Receptor-activated Ca2+ Influx via Human Trp3 Stably Expressed in Human Embryonic Kidney (HEK)293 Cells
EVIDENCE FOR A NON-CAPACITATIVE Ca2+ ENTRY*

Xi ZhuDagger §, Meisheng Jiang, and Lutz Birnbaumerpar

From the Dagger  Department of Pharmacology and Neurobiotechnology Center, Ohio State University, Columbus, Ohio 43210 and Departments of  Anesthesiology and Biological Chemistry, School of Medicine, and the par  Molecular Biology and Brain Research Institutes, UCLA, Los Angeles, California 90095

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Ca2+ release from its internal stores as a result of activation of phospholipase C is accompanied by Ca2+ influx from the extracellular space. Ca2+ influx channels may be formed of proteins homologous to Drosophila Trp. At least six non-allelic Trp genes are present in the mouse genome. Full-length human, bovine, mouse, and rat cDNAs for Trp1, 3, 4, 6 have been cloned. Expression of these genes in various mammalian cells has provided evidence that Trp proteins form plasma membrane Ca2+-permeant channels that can be activated by an agonist that activates phospholipase C, by inositol 1,4,5-trisphosphate, and/or store depletion. We have stably expressed human Trp3 (hTrp3) in human embryonic kidney (HEK)293 cells. Measurement of intracellular Ca2+ concentrations in Fura2-loaded cells showed that cell lines expressing hTrp3 have significantly higher basal and agonist-stimulated influxes of Ca2+, Mn2+, Ba2+, and Sr2+ than control cells. The increase in Ca2+ entry attributable to the expression of hTrp3 obtained upon store depletion by thapsigargin was much lower than that obtained by stimulation with agonists acting via a Gq-coupled receptor. Addition of agonists to thapsigargin-treated Trp3 cells resulted in a further increase in the entry of divalent cations. The increased cation entry in Trp3 cells was blocked by high concentrations of SKF 96365, verapamil, La3+, Ni2+, and Gd3+. The Trp3-mediated Ca2+ influx activated by agonists was inhibited by a phospholipase C inhibitor, U73122. We propose that expression of hTrp3 in these cells forms a non-selective cation channel that opens after the activation of phospholipase C but not after store depletion. In addition, a subpopulation of the expressed hTrp3 may form heteromultimeric channels with endogenous proteins that are sensitive to store depletion.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The activities of a large number of enzymes are regulated through changes of intracellular Ca2+ concentration ([Ca2+]i).1 Under resting conditions, cells keep [Ca2+]i at approximately 100 nM. A rise of the cytosolic Ca2+ triggers a cascade of Ca2+-sensitive events that are both immediate, such as secretion, contraction, and mobilization of energy resources (e.g. glycogenolysis), and long term, such as changes in the transcription of many genes. Some of the Ca2+-responsive transcription processes are known to cause proliferation and programmed cell death (1-3). In both nonexcitable and excitable cells, Ca2+ signaling pathways can be activated by a ligand binding to cell surface receptors that activate phospholipase C (PLC). These include receptors that activate heterotrimeric G proteins and receptors signaling through activation of protein tyrosine kinases. The activation of PLC leads to the production of inositol 1,4,5-trisphosphate (IP3), which binds to IP3 receptors, a class of intracellular ligand-operated Ca2+ release channels. The opening of IP3 receptors allows Ca2+ to exit from its internal storage pools, causing a rapid increase in [Ca2+]i. The increased cytosolic Ca2+ level is reduced quickly by Ca2+ pumps located on both the endoplasmic reticulum and the plasma membrane, causing [Ca2+]i to decrease. The Ca2+ signal is prolonged, however, by the opening of a set of plasma membrane Ca2+-permeant channels that allow Ca2+ to enter cells from the extracellular space, where the concentration of Ca2+ is in the millimolar range. In many, and possibly all cells, the entering Ca2+ is taken up rapidly by a storage compartment from which it is re-released in the continued presence of the triggering extracellular signal. This may, although not necessarily, be accompanied by periodic oscillations of the [Ca2+]i and allows for regulation of cytosolic as well as membrane-associated functions of the affected cells (for reviews, see Refs. 1 and 4).

Putney (5, 6) coined the term capacitative Ca2+ entry (CCE) for Ca2+ entry that is activated upon stimulation of cells with agonists that promote Ca2+ release from internal stores. After the discovery that CCE can be stimulated by mere store depletion, as occurs after inhibition of sarcoplasmic endoplasmic reticulum Ca2+-activated ATPases with thapsigargin (TG) (7, 8), channels that mediate this type of CCE have been referred to as store-operated channels. In most cells, Ca2+ entry after stimulation by agonists can be explained by activation of store-operated channels, but neither the mechanism of how the store depletion signal is transmitted to the plasma membrane nor the molecular nature of the plasma membrane channels mediating Ca2+ has been clearly identified. Moreover, although store-operated plasma membrane Ca2+-permeant channel activities have been found in many cell types (9-13), channels regulated by other mechanisms, such as a G protein, IP3, inositol 1,3,4,5-tetrakisphosphate (IP4), Ca2+, or ATP, seem to coexist with store-operated channels (14-17). The molecular basis of this diversity and the relation of store depletion-insensitive Ca2+-permeant channels to store depletion-sensitive Ca2+-permeant channels are unclear. However, it is likely that multiple Ca2+ influx channels are involved in Ca2+ entry initiated by the activation of PLC-linked receptors.

In Drosophila eyes, the light-induced current is carried by channels formed from at least two related photoreceptor-specific proteins, Trp and Trpl (Trp-like). Trp was identified by molecular cloning as a protein missing from the transient receptor potential (trp) mutant (18). Trpl was identified biochemically as a calmodulin-binding protein and was cloned by standard techniques revealing a structure that shares a high degree of homology with Trp (19). Because the phototransduction pathway in insects resembles the PLC/IP3 signaling pathway in mammalian cells, it was speculated that mammalian homologs of Trp may exist and form Ca2+ influx channels. In support of this, expression of Trpl in Sf9 cells led to development of an agonist-stimulated non-selective Ca2+-permeable ion conductance, and that of Trp led to the development of a TG-stimulated ion conductance (20-22). However, neither Trp-formed channels nor Trpl-formed channels displayed the ion permeation properties of endogenous insect CCE channels and, while forming channels, Trpl was not activated by TG treatment. More recently, work from the laboratories of Minke (23) and Montell (24) showed that Trp and Trpl can form heteromultimeric ion channels with properties that differ from those of their parental molecules, suggesting that native voltage-independent Ca2+ influx channels in insects are likely to be formed of more than one type of subunit, i.e. to be heteromultimers.

Several mammalian sequences homologous to the Drosophila Trps2 have been identified from the data base of expressed sequence tags and by reverse transcriptase-polymerase chain reaction using degenerate oligonucleotide primers based on the Drosophila sequences (25-30). In the mouse we found the existence of at least six non-allelic Trp genes that can be divided into four major types based on primary amino acid sequence similarity (26, 31). Full-length cDNAs for Trp1, 3, 4, and 6 from human, murine, rat and bovine sources have been reported (25-27, 29-32). Functional expression of human Trp1, Trp3, bovine Trp4, or mouse Trp6 in COS, Chinese hamster ovary, or human embryonic kidney (HEK)293 cells results in enhancement of either agonist-stimulated Ca2+ entry or of IP3- or TG-stimulated inward currents that are at least in part carried by Ca2+ (26, 29, 31, 32). More importantly, agonist-stimulated Ca2+ influx in murine L cells was blocked by transfection of murine Trp cDNA fragments in the antisense direction (26, 31), showing that mammalian Trp proteins are involved in agonist-stimulated Ca2+ influx.

For the present work we developed stable HEK293 cell lines expressing human Trp3 (hTrp3) and studied their Ca2+ influx properties. We report that the expression of hTrp3 caused an increase of [Ca2+]i under basal and agonist-stimulated conditions. The influx pathway formed by hTrp3 permeates Ca2+, Sr2+, and Ba2+ equally well, whereas the pathways intrinsic to HEK293 cells seem to be more Ca2+ selective. The influx due to hTrp3 can be blocked by SKF 96365, a known CCE blocker, or by verapamil, a nonspecific blocker for L-type voltage-sensitive Ca2+ channels. Although treatment with TG caused a small increase of Ca2+ influx over the basal in cell lines expressing hTrp3, a large portion of the influx pathway due to hTrp3 is not sensitive to store depletion and seems to depend primarily on the activation of PLC.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- SKF 96365 hydrochloric acid was purchased from Calbiochem. TG, U73122, (±) verapamil were from Research Biochemicals International. Carbachol, 3-amino-9-ethyl-carbozole tablets, deoxycholic acid, phenylmethanesulfonyl fluoride, soybean trypsin inhibitor, leupeptin, and Nonidet P-40 were purchased from Sigma. 35S-Express protein labeling mix (10 Ci/liter) and N-[3H]methylscopolamine (82 Ci/mmol) were purchased from NEN Life Science Products. Peptide-N-glycosidase F and endoglycosidase H were from Boehringer Mannheim. Protein A-Sepharose (4 Fast Flow) was from Pharmacia Biotech. Fura2 acetoxymethyl ester (Fura2/AM) and Pluronic F-127 were from Molecular Probes (Eugene, OR). Monoclonal antibody 12CA5 was from Babco (Berkeley, CA). All tissue culture reagents were purchased from Life Technologies, Inc. unless indicated otherwise.

cDNAs and Expression Vectors-- The wild type hTrp3 was subcloned into the mammalian expression vector pcDNA3 (26). The insert, located in between the EcoRI and the XbaI site of the polylinker of pcDNA3, contains nucleotide -15 (A of the first ATG is nucleotide 1) to 3285 of hTrp3 cDNA which includes a poly(A) tail (n = 30) and is flanked by a 36-nucleotide sequence from the Marathon cDNA adaptor (CLONTECH) on each side. To introduce an epitope for hemagglutinin (HA) at the C terminus of hTrp3, oligonucleotide A, 5'-CCCAGCATGCTGTACCCGTACGATGTTCCTGATTACGCGAGATGTGAATGATGCAGCA-3', which contains a sequence (underlined) encoding the HA epitope (YPYDVPDYA) in between codons for Leu-845 and Arg-846 at the C terminus of hTrp3 cDNA, was synthesized. A partial hTrp3 sequence was amplified by polymerase chain reaction using hTrp3 in pcDNA3 as a template and oligonucleotide A and Sp6 as primers. The polymerase chain reaction product was subcloned back into the original plasmid by ligation of an EcoRI/SphI fragment generated from hTrp3/pcDNA3, the SphI/XbaI fragment of the polymerase chain reaction product, and the EcoRI/XbaI fragment of pcDNA3. The presence of the HA epitope sequence in the hTrp3-HA/pcDNA3 plasmid was confirmed by sequencing. The rat V1aR/pcDNA3 expression vector was a gift from Dr. Mariel Birnbaumer.

Cell Lines and Cell Culture Conditions-- HEK293 cells were cultured in Dulbecco's modified Eagle's medium containing 4.5 mg/ml glucose, 10% heat-inactivated fetal bovine serum, 50 units/ml penicillin, and 50 mg/ml streptomycin. The hTrp3-HA/pcDNA3 (100 ng) was transfected into the HEK cells, plated at 2.8 × 106 cells/10-cm tissue culture dish 20 h before transfection, by the calcium phosphate/glycerol shock method (33). After 24 h, cells were harvested, diluted in medium supplemented with 400 µg/ml G418, and transferred to wells of 96-well plates at different dilutions. G418-resistant transformants were expanded into 12-well plates. To identify the clones expressing hTrp3-HA, cells were seeded in 96-well plates precoated with poly-D-lysine (100 µg/ml). Immunocytochemical staining was performed as described by Vannier et al. (34) using a monoclonal HA antibody (12CA5) as the primary antibody, anti-mouse IgG conjugated with peroxidase (Amersham) as the secondary antibody, and 3-amino-9-ethyl-carbozole as the colorant. Positive cells were stained red and were visualized through a light microscope. Eleven cell lines expressing the HA epitope were further expanded and analyzed for the expression of HA-tagged hTrp3 by immunoprecipitation with 12CA5 as described below. All cell lines synthesized an immunoprecipitated protein of approximately 100 kDa when analyzed by SDS-polyacrylamide gel electrophoresis, which corresponds to the predicted size of hTrp3. Two cell lines, HEKTrp3-9 (T3-9) and HEKTrp3-65 (T3-65) were used for further analysis. Control cells were transfected with V1aR/pcDNA3 under the same conditions, two G418-resistant clones were randomly selected and designated as control-1 (C1) and control-2 (C2). The stable cell lines were diluted twice weekly and maintained in medium supplemented with 400 µg/ml G418.

Immunoprecipitation-- Cells (2 × 106) were plated in 6-cm tissue culture dishes at least 16 h before the experiments. Cells were washed once with Hanks' balanced salt solution and then incubated in 1 ml of methionine/cysteine-free Dulbecco's modified Eagle's medium (ICN) containing 5% fetal bovine serum at 37 °C for 1 h. The medium was replaced by the same medium containing 50 µCi/ml 35S-express protein labeling mix containing [35S]Met and [35S]Cys for a desired time. When needed, the incorporated radioactivity was chased by replacing the labeling medium with 4 ml of regular Dulbecco's modified Eagle's medium and continuing incubation at 37 °C for the desired time period. At the end of the incubation, cells were rinsed twice with Dulbecco's phosphate-buffered saline solution without Ca2+ and Mg2+, scraped off from the culture dish in 1 ml of ice-cold Dulbecco's phosphate-buffered saline containing 1 mM EDTA and 0.1 mM phenylmethanesulfonyl fluoride, and pelleted by centrifugation at 5,000 rpm in a microcentrifuge at 4 °C for 5 min. Cells were then homogenized in 0.5 ml of RIPA buffer (150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.1% SDS, 50 mM Tris-HCl, pH 8.0) containing 0.1 mM phenylmethanesulfonyl fluoride, 1 µg/ml soybean trypsin inhibitor, and 0.5 µg/ml leupeptin. Immunoprecipitation was performed using the anti-HA monoclonal antibody (12CA5) as described by Innamorati et al. (35). For glycosidase treatment, cell lysate was incubated with peptide-N-glycosidase F (2 units/ml) or endoglycosidase H (10 milliunits/ml) at room temperature for 1 h before antibody (12CA5) was added. The immunoprecipitated proteins were eluted in 80 µl of 2 × Laemmli buffer (1 × = 62.5 mM Tris-HCl, 1% SDS, 10% glycerol, 10% beta -mercaptoethanol, pH 6.8) and separated by SDS-polyacrylamide gel electrophoresis in 9% acrylamide gels at 40 mA for 2 h. The gels were stained with Coomassie Blue to visualize the molecular markers, destained, and then dried for autoradiography.

Measurement of [Ca2+]i-- Changes of [Ca2+]i in individual cells were monitored after loading cells with Fura2 by fluorescence videoimaging microscopy using an Attofluor Digital Imaging and Photometry attachment of a Carl Zeiss Axiovert inverted microscope as described before (26).

Changes of [Ca2+]i in cell populations were measured using an Aminco-Bowman Series 2 luminescence spectrofluorometer (SLM Instruments, Inc.). Briefly, cells grown to confluence in 15-cm dishes were trypsinized and collected in a 50-ml capped polypropylene tube. After a centrifugation for 5 min at 400 × g and removal of the supernatant, the cell pellets were resuspended at room temperature in an extracellular solution (ECS) composed of 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 10 mM glucose, 0.1% bovine serum albumin, 15 mM Hepes, pH 7.4. Cells were incubated in ECS supplemented with 5 µM Fura2/AM and 0.05% Pluronic F-127 at 37 °C for 40 min. Cells were then washed once and resuspended in ECS at 2 × 106 cells/ml. Aliquots of 2 ml were kept in the dark at room temperature until use. Before each measurement, cells were washed twice in ECS or twice in ECS to which no CaCl2 was added if a Ca2+-free/Ca2+ readdition protocol was used. 2 ml of cell suspension was transferred to a quartz cuvette and maintained at 32 °C under continuous stirring. Changes in intracellular Fura2 fluorescence intensity were measured by alternating excitation at 340 and 380 nm at 3-s intervals and detecting emission at 510 nm. Autofluorescence of the cells at 340 and 380 nm was determined from unloaded cells of an equivalent cell density and subtracted from values obtained for the Fura2-loaded cells. Drugs were added by a small aliquot (<20 µl) of concentrated stocks (dissolved either in water or dimethyl sulfoxyide) to achieve the final concentrations. At the end of each recording, 0.1% Triton X-100 was added to the cells to determine the maximal fluorescence ratio (Rmax). This was followed by an addition of 20 mM EGTA to determine the minimal fluorescence ratio (Rmin). [Ca2+]i was calculated according to Grynkiewicz et al. (36).

In most experiments, Ca2+ entry under the basal or stimulated conditions was measured by a Ca2+-free/Ca2+ readdition protocol, in which cells were incubated in a nominally Ca2+-free ECS stimulated or not by the addition of an agonist or a store depletion drug. The stimulation normally caused a transient [Ca2+]i increase due to the release of Ca2+ from intracellular Ca2+ stores. After allowing the first [Ca2+]i peak to decrease to a steady-state level (normally 3-7 min), CaCl2 was added to give a final concentration of 1.8 mM. Typically, this led to a second [Ca2+]i increase. This extracellular Ca2+-dependent [Ca2+]i increase is assumed to be caused by Ca2+ influx.

Measurements of Influxes for Sr2+, Ba2+, and Mn2+-- A Ca2+ free/cation addition protocol, analogous to the Ca2+-free/Ca2+ readdition protocol in which Sr2+ or Ba2+ was added during the cation readdition phase, was used to study the entry of Sr2+ and Ba2+ into cells. The extent of influx of these divalent cations is expressed in the figures as changes in the ratio of 340 to 380 nm fluorescence without the estimation of their intracellular concentrations.

In a similar manner, MnCl2 was added to a final concentration of 50 µM to cells incubated in a nominally Ca2+-free ECS under either unstimulated or stimulated conditions. Fluorescence quenching was studied using the Fura2 isosbestic excitation wavelength at 360 nm and recording emitted fluorescence at 510 nm. At the end of each recording, 0.1% Triton X-100 was added to cells to determine the maximal quenching of Fura2 by Mn2+. Results of fluorescence quenching are expressed (in percent) as the amount of fluorescence decreased from the initial value (before the addition of Mn2+) divided by the maximal loss after the addition of Triton X-100.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Activity of HA Epitope-tagged hTrp3-- We showed previously (26) that transient expression of hTrp3 in COS-M6 cells results in a 2-fold increase of Ca2+ influx in response to the activation of a coexpressed M5 muscarinic receptor. To study the function and biochemical properties of hTrp3 in more detail, we decided to develop stable cell lines expressing hTrp3 at high levels. To aid the identification of cell lines expressing hTrp3, we added an HA epitope at the C terminus of hTrp3 (hTrp3-HA). The resulting cDNA construct was first transfected to COS-M6 cells, and its function on Ca2+ influx was compared with the wild type hTrp3, which was transfected in parallel in the same experiment. Ca2+ influx induced by 20 µM carbachol (CCh) was identical in cells transfected with the wild type hTrp3 or hTrp3-HA (Fig. 1). We transfected hTrp3-HA/pcDNA3 into HEK293 cells and selected 11 cell lines that expressed the HA-tagged hTrp3. Two cell lines, HEKTrp3-9 (T3-9) and HEKTrp3-65 (T3-65), expressed relatively high levels of hTrp3 and were used in studies presented in this report. Two control cell lines (C1 and C2) were obtained by transfecting cDNA for the rat V1aR into the HEK293 cells. Thus the hTrp3 cells and the control cells underwent the same type of treatment and selection processes and were kept under the same culturing conditions.


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Fig. 1.   Introduction of an HA epitope at the C terminus of hTrp3 does not alter its ability to enhance Ca2+ influx in response to stimulation of a Gq-coupled receptor. COS-M6 cells were transiently cotransfected with cDNAs encoding wild type hTrp3 (a), hTrp3-HA (b), or murine luteinizing hormone receptor (c, control) plus the rat M5 muscarinic receptor as described previously (26). Changes of [Ca2+]i in individual cells loaded with Fura2 were measured by videoimaging microscopy 40 h post-transfection as described in Zhu et al. (26). CCh at 20 µM was added at 1 min and was present throughout the experiment as indicated by the horizontal bar. A solution containing 0.5 mM EGTA was replaced by a solution containing 1.8 mM CaCl2 at 4 min. Data are averages of [Ca2+]i pooled from cells that responded to CCh from six experiments. The numbers of the cells are 93 (a), 121 (b), and 154 (c).

Glycosylation of hTrp3-- Immunoprecipitation of HA-tagged hTrp3 from metabolically labeled T3-9 and T3-65 cells showed multiple bands at around 100 kDa (bands 1, 2, 3, 4) and a haze of radioactivity above these bands (Fig. 2A). Treatment with endoglycosidase H selectively eliminated only one of the middle bands (band 2) and increased the intensity of the lower band (band 1), suggesting that band 2 is a high mannose-containing immature form of the glycosylated hTrp3. Treatment with peptide-N-glycosidase F removed bands 2-4 and the haze while greatly enhancing the intensity of band 1. Thus, bands 2, 3, 4 and the haze of radioactivity seen on the SDS-polyacrylamide gel electrophoresis represent hTrp3 at different stages of glycosylation during remodeling of the protein-attached sugar moieties, whereas band 1 represents the non-glycosylated hTrp3. In COS cells transiently transfected with hTrp3-HA, the predominant protein was the high mannose-containing immature glycosylated form (band 2), as shown by its sensitivity to the endoglycosidase H treatment (see Fig. 7 in Ref. 31). This is not surprising since transiently transfected COS cells are known to synthesize a large quantity of the exogenous proteins of which only a fraction is processed to mature cell surface form (37).


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Fig. 2.   Immunoprecipitation of hTrp3-HA from two stable HEK293 cell lines (T3-9 and T3-65) by an anti-HA monoclonal antibody, 12CA5. Cells were metabolically labeled with [35S]Met/Cys for 2 h or as indicated. Immunoprecipitation was carried out as described under "Experimental Procedures." Panel A, glycosylation analysis of hTrp3. Cell lysate was either not treated (N) or treated with endoglycosidase H (H) or peptide N-glycosidase F (F) for 60 min at room temperature before the addition of anti-HA antibody. Panel B, time course of [35S]Met/Cys incorporation into different forms of hTrp3 in T3-9 cells. Panel C, a pulse-chase experiment showing the maturation process of hTrp3 in T3-9 cells.

The maturation process of hTrp3 can be followed in a pulse-chase experiment, as shown in Fig. 2B. After 15 min of labeling with a mixture of [35S]Met and [35S]Cys, the high mannose glycosylated hTrp3 was the major form. At 30 min, the more mature forms of glycosylated hTrp3 started to appear. At 60 and 120 min, the intensities of all of the bands increased proportionally compared with that at 30 min. The haze of radioactivity became more evident. When cells were labeled for 15 min and then the labeled 35S was chased with culture medium containing unlabeled amino acids, the intensities of both the non-glycosylated and the high mannose-containing form of hTrp3 decreased as the chase time increased (Fig. 2C). Bands 3 and 4 and the haze remained after 3 h of chase. The proportions of bands 3 and 4 and the haze remained constant for up to 8 h (not shown).

Basal and Stimulated Ca2+ Influx in hTrp3-expressing HEK Cells-- Ca2+ influx activities in the T3-9 and T3-65 cells under basal or stimulated conditions were compared with that of control cells by studying extracellular Ca2+-dependent [Ca2+]i changes in Fura2-loaded cells suspended at a density of 2 × 106 cells/ml. For the most part, we show results obtained from T3-9 and C1 cells. Similar results were obtained with T3-65 cells. C1 and C2 cells did not differ in their way of handling [Ca2+]i. Fig. 3 shows traces of [Ca2+]i changes during the course of typical experiments in which Trp3 (upper traces) or control (lower traces) cells were either not stimulated (panel A) or stimulated with 200 µM CCh (panel B) or 0.2 µM TG (panel C). Ca2+ influx was studied using the Ca2+-free/Ca2+ readdition protocol described under "Experimental Procedures." CCh was used to activate the PLC pathway via an endogenous muscarinic receptor found in the HEK293 cells. Fig. 4A shows the abundance of the muscarinic receptor sites in each cell line determined according to Liao et al. (38). Similar results were obtained by stimulating cells with 100 µM ATP, which activates an endogenous purinergic receptor. To study Ca2+ influx induced by store depletion, we used 0.2 µM TG to block the endoplasmic reticulum Ca2+-ATPase, which passively depletes the internal store without increasing the production of IP3 (4). Fig. 4 summarizes results obtained for two Trp3 cells (T3-9 and T3-65) and two control cells (C1 and C2).


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Fig. 3.   Calcium influx in stable HEK293 cell line expressing hTrp3 (T3-9) or rat V1aR (control, C1). Cells grown to confluence were trypsinized, loaded with Fura2, and maintained in suspension. Changes in [Ca2+]i were measured in cell population at a density of 2 × 106 cells/ml using a SLM-Aminco-Bowman spectrofluorometer. Cells were incubated in a nominally Ca2+-free solution either untreated (panel A) or treated with 200 µM CCh (panel B) or 0.2 µM TG (panel C) as indicated. The activities of Ca2+ influx were studied by readdition of 1.8 mM Ca2+ to the solution.


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Fig. 4.   Panel A, abundance of endogenous muscarinic receptor (mAchR) in control and Trp3 HEK293 cells. The number of the receptors/cell for each cell line was determined by Scatchard analysis of the binding of N-[3H]methylscopolamine ([3H]NMS) in increasing concentrations to intact cells as described in Liao et al. (38). Data are averages ± range of results obtained from two experiments. Panel B, basal levels of [Ca2+]i in control and Trp3 cells maintained in a physiological ECS containing either no Ca2+ (open bars) or 1.8 mM Ca2+ (filled bars). Cell lines were developed, and [Ca2+]i in cell populations was measured in Fura2-loaded cells as described under "Experimental Procedures." For [Ca2+]i in Ca2+-free solutions, cells were washed twice and then resuspended in a nominally Ca2+-free ECS. For [Ca2+]i in 1.8 mM Ca2+, cells were washed twice and then resuspended in the same ECS except that 1.8 mM Ca2+ was included. Data are averages ± S.E. of results from the number of experiments indicated in parentheses. Panel C, Ca2+ readdition elicited [Ca2+]i increases in control and Trp3 cells at basal or stimulated by agonist or TG. Cells were incubated in a nominally Ca2+-free solution unstimulated (open bars) or stimulated with 200 µM CCh (filled bars) or 0.2 µM TG (shaded bars). Then Ca2+ was added to the ECS to a final concentration of 1.8 mM. Data are maximal [Ca2+]i reached within 1 min of Ca2+ addition minus the [Ca2+]i before the Ca2+ addition from each experiment; averages ± S.E. are from the number of experiments indicated in parentheses. Filled star, because of relatively low level of Ca2+ mobilization in T3-65 and C2 by CCh alone (not shown), ATP (100 µM) was included to release the internal Ca2+ maximally before readdition of extracellular Ca2+.

Although in a nominally Ca2+-free buffer, basal [Ca2+]i in Trp3 cells is not significantly different from that of control cells; when the cells are maintained in a normal physiological solution containing 1.8 mM Ca2+, the resting [Ca2+]i in Trp3 cells is 40-70% higher than that in control cells (Fig. 4B). Addition of 1.8 mM Ca2+ to the extracellular medium of cells incubated in the Ca2+-free solution causes a 75-130 nM increase of [Ca2+]i in the Trp3 cells (Figs. 3A and 4C). In control cells, the increase was less than 20 nM, of which a fraction could represent titration of Fura2 leakage or a small proportion of leaking cells in the whole population. Therefore, under non-stimulated conditions, control HEK293 cells exhibit very low Ca2+ influx, whereas Ca2+ influx channels in the Trp3 cells appear to have a higher spontaneous activity.

In control cells maintained in a nominally Ca2+-free solution, CCh caused a rapid and transient increase of [Ca2+]i because of the release from internal Ca2+ stores. Readdition of 1.8 mM Ca2+ to the medium causes [Ca2+]i to increase about 120 nM. In TG-treated cells, readdition of Ca2+ causes a larger increase in [Ca2+]i (up to 240 nM). In the Trp3 cells, the [Ca2+]i following Ca2+ readdition after CCh or TG treatment is higher than under basal conditions, increasing by about 230 and 270 nM for CCh and TG, respectively (Fig. 4C).

A low cytosolic Ca2+ level is maintained by Ca2+ pumps located on plasma membrane and endoplasmic reticulum, as well as other Ca2+-buffering systems inside cells, which actively extrude Ca2+ out of the cell or into its intracellular stores. Opening of the Ca2+ influx channels results in a rapid rise of [Ca2+]i, which in turn causes an increase in extrusion of Ca2+ mediated by the Ca2+ pumps. Therefore, at any given time, [Ca2+]i is a result of the dynamic interplay of Ca2+ pumps, intracellular Ca2+ channels (e.g. IP3 receptors), and plasma membrane Ca2+ influx channels. Upon readdition of Ca2+ to the extracellular medium, three influx activities could contribute positively to the increase of [Ca2+ ]i in CCh- or TG-stimulated Trp3 cells. The first is the basal activity mediated by Trp3. The second is influx intrinsic to the HEK cells stimulated by CCh or TG. The third is a Trp3-mediated influx stimulated by the same drugs. Because a significant fraction of Ca2+ entering from external space is removed by the Ca2+ pumps, the net increase of [Ca2+]i will not be the sum of influx arising from the three activities. Therefore, from the experiments shown in Figs. 3 and 4, it is difficult to conclude whether stimulation by CCh or TG increases a Trp3-mediated Ca2+ influx pathway or if the stimulated increase in [Ca2+]i observed in the Trp3 cells results merely from the opening of Ca2+ influx channels endogenous to the HEK cells. More difficult is to see whether store depletion causes more Ca2+ influx in Trp3 cells than in control cells because the increase in [Ca2+]i in the two Trp3 cell lines is not significantly higher than that in C2 cells (Fig. 4C). To test whether any stimulated Ca2+ influx is caused by the expression of hTrp3, we selectively blocked the endogenous Ca2+ influx with 10 µM Gd3+. As shown in Fig. 5, A, B, and E), in the presence of 10 µM Gd3+, the endogenous Ca2+ influx activated by CCh and TG decreased >85% in control cells. In contrast, addition of 10 µM Gd3+ to the Trp3 cells reduced <10% of the basal Ca2+ influx (Fig. 5E), about 15% of Ca2+ influx stimulated by CCh (Fig. 5, C and E), and 55-60% stimulated by TG (Fig. 5, D and E). Assuming that the Trp3-mediated influx pathway is insensitive to 10 µM Gd3+, we conclude that for Trp3 cells treated with CCh, a significant proportion of Ca2+ influx is mediated by Trp3, whereas in cells treated with TG, most of the Ca2+ influx is carried by the endogenous CCE channels.


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Fig. 5.   Effect of 10 µM Gd3+ on Ca2+ influx in control (panels A and B) and Trp3 cells (panels C and D). Cells were treated with either 200 µM CCh (panels A and C) or 0.2 µM TG (panels B and D) in a nominally Ca2+-free solution with or without 10 µM GdCl3 added 2 min after the application of the drugs. CaCl2 was added as indicated to a final concentration of 1.8 mM. Panel E, summary of Gd3+ effect on basal and stimulated Ca2+ influx in Trp3 and control cells. Cells were incubated in a nominally Ca2+-free solution unstimulated (open bars) or stimulated with 200 µM CCh (filled bars) or 0.2 µM TG (shaded bars). GdCl3 was added to a final concentration of 10 µM to cells 2 min before the addition of 1.8 mM Ca2+ to the external solution. Data are maximal [Ca2+]i reached within 1 min of Ca2+ addition minus the [Ca2+]i before Ca2+ addition from each experiment, averages ± S.E. from the number of experiments indicated in parentheses. Filled star, for T3-65 and C2 cells, ATP (100 µM) was included in CCh to maximally release the internal Ca2+ before readdition of extracellular Ca2+.

Although in the presence of 10 µM Gd3+, the maximal increase in [Ca2+]i in Trp3 cells in response to the addition of 1.8 mM Ca2+ under basal and TG-stimulated conditions is very similar, the rate of [Ca2+]i increase in TG-treated cells appears to be faster than in the non-stimulated cells (Fig. 6A). In fact, in both cell lines expressing hTrp3, maximal [Ca2+]i was reached within 40 s after the addition of external Ca2+, and then [Ca2+]i gradually decreased. At 1 min after the addition of Ca2+ the increase in [Ca2+]i in TG-stimulated cells was equal to that in non-stimulated cells. Then [Ca2+]i in the TG-stimulated cells continued to drop, whereas that in non-stimulated cells stayed constant. Fig. 6B shows TG-stimulated Ca2+ influx resistant to 10 µM Gd3+ in control and Trp3 cells. Date are from subtraction of [Ca2+]i changes of TG-stimulated cells from that of non-stimulated cells. Control cells have very low CCE activity when 10 µM Gd3+ is present, whereas Trp3 cells have a small and transient CCE activity that lasts for less than 1 min. Therefore, although very small, there is an increase of store-operated Ca2+ influx due to expression of hTrp3 in the HEK cells. Furthermore, TG also appears to block the basal Ca2+ influx seen in Trp3 cells. This could be due to the closing of the constitutively active hTrp3 channel or the enhancement of intracellular Ca2+ removal. Neither function has been described for TG.


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Fig. 6.   Gd3+-resistant CCE in Trp3 cells. Panel A, comparison of Ca2+ influx under basal (open symbols) and TG-stimulated conditions (filled symbols) in Trp3 (T3-9 at upper left and T3-65 at upper right) and control cells (C1 at lower left and C2 at lower right). Cells were incubated in a nominally Ca2+-free solution unstimulated or stimulated with 0.2 µM TG. GdCl3 was added to a final concentration of 10 µM to cells 2 min before the addition of 1.8 mM Ca2+ to the external solution. Data are from the same experiments shown in Fig. 5E and averages ± S.E. of [Ca2+]i obtained at 3-s intervals minus the [Ca2+]i before Ca2+ addition. Panel B, Gd3+-resistant CCE for Trp3 (circles) and control (squares) was obtained by subtracting the averages of [Ca2+]i increase in TG-stimulated cells (filled symbols in panel A) from that in unstimulated cells (open symbols in panel A).

Divalent Cation Selectivity of Influx Channels in Control and the Trp3 HEK Cells-- We compared the influx of Ba2+ and Sr2+ through the Trp3-mediated pathway in Trp3 cells and the endogenous pathway in control cells. Increasing concentrations of both Sr2+ and Ba2+ have been shown to produce a shift in Fura2 excitation wavelength spectrum similar to that produced by Ca2+, but with somewhat higher dissociation constants (39, 40). Therefore, increases in the fluorescence ratio of Fura2 obtained at excitation wavelengths of 340 and 380 nm reflect increases of intracellular concentrations of Ba2+ or Sr2+. Control or Trp3 cells were incubated in nominally Ca2+-free solutions either unstimulated or stimulated by CCh before 1.8 mM Sr2+ or Ba2+ was added into the ECS substituting for Ca2+. Fig. 7, A-D, shows that the rates of Ba2+ and Sr2+ influx into Trp3 cells are much higher than into control cells. Because Ba2+ is a poor substrate for Ca2+ pumps responsible for extrusion from the cytosol (39), a declining phase of intracellular [Ba2+] is normally not seen, as is the case with Ca2+ and Sr2+ (Fig. 7, C and D). In CCh-stimulated control cells, the initial rate of fluorescence ratio increase with Ca2+ (2.0 units/min) is at least two times faster than the rates with Sr2+ (0.6 unit/min) and Ba2+ (0.6 unit/min). In contrast, the rates of fluorescence ratio increase in Trp3 cells are very similar regardless of which divalent cation was added (5.5 for Ca2+, 6.8 for Sr2+, and 6.2 for Ba2+, in units/min). Therefore, in HEK293 cells, the endogenous influx pathway activated by CCh seems to be more selective for Ca2+ than Ba2+ and Sr2+, whereas the Trp3-mediated influx is not selective for these cations.


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Fig. 7.   Influx of Sr2+, Ba2+, and Mn2+ into Trp3 (T3-9) and the control (C1) cells under basal (left) or stimulated (right) conditions. Left, at the time point indicated, Sr2+, Ba2+, or Mn2+ was added to cells incubated in a nominally Ca2+-free solution to a final concentration of 1.8 mM, 1.8 mM, or 50 µM, respectively. Right, 200 µM CCh was added to cells incubated in a nominally Ca2+-free solution 3 min before the addition of the divalent cations.

Mn2+ can enter cells through certain types of Ca2+ influx channels and quenches Fura2 fluorescence (41, 42). The addition of 50 µM MnCl2 to cells suspended in a nominally Ca2+-free solution causes a slow but constant decrease of fluorescence, indicative of Mn2+ entry into the cells under basal conditions (Fig. 7E). The rate of Mn2+ entry into Trp3 cells was faster than into control cells. In CCh-stimulated cells, Mn2+ entry was increased in both the control and the Trp3 cells (Fig. 7F).

Effect of Ca2+ Channel Blockers-- Although not specific, SKF 96365 has been shown to block agonist-stimulated Ca2+ influx in many cells (43). Fig. 8A shows that at 25 µM, SKF 96365 inhibits completely the basal Ca2+ influx of Trp3 cells. In CCh-treated cells, the drug inhibits Ca2+ influx in both Trp3 and control cells. However, SKF 96365 seems to inhibit the Trp3-mediated influx more effectively than the endogenous influx. The residual Ca2+ influx left in the Trp3 cells is comparable to that seen in control cells. The IC50 for SKF 96365 was about 5 µM as estimated from measuring the CCh-stimulated Ba2+ influx in the Trp3 cells (Fig. 8B). Verapamil, a blocker of L-type voltage-gated Ca2+ channels, not known to affect CCE or agonist-stimulated Ca2+ entry in general, also prevents divalent cation entry via the Trp3-mediated pathway with an IC50 of about 4 µM (Fig. 8C). Both the basal and the stimulated cation influx in Trp3 cells can be blocked by high concentrations of Ni2+ (6 mM), La3+ (150 µM), and Gd3+ (200 µM).


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Fig. 8.   Ca2+ influx mediated by hTrp3 is blocked by SKF 96365 and verapamil. Panel A, basal influx of Ca2+ in the Trp3 cells (T3-9) is blocked completely by 25 µM SKF 96365, added 3 min before reintroduction of 1.8 mM Ca2+. Panel B, effect of 25 µM SKF 96365 on CCh-stimulated Ca2+ influx in the Trp3 cells. SKF was added 1 min before the reintroduction of 1.8 mM Ca2+. Panel C, effect of 25 µM SKF 96365 on CCh-stimulated Ca2+ influx in control cells (C1). The experiment was performed as in panel B. Panel D, dose-response effect of SKF 96365 on Ba2+ influx stimulated by CCh in the Trp3 cells. Cells were treated with 200 µM CCh in a nominally Ca2+-free solution for 5 min. SKF 96365 was added to final concentrations as indicated 1 min before the addition of 1.8 mM BaCl2. Panel E, dose-response effect of ± verapamil on Ba2+ influx stimulated by CCh in the Trp3 cells. The experiment was performed as in panel D.

PLC-dependent and Store Depletion-insensitive Cation Influx in Trp3 Cells-- To explore further the activation mechanism of Trp3-mediated Ca2+ influx, we tested whether depleting internal stores with TG would prevent agonist-stimulated [Ca2+]i increase. In control cells maintained in a nominally Ca2+-free solution, the addition of 200 µM CCh after incubating cells with 200 nM TG for 6 min caused no significant [Ca2+]i increase (not shown). In the Trp3 cells, a small rise in [Ca2+]i (27 ± 6 nM net increase at the peak, n = 4) was induced by CCh after cells had been incubated with TG for 6 min. However, when the cells were treated with TG in a medium containing 1.8 mM Ca2+ and the endogenous influx pathway was blocked by 10 µM Gd3+, the addition of CCh caused a transient increase of [Ca2+]i of 105 ± 8 nM (n = 11) in Trp3 cells (Fig. 9A). A similar increase of [Ca2+]i was not seen in control cells. In the experiment shown in Fig. 9B, the endogenous Mn2+ influx stimulated by either CCh or TG was blocked by 10 µM Gd3+. Addition of CCh to Trp3 cells pretreated with TG caused an increase in the rate of Mn2+ entry, which was not seen in control cells (right panel). The rate of Mn2+ entry under these conditions (TG followed by CCh) was not significantly different from the rate of entry observed in Trp3 cells treated only with CCh (left panel). In similar experiments, Ba2+ or Sr2+ was added to the TG-treated cells. Stimulation by CCh in the presence of extracellular Ba2+ or Sr2+ caused additional influx of these cations in Trp3 cells (Fig. 9, C and D) but not in control cells. These results suggest that in HEK cells expressing hTrp3, not only does receptor stimulation activate the influx of divalent cations better than store depletion, but signals from store depletion do not prevent or occlude the Trp3-mediated influx activated via receptor stimulation. Most of the influx activity that resulted from overexpression of hTrp3 alone in the HEK293 cells is thus activated by a mechanism other than store depletion.


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Fig. 9.   Influx of divalent cations through the hTrp3-mediated pathway can be activated in cells previously depleted of internal Ca2+ stores. Fura2-loaded HEK293 cells expressing hTrp3 (T3-9) or control cells (C1) were treated with 0.2 µM TG either in a solution containing 1.8 mM Ca2+ (panel A) or in a nominally Ca2+-free solution (panels B-D). Panel A, in the presence of 1.8 mM extracellular Ca2+, the endogenous Ca2+ influx was blocked by the addition of 10 µM Gd3+. Stimulation by 200 µM CCh caused a transient increase of [Ca2+]i only in the Trp3 cells. Panel B, Mn2+ entry in the presence of 10 µM Gd3+ recorded by Fura2 fluorescence quenching using the Fura2 isosbestic excitation wavelength at 360 nm and emission at 510 nm. Panel B, left, 50 µM MnCl2 was added as indicated 3 min after the cells had been incubated with 200 µM CCh in a nominally Ca2+-free solution. Under these conditions, CCh only increased the rate of Mn2+ entry over the basal (not shown) rate in the Trp3 cells but not the control cells. Panel B, right, 50 µM MnCl2 was added as indicated 3 min after cells had been incubated with 0.2 µM TG. 200 µM CCh was added 4 min later as indicated. An increase of Mn2+ entry was seen in the Trp3 cells. In panels C and D, a similar protocol was used. Ba2+ (panel C) or Sr2+ (panel D) was added to TG-treated cells to a final concentration of 1.8 mM. CCh (200 µM) was added 4 min later. The increase of Fura2 fluorescence ratio in response to CCh seen in Trp3 cells was caused by the entry of Ba2+ or Sr2+.

The signaling cascade initiated by an agonist includes the activation of its receptor, of a G protein, of PLC, and thus the production of IP3 and the release of Ca2+ from its internal stores. Although store depletion does not appear to be the cause for agonist-activated Ca2+ influx through the Trp3-mediated pathway, activation of PLC seems to be necessary. When 15 µM U73122, an inhibitor of PLC (44), was used, not only Ca2+ mobilization (not shown) but also Ca2+ and Ba2+ influx stimulated by CCh was blocked (Fig. 10). Basal Ca2+ influx in the Trp3 cells was little affected by the treatment of U73122. Therefore, either the production of IP3 or the active form of the PLC is required for the activation of the agonist-stimulated Trp3-mediated influx pathway.


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Fig. 10.   CCh-activated influx of Ca2+ (upper panel) and Ba2+ (lower panel) via hTrp3 is prevented by a phospholipase C inhibitor, U73122. HEKTrp3-9 cells were used. U73122 was added to the ECS to a final concentration of 15 µM 3 min before the addition of 0.2 µM TG. Experiments were performed as described in Fig. 9, A and C. Note that the store depletion-insensitive influx activated by CCh as seen in Fig. 9, A and C, is blocked by the PLC inhibitor.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Calcium mobilization from internal stores and subsequent Ca2+ entry from extracellular space are the two major components of Ca2+ signaling following activation of PLC by cell surface receptors. It has long been established that Ca2+ is released from its internal stores via IP3 receptors in response to the increase of IP3 production (1). Only recently has evidence accumulated showing that Ca2+ influx can occur via plasma membrane channels formed of Trp homologs (26, 29, 32). The two Drosophila photoreceptor Trp proteins have different functional features. DTrp was found to form a Ca2+ influx channel activated either by IP3 or by store depletion with TG, when expressed in insect Sf9 cells (22), Xenopus oocytes (23, 28), or 293T cells (24). On the other hand, DTrpl forms a non-selective cation channel that is activated by stimulation of Gq-coupled receptors but is not sensitive to store depletion (20, 21, 24). In addition, DTrpl also displayed significant basal activity (20). By comparing the amino acid sequences of the mammalian Trp proteins with that of Drosophila Trp or Trpl, it is difficult to predict whether any of the mammalian Trps would functionally resemble DTrp or DTrpl. A recent report by Philipp et al. (29) showed that bovine Trp4 transiently expressed in HEK293 cells forms a channel that is relatively more selective for Ca2+ than for monovalent cations and is activated to the same extent by either IP3 or TG, suggesting that Trp4 may functionally resemble the DTrp. Zitt et al. (32) also reported that hTrp1 expressed in Chinese hamster ovary cells can be activated by store depletion treatment, although the channel formed in this case is non-selective. Thus, hTrp1 seems to resemble partly both DTrp and DTrpl. Experiments to be reported elsewhere3 indicate that the stable expression of hTrp3 in the HEK293 cells gives rise to a novel Ca2+-permeant cation influx current that is spontaneously active, non-selective, and can be further stimulated by activation of Gq-coupled receptors. In the present experiments, the majority of the increased Ca2+ influx due to hTrp3 is neither activated by store depletion alone nor occluded by store depletion with TG. Thus, hTrp3 functionally resembles Drosophila Trpl.

Ca2+ influx mediated by channels insensitive to store depletion have been shown to coexist with the store depletion-activated pathway in many systems. Orrenius and colleagues (42, 46, 47) reported that in cells such as hepatocytes, T lymphocytes, adrenal glomerulosa cells, fibroblasts, platelets, and anterior pituitary cells, Ca2+ influx activated by emptying the agonist-sensitive stores could not completely mimic the influx activated by receptor agonists. More importantly, the store-insensitive cation influx in hepatocytes was less selective for Ca2+ than influx activated by TG. Clementi et al. (48) also reported that stimulation of PC12 cells with carbachol activated two Ca2+ influx responses, of which one was store-dependent and the other was directly dependent on receptor activation. Work by Montero et al. (49) showed that in differentiated HL60 cells, N-formyl-leucyl-phenylalanine activated an additional Ca2+ influx after the internal Ca2+ store had been completely emptied by TG. In electrophysiological studies, multiple types of Ca2+-conducting currents bearing distinct characteristics are often observed by investigators using carefully designed protocols. In mast cells, Ca2+ enters through both a Ca2+ release-activated current (ICRAC) and a 50-picosiemen channel that is activated more directly by receptor/G protein coupling (16, 50). In A431 epithelial cells, up to four different types of Ca2+-permeant channels could be involved in Ca2+ influx following the activation of G protein (shown by the action of GTPgamma S) or perfusion of IP3 (14). In addition, IP3 was found to activate plasma membrane channels in B lymphocytes (51) and substance P receptor-transfected Chinese hamster ovary cells (52). Moreover, IP3 may activate the same channel that is sensitive to store depletion. In inside-out patches of vascular endothelial cells, IP3 was shown to modulate the activity of a Ca2+ influx channel which is indistinguishable from that activated by treatment with 2',5'-di(tert-butyl)1,4-benzohydroquinone, i.e. by store depletion (17). Thus, under physiological conditions, it is likely that after receptor activation, Ca2+ enters through multiple Ca2+-permeant channels. The relative contribution of each influx pathway is likely to differ depending on the cell type and probably the type and the degree of the stimulation. An estimate has been made for rat mast cells in which, under physiological conditions, the amount of Ca2+ conducted by ICRAC is three times of that carried by the 50-picosiemen channel (16, 53).

The activation mechanism of agonist-stimulated Ca2+ influx via hTrp3 is unclear. Controversy exists with respect to the mechanism of activation of the Drosophila Trpl expressed in the Sf9 cells. Dong et al. (54) reported that DTrpl is activated by intracellular perfusion with IP3, whereas Obukhov et al. (55) found that in excised patches, DTrpl is activated by the active forms of the alpha  subunits of the G protein G11 and Gq, but not by IP3. The fact that agonist-stimulated store-insensitive Ca2+ influx via hTrp3 can be prevented by an inhibitor of PLC suggests that the channel formed by hTrp3 is activated by a factor downstream of PLC activation, and upstream of store depletion. Since our experiments were carried out under conditions in which store depletion occurred either before or upon the addition of the agonist, we cannot rule out the possibility that Ca2+ mobilization is also a necessary step for agonist-induced Ca2+ influx through Trp3. If that is the case, stimulation of hTrp3 would require both store depletion and the generation of this other factor downstream of PLC activation. The likely candidates for this factor are IP3 and its derivatives, for instance IP4. According to a so-called conformational coupling model (56), it is also possible that the channel is activated through direct interaction between the channel and the activated IP3 receptor. Less likely, although not impossible, is that the channels could be stimulated by the activated PLC itself. A more recent study by Zitt et al. (45) showed that hTrp3 may be activated by Ca2+. However, treatment with TG also induces an increase in [Ca2+]i. This increase does not seem to activate Ca2+ influx mediated by Trp3 as well as that stimulated by receptor activation, suggesting that factors more than just intracellular Ca2+ are involved in activating Trp3. The 50-picosiemen channel found in mast cells is not activated by IP3 but instead requires the activation of receptor/G protein system (16). Thus, whether hTrp3 expressed in the HEK293 cells forms a channel that resembles any of the Ca2+ influx channels found in native tissues or isolated primary cells remains to be elucidated.

Because of some homology between the last four putative transmembrane segments of Trp and those of the subdomains of voltage-gated Ca2+ and Na+ channels, it has been proposed that a Trp-based channel may be a tetramer (19). Based on our finding that the mammalian genome contains at least six Trp genes, we speculated that a channel assembled by Trps can be either homotetrameric or heterotetrameric and that this could be a mechanism to create functionally diverse Ca2+-permeant channels, including store depletion-activated, store depletion-insensitive, and channels activated by both store depletion and independently by agonists (31). In the stable HEK293 cell lines, a homotetrameric hTrp3 may have become the predominant influx channel formed because of overexpression of this protein. However, it may not represent any of the native Ca2+ influx channels present in this or other cell types. Because the cDNA for hTrp3 was isolated from HEK293 cells (26), we believe that there is endogenous hTrp3 protein in these cells. However, we do not know whether channels formed by hTrp3 alone are present in the native HEK293 cells because a component of Ca2+ influx resembling that expressed in the Trp3 cell lines is either missing or too small to be detected in the control cells. One possibility is that the endogenous hTrp3 in the HEK293 cells plays a very minor role in Ca2+ influx in response to agonist stimulation. The other possibility is that hTrp3 and other endogenous Trp proteins form heterotetrameric channels that are responsible for agonist and store depletion-activated Ca2+ entry in these cells. Evidence for the formation of a heteromultimeric Trp-based Ca2+ influx channel was recently obtained by Gillo et al. (23) who observed that coexpression of Drosophila Trp and Trpl in Xenopus oocytes leads to the appearance of a channel with an ion selectivity and La3+ sensitivity different from those seen in oocytes expressing either protein alone. Interestingly, the new channel is activated to the same extent either by IP3 or by TG, even though one of its components, DTrpl, is not sensitive to store depletion. More recently, Montell and colleagues (24) demonstrated that the N termini of DTrp and DTrpl interact with each other both in vitro and in vivo. Coexpression of the two proteins in 293T cells gave rise to a store depletion-sensitive cation influx channel that had features from both DTrp and DTrpl. Interestingly, cells expressing both DTrp and DTrpl did not have an increased basal inward current as found in cells expressing DTrpl alone. The authors proposed that DTrpl in Drosophila photoreceptors does not form homomultimers by itself, and its spontaneous activity is prevented by forming heteromultimeric channels with DTrp or other Trp-related proteins. A similar conclusion may be drawn for hTrp3 since it behaves very similarly to DTrpl when expressed alone. Because Drosophila Trp proteins can form store-operated heteromultimeric channels even if only one subunit is capable of detecting the signal from store depletion, it is possible that the exogenously expressed hTrp3 in the stably transfected cell lines also forms heteromultimers in combination with the endogenous Trp proteins, of which some can be activated by store depletion. This would explain the small but significant Gd3+-resistant TG-stimulated Ca2+ influx in Trp3 cells (Fig. 6C). The smaller TG-stimulated increase of Ca2+ influx than the CCh-stimulated increase found in transiently transfected COS cells (26) can also be explained this way. On the other hand, the contribution of hTrp3-containing heteromultimeric channels to TG-stimulated Ca2+ influx could be more significant than that being revealed by the protocol shown in Fig. 6 if these channels are more sensitive to Gd3+ than the homomultimeric channel formed by hTrp3. This is even more so when the new channels formed cannot be distinguished from the native Ca2+ influx channels present in the HEK293 cells by Ca2+ channel blockers. Therefore, although our results show that overexpression of hTrp3 in HEK293 cells gives rise to a cation entry pathway insensitive to regulation by a store-operated manner, we cannot at this point rule out the possibility that Trp3 is involved in forming native store-operated channels in HEK293 and other cells. Other Trp-unrelated proteins may also participate in the formation of the Ca2+ influx channels. It is yet to be firmly established that Trps are the true or the only subunits of store- or agonist-stimulated Ca2+ entry channels, as this will require purification of the channel complexes followed by reconstitution into either vesicles or planar lipid bilayers and analysis of channel activity. Further, even if they were channel subunits, other auxiliary proteins are expected to be required for the formation the native channel(s).

In conclusion, Ca2+ influx following the activation of the PLC/IP3 signaling pathway is an important phenomenon. Channels with different properties have been described in various cell types (for review, see Refs. 3 and 53). These include the store-operated channels that differ both in conductance and ion selectivity. These also include channels that do not appear to be store-operated. The presence of six Trp homologs in mammals and the possibility that they form heteromultimeric channels provide a plausible explanation for the tissue- and cell-specific behavior of Ca2+ influx pathways (and channels) found in different systems.

    ACKNOWLEDGEMENTS

We thank Drs. G. Innamorati, H. Sadeghi, B. Vannier, N. Qing, M. Birnbaumer, and E. Stefani for technical advice.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants GM54235 (to X. Z.) and HL45198 (to L. B.).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.

§ To whom correspondence should be addressed: Dept. of Pharmacology, Ohio State University, 206 Rightmire Hall, 1060 Carmack Rd., Columbus, OH 43210.

1 The abbreviations used are: [Ca2+]i, intracellular Ca2+ concentration; PLC, phospholipase C; IP3, inositol 1,4,5-trisphosphate; CCE, capacitative Ca2+ entry; TG, thapsigargin; IP4, inositol 1,3,4,5-tetrakisphosphate; HEK, human embryonic kidney; HA, hemagglutinin; V1aR: vasopressin receptor type 1a; C1 and C2, control-1 and control-2 cells, respectively; ECS, extracellular solution; CCh, carbachol; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate.

2 The nomenclature of mammalian Trps is used according to Zhu et al. (26). Thus, the human sequence that appeared in Wes et al. (27) and Zitt et al. (32) is hTrp1; the partial murine sequence published by Petersen et al. (28) and the full-length bovine and rat sequences reported by Philipp et al. (29) and Funayama et al. (30), respectively, are Trp4.

3 R. S. Hurst, X. Zhu, G. Boulay, E. Stefani, and L. Birnbaumer, submitted for publication.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

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