Ca2+-dependent
K+ channels have been identified in many cell
types and have now been cloned (40). Background Ca2+-activated K+ channels have also been
recorded, but they have not yet been cloned in mammals. They are
particularly well expressed in sensory neurons of the nodose ganglion,
where they contribute to the lack of spontaneous activity observed in
these cells (41). Background Ca2+-activated K+
channels have been cloned in plant cells and correspond to a two-P
domain K+ channel, which is activated by cytosolic
Ca2+ via two EF-hand domains (42). A reasonable assumption
would then be that the mouse channel subunit KCNK6, which comprises an
EF-hand domain, would also be a background channel regulated by
internal Ca2+. Sequences of the human subunits KCNK7-A, -B,
and -C do not contain an EF-hand domain, but an indirect
Ca2+ regulation would also be possible in that case through
the Ca2+-calmodulin kinase site.
Electrophysiological experiments have revealed that neither KCNK6 nor
KCNK7 can generate channel activity by themselves in Xenopus
oocytes or transfected COS-7 cells. Reasons for this lack of expression
have been particularly analyzed for KCNK6. Biochemical experiments
showed KCNK6 protein expression and, as for TWIK-1 (11), subunit
dimerization in transfected cells, indicating that the absence of
channel activity was not due to a lack of expression or dimerization,
or to a degradation of the protein. Immunocytolocalization on
transfected COS-7 cells has shown that the absence of current is due to
the fact that KCNK6 is unable to reach the plasma membrane. A first
interpretation of this result would be that KCNK6 is an intracellular
channel. If this were the case, then the putative K+
channel subunit would be expected to carry one of the signals for
intracellular retention on its cytoplasmic regions. One such possible
signal is indeed observed in a KCNK6 region that overlaps with the
EF-hand region. However, suppression of this hypothetical retention
signal by deletion did not confer access to the plasma membrane.
Chimeras formed with KCNK6 and cytoplasmic elements of TREK1 or TASK1
channels (which have an easy access to the cell surface) also failed to
reach the plasma membrane and to form electrophysiologically recordable
channels. Another possibility, which is at present the most probable
one, would be that KCNK6 needs a partner to reach the plasma membrane.
However, this partner remains to be identified, and is not present in
the heterologous expression systems used. An association between
partner channel subunits essential for the expression of channel
activity has been observed with G-protein-gated inward rectifiers (43,
44), with some of the voltage-sensitive K+ channels (17,
19, 45-47), or with epithelial Na+ channels (48-50). The
required association can also take place between channel subunits and
other types of subunits, themselves acting as chaperones (51, 52) or
carrying other types of functions such as for the ATP-sensitive
K+ channels (53, 54) or for the channel generating the I-Ks cardiac K+ current (55-57).
KCNK6 is mainly expressed in tissues such as eyes, lung, and stomach.
It is also significantly expressed in the embryo. The highest level of
expression is found in the eyes, where in situ hybridization
and immunohistochemistry showed that KCNK6 is only expressed in
ganglion cells and in some neurons of the inner nuclear layer. This
very restricted localization probably explains the absence of detection
by Northern blot. In the mammalian retina, the first spontaneous
Ca2+ waves are observed at postnatal day 2 and are thought
to result from Ca2+ influx associated with burst of action
potentials seen in ganglion cells at this developmental time (58-60).
The early appearance of the KCNK6 channel in development, the fact that
it has a Ca2+-binding domain probably conferring
Ca2+ sensor properties, and its selective expression in
ganglion cells suggest that this channel could play a role in the
modulation of the electrical signal in the retina.
The human channel subunit KCNK7 is very close in structure from KCNK6
(80% of identity on the core channel), but it does not contain an
EF-hand domain. KCNK7 RNA is alternatively spliced by an exon skipping
mechanism, which generates five different mRNAs in the human brain.
These five mRNAs present three alternative stop codons (named A, B,
and C) and code for three subunits named KCNK7-A, -B, and -C. KCNK7-B
and -C subunits are truncated in the C-terminal sequence. The mRNA
coding for these two short forms represent about 50% of the total
KCNK7 mRNA. Interestingly, KCNK7-B and -C have a very short M4
domain with a potential anchor at the C-terminal extremity
(myristoylation site and amidation site, in KCNK7-B and KCNK7-C,
respectively). Short TMDs are considered as important factors for the
ER retention (27, 28, 61). Plasma membrane proteins generally have
longer TMDs than Golgi membrane proteins (62). Therefore, the short M4
domain of KCNK7-B and -C could confer an intracellular localization.
The structures of the second P region (P2) of the different mammalian
two-P domain channels are compared in Fig.
8. An important element of the signature
of K+ channel function has long been recognized as being
the P domain GYG sequence (63). This sequence is found in most
voltage-sensitive, Ca2+-sensitive, and inward rectifier
K+ channels (3, 64, 65). In the two-P domain channels, the canonical GYG structure is replaced by a GFG (TREK1, TASK1, TASK2, and
TRAAK) or by a GLG (TWIK1 and KCNK6). A tryptophan residue (Trp213 in TWIK-1) is conserved in TWIK1, TREK1, TRAAK1,
TASK1, and TASK2, but it is replaced by a cysteine residue in KCNK6 and
KCNK7. In addition, two adjacent residues, a tyrosine and a valine
(Tyr234-Val235 in TWIK-1), which are also
present in all other members of the family, are replaced by a
leucine-leucine sequence (Leu221-Leu222 in
KCNK6) in KCNK6 and KCNK7. KCNK7 is unique, since a glutamic acid
residue (Glu219 in KCNK7) is found instead of the strictly
conserved glycine residue that is present in the filter part of all of
the other members of the K+ channel family. Moreover, a
negatively charged residue (Asp or Glu residues in the different
members of this family) is always found at a 13-amino acid distance of
the G(F/L)G sequence filter but is replaced by a glycine
(Gly204) in KCNK7. Interestingly, the comparison with the
recently published tertiary structure of the selectivity filter of the
Kcsa K+ channel (3) reveals that the side chains of the
amino acids found in the ion filter region, Glu219 and
Leu221-Leu222 in KCNK7, are close in space to
the side chains of residues observed in the pore helix region,
Gly204 and Cys200, respectively (see model in
Fig. 8), suggesting an adaptation of the pore structure.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
of
each sample was used as template for PCR amplification (Taq
DNA polymerase, Life Technologies) using KCNK6 (nucleotides
12-31, 5'-GAAACCATGGGCCCGATACC-3', and 366-385,
5'-CAAGGGCTGCATAGACCACA-3') or glyceraldehyde-3-phosphate dehydrogenase
(CLONTECH) primers. KCNK6-amplified fragments were transferred onto nylon membranes and then probed with a
32P-labeled internal primer (bases 302-323,
5'-GCATCCTCACCACCACCGGTTA-3').
20 °C.
Specific antisense cRNA probes were generated with T7-RNA polymerase
(Boehringer), by in vitro transcription using
[33P]D-UTP (3000 Ci/mmol, ICN
Radiochemicals), from a BamHI-linearized pBluescript
SK-plasmid containing an 800-base pair fragment from the
3'-untranslated sequence of KCNK6. Sections were treated using standard
procedures (17). For control experiments, adjacent sections were
hybridized with a corresponding sense riboprobe or digested with RNase
before hybridization.
70 °C. For each
retina, three sections were placed on 3-aminopropylethoxysilane-coated
slides. Frozen sections were permeabilized with 0.3% Tween 20 in PBS
for 3.5 min at room temperature followed by two rinses in PBS. Sections
were then incubated with a blocking solution (1% goat serum/PBS) for
5 h, and then the primary antibody (1:400) was added overnight.
After three rinses in PBS, the second goat anti-rabbit IgG biotinylated
antibody (1:330) was incubated for 1 h and amplified with the
Vectastain Elite ABC kit from Vector Laboratories. The antigen-antibody
complexes were visualized by 3,3'-diaminobenzidine staining using the
Vector Laboratories Kit. Control for the specificity of KCNK6
immunostaining was done by preabsorption of the primary antibodies
(1:400) with the GST-KCNK6 fusion protein. All sections were washed
three times in PBS and one time in distilled water and then mounted
with Entellan (Merck).
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-subunit (named KCNK6). The corresponding full-length
cDNA was obtained by RACE and sequenced. The coding sequence was
then reamplified from both mouse brain and lung cDNAs to verify the
sequence and to exclude all possible PCR errors. The KCNK6 cDNA
contains an open reading frame of 1008 base pairs and codes for a
protein of 335 amino acids (Fig.
1A) with a calculated
molecular mass of 35 kDa. KCNK6 is more related to TWIK-1 than to the
other two-P domain K+ channels (40% of amino acid identity
(Fig. 1C)). Like all of these channels, KCNK6 displays four
potential transmembrane segments (M1-M4) and two P domains (P1 and P2,
P for pore-forming domain) (Fig. 1, A and B).
Another important characteristic is the presence of an extended
extracellular loop between M1 and P1 that contains a cysteine residue
at a position (Cys57) analogous to the cysteine residue
(Cys69) involved in the disulfide-bridged homodimerization
of TWIK-1 (11). The protein sequence of KCNK6 contains consensus sites for N-linked glycosylation (residue 82) and phosphorylation
by protein kinase C (residue 3) and casein kinase II (residues 255, 274, 276, 308, and 327). Three potential Src homology 3 binding motifs
are present in the C-terminal part of the protein (minimal consensus
sequence PXXP (21, 22)). Computer-based analysis also
indicates the very interesting presence of a potential
Ca2+-binding site (EF-hand motif) at its C terminus (Fig.
1D). This region includes the complete EF-hand loop as well
as the first residue that follows the loop, which is always
hydrophobic. Moreover, as in a classical EF-hand domain (23-25), the
12 residues loop of this region of KCNK6 are flanked on both sides by
-helical structures (nnPredict program; data not shown).
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Fig. 1.
A, alignment of the mouse amino acid
sequences of KCNK6 (accession number AF110521) with mouse TWIK, TREK,
TRAAK, and human TASK-1. The four transmembrane segments, M1-M4, and
the two pore-forming regions P1 and P2 are underlined.
Potential cytoplasmic sites for protein kinase C ( ) and casein
kinase II (
) are shown. On the cytoplasmic C terminus, potential Src
homology 3 binding motif (PXXP) and EF-hand
Ca2+-binding domain are underlined. The
conserved N-glycosylation site (Y) on the
extracellular loop M1-P1 are shown. Identical residues are enclosed in
solid boxes, while conservatively related residues are in
shaded boxes. B, topological model for KCNK6 deduces from
hydropathy analysis. As in A, the potential sites and
putative domains are indicated on topological model. C,
proposed dendrogram for the extended two-P domain K+
channel family. The percentage indicates the identity between the
-subunits. D, details of the consensus pattern of EF-hand
Ca2+-binding domain (identified with the Prosite program)
and alignment of this EF-hand motif of KCNK6 with the EF-hands from
calmodulin from Pneumocystis carinii (CaM), with L-type
Ca2+ channel (L-Type CaC), and with the two
EF-hands of outward rectifying two-P domain K+ channel from
Arabidopsis thaliana (KCO1-EF1 and
KCO1-EF2). The residues denoted by
X, Y, Z,
Y,
X, and
Z are involved in coordinating
Ca2+. X, any amino acid; braces,
excluded amino acid; brackets, conserved amino acid.
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Fig. 2.
A, tissue distribution analysis of KCNK6
mRNAs by reverse transcription-PCR in mouse. B, in
situ hybridization dark field photomicrograph; C,
immunohistolocalization on a vertical section of mouse retina.
INL and ONL, inner and outer nuclear layers;
IPL, inner plexiform layer; RGC, retinal ganglion
cell layer. The arrows indicate regular punctual staining in
the distal region of INL. Scale bar,
80 µm.
1
2 subunits of G
proteins, which lead to activation of G-protein-gated inward rectifier
(26), as well as coexpression with the closely related two-P domain
subunit TWIK-1, but again without success. Constructions with pCI and
pRc/CMV expression vectors were also tested with the same negative results.
-mercaptoethanol), two bands were detected with molecular mass of 44 and 40 kDa. A similar
blot profile was revealed using affinity-purified anti-KCNK6 antibodies
(Fig. 3B). In order to
demonstrate the specificity of the anti-KCNK6 antiserum and to justify
its use to analyze the expression of the KCNK6 protein in transfected
cells, we checked that no signal was obtained from control COS-7 cells
expressing the TWIK-1 protein. This expression of the human TWIK-1
protein was verified in the same experiment with the affinity-purified rabbit antibodies directed against the C-terminal part of TWIK-1 (11)
(data not shown). The masses of 37 and 40 kDa are in good agreement
with the theoretical values of 35 and 37 kDa for KCNK6 and its tagged
form, respectively. As for the TWIK-1 subunit (11), the observation
that the band moving at 77 kDa gives rise to a band moving at 37 kDa in
the presence of a reducing agent strongly suggests that KCNK6 can
self-associate via a disulfide bond to form a homodimer. The masses of
41 and 44 kDa for KCNK6 and its tagged form suggest, as previously
demonstrated for TWIK-1 (11), the existence of glycosylated forms. All
of these results were reproduced in Sf9 cells (data not
shown).
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Fig. 3.
A and B, microsomal proteins
of TWIK-1-, N-terminally tagged KCNK6-, or KCNK6-expressed COS-7 cells
were analyzed in nonreducing ( ME:
) or reducing
(
-ME: +) conditions. Immunodetection was performed either
with the commercial mouse anti-Tag antibody M2 (A) or with
the rabbit anti-KCNK6 antiserum (B). C-E,
immunofluorescence localization of the KCNK6 subunit on permeabilized
COS-7 cells. Immunocytolo- calization was realized on cells
expressing native KCNK6 subunit (C) with anti-KCNK6
polyclonal antibodies. For comparison, D shows the plasma
membrane expression of the two-P domain K+ channel TRAAK.
Negative controls for anti-KCNK6 polyclonal antibodies (E)
were performed on cells expressing TWIK-1 subunit. Anti-KCNK6
antibodies were visualized using a goat anti-rabbit
IgG-tetramethylrhodamine isothiocyanate-conjugated secondary
antibody (red de- tection), and anti-TRAAK antibodies
were detected using a goat anti-rabbit IgG-fluorescein
isothiocyanate-conjugated secondary antibody (green
detection).
C274/296
deletion mutant (Fig. 4) also failed to
create access of KCNK6 to the plasma membrane. The immunolocalization
of this mutant displays a pattern identical to that of the wild type
subunit, and no current could be recorded in electrophysiological
experiments. Other deletion mutants (KCNK6
C296, KCNK6
C282,
KCNK6
C273) corresponding to the C terminus domain, where retention
signals are generally localized, were constructed and expressed (Fig.
4). Again, the intracellular localization of these mutants was the same
as for native KCNK6, and these mutants were electrophysiologically
inactive. In order to suppress all possible retention signals on the
KCNK6 cytoplasmic parts, chimeras have been prepared between KCNK6 and
intracellular parts of the two-P domain TASK1 or TREK1 channels, which
by themselves reach the plasma membrane (5, 9). The KCNK6/TASK-C and
KCNK6/TREK-loop chimeras presented in Fig. 4 were produced and
expressed. Again, the same characteristic ER lacy pattern was observed
by immunocytolocalization of these chimeras, and no current was
recorded. Taken together, these results show that the KCNK6 subunit
remains inside of the cell but that it is probably not specifically
expressed in the ER, since this subunit does not contain specific
retention signals. The results suggest that KCNK6 necessitates an
association with a partner to reach the plasma membrane. This possible
partner seems to be absent in the expression systems used.
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Fig. 4.
Construction of chimeras and deletion mutants
of KCNK6. A schematic diagram of wild-type, EF-hand deleted,
C-terminal truncated, and chimerical channels is shown. The sites of
deletion and the junction in the chimeras are indicated by the amino
acid position.
-helical
structure (about 16 residues as deduced from the nnPredict program and
hydropathy analyses (data not shown)) in comparison with the equivalent
domains of the other K+ channels.
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Fig. 5.
Sequence alignment of mouse KCNK6 with human
KCNK7 subunit (KCNK7-A, accession number AF110522) and its shorter
splicing forms (KCNK7-B and KCNK7-C, accession numbers AF110523 and
AF110524, respectively). In addition to the potential sites
indicated in Fig. 1A, we show the cytoplasmic sites for
Ca2+-calmodulin kinase II ( ), N-myristoylation (
),
and amidation (
). For comparison, the potential sites from KCNK6 are
also reported.
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Fig. 6.
A, characterization of the exon skipping
on the 3'-end of KCNK7 mRNA. B, agarose gel
electrophoresis of PCR products obtained with primers F and R (see
"Experimental Procedures"). This PCR was performed on human brain
cDNA with (line 1) or without reverse
transcriptase (line 3) and on human genomic DNA
(line 2). The Southern blot (C) from
line 1 is performed with probe 1 to realize a
relative quantification of the different splicing forms by scanning
(Tina program), and with probe 2 to show the skipped exon into the
smaller band. The four bands in line 1 correspond
to five splicing forms deduced from the sequencing of the different PCR
products (D).
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Fig. 7.
Schematic diagram of human chromosome 11 and
chromosomal location of human KCNK7 in the
context of the Whitehead framework map. The Genebridge 4 radiation
mapping panel (Research Genetics, Inc.) was used to map
KCNK7 at 6.4 cRays from the framework marker WI-1409 with a
logarithm of odds score for linkage of >21. Although radiation hybrid
maps are not anchored to the cytogenetic maps, the most likely location
of KCNK7 is 11q13.
DISCUSSION
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ABSTRACT
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DISCUSSION
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Fig. 8.
Top, alignment of the second pore region
of mammalian two-P domain channels. The arrows indicate the
singular amino acids found in the KCNK6 and/or KCNK7 pore domain.
Junctions between arrows indicate the regions that are
assumed to be close to each other in the tertiary structure (3).
Bottom, pore model for the two-P domain channels, KCNK6 and
KCNK7, based on the tertiary structure of the selectivity filter of the
Kcsa K+ channel (3).
There is a growing number of pathological situations associated with
mutations in diverse ion channel genes (66-75), and it would then be
not very surprising if diseases were also associated with the two-P
domain class of channel subunits. The human KCNK7 gene has been mapped
to the 11q13 region of the human genome near the markers WI-1409. Four
different diseases are associated with this genome region: the Best's
vitelliform dystrophy, which is an autosomal dominant juvenile onset
macular degeneration and associated with an electrophysiological
abnormality and a degeneration of the retinal pigment epithelium (76);
the Bardet-Biedl syndrome, which combines mental retardation, obesity,
hypogenitalism, and progressive retinal pigmentary dystrophy (77); the
insulin-dependent diabetes mellitus named IDDM4 (78); and
the cerebellar ataxia named CLA1 (79).
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ACKNOWLEDGEMENTS |
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We are grateful to M. Jodar, G. Jarretou, and F. Aguila for expert technical assistance; to V. Briet and Y. Benhamou for secretarial assistance; and to M. Ettaiche, R. Waldmann, A. Patel, E. Honoré, N. Voilley, J. De Weille, M. Fink, and J. Barhanin for fruitful discussion.
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FOOTNOTES |
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* This work was supported by 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) AF110521 (KCNK6), AF110522 (KCNK7-A), AF110523 (KCNK7-B), and AF110524 (KCNK7-C).
To whom correspondence should be addressed: Institut de
Pharmacologie Moléculaire et Cellulaire, CNRS, 660 route des
Lucioles, Sophia Antipolis, 06560 Valbonne, France. 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.
2 M. Salinas, R. Reyes, F. Lesage, M. Fosset, C. Heurteaux, G. Romey, and M. Lazdunski, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: EST, expressed sequence tag; RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; GST, glutathione S-transferase; ER, endoplasmic reticulum.
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