From Elan Pharmaceuticals,
Menlo Park, California 94025 and the § Department of
Anesthesia and Perioperative Care, School of Medicine, University of
California, San Francisco, California 94143-0542
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ABSTRACT |
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Potassium channels are found in all mammalian
cell types, and they perform many distinct functions in both excitable
and non-excitable cells. These functions are subserved by several
different families of potassium channels distinguishable by primary
sequence features as well as by physiological characteristics. Of these
families, the tandem pore domain potassium channels are a new and
distinct class, primarily distinguished by the presence of two
pore-forming domains within a single polypeptide chain. We have cloned
a new member of this family, TWIK-2, from a human brain cDNA
library. Primary sequence analysis of TWIK-2 shows that it is most
closely related to TWIK-1, especially in the pore-forming domains.
Northern blot analysis reveals the expression of TWIK-2 in all human
tissues assayed except skeletal muscle. Human TWIK-2 expressed
heterologously in Xenopus oocytes is a non-inactivating
weak inward rectifier with channel properties similar to TWIK-1.
Pharmacologically, TWIK-2 channels are distinct from TWIK-1 channels in
their response to quinidine, quinine, and barium. TWIK-2 is inhibited
by intracellular, but not extracellular, acidification. This new clone
reveals the existence of a subfamily in the tandem pore domain
potassium channel family with weak inward rectification properties.
Potassium (K+) channels are the most diverse class of
ion channels discovered. In humans over 50 distinct channels have been identified in both excitable and non-excitable cell types. These channels are involved in the control of a variety of cellular functions, including neuronal firing, neurotransmitter and hormone secretion, and cellular proliferation. All of these channels contain a
pore-forming (P)1 domain with
a sequence motif common to all K+ channels, the tripeptide
sequence G(Y/F)G, found in this domain. The residues immediately
adjacent to either side of this motif are also well conserved. It is
believed that four of these P domains contribute to the formation of a
functional K+-conductive pore (1-3).
Analysis of the K+ channels in the Caenorhabditis
elegans genome suggests eight distinct families of K+
channels (4). These channels contain 2, 4, or 6 transmembrane domains
and 1 or 2 P domains, with both termini oriented toward the cytoplasm.
The presence of other features in the primary sequence differentiates
the families into distinctive functional subtypes. For example, among
the six transmembrane domain channels, members of the Kv
channel family have a charged S4 transmembrane domain characteristic of
voltage-gated channels. K+ channels with only two
transmembrane domains surrounding a single P domain are all inward
rectifiers (Kir). Mammalian homologues of each of these classes
have been identified.
The most recently discovered class of potassium channels is the tandem
pore domain potassium (Kt) channel family. Kt channels
have four putative transmembrane domains and two P domains. The P
domains are separated by the second and third transmembrane domains.
Although all these Kt channels have a conserved core region
between M1 and M4, the amino- and carboxyl-terminal domains are quite
diverse. Kt channels represent the most abundant class of
K+ channels in C. elegans, with at least 39 distinct members (5). Four mammalian Kt channels have been
cloned to date. All the mammalian channels in this family exhibit
nearly instantaneous, non-inactivating K+ currents. The
first mammalian Kt channel discovered was the weak inward
rectifier TWIK-1 (6). Although TWIK-1 is sensitive to some classical
K+ channel blockers (e.g. Ba2+), it
is relatively insensitive to others (e.g. tetraethylammonium and 4-aminopyridine). TWIK-1 activity is indirectly inhibited by
acidification and stimulated by the phorbol ester PMA. The second pore
domain, P2, has an atypical sequence, GLG, often seen in the P2 domains
of Kt channels in C. elegans. Northern blot analysis
shows that TWIK-1 mRNA is widely expressed.
We report here the discovery of a new Kt channel cloned from
human brain. We have named this channel TWIK-2 on the basis of three
criteria. First, TWIK-2 shares significant sequence homology to TWIK-1,
especially in the P domains. Second, the tissue distribution of TWIK-2
is similar to that of TWIK-1. Finally, the TWIK-2 channel is a weak
inward rectifier whose electrophysiologic properties are similar to
those of TWIK-1.
Molecular Cloning of TWIK-2--
The basic local alignment
search tool (7) was used to identify human expressed sequence tag
entries similar to the mammalian Kt channels TWIK-1, TREK-1,
and TASK. The translation product of one of the returned ESTs,
AA604914, is most closely related to human TWIK-1 (E = 3 × 10
The sequence reported for AA604914 was used to isolate cDNA clones
using a modified solution capture method (8). Briefly, three
oligonucleotides were designed, with two unmodified oligonucleotides flanking a third, biotinylated 30-mer oligonucleotide (Keystone, Camarillo, CA). These oligonucleotides were incubated with a
NaOH-denatured human brain SuperScript cDNA library (Life
Technologies, Inc.). The solution was neutralized and the hybridization
proceeded overnight at 40° C. The annealed plasmid-biotinylated
oligonucleotide complexes were isolated with streptavidin-coated
magnetic beads (Dynal, Lake Success, NY). The plasmids were eluted from
the beads and were used to transform bacteria. The resultant bacterial
colonies were transferred onto Hybond-N filters (Amersham Pharmacia
Biotech) and probed with a radiolabeled oligonucleotide derived from
AA604914. After extensive washing in 2× SSPE, 0.5% SDS, the filters
were exposed to BioMax film (Amersham Pharmacia Biotech). Individual bacterial clones were picked from the original dish, and the plasmid was recovered from overnight minicultures. Bidirectional sequencing demonstrated an ORF that contained the nucleotide sequence found in EST
AA604914. Sequence analysis and alignments were performed with the
LaserGene suite (DNAStar, Madison, WI),
National Center for Biotechnology Information resources2
and the Prosite internet
resource.3
The TWIK-2 cDNA was subcloned into a Xenopus oocyte
expression plasmid, pOX, a generous gift from Dr. Tim Jegla (Stanford University). This vector contains the 5'- and 3'-untranslated regions
of the Xenopus Northern Blot Analysis--
A 510-base pair fragment was
amplified from the TWIK-2 sequence by the polymerase chain reaction and
was used to probe a human multiple tissue Northern blot
(CLONTECH, Palo Alto, CA). The cDNA fragment
was labeled with Transcript Preparation and Oocyte
Electrophysiology--
Transcripts were synthesized from linearized
cDNA templates with T3 RNA polymerase (mMessage mMachine; Ambion,
Austin, TX). Defolliculated Xenopus laevis oocytes were
injected with 1-15 ng of cRNA. Standard methods for oocyte preparation
and maintenance were used (10). One to four days after injection,
two-electrode voltage clamp recordings were performed at room
temperature. Voltage pulse protocols were applied from a holding
potential of
Signals were filtered with an 8-pole low-pass Bessel filter (Frequency
Devices, Haverhill, MA) set at a 20-100-Hz cut-off prior to sampling
at 40-1000 Hz. To quantify responses, leakage currents of
water-injected oocytes were averaged and subtracted from currents of
cRNA-injected oocytes. Relative response is defined as current measured
for the Primary Structure of TWIK-2--
We analyzed eight positive clones
from our solution hybridization screen. Six of the eight clones
contained inserts of 2.5-2.7 kb. Two of these positives were
sequenced. They contained an identical ORF of 939 base pairs encoding a
313-amino acid polypeptide with primary sequence features of a novel
human Kt channel (Figs. 1 and
2; GenBankTM accession number
AF117708). The gene encoded by this ORF was named TWIK-2 (see below).
The predicted molecular mass of TWIK-2 is 33,751 daltons. Analysis of
this clone reveals two upstream in-frame termination codons and an
adequate translation initiation sequence. Two distinct P domains are
located between pairs of transmembrane domains identified by hydropathy
analysis (Kyte/Doolittle). This core region is preceded by a 3-amino
acid amino-terminal domain and followed by a 57-amino acid
carboxyl-terminal domain. The arrangement of P and transmembrane
domains, coupled with the lack of an amino-terminal signal sequence and
a large domain between transmembrane domain M1 and the first P domain,
suggests a transmembrane topology identical to that of other Kt
channels.
Of the known mammalian Kt channels, TWIK-2 has structural
features most similar to TWIK-1. In TWIK-1, a specific cysteine residue
found in the M1-P1 loop (Cys-69) is implicated in the formation of
homodimers by extracellular disulfide bond formation (11). A similarly
placed cysteine is found in TWIK-2 (Cys-53). TWIK-2 has two
N-glycosylation consensus sequences (Asn-79 and Asn-85; Fig.
2), whereas TWIK-1 has a single glycosylation site in the M1-P1 loop.
Two potential phosphorylation sites have been identified in TWIK-2
(Fig. 2). Like TWIK-1, TWIK-2 has a single casein kinase II recognition
sequence (Ser-304) but lacks a Ca+2-calmodulin kinase
phosphorylation site. TWIK-2 has a potential PKC phosphorylation site
(Ser-158) that is located in the putative M2-M3 cytoplasmic loop. In
TWIK-1 this PKC site is also found in the M2-M3 loop at residue
Thr-161. We have named the new gene TWIK-2 because of its
greatest overall sequence similarity with TWIK-1 compared with other
Kt channels (53.8 versus 24-33%). In addition, 19 consecutive amino acids in its P2 domain are identical with amino acids
within the P2 domain in TWIK-1. The P1 and P2 domains of TWIK-2 are
more closely related to their counterparts in TWIK-1 (72 and 88% amino
acid identity, respectively) than any other Kt channel (Fig.
3, A and B).
Expression Pattern of TWIK-2--
To determine the expression
pattern of TWIK-2 in human tissues, a human multiple tissue Northern
blot was hybridized with a cDNA probe derived from the TWIK-2
nucleotide sequence. A 2.6-kb species, similar in size to the
full-length TWIK-2 cDNA described above, was present in a variety
of tissues, primarily in placenta, heart, colon, and spleen (Fig.
4). Lower levels of TWIK-2 mRNA could
be detected in peripheral blood leukocytes, lung, liver, kidney, and
thymus, with the lowest detectable levels found in brain. Two
additional species, 6.8 and 1.35 kb, were also detected. The relative
expression levels for these two species in these tissues closely
paralleled the level of the 2.6-kb species. Significant TWIK-2
expression was also detected in pancreas (data not shown). TWIK-2 could
not be detected in skeletal muscle by Northern blot analysis. Although
the hierarchy of expression levels in these tissues is different for
TWIK-1 and TWIK-2, TWIK-2 is expressed in the tissues in which TWIK-1
expression has been detected (6).
Electrophysiology--
Electrophysiologic properties of
heterologously expressed TWIK-2 channels were measured in
Xenopus oocytes. TWIK-2 currents activated instantaneously,
did not inactivate, and deactivated within the resolution of
two-electrode voltage clamp (Fig. 5, A and B). Weak inward rectification of TWIK-2
currents was observed (Fig. 5C). However, unlike both human
and mouse TWIK-1 (6, 12), TWIK-2 currents did not saturate at
depolarized potentials in either FR (Fig. 5, A and
C) or ND96 (data not shown). The slope of the plot of
reversal potential versus K+ concentration was
53 ± 3 mV per 10-fold change in K+ concentration
(Fig. 5C).
Pharmacology--
Pharmacologic modulators of other cloned tandem
pore domain potassium channels were examined, in addition to
non-selective potassium channel inhibitors. To test the sensitivity of
TWIK-2 currents to changes in extracellular pH, oocytes expressing
TWIK-2 were incubated in FR whose pH was titrated with either HCl or NaOH. TWIK-2 currents were not sensitive to extracellular pH over the
range of pH 5.6 to 8.4 (n = 4-6 oocytes at each
extracellular pH, data not shown). However, pharmacologic treatments (1 mM 2,4-dinitrophenol or 100% carbon dioxide) known to
lower intracellular pH (13) reduced TWIK-2 currents substantially (Fig.
6A).
TWIK-2 currents were not inhibited by quinidine (100 µM,
n = 5) or quinine (100 µM,
n = 4, data not shown). TWIK-1 currents are inhibited
with these treatments (6). TWIK-2 currents were also relatively
insensitive to barium (Fig. 6B). Zinc (100 µM) slightly inhibited TWIK-2 currents (
Unlike TREK-1 currents, TWIK-2 currents were not inhibited by
N-methyl D-glucamine substitution for sodium in
frog Ringer's solution (n = 5, data not shown). The
following pharmacologic treatments had no or only minimal effect on
TWIK-2 currents (data not shown): 4-aminopyridine (1 mM,
n = 3), cesium chloride (1 mM,
n = 3), ethanol (17 mM, n = 3), n-octanol (1 mM, n = 3),
arachidonic acid (10 µM, n = 6),
histamine (100 µM, n = 2), isoflurane
(500 µM to 1 mM, n = 6),
bupivacaine (10 µM, n = 3),
tetraethylammonium (10 mM, n = 6),
acetylcholine (1 µM, n = 6), secretin (1 nM, n = 4), apamin (1.0 µM,
n = 4), glibenclamide (30 µM,
n = 5), magnesium (5 mM, n = 5), and gadolinium (100 µM, n = 3).
Changes in perfusate osmolality (200-400 mmol/kg) from addition of
sucrose had minimal effect (<10%, n = 3) on TWIK-2 currents.
We describe the cloning and functional expression of a new member
of the mammalian Kt family of channels, TWIK-2. This new
channel is the smallest of the Kt channels yet cloned but
possesses structural features that define this family of potassium
channels, i.e. two P domains, each bounded by a pair of
transmembrane domains. For each of the P domains, TWIK-2 is most
closely related to TWIK-1. The P2 domain in TWIK-2 is strikingly homologous to the P2 domain in TWIK-1 (Fig. 3B). Only TWIK-1
and TWIK-2 have a GLG sequence motif in the second P domain; all of the
other cloned mammalian Kt channels have a GFG sequence in this position.
Besides the high primary sequence similarity of TWIK-2 to TWIK-1, we
also found physiologic and pharmacologic similarities between the two
channels. Both channels show weak inward rectification and have similar
current-voltage relationships. Like TWIK-1 channels, TWIK-2 channels
are inhibited by intracellular acidity. The only Kt channels
inhibited by intracellular acidity are TWIK-1 (6) and TOK1 (14), an
outwardly rectifying Kt channel from Saccharomyces
cerevisiae (14, 15). In contrast to TOK1 and TWIK-1, TWIK-2 is not
sensitive to barium. Although our data suggest a role for modulation of
TWIK-2 by PKC, the effect we observed in oocytes was small. Treatment
of oocytes expressing TWIK-1 with the phorbol ester PMA has been shown
to activate TWIK-1 channel activity, presumably by activating PKC (6).
The single PKC site in TWIK-1 is located within the M2-M3 loop, but
mutation of this site (Thr-161) to an alanine residue did not alter the PMA sensitivity of TWIK-1. The PKC site in TWIK-2 is in the same loop.
Therefore, like TWIK-1, the sensitivity of TWIK-2 to PMA may reflect an
indirect effect of PKC on the channel.
TWIK-2 is distinct from the other members of the Kt family. The
second cloned mammalian Kt channel, TREK-1, is an outward
rectifier Kt channel (16). Expressed in many brain regions and
the lung, as well as in kidney, heart, and skeletal muscle, this
channel, unlike TWIK-2, is insensitive to acidification. TASK, the
first mammalian K+ channel found to satisfy all the
characteristics of a background channel, is a voltage-insensitive open
rectifier whose activity is inhibited by extracellular acidification
(10, 17). Most recently, a fourth mammalian Kt channel, TRAAK,
was reported (18). This open rectifier also has characteristics of a
background K+ conductance and is expressed only in the
central nervous system, including the retina and spinal cord.
Like TWIK-1, TWIK-2 has a widespread distribution. Of the tissues
examined, TWIK-2 expression was absent from skeletal muscle only.
TWIK-2 is abundantly expressed in pancreatic tissue, and heterologous
TWIK-2 currents resemble physiologic base-line potassium channels that
maintain the resting membrane potential of acinar cells (19-21). Like
the physiologic base-line potassium channels expressed in pancreatic
acinar cells, TWIK-2 is inhibited by intracellular acidity but is not
sensitive to a number of potassium channel inhibitors, including
tetraethylammonium, Ba2+, and 4-aminopyridine (20). In
addition, barium-insensitive potassium currents have been described in
the kidney (22-24). Weak inward rectifiers have also been reported in
hepatocytes (25) as well as in eosinophils (26).
TWIK-2 is a weak inward rectifier whose zero current potential follows
the reversal potential for K+. For some potassium channels,
the primary sequence provides clues to potential mechanisms of inward
rectification. Intracellular polyamines and magnesium control the
degree of inward rectification of Kir channels by interacting
with acidic residues within the second transmembrane domain (27, 28).
Although the degree of inward rectification of TWIK-1 is known to
depend on intracellular magnesium levels, in general the mechanisms of
rectification within the tandem pore domain family remain unknown.
One area of future study is the formation of Kt channel dimers.
TWIK-1 forms homodimers via a disulfide bridge between subunits
involving identical extracellular cysteine residues (Cys-69) found in
the M1-P1 linker region (11). TWIK-2 has a similarly placed cysteine
residue, Cys-53. A TWIK-2 mutant in which Cys-53 has been mutated into
an alanine residue did not show channel activity after heterologous
expression in oocytes (data not shown). TWIK-2 may therefore form
functional homodimeric channels via a disulfide bridge. Northern blot
analysis of TWIK-2 expression demonstrates significant overlap with the
pattern of TWIK-1 expression. It is therefore possible that
TWIK-1/TWIK-2 heterodimers may form in those tissues, suggesting a
mechanism for further functional diversity within the tandem pore
domain K+ channel family.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
EXPERIMENTAL PROCEDURES
5) and human TASK (E = 2 × 10
5) by BLAST-X analysis. No overlapping EST clones
were found by BLAST-N analysis.
-globin gene flanking the insert (9).
-[32P]dCTP (Amersham Pharmacia
Biotech) with the Rediprime labeling kit (Amersham Pharmacia Biotech)
and hybridized to the Northern blot membrane with the high efficiency
hybridization system (Molecular Research Center, Cincinnati, OH) under
conditions specified by the manufacturer. The washed blot was exposed
to film (Amersham Pharmacia Biotech).
80 mV with 1-s voltage pulse steps ranging from
140 to
+40 mV in 20 mV increments, with 1.5-s interpulse intervals. Except
where noted, all two-electrode voltage clamp experiments were performed
with frog Ringer's solution (FR, composition in mM: 115 NaCl, 2.5 KCl, 1.8 CaCl2, 10 HEPES, pH 7.6), high potassium
frog Ringer's solution (HK, composition in mM: 2.5 NaCl,
115 KCl, 1.8 CaCl2, 10 HEPES, pH 7.6) or ND96 (composition
in mM: 96 NaCl, 2.0 KCl, 1.8 CaCl2, 1.0 MgCl2, 5 HEPES, pH 7.6) as perfusate. Recordings were
obtained in a 25-µl recording chamber at flow rates of 1-4 ml/min.
Water-injected oocytes were used as controls, undergoing the same
treatments as transcript-injected oocytes. Except where noted, at least
three oocytes were assayed for each experimental condition reported.
80 to +40 mV pulse during the treatment condition compared
with control.
RESULTS
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Fig. 1.
Nucleotide sequence of TWIK-2. The
GenBankTM sequence of EST, AA604914, is
underlined.
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Fig. 2.
Amino acid sequence of TWIK-2. The P
domains (underlined), potential glycosylation sites (*), and
potential phosphorylation sites (boxed) described in the
text are indicated. The putative transmembrane domains are
highlighted.
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Fig. 3.
Kt channel pore domain
alignments. P regions from the indicated Kt channels were
aligned as in Fig. 2. A, P1 regions. B, P2
regions. Highlighted residues indicate identity with the
TWIK-1 sequence.
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Fig. 4.
Northern blot analysis. Human multiple
tissue Northern blots were probed with radiolabeled cDNA derived
from TWIK-2 as described under "Experimental Procedures."
PBL, peripheral blood leukocytes; S INTESTINE,
small intestine; Sk MUSCLE, skeletal muscle.
Markers represent migration of RNA standards of the
indicated sizes (kb).
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Fig. 5.
Biophysical properties of TWIK-2 currents in
Xenopus oocytes studied with two-electrode voltage
clamp. Whole cell currents of TWIK-2 cRNA-injected oocytes
perfused with either frog Ringer's solution (FR,
A) or high potassium frog Ringer's solution (115 mM K+; HK, B). Voltage
pulses (1-s duration) are from 120 to +40 mV from a holding potential
of
80 mV (inset). Data also are shown for control
water-injected oocytes. C, shows average current-voltage
curves of TWIK-2-injected oocytes (n = 6) at different
levels of extracellular potassium and a plot of the reversal potentials
obtained at these varying extracellular potassium concentrations.
Error bars represent S.E.
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Fig. 6.
Acid and barium sensitivity of TWIK-2.
A, intracellular pH sensitivity of TWIK-2. Mean
current-voltage curves of TWIK-2-injected oocytes in the presence or
absence of carbon dioxide (100%, n = 5) or
2,4-dinitrophenol (DNP) (1 mM, n = 6) perfused with high potassium frog Ringer's solution
(HK). Currents from control water-injected oocytes were
unchanged by these treatments. B, effect of barium of TWIK-2
currents. Mean current-voltage curves of TWIK-2-injected oocytes in the
presence or absence of barium (1 mM, n = 5). Barium dose-response data are shown in the bar graph of
B. Currents ( 140 mV pulse in HK) have been normalized to
control currents. 2,4-Dinitrophenol (DNP) was applied for
6-10 min prior to application of the voltage pulse. Other treatments
were applied for 2 min prior to pulse protocol. Error bars
represent S.E. (n values shown above).
10 ± 1%, n = 3, p = 0.005 by paired t test). Currents
observed in TWIK-2-injected oocytes were slightly larger after
treatment with the PKC activator PMA (50-100 nM; 24 ± 3%; n = 5, p = 0.001 by paired
t test). Pharmacologic treatments known to activate protein
kinase A (forskolin, 10 µM with
3-isobutyl-1-methylxanthine, 1 mM, n = 4; or 8-bromo-cyclic AMP, 300 µM, n = 4)
had no effect on TWIK-2 currents (data not shown).
DISCUSSION
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ACKNOWLEDGEMENTS |
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We thank Ruth Cong and Shannan Downey for excellent technical assistance.
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FOOTNOTES |
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* This work was supported in part by Grants GM51372 and GM57740 from the National Institutes of Health.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) AA117708.
¶ To whom correspondence should be addressed: Dept. of Anesthesia, School of Medicine, 513 Parnassus Ave., Box 0542, Rm. S261, University of California, San Francisco, CA 94143-0542. Tel.: 415-476-5200; Fax: 415-476-8841; E-mail: spyost{at}itsa.ucsf.edu.
2 Available at the following on-line address: www.ncbi.nlm.nih.gov.
3 Internet resource found on the ExPASy server at expasy.hcuge.ch.
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ABBREVIATIONS |
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The abbreviations used are: P, pore-forming; Kv, voltage-gated K+ channel; Kir, inward rectifier K+ channels; Kt, tandem pore domain K+ channels; TWIK, tandem pore domain weak inward rectifying K+channel; PMA, phorbol 12-myristate 13-acetate; BLAST, basic local alignment search tool; EST, expressed sequence tag; ORF, open reading frame; kb, kilobases, PKC, protein kinase C.
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