The Membrane Topology of Human Transient Receptor Potential 3 as Inferred from Glycosylation-scanning Mutagenesis and Epitope Immunocytochemistry*

Brigitte VannierDagger , Xi Zhu, Darren Brown, and Lutz Birnbaumer

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

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha 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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

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 Delta 405, Delta 418, and Delta 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 Delta 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% beta -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.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

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).

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.

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 Delta 405, Delta 418, and Delta 562 hTrp3 mutants. In addition, we prepared the double mutant Delta 405/Delta 418 hTrp3. Mutants Delta 405, Delta 418, Delta 405/Delta 418, and Delta 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 Delta 405 and Delta 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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Table I
Oligonucleotides used as primers to inactivate endogenous glycosylation sites and to insert new ones


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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."

The Delta 418 and Delta 405/Delta 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 Delta 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.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 right-arrow 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. open circle , endogenous glycosylation sites; bullet , glycosylation sites inserted by site-directed mutagenesis into Delta 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.

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.

    FOOTNOTES

* Supported in part by National Institutes of Health Grant HL-45198 (to L. B.) and GM-54235 (to X. Z.).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.

Dagger Recipient of a fellowship from the Association pour la Recherche Contre le Cancer (ARC).

1 The abbreviations used are: IP3, inositol 1,4,5- trisphosphate; H1-H7, hydrophobic regions of Trp proteins; HA, hemagglutinin antigen; RIPA, radioimmunoprecipitation assay buffer; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; TM, transmembrane; Trp, transient receptor potential; hTrp3, human Trp 3; PNGase F, peptide N-glycosidase F; Endo H, endoglycosidase H.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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