Muscarinic Acetylcholine Receptor Regulation of TRP6 Ca2+ Channel Isoforms

MOLECULAR STRUCTURES AND FUNCTIONAL CHARACTERIZATION*

Lei ZhangDagger and David Saffen

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



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-TRP6ADelta 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.

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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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).


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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)).

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).


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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.

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).


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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).

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).


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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).

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


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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).

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.


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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.

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.


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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).

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


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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).

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.


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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).

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).


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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.

Properties of the Constructs TRP6ADelta 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 TRP6ADelta and TRP6C+. As shown in Fig. 11A, TRP6ADelta 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 rTRP6ADelta 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, TRP6ADelta 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 TRP6ADelta and TRP6C+. A, comparison of the structures of TRP6A, TRP6C, and constructs TRP6ADelta 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.

To test the functional importance of the 735-802 segment, we measured carbachol- and OAG-stimulated Ba2+ influx in pBOS-TRP6ADelta - or pBOS-TRP6C+-transfected COS cells. As shown in Fig. 12A, cells expressing TRP6ADelta 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 TRP6ADelta 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 TRP6ADelta and TRP6C+. COS cells were transfected with pEGFP-N1, pBOS-M5, and pBOS-TRP6ADelta (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

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 TRP6ADelta , which is rTRP6A deleted for this segment, and TRP6C+, which is rTRP6C with this fragment added on. As expected, TRP6ADelta 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.

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.

    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).

Dagger 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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DISCUSSION
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