From the Departments of Anesthesiology, Biological Chemistry and
Molecular, Cell, and Developmental Biology and the Molecular Biology
and Brain Research Institutes, University of California,
Los Angeles, California
Transient receptor potential (Trp) proteins form
ion channels implicated in the calcium entry observed after stimulation
of the phospholipase C pathway. Kyte-Doolittle analysis of the amino acid sequence of Trp proteins identifies seven hydrophobic regions (H1-H7) with potential of forming transmembrane segments. A limited sequence similarity to voltage-gated calcium channel
1 subunits lead
to the prediction of six transmembrane (TM) segments flanked by
intracellular N and C termini and a putative pore region between TM5
and TM6. However, experimental evidence supporting this model is
missing. Using human Trp 3 to test Trp topology, we now confirm the
intracellular nature of the termini by immunocytochemistry. We also
demonstrate presence of a unique glycosylation site in position 418, which defines one extracellular loop between H2 and H3. After removal
of this site and insertion of ten separate glycosylation sites, we
defined two additional extracellular loops between H4 and H5, and H6
and H7. This demonstrated the existence of six transmembrane segments
formed of H2-H7. Thus, the first hydrophobic region of Trp rather than
being a transmembrane segment is intracellular and available for
protein-protein interactions. A site placed in the center of the
putative pore region was glycosylated, suggesting that this region may
have been luminal and was reinserted into the membrane at a late stage
of channel assembly.
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INTRODUCTION |
Activation of a Gq protein-coupled receptor leads to the
production of inositol 1,4,5-trisphosphate
(IP3)1 via
phospholipase C and subsequently to a biphasic increase of intracellular Ca2+ concentration. The first phase is due to
the release of Ca2+ from intracellular stores. The second
depends on extracellular Ca2+ that regulates cellular
effector systems and replenish the stores. We refer to this form of
Ca2+ entry as capacitative calcium entry, a term originally
coined by Putney (1, 2). Recently, cDNAs coding for Trp proteins, a
family of mammalian proteins homologous to Drosophila Trp
and Trp-like have been cloned (3, 4) and shown to encode ion channels
that participate in capacitative calcium entry (5, 6).
Although the functional aspects of mammalian and Drosophila
Trp proteins have been studied (5-8), their transmembrane topology has
not been addressed by methods other than computer-based predictions of
hydrophobicity with the recognition that they exhibit a limited sequence similarity to portions of voltage-gated Ca2+.
Hydrophobicity analysis shows the existence of seven regions (H1-H7)
able to traverse the plasma membrane. We found similarity to
Ca2+ channels in regions H6 and H7 and the intervening
segment that is thought to contribute to the pore structure (4).
The present study addresses the transmembrane topology of one of the
Trp proteins, human transient receptor potential 3 (hTrp3), as seen
after transient expression in COS cells. hTrp3 is predicted to be a
protein of 848 amino acids with the seven hydrophobic regions mentioned
above and six endogenous NX(S/T) consensus glycosylation (6). Previous studies from our laboratory in which hTrp3, tagged at the
C terminus with the hemagglutinin antigen (HA) epitope, was
immunoprecipitated from extracts of metabolically labeled HEK cells and
analyzed by SDS-PAGE and autoradiography, showed that hTrp3 migrates as
a doublet of ~97-100 kDa (9). Digestion with peptide N glycosidase F
(PNGase F) and endoglycosidase H (Endo H) indicated that the upper band
of the doublet corresponded to a mature, Endo H-insensitive and
endoglycosidase F-sensitive form of hTrp3, whereas the lower band was
an immature Endo H-sensitive form (9). A similar experiment in COS
cells, showed only the Endo H sensitive form (9).
These initial results showed that at least one of the six putative
sites present in the hTrp3 protein is available to the glycosylation
machinery of COS and HEK cells. Below, we identify the location of the
glycosylated site. After sequential introduction of consensus
glycosylation sites into a Trp from which the endogenous glycosylated
site had been removed by site-directed mutagenesis, we then show which
of the hydrophobic regions span the membrane and thus form
transmembrane segments (TMs). Glycosylation scanning mutagenesis has
been used to elucidate topologies of several proteins, including the
cystic fibrosis transmembrane conductance regulator (10) and a
potassium channel, ROMK1 (11). Localization of N and C termini on the
cytoplasmic side of the membrane allowed us to assign the direction in
which the transmembrane segments span the membrane.
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EXPERIMENTAL PROCEDURES |
cDNA Constructs--
cDNAs encoding hTrp3 (U47050)
carrying the primary antigenic epitope YPYDVPDYA of the
Haemophilus influenza virus hemagglutinin antigen (HA) at
its N or C termini were constructed by introducing into pcDNA3
carrying the hTrp3 on reading frame the nucleotide sequence 5'TAC CCG
TAC GAT GTT CCT GAT TAC GCG immediately after the ATG initiation codon
or the TGA stop codon. The resulting plasmids were called
pcD-HA(N)-hTrp3 and pcD-HA(C)-hTrp3. All glycosylation mutations were
introduced into pcD-HA(N)-hTrp3. Consensus glycosylation sites at
positions 405, 418, and 562 (numbers correspond to the position of the
asparagine of the consensus glycosylation motifs) were removed to give
the
405,
418, and
562 forms of hTrp3 by replacing the
corresponding asparagines by the amino acids shown in Fig. 1 using the
oligonucleotides listed in Table I. We used a two-step PCR approach in
which two initial PCR fragments with overlapping ends encoding the
desired mutations were used as primers in a second step PCR to create extended BstEII/HpaI fragments of 1909 base pairs
with the desired mutation. The BstEII- and
HpaI-digested PCR fragments were then cloned into
BstEII- and HpaI-digested pcD-HA(H)-hTrp3. The
sense strand primers for the first round PCR are listed in Table I. All
the other mutations were introduced into pcD-HA(N)-hTrp3 by standard
site-directed mutagenesis using a commercially available in
vitro mutagenesis kit (QuickChange, Stratagene), primers listed in
Table I and the
418 hTrp3 mutant as template.
Metabolic Labeling of COS Cells Expressing the Wild Type or
Mutant Forms of hTrp3, Immunoprecipitation, and Glycosidase
Treatments--
COS-M6 cells were maintained under subconfluent
conditions at 5% CO2 in Dulbecco's minimum essential
medium containing 4.5 mg/ml D-glucose, 10%
heat-inactivated fetal bovine serum, 50 units/ml penicillin, and 50 units/ml streptomycin at 37 °C. 24 h prior to transfection,
cells were seeded into fresh 10-cm Petri dishes at a density of
106 cells/plate. Cells were then transfected with 5 µg
plasmid DNA/10-cm dish using the DEAE-dextran method as described in
Sambrook et al. (12). After 48 h, the cells were rinsed
with Hank's balanced salt solution (Life Technologies, Inc.), overlaid
for one hour with methionine-free Dulbecco's minimum essential medium
(ICN), and labeled for 90 min with 1 ml of the same medium containing 50 µCi of 35S-Express Protein labeling mixture (NEN Life
Science Products). After rinsing, the cells were scraped from the
plates and collected by centrifugation at 2000 × g for
5 min at 4 °C. The cell pellet from each plate was lysed by addition
of 500 µl RIPA buffer (150 mM NaCl, 50 mM
Tris-HCl, pH 8.0, 0.5 mM EDTA, 1% Nonidet P-40, 0.5%
deoxycholic acid, 0.1% SDS) containing protease inhibitors (0.1 mM phenylmethylsulfonyl fluoride, 1 µg/ml soybean trypsin inhibitor, 0.5 µg/ml leupeptin). Disruption of the cells was ensured by drawing the mixture several times into a 1-ml syringe fitted with a
25-gauge needle. The lysate was cleared by centrifugation at
13,500 × g for 10 min at 4 °C, and the supernatant
was divided into aliquots of 200 µl. One aliquot was used as control,
the other was digested with 0.2 unit of PNGase F (Boehringer Mannheim , 0.2 unit/µl) or 5 units of endoglycosidase H (Boehringer Mannheim, 0.1 unit/µl) for 2 h at room temperature.
Monoclonal antibody 12CA5 (ascites fluid diluted 1:100) and 50 µl of
a 50% (v/v) slurry of protein A-Sepharose, prewashed with RIPA buffer,
were added and incubated for either 4 h at room temperature or
overnight at 4 °C. The beads were centrifuged (1,000 × g, 2 min), washed three times with 1 ml of RIPA buffer, and recovered each time by centrifugation. Proteins were eluted with 80 µl of 1.5 × Laemmli buffer containing 10%
-mercaptoethanol. The samples were analyzed by SDS-PAGE in 9% polyacrylamide gels, followed by autoradiography of the dried gel slabs.
Immunocytochemical Localization of HA Epitope-tagged hTrp3
Expressed on COS-M6 Cells--
To determine the epitope orientation of
HA-tagged protein on the plasma membrane, 2 µg of cDNA was
transfected into COS-M6 cells as described in Zhu and Birnbaumer (13).
One day after transfection, the cells were trypsinized and seeded at
about 2,000 cells/well onto two sets of 96-well plates. Immunochemical
staining with a monoclonal HA antibody, 12CA5 (Babco, Berkeley, CA) was performed one day later. For one set, cells were washed three times
with 100 µl of Dulbecco's phosphate-buffered saline (D-PBS) solution
without Ca2+ and Mg2+ at room temperature. 100 µl of ice-cold 4% paraformaldehyde in D-PBS was then added to fix
and permeabilize the cells for 15 min. Immunocytochemical staining of
the intact cells was performed as described by Vannier et
al. (14).
All incubations were carried out at room temperature at 100 µl of
solution per well. Cells were first incubated in buffer A (3% bovine
serum albumin, 0.2% Triton X-100 in D-PBS) for 1 h and then 10 min in buffer A with 0.3% hydrogen peroxide. After washing once in
D-PBS, cells were incubated in buffer A with the primary antibody,
12CA5 (1:200 dilution) for 1 h. The cells were then washed three
times with D-PBS and incubated in the secondary antibody, anti-mouse
IgG conjugated with peroxidase (Amersham Pharmacia Biotech), at a
1:1000 dilution in buffer A for 1 h.
The cells were washed three times and incubated for 30 min with
3-amino-9-ethyl-carbazole (AEC, Sigma) following the protocol of the
manufacturer. Positive cells were stained red and visible through a
light microscope. In this case, the anti-HA antibody reached both the
inside and outside of the plasma membrane. All cells expressing the
epitope-tagged protein are stained.
For the other set, the cell culture medium was replaced with 100 µl
of fresh medium containing 12CA5 monoclonal antibody (1:200 dilution),
and the incubation continued for two hours in the cell culture
incubator. The cells were then washed three times with D-PBS and fixed
with 4% paraformaldehyde for 15 min. Immunocytochemical staining was
performed as above without incubation with the primary antibody. In
this case, since the anti-HA antibody was used when the cells were
still intact, only the epitope that was exposed to the external part of
the cells should show positive staining.
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RESULTS |
N and C Termini of hTrp3 Are Intracellular--
All Trp protein
models place the N and C termini in the cytoplasm.
Co-immunoprecipitation of Drosophila Trp with INA-D, the presence of ankyrin repeats in the N termini and of consensus calmodulin binding sites in the C termini of Drosophila Trp
and Trp-like are consistent with their cytoplasmic location. However, we found no data in the literature that would substantiate this assumption. We therefore expressed both the HA(N)- and HA(C)-hTrp3, as
well as an N-terminally tagged vasopressin receptor, HA(N)-V2R, in COS
cells and tested for accessibility of the HA epitope to an HA
monoclonal antibody under conditions where the cell's integrity was
either preserved (extracellular staining) or destroyed by fixation with
paraformaldehyde (intracellular plus extracellular staining) as
described under "Experimental Procedures."
As shown in Fig. 1, even though
expression of the V2 vasopressin receptor tagged with the HA epitope at
its N terminus yielded a positive reaction when subjected to
"extracellular" staining, neither the hTrp3 with the epitope on the
N terminus nor hTrp3 with the HA epitope at the C terminus gave a
positive reaction for extracellular HA epitope. We verified that the
cells expressed the constructs by applying and obtaining a positive
reaction with the whole cell staining test. This test showed that Trp
was expressed throughout the cell including the lamellopodia, which due
to their thin and transparent nature are not visible by simple light
microscopy when they are not stained.

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Fig. 1.
Immunocytochemical test for cell surface
expression of hTrp3 tagged at its N or C termini with the HA epitope.
A, whole cell (left panels) and extracellular
(right panels) staining of COS cells transfected with
pcD-HA(N)-hTrp3 or pcD-HA(C)-Trp3. B, same as in
A but after transfection of the V2 vasopressin receptor
tagged at its extracellular N terminus with the HA epitope (kindly
provided by Mariel Birnbaumer).
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These results validated the previous assignments of a cytosolic
orientation to the N and C termini of Trp proteins (6, 15). This is
especially important as it was recently shown that Ca2+-
and voltage-dependent K+ channels
(Kv(Ca)) have an extracellular N terminus and traverse the
plasma membrane seven times instead of six times as had been presumed
on the basis of its homologous relation to Shaker type K+
channels and the four hydrophobic repeats that form up voltage-gated Ca2+ and Na+ channels (16).
Transmembrane Disposition of hTrp3--
N-glycosylation
of proteins is co-translational and occurs on the luminal side of the
endoplasmic reticulum, which corresponds to the extracellular side of
the cells once the protein is transported to the plasma membrane.
Therefore, only extracellular sites of a transmembrane protein targeted
to the plasma membrane are glycosylated. Amino acid sequence analysis
of hTrp3 showed that it contained seven hydrophobic regions (H1-H7)
between amino acids 350 and 680. In addition, it also has six consensus
NX(S/T) motifs for N-glycosylation. One with Asn
at position 339 is located prior to H1; three at positions 405, 418, and 562 are in stretches that link hydrophobic regions and could
constitute loops between TM segments; and the remaining two at
positions 657 and 673 are either within H7 or at the presumed
TM-cytosol interface (Fig. 2,
A and B).

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Fig. 2.
Schematic representation of the hydrophobic
profile of hTrp3 as determined by Kyte-Doolittle analysis of the amino
acid sequence and summary of placement of natural and artificially
inserted consensus glycosylation sites. A, Kyte-Doolittle
plot of hTrp3 hydropathy. Hydrophobic regions H1-H7 and the putative
pore region (PP) are highlighted. B, positions of
consensus glycosylation sites. Black endogenous sites;
open triangles denote glycosylation sites inserted by
site-directed mutagenesis. Numbers indicate the position of
the Asn residue of the NX(S/T) motifs.
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Since we had previously shown that hTrp3 is glycosylated (9), we first
determined where it is glycosylated. Using the construct of hTrp3
tagged with an HA epitope at the N terminus, a first series of mutants
was produced where the endogenous glycosylation sites with Asn at
positions 405, 418, or 562 were changed to Asp, Gly, or Val,
respectively, as shown in Table I giving
the
405,
418, and
562 hTrp3 mutants. In addition, we prepared
the double mutant
405/
418 hTrp3. Mutants
405,
418,
405/
418, and
562 were expressed in COS cells, labeled with
[35S]methionine, immunoprecipitated before and after
treatment with PNGase F, and analyzed by SDS-PAGE (Fig.
3). For wild type hTrp3 and mutants
405 and
562, the major band detected was sensitive to digestion
by PNGase F and also by Endo H as previously shown (9). This band
therefore corresponds to a glycosylated immature form of the protein. A
minor band that corresponds to the immature nonglycosylated protein was
also observed under the 90-min labeling condition used in these
experiments. Since glycosylation is a co-translational event, the
detection of a fraction of the hTrp3 as a nonglycosylated protein plus
the remainder of the protein as a form sensitive to Endo H indicates
that COS cells cannot fully process the hTrp3 protein. Such
observations have also been reported for the Shaker B K+
channel expressed in Sf9 cells (17). Although it was not
biochemically detectable, the fully matured hTrp3 is very likely also
expressed in COS cells, as there is synthesis of an active cation
influx pathway in cells transfected with the full-length hTrp3
(17).

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Fig. 3.
SDS-PAGE analysis of the glycosylation state
of the wild type and mutant forms of HA-tagged hTrp3. Wild type
and mutant HA(N)-hTrp3 were expressed in COS-M6 cells, labeled with
[35S]methionine, extracted with RIPA buffer, treated in
the absence and presence of PNGase F, immunoprecipitated with 12CA5
monoclonal antibody, and analyzed by SDS-PAGE and autoradiography as
described under "Experimental Procedures."
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The
418 and
405/
418 mutants were not sensitive to PNGase F and
migrated with the same mobility of the wild type hTrp3 digested with
PNGase F (Fig. 3A). This indicated that Asn-418 is the site glycosylated in hTrp3 and that contrary to the model previously proposed by us (6), the loop between H2 and H3 where this site is
located is extracellular rather than intracellular. Position 405, although close to 418 was not glycosylated, indicating that it is
either too close to the membrane to be available to the glycosylation
machinery or that it is located in a transmembrane region.
The
418 mutant cDNA was used as a starting point for the
construction of the subsequent mutants in which single glycosylation sites were inserted into each of the putative extra- and intracellular loops (Fig. 2B). To facilitate identification of the mutant
clones, the nucleotide composition of the insert was chosen so as to
not only introduce a NX(S/T) coding sequence but to also
create in each instance a new EcoRI restriction site
(GAATTC, Table I).
Figs. 3, B and C, show that mutants with
consensus glycosylation sites at positions 379 (between H1 and H2), 457 (between H3 and H4), 570 (between H5 nd H6), and 696 (immediately after H7) were unaffected by PNGase F treatment and migrated as single bands
of the same apparent size as the nonglycosylated wild type hTrp3. This
indicated that these mutants are not glycosylated. A doublet,
characteristic of the glycosylated hTrp3 in COS cells, was observed for
mutant 509 (insertion of a site between H4 and H5). None of four
insertion mutants, two in positions 604 and 610 located in the linker
connecting H6 to the putative pore and two in positions 637 and 644 located in the linker connecting the putative pore to H7, were
glycosylated even though each of the mutant proteins was expressed as
shown by immunoprecipitation of the corresponding metabolically labeled
bands (data not shown).
These results along with the glycosylation of the wild type hTrp3 at
position 418 showed that hTrp3 had a transmembrane topology that is
consistent with H1 being intracellular and H2-H7 forming six TM
segments. The data so far indicated the existence of four transmembrane
segments formed of H2, H3, H4, and H5 but gave no indication as to the
transmembrane orientation (or lack of thereof) of H6 and H7.
The failure of mutants to be glycosylated in positions corresponding to
the linkers surrounding the putative pore region could be either
because of unlucky choice of the insertions creating unfavorable
conformations or because the stop-transfer and membrane-anchor signals
that delimit transmembrane regions are closer to the putative pore than
thought. This would place the glycosylation motifs within the membrane
and make them unaccessible to the glycosylation machinery.
We thus made an additional mutant in which we placed the glycosylation
motif in the middle of the putative pore, at position 621. As shown in
Fig. 3C, hTrp3 with a glycosylation site in the putative pore is
glycosylated and indicates that H6 and H7 traverse the plasma
membrane.
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DISCUSSION |
The results obtained in this study through glycosylation mutants
indicate that Trp proteins indeed have six transmembrane segments (Fig.
4), of which the last two are connected
by a large loop. Although data to this effect are not yet available,
the likelihood of it being the pore is predicated on the sequence homology between TM5-TM6 loops of Trps and the S5
S6 loops of voltage-gated Ca2+ channels (cf. Fig. 5 of Ref.
4).

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Fig. 4.
Proposed model of the topology of hTrp3 based
on experiments presented in this report. , endogenous
glycosylation sites; , glycosylation sites inserted by site-directed
mutagenesis into 418 HA(N)-hTrp3. All mutant proteins were expressed
and tested for glycosylation. Glycosylated sites, deduced from the
results shown in Fig. 3, are highlighted by a the tree-like
ideogram. Cytosolic orientation of N and C termini is based on the
immunocytochemical results shown in Fig. 1.
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Ours are not the first experiments showing glycosylation of a pore
region, as the pore region of the ROMK1 K+ channel has been
shown to be susceptible to glycosylation when suitably mutated (11).
Once assembled, the pore regions are presumed to be intramembranous.
Their susceptibility to glycosylation indicates that the "pores" at
one point in time were luminal and suggests that their insertion into
the lipid bilayer is a late event in the maturation of the channel.
Further experiments in which single amino acids of the putative pore
formed by the H6 to H7 linker are changed followed by determination of
changes in ion selectivity will be needed to confirm that the linker
indeed contributes to the formation of the pore of the channels formed by Trp proteins. The present study identifies on an experimental basis
which of the seven hydrophobic domains of Trp proteins traverse the
plasma membrane in which direction. Contrary to previous assumptions, the H1 domain is not a TM segment, whereas the much shorter H3 is a TM
segment.
The delineation of six transmembrane regions formed of hydrophobic
regions 2, 3, 4, 5, 6, and 7 clearly confirms at the topological level
the relatedness of Trp channels to other channels formed of units that
traverse the membrane six times. In addition by analogy, the data
suggest that Trp channels should be tetrameric in nature, as are
voltage-gated K+ channels, Ca2+, and
Na+ channels, which are concatenated tetramers (18).
In a recent work the Drosophila Trp and Trp-like have been
shown by co-immunoprecipitation to form heteromultimers as well as
homomultimers (19). The production of chimeras between Trp or Trp-like
and the Shaker B channel demonstrated that these interactions not only
occur in intact cells but may have a functional significance in
Drosophila. Formation of heteromultimers between Trp3 and
Trp1 was also shown in this study, but no functional data were
presented. Although Trp3 and Trp1 are both expressed in the brain, we
do not know if hTrp1 and hTrp3 are expressed in the same cells. Further studies will be needed not only to demonstrate that functional multimers of Trp are formed in mammalian cells but also to determine the composition and stoichiometry of these complexes.