From the Department of Neurochemistry, Faculty of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan
Received for publication, September 29, 2000, and in revised form, December 22, 2000
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ABSTRACT |
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In this study, we report the molecular cloning of
cDNAs encoding three distinct isoforms of rat (r) TRP6
Ca2+ channels. The longest isoform, rTRP6A, contains
930 amino acid residues; rTRP6B lacks 54 amino acids (3-56) at the N
terminus, and rTRP6C is missing an additional 68 amino acids near the C terminus. Transient transfection of COS cells with expression vectors
encoding rTRP6A or rTRP6B increased Ca2+ influx and gave
rise to a novel Ba2+ influx after activation of
M5 muscarinic acetylcholine receptors. By contrast, passive
depletion of intracellular Ca2+ stores with thapsigargin
did not induce Ba2+ influx in cells expressing rTRP6
isoforms. Ba2+ influx was also stimulated in
rTRP6A-expressing cells after exposure to the diacylglycerol analog,
1-oleoyl-2-acetyl-sn-glycerol (OAG), but rTRP6B-expressing
cells failed to show OAG-induced Ba2+ influx. Expression of
a rTRP6 N-terminal fragment of rTRP6B or rTRP6A antisense RNA blocked
M5 muscarinic acetylcholine receptor-dependent Ba2+ influx in COS cells that were transfected with rTRP6
cDNAs. Together these results suggest that rTRP6 participates in
the formation of Ca2+ channels that are regulated by a
G-protein-coupled receptor, but not by intracellular Ca2+
stores. In contrast to the results we obtained with rTRP6A and rTRP6B,
cells expressing rTRP6C showed no increased Ca2+ or
Ba2+ influxes after stimulation with carbachol and also did
not show OAG-induced Ba2+ influx. Glycosylation analysis
indicated that rTRP6A and rTRP6B are glycosylated in COS cells, but
that rTRP6C is mostly not glycosylated. Together these results suggest
that the N terminus (3-56 amino acids) is crucial for the activation
of rTRP6A by diacylglycerol and that the 735-802 amino acid segment
located just downstream from the 6th transmembrane segment may be
required for processing of the rTRP6 protein.
Cytosolic Ca2+ signals play important roles in the
development, function, and death of cells (1, 2). Increases in
intracellular Ca2+ typically result from the release of
Ca2+ from intracellular stores and/or from an increased
influx of extracellular Ca2+ (3). In many types of cells,
stimulation of seven-transmembrane receptors that couple to the
production of inositol 1,4,5-triphosphate is followed by a biphasic
increase in intracellular Ca2+. An initial transient
increase is due to the opening of inositol 1,4,5-triphosphate-activated
Ca2+ channels that release the Ca2+ from the
endoplasmic reticulum. This is followed by a sustained increase in
Ca2+ due to the influx of Ca2+ across the
plasma membrane (4-6). Calcium influx that is triggered by the
emptying of intracellular stores was first termed capacitative calcium
entry by Putney (7). The molecular identification of Ca2+
channels mediating capacitative calcium entry is currently a topic of
wide interest.
The molecular cloning of cDNAs encoding Ca2+ channels
that function in the visual system of Drosophila (d), the
transient receptor potential
(dTRP)1 (8) and dTRP-like
(dTRPL) (9) channels, provided the first two candidates for
capacitative calcium entry channels. dTRP was shown to form
Ca2+-permeable channels that are activated by store
depletion, whereas dTRPL forms nonselective cation channels that are
constitutively active and insensitive to store depletion when expressed
in Sf9 cells (10, 11). Coexpression of dTRP and dTRPL was
also shown to give rise to a store-operated current that is distinct
from that produced by either dTRP or dTRPL alone (12). Furthermore, dTRP and dTRPL interact directly, suggesting that they are likely to
form heteromeric channels (12, 13).
A search for mammalian homologues of the dTRP and dTRPL genes has
yielded seven TRP homologues, TRP1-7, which are differentially expressed in various tissues. The functional characteristics of channels encoded by these genes have been studied primarily in heterologous cell systems (14-32). Based on these studies, the seven
mammalian TRP homologues can be divided into two groups: TRP1/2/4/5,
which are thought to form channels activated by Ca2+ store
depletion, and TRP3/6/7, which are thought to be activated by
Gq-coupled receptors independently of Ca2+
stores (14, 20, 33, 34). There are still controversies, however,
concerning the mechanisms of regulation of individual TRP channels
(23-27, 29, 34, 35). For example, TRP6 has been reported to be both a
G-protein receptor-coupled, Ca2+ store-independent channel
(25, 26) and a Ca2+ store-operated channel (27). Recently,
human (h) TRP6, hTRP3 (26), and mouse (m) TRP7 (28) have been reported
to be activated by diacylglycerol (DAG), but the activation pathway and
domain within the TRP proteins required for activation are still unclear.
To study the mechanisms of regulation of TRP6, we used a RT-PCR
strategy to clone the rat (r) TRP6 cDNA. This approach yielded cDNA encoding three distinct isoforms of TRP6. When expressed in
COS cells, rTRP6A and rTRP6B form Ca2+ channels that couple
to M5 muscarinic acetylcholine receptors (mAChR) and are
regulated independently of intracellular Ca2+ stores. The
longest isoform, rTRP6A, is activated by the DAG analog,
1-oleoyl-2-acetyl-sn-glycerol (OAG), but rTRP6B is not, suggesting that amino acids present in the N terminus of rTRP6A are
crucial for activation by DAG but are not required for activation by
carbachol. Finally, the apparent inability of the shortest TRP6
isoform, rTRP6C, to undergo glycosylation and form functional channels
suggests that a domain located on the cytoplasmic side of the sixth
transmembrane segment may be required for processing of the rTRP6 protein.
Molecular Cloning--
rTRP6A cDNA was cloned from rat lung
total RNA, and rTRP6B and C cDNAs were cloned from PC12D cell (36)
total RNA using a RT-PCR approach. The forward primer for rTRP6A was
5'-CCAGGCACTTGCCATGAGCCAGAG-3'; the forward primer for rTRP6B and C was
5'-ATGAGCCGGGGTAATGAAAACAGAC-3', and the common reverse primer was
5'-CCAATCGATCTATCTGCGGCTTTCC-3'. The RT-PCR products were isolated from
1% agarose gels and cloned in pGEM-T Easy (Promega). TRP6 cDNAs
were excised from this vector by digesting with NotI and
SpeI and subcloned between the NotI and
SpeI sites of pEF-BOS-SK, a derivative of the mammalian
expression vector pEF-BOS (37) that contains the multiple cloning site of pBluescript-SK (Stratagene). The resulting plasmids were designated pBOS-TRP6A, -B, and -C. An antisense expression vector,
pBOS-TRP6A-antisense, was constructed by subcloning a
NotI/SpeI DNA fragment encoding rTRP6A in
pEF-BOS-KS. An expression vector encoding the TRP6B N-terminal fragment
fused to green fluorescent protein (GFP), pEGFP-TRP6B-N, was
constructed by cloning a DNA fragment encoding amino acid residues
1-301 of rTRP6B between the EcoRI and XmaI sites
of pEGFP-N2 (CLONTECH). pBOS-TRP6A Cell Culture and Transfection--
COS-7 cells were cultured in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum. Confluent cultures were replated in 12-well plates at a
density of 5 × 104 cells/well. After 24 h, the
medium was changed, and 100 µl of transfection mixture was added to
each well. Transfection mixtures contained 500 ng of the M5
muscarinic receptor expression plasmid pBOS-M5 (38), 1 µg
of rTrp6 cDNA, 50 ng of the GFP expression plasmid pEGFP-N1
(CLONTECH), and 1.5 µl of
LipofectAMINETM 2000 (Life Technologies, Inc.) in 100 µl
of Opti-MEM I (Life Technologies, Inc.). After 24 h at 37 °C,
the transfection mixtures were removed, and normal medium was added to
the cells.
Measurement of Ca2+ and Ba2+
Influxes--
Two days after transfection, cells were loaded with fura
2-AM (Wako, 2 µM in Krebs-Ringer-HEPES (KRH) buffer: 6 mM Hepes-NaOH, 125 mM NaCl, 5 mM
KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4·7H2O, 2 mM CaCl2·2H2O, 6 mM glucose, pH 7.4)
for 90 min at room temperature in the dark. Changes in intracellular
Ca2+ and Ba2+-dependent
fluorescence were measured in individual cells or small groups of cells
by fluorescence videomicroscopy using the Argus-50/Ca system (Hamamatsu
Photonics K.K). Fura-2 loaded cells were stimulated with 340 nm and 380 nm of light at 1.4-s intervals, and fluorescence was recorded at 510 nm. Data were calculated using Microsoft Excel 98. Artifacts associated
with the addition of drugs were graphically removed from fluorescence traces.
Generation of Polyclonal Anti-TRP6 Antibodies--
A 17-amino
acid peptide (914KLGERLSLESKQEESRR930) and a
12-amino acid peptide (788QGHKKGFQEDAE799) were
used as the antigen peptides for the production of antiserum in rabbits
(Biologica Co., Nagoya, Japan) and were designated as anti-TRP6-C
terminus and anti-TRP6 (788), respectively. These TRP6 antibodies
were purified by affinity chromatography (SulfoLink kit, Pierce) using
the antigen peptide as described in Harlow and Lane (39).
Western Blots--
Cells were cultured and transfected as
described above. Two days after transfection, cells were rinsed once
with phosphate-buffered saline and then lysed by the addition of
radioimmune precipitation lysis buffer (150 mM NaCl, 1.0%
Nonidet P-40 (nonylphenoxypolyethoxyethanol), 0.5% deoxycholate, 0.1%
SDS, 50 mM Tris, pH 8.0, 2 µM leupeptin, 1 mM phenylmethanesulfonyl fluoride, 10 µg/ml aprotinin).
The mixture was sonicated in the cold and cleared by centrifuging at
5,000 rpm for 5 min. The samples were mixed with the same volume of 2×
SDS sample buffer (100 mM Tris, pH 6.8, 200 mM
dithiothreitol, 4% SDS, 0.2% bromphenol blue, 20% glycerol) and
resolved by standard SDS-polyacrylamide gel electrophoresis (8%
acrylamide resolving gel). Proteins were electrophoretically
transferred to polyvinylidene difluoride membranes (0.2 µm,
FluorotransTM, Pall Corp.). After the transfer, membranes
were blocked overnight at 4 °C with phosphate-buffered saline
containing 5% powdered skim milk and 0.05% Tween 20 (polyoxyethylene
sorbitan monolaurate). The membranes were then exposed to anti-TRP6
antibody (650 µg/ml, 1:500 dilution) in phosphate-buffered saline
(containing 0.5% powdered skim milk and 0.05% Tween 20) for 1 h
at room temperature. The membranes were washed three times with the
above buffer and incubated in buffer containing anti-rabbit IgG
antibodies cross-linked with horseradish peroxidase (Jackson Immuno
Research Laboratories, Inc.; 1:2000 dilution) for 1 h at room
temperature. Membranes were washed three times, and TRP6 bands were
visualized by staining with a freshly prepared solution containing 0.25 mg/ml 3,3'-diaminobenzidine (Wako), 0.01%
H2O2, 0.04% NiCl2, and 50 mM Tris-Cl, pH 7.5.
Deglycosylation--
Cell lysates were treated with
endoglycosidase H (0.05 units, Roche Molecular Biochemicals) or
peptide N-glycosidase F (5 units, Roche Molecular
Biochemicals) overnight at 4 °C with rotation. The samples were then
mixed with an equal volume of 2× SDS sample buffer and resolved by
SDS-polyacrylamide gel electrophoresis and Western blots as described above.
Primary Structures of Rat TRP6 Isoforms--
Using a RT-PCR
strategy, we cloned cDNAs encoding three rat TRP6 isoforms: rTRP6A
from rat lung and rTRP6B and C from PC12D cells. rTRP6A is composed of
930 amino acids, and rTRP6B and C contain 876 and 808 amino acids,
respectively. Compared with rTRP6A, rTRP6B is missing 54 amino acids at
the N terminus, and rTRP6C is missing an additional 68 amino acids near
C terminus (Fig. 1A and
B). Hydropathy analysis suggests that rTRP6 has six
hydrophobic transmembrane domains and one pore-forming region, located
between the fifth and sixth transmembrane segments. Both N- and
C-terminal regions contain a large percentage of hydrophilic amino acid
residues, and the N terminus of rTRP6A-C is predicted to contain three
ankyrin-like repeats (Fig. 1C).
Expression and Distribution of rTRP6--
When expressed in COS
cells, rTRP6A, -B, and -C proteins of apparent molecular masses 107, 98, and 87 kDa, respectively, were detected using an antibody that
recognizes the rTRP6 C terminus. By contrast, COS cells transfected
with the empty expression vector, pBOS-SK, produced no detectable TRP6
protein (Fig. 2A,
left). The specificity of the rTRP6-C terminus antibody is
demonstrated by the fact that preincubation with the TRP6-C-terminal
oligopeptide completely blocked the appearance of TRP6 bands in the
Western blots (Fig. 2A, right). Endogenous rTRP6
proteins were found to be widely expressed in rat brain (Fig.
2B, left). The rTRP6 protein expressed in brain
roughly comigrated with rTRP6A expressed in COS cells (Fig.
2B, right).
Functional Characterization of rTRP6A, -B, and -C--
To study
the roles of the three rTRP6 isoforms in calcium signaling, rTRP6A, -B,
or -C cDNAs or pBOS-SK were transiently expressed in COS cells
along with expression vectors for M5 muscarinic
acetylcholine receptor and GFP. An expression vector encoding
M5 mAChR, which couples to Gq (38), was used to
study the role of rTRP6 in carbachol-stimulated Ca2+ entry.
Cells were loaded with fura-2 in KRH (containing 2 mM CaCl2), and extracellular Ca2+ was chelated by
adding 4 mM EGTA just before making the measurements. The
addition of 500 µM carbachol to these cells induced a
rapid rise in cytosolic Ca2+, which decreased to basal
levels within 2-3 min (Fig. 3,
A-D). This transient increase in Ca2+
corresponds to the rapid release of Ca2+ from internal
stores and subsequent expulsion of Ca2+ from the cell. The
addition of 4 mM CaCl2 to the extracellular solution induced a second increase in cytosolic Ca2+ due to
the influx of extracellular Ca2+ (Fig. 3, A-D).
Compared with the control cells, the Ca2+ increases due to
influx were much larger in cells expressing rTRP6A or -B, but cells
expressing rTRP6C showed no significant differences in Ca2+
influx compared with the control (Fig. 3E).
To further characterize the rTRP6 channels, we examined the ability of
carbachol to stimulate Ba2+ influx in COS cells expressing
individual rTRP6 isoforms. Ba2+ is frequently used as a
substitute for Ca2+ since its entry into the cell can also
be monitored by measuring increases in fura-2 fluorescence. Previously,
we found that endogenously expressed store-operated Ca2+
channels in neuronal PC12D cells are relatively impermeable to Ba2+ ions.2
pBOS-SK-transfected COS cells in nominally Ca2+-free KRH
buffer did not show any Ba2+ influx after stimulation with
carbachol and subsequent addition of 200 µM
BaCl2 (Fig. 4A).
By contrast, significant carbachol-stimulated Ba2+ influx
was observed in TRP6A- or -B-transfected cells (Fig. 4, B
and C). COS cells expressing rTRP6C, the shortest isoform,
showed no Ba2+ influx (Fig. 4D).
To determine whether the regulation of rTRP6 is dependent on the
depletion of intracellular Ca2+ stores, the effects of
thapsigargin were examined. Thapsigargin indirectly causes the release
of Ca2+ stored in the endoplasmic reticulum by irreversibly
inhibiting the sarco/endoplasmic reticulum calcium pump, which
functions to fill these stores (40). In nominally Ca2+-free
medium, the addition of 1 µM thapsigargin depleted the
intracellular Ca2+ stores, resulting in a transient
increase in cytosolic Ca2+. Ba2+ influx was not
observed in control cells or in rTRP6A-, -B-, or -C-transfected cells
after the addition of 200 µM BaCl2 to the
medium (Fig. 5, A-D). Similar
results were obtained using 100 nM thapsigargin to deplete
intracellular Ca2+
stores.3
Inhibition of Ba2+ Influx by Expression of the rTRP6B
N-terminal Fragment or rTRP6A Antisense RNA--
Coexpression of
rTRP6B-N (encoding the N-terminal 1-301 amino acid residues of rTRP6B)
with rTRP6A totally blocked carbachol-stimulated Ba2+
influx (Fig. 6, A and
B). A similar inhibitory effect was observed when pBOS-TRP6A
antisense expression vector was cotransfected with the rTRP6B
expression vector (Fig. 6, C and D). In Fig.
6E, Western blots show that rTRP6B protein levels are
significantly reduced in cells cotransfected with the rTRP6A antisense
expression vector.
These data suggest that expression of rTRP6A or -B causes an increase
in Ca2+ influx compared with the control cells. To obtain
additional evidence for this model, we examined the effects of rTRP6A
antisense RNA expression on Ca2+ influx. Fig.
7 shows that in nominally
Ca2+-free medium, carbachol-stimulated Ca2+
influx was reduced to control levels when rTRP6A antisense RNA was
coexpressed with rTRP6A.
Effects of OAG and Phorbol 12-Myristate 13-Acetate (PMA) on
Ba2+ Influx--
Recent studies show that hTRP3 and hTRP6
(26) and also mTRP7 (28) can be directly activated by the analogs of
diacylglycerol. We therefore tested the effects of a membrane-permeable
diacylglycerol analog, OAG, on Ba2+ influx in COS cells
expressing rTRP6. Cells in nominally Ca2+-free medium were
exposed to 100 µM OAG and then 200 µM
BaCl2. No Ba2+ influx was observed in
pBOS-SK-transfected control cells (Fig. 8A), but significant
stimulation of the influx of Ba2+ by OAG was observed in
rTRP6A-expressing cells (Fig. 8B). By contrast, there was no
OAG-induced Ba2+ influx in rTRP6B- or -C-transfected cells
(Fig. 8, C and D). Pretreatment of cells with 500 nM PMA, a PKC activator, abolished OAG-activated,
rTRP6A-mediated Ba2+ influx (Fig. 8F), whereas
pretreatment with PMA alone had no effect on Ba2+ influx
(Fig. 8E). PMA also failed to stimulate Ba2+
influx when added just before the addition of
BaCl2.3 Similar results were obtained using 100 nM PMA.3
Effects of PMA and GF 109203X on M5 Receptor-mediated
Ba2+ Influx--
To further investigate the involvement of
PKC in M5 receptor-mediated Ba2+ influx, we
examined the effects of the phorbol ester PMA and the PKC inhibitor GF
109203X (41, 42). Pretreatment of COS cells expressing rTRP6A and
M5 receptor with PMA blocked carbachol-activated, rTRP6A-mediated Ba2+ influx (Fig.
9, A and B). GF
109203X alone did not significantly affect carbachol-induced
Ba2+ influx (Fig. 9C), but pretreatment of the
cells with GF 109203X before PMA abolished the inhibitory effect of
phorbol ester (Fig. 9D). These results indicate that PMA
inhibits carbachol-activated Ba2+ influx by activating
PKC.
Glycosylation Analysis of rTRP6--
The predicted amino acid
sequence of rTRP6A contains nine consensus NX(S/T) motifs
for N-glycosylation (Fig.
10A). Because only sites
that are exposed to the extracellular side of the membrane are expected
to be glycosylated, the topological model for rTRP6 depicted in Fig. 1
suggests that only Asn at position 711 is a candidate for
glycosylation. To analyze whether rTRP6 is glycosylated in COS cells
and to provide evidence that rTRP6 proteins are expressed on the
surface of plasma membrane, we treated the cell lysates with two
glycosidases, endoglycosidase H and peptide N-glycosidase F
before performing the Western blot analysis. Treatment with endoglycosidase H, an enzyme that cleaves the N-glycosidic
bond between the first and second GlcNAc residue of high
mannose-containing, immature glycoproteins did not change the pattern
of rTRP6A significantly, but eliminated two sharp bands located just
above the rTRP6B band with the highest mobility. Treatment with peptide
N-glycosidase F, an enzyme that cleaves the
N-glycosidic bond between the sugar chain and asparagine of
the mature and immature forms of glycoproteins, almost totally
eliminated the hazy material between 83 and 175 kDa and increased the
intensities of rTRP6A and -B high mobility bands, but did not change
the appearance of the rTRP6C band (Fig. 10B).
Properties of the Constructs TRP6A
To test the functional importance of the 735-802 segment, we measured
carbachol- and OAG-stimulated Ba2+ influx in pBOS-TRP6A In the present study, we cloned three isoforms of rat TRP6,
designated rTRP6A, -B, and -C. These isoforms may result from alternative RNA splicing as suggested for other TRP subtypes (15-20, 28, 45). rTRP6A closely resembles human TRP6 (26;
GenBankTM accession number AF080394) and mouse TRP6
(25; GenBankTM accession number U49069), with nucleic acid
identities of 88.38 and 94.41%, respectively. rTRP6B closely resembles
a second mouse isoform of TRP6 (unpublished sequence;
GenBankTM accession number AF057748) with a nucleic
acid identity of 94.83%. rTRP6C is a novel isoform. Mizuno et
al. (27) also report the cloning of TRP6 from rat brain. Their
sequence is similar to our rTRP6A cDNA, except that it contains an
insertion of a G residue at position of 1210 and a deletion of a
C residue at position 1326, resulting in a shift in the reading frame
in this region compared with our sequences and also with respect to the mouse and human TRP6 sequences. Previous studies found that mouse TRP6
mRNA is expressed at low levels in brain (25). Using Western blot
analysis, we found that rTRP6 protein is expressed throughout the
brain, including the cortex, cerebellum, hippocampus, brain stem, and
midbrain regions (Fig. 2B).
In COS cells, transient expression of rTRP6A and -B resulted in a 2.5- and a 1.5-fold increase in Ca2+ influx compared with
control cells when Ca2+ was added back to cultures
stimulated with carbachol in the absence of extracellular
Ca2+. By contrast, rTRP6C-transfected cells showed no
significant Ca2+ increase compared with the control
cells in the same assay (Fig. 3).
To date, seven mammalian homologues (Trp1-7) of the
Drosophila Trp and Trpl genes have been isolated (15-28).
There can be divided into two groups based upon whether or not they can
be activated by store depletion (14). The mechanism by which the TRP6
channels are regulated, however, is still unresolved. Boulay et
al. (25) found that mouse TRP6 functions as a G-protein-coupled receptor-regulated Ca2+ store-independent channel when
expressed in COS cells (25). By contrast, Mizuno et al. (27)
reported that rat TRP6 is a thapsigargin-sensitive and Ca2+
store-regulated channel in COS cells. In our study, COS cells expressing rTRP6A or -B showed significant Ba2+ influx in
response to carbachol stimulation, whereas rTRP6C or control vector
pBOS-SK-transfected cells did not show any Ba2+ influx
(Fig. 4). By contrast, thapsigargin failed to induce any Ba2+ influx in rTRP6A-, -B-, or -C-transfected cells (Fig.
5). Together these data indicate that rTRP6 functions as a
receptor-activated, Ca2+ store-independent Ca2+
channel in COS cells.
Hofmann et al. (26) first showed that human TRP6 and TRP3
are activated by DAG analogs, and Okada et al. (28) observed a similar activation for mouse TRP7. In our experiments, OAG induced significant Ba2+ influx in rTRP6A-transfected cells (Fig.
8B), but not in control cells (Fig. 8A).
Interestingly, cells expressing rTRP6B failed to show OAG-induced
Ba2+ influx (Fig. 8C). rTRP6B is missing a
54-amino acid (3-56) segment that is present at the N terminus of
rTRP6A, suggesting that these amino acids are crucial for activation by
DAG but are not required for rTRP6 activation by mAChR (Fig.
4B). Together these results imply that activation of rTRP6
after stimulation of mAChR is mediated by DAG-dependent and
DAG-independent pathways. Further study will be needed to elucidate the
role of the N-terminal 54-amino acid segment in the activation of
rTRP6A by DAG.
In addition to its ability to activate Ba2+ influx through
rTRP6A, OAG is also known to activate PKC (46). To determine whether PKC activation is correlated with Ba2+ influx, we examined
the effects of another PKC activator, PMA, on Ba2+ influx.
Pretreatment with PMA did not induce Ba2+ influx (Fig.
8E), but instead blocked OAG-induced Ba2+ influx
in rTRP6A-expressing cells (Fig. 8F). These data indicate that DAG-induced Ba2+ influx does not involve the
activation of protein kinase C but, rather, that activation of PKC
blocks the activation of the rTRP6A channel. Pretreatment of cells with
PMA also blocked carbachol-activated, rTRP6A-mediated Ba2+
influx (Fig. 9, A and B). This inhibition is
mediated by PKC since pretreatment of the cells with GF 109203X before
PMA abolished the inhibitory effects of this phorbol ester (Fig.
9D).
If DAG can both activate and inhibit TRP6 channels, one might expect
that pretreatment of the cells with GF 109203X alone would potentiate
carbachol-induced Ba2+ influx. Pretreatment with GF
109203X, however, did not significantly change the rate or extent of
carbachol-stimulated Ba2+ influx (Fig. 9C). This
result suggests that DAG produced after activation of mAChR might
stimulate Ba2+ influx more efficiently (or perhaps more
rapidly) than it activates PKC. Activation by OAG and inhibition by PMA
has also been reported for mTRP7 (28). Clearly, further study is needed
to elucidate the mechanisms underlying the regulation of TRP
Ca2+ channels by DAG.
Previous studies from Birnbaumer and co-workers showed that human TRP3
proteins expressed in HEK293 (30) and COS cells (31, 32) and mouse TRP6
proteins in COS cells (25) are glycosylated. In our study, we also
found that rat TRP6 proteins are glycosylated when expressed in COS
cells. rTRP6A is present mostly in the mature glycosylated form,
whereas rTRP6B contains both mature glycosylated and immature and
unglycosylated forms. By contrast, most of the rTRP6C is not
glycosylated (Fig. 10B), suggesting that rTRP6C may not be
processed normally. This may explain why rTRP6C did not respond to
stimulation with carbachol and OAG.
The glycosylation results led us to examine the importance of the 68 amino acids (735-802 segment) missing from the rTRP6C, which is
located on the cytosolic side immediately adjacent to the sixth
transmembrane domain. To determine the function of this segment, we
constructed TRP6A In summary, we have cloned three distinct rTRP6 isoforms and
demonstrated that rTRP6 contributes to the formation of
G-protein-coupled receptor-regulated store-depletion-independent
Ca2+ channels when expressed in COS cells. We have also
identified two domains that are important for the function of rTRP6
channels: the N terminus (3-56 amino acids), which is crucial for the
activation of rTRP6A by DAG, and the 735-802 segment located just
downstream from the sixth transmembrane segment, which may be required
for processing of the rTRP6 protein.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was
constructed by digesting pBOS-TRP6A and pBOS-TRP6C with XhoI
and SpeI and ligating the small
XhoI/SpeI fragment from pBOS-TRP6C to the large
fragment of pBOS-TRP6A. In the same way, pBOS-TRP6C+ (which
has the same primary structure as pBOS-TRP6B) was constructed by
ligating the small XhoI/SpeI fragment from
pBOS-TRP6A to the large fragment of pBOS-TRP6C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Primary structures of rat TRP6 isoforms.
A, amino acid sequences of three rat TRP6 isoforms were
aligned using the ClustalW program (43). Underlines indicate
the peptides used for production of anti-TRP6 antibodies. B,
schematic comparison of TRP6A, -B, and -C. MS,
methionine-serine. The black regions indicate sequences
deleted in TRP6B and/or TRP6C. C, Kyte and Doolittle
hydropathy (DNA Strider 1.2) analysis of TRP6A using a window size of 9 amino acids (aa). The striped bars indicate the
ankyrin repeats, the open bars indicate the putative
transmembrane segments (S1-S6), and the black bar indicates
the putative pore region (P) between S5 and S6 (SMART
prediction tool (44)).
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Fig. 2.
Western blots showing the
expression of rTRP6. A, extracts were prepared from
transfected COS cells. Left, TRP6 isoforms expressed in COS
cells; right, bands blocked by preincubation of the TRP6
antibody with the antigen peptide. B, expression of
endogenous rTRP6 in various regions of rat brain (left) and
exogenously expressed rTRP6 in COS cells (right). Tissues
were homogenized in 1× SDS sample buffer (minus dithiothreitol), and
the extracts were centrifuged at 5,000 rpm to remove tissue debris.
Protein concentrations were determined using the BCA method (Pierce).
Samples were boiled for 5 min before loading onto a SDS-polyacrylamide
electrophoresis gel (14 µg of total protein were loaded in each
lane). The positions of the 175-, 83-, 62-, and 47.5-kDa
protein size markers (New England Biolabs) are indicated. The higher
molecular weight materials that stained with anti-TRP6 Ab represent
aggregated TRP6 protein, which formed when samples were boiled.
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Fig. 3.
Carbachol induces Ca2+ influx in
M5 mAChR- and TRP6-expressing COS cells. COS cells
were transfected with pEGFP-N1, pBOS-M5, and pBOS-SK
(A), pEGFP-N1, pBOS-M5, and pBOS-TRP6A
(B), pBOS-TRP6B (C), or pBOS-TRP6C
(D). Ca2+-dependent changes in
fluorescence were measured in fura-2 loaded COS cells in KRH
(containing 2 mM CaCl2) using the Argus-50/Ca
system. EGTA (4 mM) was added to the cells to chelate
extracellular Ca2+ before measurements. Cells were exposed
to 500 µM carbachol (Carb) and 4 mM CaCl2 at the times indicated by the
arrows. Expression of GFP was used as a marker for cells
that had taken up plasmids. The traces shown are the
averages of seven measurements performed on groups of GFP fluorescent
cells (3-10 cells/group). E, mean amplitudes of
carbachol-induced Ca2+ influx in control and
TRP6-expressing cells. The amplitudes were obtained by subtracting the
base-line 340-nm/380-nm ratio from the 340-nm/380-nm ratio measured at
the highest point of Ca2+ influx. Asterisks (*)
above the bars indicate the statistical significance of
differences (p < 0.01) compared with the control (by
Bonferroni's t test).
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Fig. 4.
Carbachol induces Ba2+ influx in
M5 mAChR- and TRP6-expressing COS cells.
Ca2+- and Ba2+-dependent changes in
fluorescence were measured in fura-2-loaded COS cells in nominally
Ca2+-free KRH. COS cells were transfected with pEGFP-N1,
pBOS-M5, and pBOS-SK (A), pEGFP-N1,
pBOS-M5, and pBOS-TRP6A (B), pBOS-TRP6 B
(C), or pBOS-TRP6C (D). Cells were exposed to 500 µM carbachol and 200 µM BaCl2
at the times indicated by the arrows. The traces
shown are the averages of 7 measurements of GFP fluorescent cells
(3-10 cells/measurement).
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Fig. 5.
Thapsigargin does not induce Ba2+
influx in COS cells expressing TRP6 isoforms. COS cells were
transfected with pEGFP-N1, pBOS-M5, and pBOS-TRP6A
(A), pBOS-TRP6B (B), or pBOS-TRP6C
(C). Changes in fluorescence were measured in fura-2-loaded
COS cells in nominally Ca2+-free KRH. 1 µM
thapsigargin and 200 µM BaCl2 were added at
the times indicated by arrows. The traces shown
are the averages of 7 measurements of GFP fluorescent cells (3-10
cells/measurement).
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Fig. 6.
Expression of the TRP6 N-terminal
fragment or TRP6 antisense RNA blocks M5 receptor-activated
Ba2+ influx. COS cells were transfected with pEGFP-N1,
pBOS-M5, and pBOS-TRP6A (A), pBOS-TRP6A and
pBOS-TRP6B-N (B), pBOS-TRP6B (C), or pBOS-TRP6B
and pBOS-TRP6A antisense (D). Changes in fluorescence were
measured in fura-2-loaded COS cells in nominally Ca2+-free
KRH; 500 µM carbachol and 200 µM
BaCl2 were added at the times indicated by the
arrows. The traces shown are the averages of 7 measurements of GFP fluorescent cells (3-10 cells/measurement).
E, Western blots showing the effect of cotransfection of
pBOS-TRP6A antisense on the synthesis of TRP6 protein. Positions of the
175-, 83-, 62-, and 47.5-kDa protein size markers are indicated
(lane 1). COS cells transfected with pBOS-TRP6B (lane
2), pBOS-TRP6B and pBOS-TRP6A antisense (lane 3), or
pBOS-SK (lane 4) are shown. Western blots (14 µg of total
cellular protein/lane) were probed with anti-TRP6-C terminus
Ab.
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Fig. 7.
Expression of TRP6 antisense RNA blocks
carbachol-stimulated Ca2+ influx. COS cells were
transfected with pEGFP-N1, pBOS-M5, and pBOS-SK
(A), pEGFP-N1, pBOS-M5, and pBOS-TRP6A
(B), or pEGFP-N1, pBOS-M5, pBOS-TRP6A, and
pBOS-TRP6A-antisense (C).
Ca2+-dependent changes in fluorescence were
measured in fura-2-loaded COS cells in KRH (containing 2 mM
CaCl2). EGTA (4 mM) was added to the cells to
chelate extracellular Ca2+ before measurements. Cells were
exposed to 500 µM carbachol and 4 mM
CaCl2 at the times indicated by the arrows. The
traces shown are the averages of 7 measurements of GFP
fluorescent cells (3-10 cells/measurements). D, mean
amplitudes of carbachol-induced Ca2+ influx in control and
TRP6-expressing cells. The amplitudes were obtained by subtracting the
base-line 340-nm/380-nm ratio from 340-nm/380-nm ratio measured at the
highest point of Ca2+ influx. The asterisk (*)
above the bar indicates the statistical significance of
difference (p < 0.01) compared with the control (by
Bonferroni's t test).
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Fig. 8.
Effects of OAG and PMA on Ba2+
influx in TRP6-expressing COS cells. COS cells were transfected
with pEGFP-N1, pBOS-M5, and pBOS-SK (A),
pEGFP-N1, pBOS-M5, and pBOS-TRP6A (B,
E, and F), pBOS-TRP6B (C), or
pBOS-TRP6C (D). Ba2+-dependent
changes in fluorescence were measured in fura-2-loaded COS cells in
nominally Ca2+-free KRH. Cells were exposed to 100 µM OAG and 200 µM BaCl2 at the
times indicated by the arrows. PMA (500 nM) was
added to the cells 5 min before measurement. The traces
shown are the averages of 7 measurements of GFP fluorescent cells
(3-10 cells/measurement).
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Fig. 9.
Effects of PMA and the PKC
inhibitor GF 109203X on M5 receptor-activated
Ba2+ influx in TRP6A-expressing COS cells.
Ba2+-dependent changes in fluorescence were
measured in fura-2-loaded COS cells transfected with pEGFP-N1,
pBOS-M5, and pBOS-TRP6A. Cells in nominally
Ca2+-free KRH were exposed to 500 µM
carbachol (Carb) and 200 µM BaCl2
at the times indicated by the arrows. PMA (100 nM) was added to the cells 5 min before measurements
(B and D). Cells were treated with 2 µM GF 109203X for 15 min before measurements
(C and D). Traces shown are the
averages of 7 measurements of GFP fluorescent cells (3-10
cells/measurement).
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Fig. 10.
Glycosylation analysis of
TRP6-expressing COS cells. A, the diagram shows the
location of consensus NX(S/T) glycosylation sites in TRP6A,
with the numbers referring to the positions of asparagines
in the NX(S/T) motifs. Only the site highlighted with a
black background is predicted to be exposed to the outside
of the cell. B, Western blot shows the effects of treatment
of TRP6 proteins with glycosidases. COS cells were transfected with
pBOS-TRP6A, -B, -C, or pBOS-SK as indicated. Cell lysates were
incubated overnight at 4 °C without (control) or with
0.05 units of endoglycosidase H, an enzyme that cleaves the
N-glycosidic bond between the first and second GlcNAc
residue of high mannose-containing immature glycoproteins
(+Endo.H) or with 5 units of peptide
N-glycosidase F, an enzyme that cleaves the
N-glycosidic bond between the sugar chain and asparagine of
the mature and immature forms of glycoproteins (+N-Gly.F).
SDS-polyacrylamide gel electrophoresis and Western blots were performed
as described under "Experimental Procedures." The positions of the
175-, 83-, 62-, and 47.5-kDa protein size markers are indicated.
and
TRP6C+--
The results in Fig. 10 show that cells
expressing rTRP6C have very low levels of the mature glycosylated
protein. This suggested that the 735-802 segment missing in rTRP6C is
crucial for processing of the rTRP6 protein. To test this hypothesis,
we prepared the constructs TRP6A
and TRP6C+. As shown in
Fig. 11A, TRP6A
is
equivalent to rTRP6A but is missing the 735-802 segment, and
TRP6C+ is equivalent to rTRP6C with the addition of the
735-802 segment (TRP6C+ is structurally the same as
rTRP6B). Fig. 11B shows the results of Western blot analysis
using the anti-TRP6-C terminus Ab. As shown in the figure, cells
expressing rTRP6A
lost most of the hazy material in the region
between 83 and 175 kDa compared with rTRP6A. By contrast, cells
expressing TRP6C+ contained more of this hazy material
compared with rTRP6C-expressing cells and showed the same pattern with
rTRP6B. Fig. 11C shows the Western blot result obtained by
probing with an antibody that recognizes a 12-amino acid peptide
located within the 735-802 segment region. As expected, TRP6A
was
not recognized by anti-TRP6 (788) Ab. By contrast,
TRP6C+ was recognized by anti-TRP6 (788) Ab and showed
the same pattern as rTRP6B (Fig. 11C).
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Fig. 11.
Western blots showing the expression of
constructs TRP6A and TRP6C+.
A, comparison of the structures of TRP6A, TRP6C, and
constructs TRP6A
and TRP6C+. The white and
striped bars represent the segments encoding the N-terminal
fragment (3-56 amino acids) and the 735-802 segment, respectively,
and the dotted line represents the vector. B and
C show Western blot results. Extracts were prepared from
transfected COS cells as described under "Experimental Procedures."
TRP6 bands were probed with anti-TRP6-C terminus Ab (B) or
anti-TRP6 (788) Ab (C). The positions of the 175-, 83-, 62-, and 47.5-kDa protein size markers are indicated.
-
or pBOS-TRP6C+-transfected COS cells. As shown in Fig.
12A, cells expressing TRP6A
failed to show Ba2+ influx response to carbachol
stimulation. By contrast, cells expressing TRP6C+ showed
significant Ba2+ influx, whereas the original
rTRP6C-expressing cells did not (Fig. 12B and Fig.
4D). There was also no Ba2+ influx observed
after stimulation with OAG in either TRP6A
or TRP6C+-expressing cells (Fig. 12, C and
D).
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Fig. 12.
Effects of carbachol and OAG on
Ba2+ influx in COS cells expressing
TRP6A and TRP6C+. COS cells
were transfected with pEGFP-N1, pBOS-M5, and pBOS-TRP6A
(A and C) or with pEGFP-N1, pBOS-M5,
and pBOS-TRP6C+ (B and D).
Ba2+-dependent changes in fluorescence were
measured in fura-2-loaded COS cells in nominally Ca2+-free
KRH. Cells were exposed to 500 µM carbachol, 100 µM OAG, and 200 µM BaCl2 at the
times indicated by the arrows. The traces shown
are the averages of 7 measurements of GFP fluorescent cells (3-10
cells/measurement).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, which is rTRP6A deleted for this segment, and
TRP6C+, which is rTRP6C with this fragment added on. As
expected, TRP6A
did not show glycosylation characteristic of the
mature forms and also failed to show Ba2+ influx response
to the carbachol stimulation (Fig. 11B and Fig. 12A). By contrast, TRP6C+ did express mature
glycosylated forms compared with rTRP6C, showing a pattern similar to
rTRP6B (to which it is structurally identical). As for rTRP6B,
significant Ba2+ influx was observed in
TRP6C+-expressing cells after stimulation with carbachol
(Fig. 11B and Fig. 12B). The first 35 amino acids
in the 735-802 segment
(735DDADVEWKFARAKLWFSYFEEGRTLPVPFNLVPSP769)
together with the sixth transmembrane segment are highly conserved in
TRPs from Drosophila, Caenorhabditis elegans, and
mammals, suggesting the potential functional importance of this domain. By contrast, the last 33 amino acids of this fragment
(770KSLLYLLLKFKKWMSELIQGHKKGFQEDAEMNK802)
are only conserved among TRP6s from different species. Expression of
mRNA encoding rTRP6C can be detected at low levels in brain and
PC12D cells by RT-PCR,3 but the function of this isoform
remains unknown.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Tatsuya Haga for helpful discussions and critical reading of the manuscript and Dr. Tomoyuki Takahashi and Fei-fan Guo for helpful discussions. We also thank Dr. Tom Bonner (National Institutes of Health) for the human M5 mAChR cDNA, Atsushi Fukuzaki for pBOS-M5, and Koichiro Inaki for pBOS-SK and pBOS-KS.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant-in-aid for Scientific Research 07279107 in Priority Areas on Functional Development of Neural Circuit by the Ministry of Education, Science, Sports, and Culture of Japan and by grants from the Japan Society for Promotion of Science (Research for Future Program) and the Japan Science and Technology Corp. (CREST).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) AB051212 (rTRP6A), AB051213 (rTRP6B), and AB051214 (rTRP6C).
To whom correspondence should be addressed. Tel.: 81-3-5841-3561;
Fax: 81-3-3814-8154; E-mail: zhanglei@m.u-tokyo.ac.jp.
Published, JBC Papers in Press, January 12, 2001, DOI 10.1074/jbc.M008914200
2 L. Zhang, F-F. Guo, and D. Saffen, unpublished observations.
3 L. Zhang and D. Saffen, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: TRP, transient receptor potential; mAChR, muscarinic acetylcholine receptor; TRPL, TRP-like; DAG, diacylglycerol; OAG, 1-oleoyl-2-acetyl-sn-glycerol; GFP, green fluorescent protein; KRH, Krebs-Ringer-HEPES; PMA, phorbol 12-myristate 13-acetate; RT-PCR, reverse transcriptase-polymerase chain reaction; PKC, protein kinase C; Ab, antibody.
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