The role of endogenous human Trp4 in regulating carbachol-induced calcium oscillations in HEK-293 cells.

We utilized 2-aminoethyoxydiphenyl borane, an agent that blocks store-operated Ca(2+) entry, as well as an antisense approach to characterize endogenous Ca(2+) entry pathways in HEK-293 cells. The thapsigargin- and carbachol-induced, but not the 1-oleolyl-2-acytyl-sn-glycerol (OAG)-induced, entry was blocked by 2-aminoethyoxydiphenyl borane. Both reverse transcriptase-PCR and Western blot analyses demonstrated endogenous expression for HTRP1, HTRP3, and HTRP4 and specific suppression of mRNA levels and Trp protein levels in cells stably expressing antisense constructs. Expression of HTRP4 antisense inhibited 35% of the carbachol (CCh)-stimulated Ba(2+) entry and 46% of the OAG-stimulated Sr(2+) entry but in contrast had no effect on the thapsigargin-stimulated Ba(2+) or Sr(2+) entry. HTRP3 antisense reduced, while HTRP1 antisense had no effect on, OAG-induced Sr(2+) entry. Of greater importance, HTRP4 antisense expression, but not HTRP3 antisense expression, blocked the sustained Ca(2+) oscillations produced by low doses of CCh (15 microm), arguing that receptor-stimulated rather than store-operated channels are involved in these sustained oscillations. HTRP4 antisense also inhibited 75% of the arachidonic acid-induced Ca(2+) entry. In summary, these data suggest that HTRP4 proteins in HEK-293 cells, differing from HTRP3 and HTRP1 proteins, do not serve as functional subunits of store-operated channels but do function as subunits for CCh- and OAG-stimulated channels. Furthermore, evidence is provided for the first time for the involvement of a Trp isoform (HTRP4) in the formation of the channel responsible for both arachidonic acid-induced Ca(2+) entry and the Ca(2+) entry needed to sustain long term Ca(2+) oscillations induced by low doses of carbachol.

We utilized 2-aminoethyoxydiphenyl borane, an agent that blocks store-operated Ca 2؉ entry, as well as an antisense approach to characterize endogenous Ca 2؉ entry pathways in HEK-293 cells. The thapsigargin-and carbachol-induced, but not the 1-oleolyl-2-acytyl-sn-glycerol (OAG)-induced, entry was blocked by 2-aminoethyoxydiphenyl borane. Both reverse transcriptase-PCR and Western blot analyses demonstrated endogenous expression for HTRP1, HTRP3, and HTRP4 and specific suppression of mRNA levels and Trp protein levels in cells stably expressing antisense constructs. Expression of HTRP4 antisense inhibited 35% of the carbachol (CCh)-stimulated Ba 2؉ entry and 46% of the OAG-stimulated Sr 2؉ entry but in contrast had no effect on the thapsigargin-stimulated Ba 2؉ or Sr 2؉ entry. HTRP3 antisense reduced, while HTRP1 antisense had no effect on, OAG-induced Sr 2؉ entry. Of greater importance, HTRP4 antisense expression, but not HTRP3 antisense expression, blocked the sustained Ca 2؉ oscillations produced by low doses of CCh (15 M), arguing that receptor-stimulated rather than store-operated channels are involved in these sustained oscillations. HTRP4 antisense also inhibited 75% of the arachidonic acid-induced Ca 2؉ entry. In summary, these data suggest that HTRP4 proteins in HEK-293 cells, differing from HTRP3 and HTRP1 proteins, do not serve as functional subunits of store-operated channels but do function as subunits for CCh-and OAG-stimulated channels. Furthermore, evidence is provided for the first time for the involvement of a Trp isoform (HTRP4) in the formation of the channel responsible for both arachidonic acid-induced Ca 2؉ entry and the Ca 2؉ entry needed to sustain long term Ca 2؉ oscillations induced by low doses of carbachol.
It is widely accepted that Ca 2ϩ serves as an essential signaling molecule, and for that reason, the level of intracellular calcium ([Ca 2ϩ ] i ) is strictly controlled. This control extends beyond regulating static levels of Ca 2ϩ to include regulation of Ca 2ϩ levels in subregions of the cell and regulation of the frequency and amplitude of Ca 2ϩ oscillations within the cell. This multifaceted regulation is required for countless cellular functions including muscle contraction, protein secretion, cell proliferation, and apoptosis (1). Mutations inducing drastic alterations in intracellular Ca 2ϩ homeostasis are most likely not compatible with life (2).
In most cells, there are two sources of Ca 2ϩ that can be tapped in order to modify resting Ca 2ϩ levels, the intracellular Ca 2ϩ stores and the extracellular space. Intracellularly, Ca 2ϩ is released from the sarco-and endoplasmic reticulum stores via two types of Ca 2ϩ channels: inositol 1,4,5-trisphosphate receptors and ryanodine receptors. Cytosolic Ca 2ϩ is taken up into the sarco-and endoplasmic reticulum by one of the three sarco-or endoplasmic reticulum Ca 2ϩ -ATPase Ca 2ϩ -pumps. Extracellular Ca 2ϩ can enter the cell via cation channels in the plasma membrane (1), and in nonexcitable cells it is well documented that depletion of internal Ca 2ϩ stores leads to the activation of store-operated channels (SOCs) 1 in the plasma membrane. This allows Ca 2ϩ to enter the cell on a continual basis during times of elevated inositol 1,4,5-trisphosphate levels, resulting in sustained elevations of cytosolic Ca 2ϩ levels. When inositol 1,4,5-trisphosphate levels decline, the Ca 2ϩ entry persists only until the internal Ca 2ϩ stores are refilled, at which time the SOCs turn off (3). The net result is that, at high doses of agonist, one normally sees a biphasic Ca 2ϩ response made up of a high transient peak representing release of internal Ca 2ϩ stores followed by a long sustained plateau phase that results from Ca 2ϩ entry.
Sometimes changes in [Ca 2ϩ ] i in response to receptor agonists can be far more complex. The signal patterns can include repetitive oscillations or waves of elevated [Ca 2ϩ ] i . The functional importance of Ca 2ϩ oscillations may relate to regulation of kinases and phosphatases, energy metabolism, secretion, and certain transcriptional events (1,4). Oscillation of Ca 2ϩ often occurs at low concentrations of agonist that generates levels of InsP 3 , which release submaximal amounts of Ca 2ϩ but sufficient amounts to trigger the positive feedback effect of Ca 2ϩ on the release of store Ca 2ϩ via inositol 1,4,5-trisphosphate receptors. In many instances Ca 2ϩ influx is required in order to sustain [Ca 2ϩ ] i oscillations (4 -7).
In the Drosophila eye, photoactivation of rhodopsin leads to the opening of the light-sensitive cation influx channels Trp (transient receptor potential) and Trpl (Trp-like) (8). When expressed in a heterologous cell system, Trp channels can be activated by depletion of Ca 2ϩ from the internal stores (9). Therefore, SOCs may be functionally related to Trp proteins. To date, seven mammalian Trp homologs (short isoforms) have been cloned, and they vary significantly in their channel behavior and mode of activation (10). Human Trps are found at highest levels in brain, heart, testis, and ovary; they are ϳ450 amino acids shorter than Drosophila Trp; they have six-transmembrane domain regions; and regions in the N-terminal domain have similarity to domains found in ankyrin and in the C terminus similar to dystrophin (11,12). Although, when expressed in a heterologous cell system, the Drosophila Trp channel appears to be store-operated, there is considerable evidence to suggest that Trp is regulated by a different mechanism during the phototransduction process (13). A similar controversy has arisen from numerous studies where various mammalian Trp homologs have been overexpressed in various cell systems; results suggest that mammalian Trps either may (14 -18) or may not (19 -21) respond to store depletion. For example, if we focus on the overexpression studies for the Trp4 isoform, there are significant disagreements concerning whether the channel is regulated by store depletion. The initial reports on bovine Trp4 described it as a capacitative calcium channel, since its overexpression in HEK-293 cells (16) or Chinese hamster ovary cells (22) led to channel activity that could be stimulated by depletion of Ca 2ϩ stores with thapsigargin. A role for rat Trp4 in capacitative calcium entry was suggested by the demonstration of a potentiated capacitative calcium entrymediated chloride current in oocytes expressing rat Trp4 (23) and was further supported by the observation that expression of a Trp4 antisense significantly reduced calcium release activated channel-like currents in adrenal cells (24). However, two recent studies argue that Trp4 does not participate in forming store-operated calcium channels. The expression of murine Trp4 in HEK-293 cells was reported to have no effect on channel activity following depletion of intracellular calcium stores but was reported to enhance the channel activity in response to activation of G q -coupled receptors or receptor tyrosine kinases (25). The expression of HTRP4 in HEK-293 cells was reported to have no effect on barium entry stimulated by calcium store depletion, supporting the findings for murine Trp4. However, in contrast to results for murine Trp4, the HTRP4 produced a constitutively active channel which was not stimulated by either phospholipase C-linked receptor activation or by OAG (26). Given these dramatically different results from previous Trp4 overexpression studies, there is a compelling need to utilize alternative approaches to help resolve the role of Trp4 in the regulation of capacitative calcium entry.
Similar discrepancies in findings between laboratories exist for Trp1 (14,15,(27)(28)(29), and Trp3 (14, 20, 30 -35). One can think of a number of theoretical reasons for overexpression studies to produce results that may not reflect the role of endogenous Trps in native Ca 2ϩ channel activity. The endogenous channels may be heterotetramers, and the channels formed in the overexpression studies may be homotetramers due to their high expression level, or the expressed Trp may be unable to form the appropriate heterotetramer channels due to the lack of the appropriate Trp isoforms in the cell being tested. We have also published data on clone-to-clone variation within the HEK-293 cell population that points out the potential pitfalls of interpreting data based on studies with a small number of stable clones expressing Trps (36). To circumvent many of the problems of overexpression studies, we have used an antisense approach to investigate these questions.
In a previous study, we reported that HEK-293 cells express mRNA for HTRP1, HTRP3, HTRP4, and HTRP6, and evidence was presented that HTRP1 and HTRP3 are involved in mediating store-operated Ca 2ϩ entry (32). In the present study, we attempt to determine whether multiple Ca 2ϩ entry pathways are mediated by endogenous HTRPs in HEK-293 cells. We have used the inhibitor, 2-aminoethyoxydiphenyl borane (2-APB), to analyze and compare Ca 2ϩ entry induced by thapsigargin, car-bachol, and OAG in HEK-293 cells. We also have generated antisense constructs for HTRP1, HTRP3, and HTRP4 and stably expressed them in HEK-293 cells. We provide evidence that in HEK-293 cells endogenous HTRP4 proteins do not participate in the formation of cation channels regulated by store depletion with thapsigargin but do participate in channel activity activated by CCh or OAG. More importantly, we provide evidence that HTRP4 proteins are involved in the formation of channels responsible for both the arachidonic acid-induced Ca 2ϩ entry and the Ca 2ϩ entry needed to sustain long term Ca 2ϩ oscillations in HEK-293 cells in response to low doses of CCh.
Isolation of cDNA-Encoding Fragments of Trps by RT-PCR-The poly(A)-RNA was isolated from HEK-293 cells using guanidinium thiocyanate extraction followed by an oligo(dT) binding method (QuickPrep Micro mRNA Purification Kit; Amersham Biosciences). The extracted mRNA was then treated with DNase (Invitrogen). The first strand cDNA was reverse transcribed using an oligo(dT) primer, and it was then amplified directly using PCR (SuperScript Preamplification System; Invitrogen). Sequences of primers to amplify specific regions of HTRP1, HTRP3, and HTRP4 as well as human ␤ actin are described in Table I. Sequence similarity analysis was performed using Seglab software by Genetics Computer Group. We used the inner primers to amplify fragments of endogenous HTRP cDNA in normal HEK-293 cells for the productions of the sense and antisense constructs and the outer primers to amplify HTRP cDNA fragments in HEK-293 cells in which sense or antisense constructs were stably expressed. The hot start PCR was performed with Taq polymerase; the initial denaturation was at 94°C for 5 min, followed by 30 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C for 30 s. A final extension was performed at 72°C for 3 min. The PCR conditions were optimized to 30 cycles of annealing at 55°C for endogenous HTRP1 and HTRP3 and to 35 cycles of annealing at 53°C for endogenous HTRP4, using the inner primers. For the outer primers, the PCR was cycled 30 times, and the annealing was at 60°C for all HTRP isoforms. For the experiments to test for mRNA expression for the ␣ and ␤ isoforms of HTRP4, the PCR was cycled 30 times, and the annealing was performed at 52.5°C. The PCR mixture (48 l) consisted of ϳ22% of first stand cDNA as a template, 1 l of 100 pM solution of each primer, 5 l of 25 mM MgCl 2 , 1 l of 10 mM dNTP mix, and 2.5 units of Taq DNA polymerase (Invitrogen). For a positive control and to determine whether equal amounts of mRNA were used for each condition, we used a human ␤-actin primer, and a negative control without RT was performed alongside all experimental samples.
The cDNA fragments of HTRP1 (369 base pairs), HTRP3 (323 base pairs), and HTRP4 (412 base pairs) from RT-PCRs were separated by electrophoresis in a 3% GTG-agarose gel. The corresponding bands were cut out of gels, extracted (QIAEX II Gel Extraction 150), and subcloned into an eukaryotic TA cloning vector pCR3.1 that accepts products in both the forward and reverse directions (eukaryotic TA cloning kit, bidirectional; Invitrogen). The clones were selected randomly, and the cDNAs were purified (Qiagen Plasmid Maxi Kit 25) and sequenced. The antisense constructs encoding HTRP1, HTRP3, and HTRP4 were expressed in HEK-293 cells, whereas the corresponding short sense constructs were expressed for the controls.
Stable Transfection of Antisense HTRP1, HTRP3, and HTRP4 Constructs-HEK-293 cells were transfected with sense or antisense constructs using the Ca 2ϩ phosphate method. G418-resistant transformants were selected using 400 g/ml G418. In total, three antisense cell lines (HTRP1AS, HTRP3AS, and HTRP4AS) and three sense cell lines (HTRP1S, HTRP3S, and HTRP4S) were established. For each cell line expressing sense or antisense constructs, all of the surviving clones (ϳ200) were pooled together to generate a cell line stably expressing one of the Trp construct(s). This was done in order to avoid the problems inherent with using small numbers of selected clones to compare the effects of gene transfection. In a previous publication, we reported significant clone-to-clone variation of SOC activity within the parent HEK-293 cell population, and, utilizing Monte Carlo analysis, we estimated the probability that this clone-to-clone variation would lead to misinterpretations of the effect of Trp channel expression (36). Cells up to passage 30 were used for Ca 2ϩ imaging.
Immunoblotting-Western blots of endogenous HTRPs were successful only after extensive characterization of HTRP antibodies (Alomone Laboratories, Jerusalem, Israel). We tested various cell lysis buffers, sample heating temperatures, support membranes, wash buffers, and antibody incubation times on samples of total cell protein, cell membrane protein, and immunoprecipitated tagged Trp protein prepared from HEK-293 cells overexpressing Trp proteins. Initial Western blot experiments were performed with anti-tag antibodies, whereas subsequent experiments compared results with anti-Trp antibodies with those obtained with anti-tag antibodies. Conditions were considered optimum when we could see clear distinct bands of the appropriate molecular weight on Western blots of Trp-expressing cells and the intensity of these bands was in great excess of corresponding bands in the lane for control HEK-293 cells. Since control samples of HEK-293 total cell protein run on minigels often did not show endogenous Trp proteins, we scaled up the procedure in order to routinely observe endogenous Trp proteins. We went to a large gel format (16 ϫ 16 cm) so that we could load more protein, and we included a protein precipitation and wash step in order to reduce the amount of contaminants associated with loading a larger protein sample onto the gels. The precipitation and wash step was found to greatly reduce the background of the Western blots. Optimum blocking and wash conditions for Westerns with HTRP4 antibodies were slightly different from those for HTRP1 and HTRP3 antibodies (see below).
Cells were lysed in modified radioimmune precipitation buffer (10 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 1% sodium deoxycholate, 2 mM EDTA). Protease Arrest (Geno Technology, Inc.) was added to prevent proteolysis. A quantitative precipitation of all of the proteins in the total cell extract was done with PAGE-Perfect (Geno Technology), and the protein precipitate was then washed to remove agents that would interfere with running high quality gels. Precipitated protein pellets were mixed with 2ϫ Laemmli buffer (plus 100 mM dithiothreitol) and heated for 30 min at 70°C, and sample pairs (antisense cells versus control cells) containing equal amounts of protein (200 g for HTRP1 and HTRP3 and 400 g for HTRP4) were loaded on a regular size gel (16 ϫ 16 cm, 7.5% SDS-PAGE). Electrophoresis was performed overnight, and then proteins were electrotransferred onto Immobilon membranes (Millipore Corp.). For blots with anti-HTRP1 and anti-HTRP3, the membranes were blocked with 5% milk solution in TBS-T (0.1% Tween 20) (for HTRP4 Western blots, the Tween 20 was omitted from the TBS solution) for 1 h and were then incubated with primary antibodies raised against the individual HTRPs (Alamone Laboratories) overnight at room temperature. The antibodies were diluted 1:200 in the blocking solution. Membranes were washed four times for 15 min each with TBS-T (for Trp4 Western blots, three times for 15 min each with TBS and one time for 15 min with TBS-T), incubated for 30 min at room temperature with secondary anti-rabbit antibody (1:10000 in 5% milk/TBS-T (TBS for HTRP4 Western blots)), washed under the same conditions, and developed with SuperSignal Chemiluminescent Substrate (Pierce).
Ca 2ϩ Imaging-[Ca 2ϩ ] i concentration was measured in cells loaded with the fluorescent indicator fura-2. Transfected cells were plated onto 25-mm coverslips 1 day before the experiment. On the next morning, cells were washed twice with a HEPES-buffered HBSS (HHBSS); loaded for 30 min with 5 M fura-2/AM that was dissolved in HHBSS supplemented with 1 mg/ml bovine serum albumin, 0.025% pluronic F127; and then unloaded in HHBSS for another 30 min. The coverslips were mounted as the bottom of a chamber that was placed on the stage of a Nikon Diaphot inverted epifluorescence microscope equipped with a Zeiss Fluor ϫ10 (or ϫ20 for single cell analysis) objectives. Cells in the chamber were perfused via an eight-channel syringe system (Anspec). A suction pipette maintained a constant volume of solution (ϳ0.5 ml) in the chamber.
An InCyt IM2 TM fluorescence imaging system (dual wavelength fluorescence imaging system; Intracellular Imaging Inc., Cincinnati, OH) was used to measure [Ca 2ϩ ] i during the experiment. Excitation light from a xenon light source was alternately passed through 340 and 380 nm narrow pass filters mounted in a Sutter filter wheel (Lambda 10-C).
The 510 nm emissions were captured by a cooled CCD camera (Cohu 4915). The images were transmitted to a computer (Intel Pentium Pro 500-MHz based PC) and processed with the imaging software InCyt IM2 TM 4.6. [Ca 2ϩ ] i was calculated by measuring the ratio of the two emission intensities for excitation at 340 and 380 nm. Calcium standard solutions, which were prepared with fura-2 potassium salt, were used to create a graph of fluorescence ratio (F 340 /F 380 ) as a function of Ca 2ϩ concentration (in nM). This graph was then used to convert fluorescence ratios in an experiment to calcium concentrations.
In experiments in which Ba 2ϩ and Sr 2ϩ influx were measured, the data are reported as the 340/380 ratio (R340/380), since the Ba 2ϩ and Sr 2ϩ calibration curves for fura-2 differ from the calibration curve for Ca 2ϩ .
During most experiments, an average response of ϳ800 cells from a single field on each coverslip was represented as one trace. The level of store-operated Ca 2ϩ entry in the cells expressing Trp sense or antisense constructs was obtained by subtracting the slope of the Ba 2ϩ leak (before stimulation) from the slope of Ba 2ϩ influx (after stimulation) for each coverslip. In Ca 2ϩ oscillation experiments, responses of single cells were reported.
Nominally Ca 2ϩ -free HBSS was prepared by stirring Ca 2ϩ -free, Mg 2ϩ -free, and HCO 3 Ϫ -free HBSS with Chelex-100 beads. After filtering out the Chelex-100 beads, MgCl 2 was added to a final concentration of 1 mM.

Effects of 2-APB on Ca 2ϩ
Signaling Induced by Thapsigargin, Carbachol, and OAG-The membrane-permeant inhibitor of the InsP 3 receptor (37), 2-APB, has been utilized in several recent studies to address the question of whether a direct interaction occurs between the inositol 1,4,5-trisphosphate receptor and overexpressed HTRP channels. However, recent reports suggest that 2-APB may be directly inhibiting the store-operated channels (38,39). Given the lack of a well defined mechanism of action, we set out simply to determine whether 2-APB could help distinguish between the various endogenous Ca 2ϩ entry pathways in our HEK-293 cells.
To investigate thapsigargin-stimulated Ca 2ϩ entry, cells in HBSS were switched to a Ca 2ϩ -free HBSS medium and allowed to establish a new base line, and then the cells were treated with thapsigargin (1 M). As expected, in the absence of Ca 2ϩ , thapsigargin induced a initial transient Ca 2ϩ peak that reflects the depletion of intracellular stores. The level of [Ca 2ϩ ] i subsequently declined, suggesting the removal of [Ca 2ϩ ] i from the cell by the plasma membrane Ca 2ϩ -ATPase. After [Ca 2ϩ ] i returned to basal levels, Ba 2ϩ (5 mM) was added into Ca 2ϩ -free medium, and the initial slope of Ba 2ϩ entry was taken to indicate the level of store-operated Ca 2ϩ entry (SOCE) (Fig.  1A). In unstimulated cells in Ca 2ϩ -free medium, 2-APB (100 M) slightly elevated Ca 2ϩ above basal levels ( Fig. 1B), possibly through its inhibitory effect on Ca 2ϩ -ATPase in the endoplasmic reticulum (37). As reported for cells overexpressing HTRP3 (40), we observed that 2-APB produced a concentration-dependent inhibition of thapsigargin-stimulated Ba 2ϩ entry ( Fig.  1B). At 75 M 2-APB, there was an ϳ50% reduction (n ϭ 3) of Ba 2ϩ entry, whereas at 100 M 2-APB, a higher level of inhibition (75%) was observed (n ϭ 3).
We used a similar protocol to investigate the effect of 2-APB on carbachol-induced Ca 2ϩ entry. In the absence of Ca 2ϩ , carbachol (100 M) induced a large, transient increase in [Ca 2ϩ ] i , indicating Ca 2ϩ release from intracellular stores following InsP 3 receptor activation. As the cytosolic Ca 2ϩ returned to basal levels, Ba 2ϩ (5 mM) was added into the bath, and receptor-stimulated Ba 2ϩ entry was observed (Fig. 1C). In a series of subsequent experiments, we applied 2-APB (100 M) to the bath for 3 min prior to carbachol stimulation. Following a 3-min preincubation with 2-APB (100 M), carbachol-induced Ba 2ϩ entry was dramatically reduced (Fig. 1D), a finding consistent with the inhibitory effect of 2-APB reported originally in cells overexpressing HTRP3 (40). Interestingly, under these conditions, the Ca 2ϩ release from internal stores was not significantly altered (Fig. 1D). With longer periods of preincubation with 2-APB (100 M), the transient Ca 2ϩ release induced by carbachol was significantly or completely inhibited (data not shown).
We next sought to assess the effect of 2-APB on OAG-induced Ca 2ϩ entry. Since previous studies investigating OAG activation of overexpressed, human Trp proteins monitored Sr 2ϩ entry, we measured Sr 2ϩ entry in the presence of 100 M OAG and the presence or absence of 2-APB (100 M). As noted for the Ca 2ϩ data in Fig. 1B, there was a dose-dependent effect of 2-APB on basal Sr 2ϩ levels (Fig. 1F). However, 2-APB, regardless of dose used, had no effect on the large slope of OAGinduced Sr 2ϩ entry (Fig. 1, E and F). The data suggest that the cation entry pathway activated by OAG is distinct from cation entry pathways activated either by store depletion with thapsigargin or by CCh stimulation of HEK-293 cells.
Level of Expression of mRNA for Trp Homologs in HTRP1AS, HTRP3AS, and HTRP4AS Cells-Our previous studies indicated that HEK-293 cells express mRNA for HTRP1, HTRP3, HTRP4, and HTRP6 (32). To evaluate the role of HTRP1, HTRP3, and HTRP4 in mediating thapsigargin-, CCh-, and OAG-stimulated Ca 2ϩ entry, we made short antisense cDNA constructs specific for HTRP1, HTRP3, and HTRP4 that we stably transfected into HEK-293 cells. This allowed us to generate three cell lines, i.e. HTRP1AS, HTRP3AS, and HTRP4AS. Stable transfection of short sense cDNA constructs (300 -400 nucleotides) for each of these Trp homologs was used as a control (i.e. HTRP1S, HTRP3S, and HTRP4S). As we described previously, we mixed all of the surviving clones (ϳ200) to generate heterogeneous populations of transfected cells for each cell line; therefore, the large cell-to-cell variations of SOCE we previously reported in the parent HEK-293 popula-tion should have no impact on our interpretations (36).
We extracted poly(A) RNAs from all cell lines mentioned above, reverse transcribed first strand cDNAs, and then specifically amplified the appropriate cDNAs via PCR using primers specific for regions just outside the sequence of the sense and antisense constructs (outer primers, Table I). This was done so as not to amplify the expressed sense and antisense constructs along with the regions from the endogenous mRNA. As shown in Fig. 2, expression of HTRP1, HTRP3, and HTRP4 mRNA was detected in HTRP1S, HTRP3S, and HTRP4S cells, respectively. Since a previous report has shown alternative splice variants of HTRP4 (41), we used a different set of primers to determine whether HTRP4 mRNA expressed was for the ␣ or the ␤ isoform. We detected both isoforms, with the ␣ isoform being the more abundant species (the ␤ isoform was about 20% of the level of the ␣ isoform; data not shown). In addition, the data in Fig. 2 show that the expression of the mRNA for the specific Trp homolog was significantly reduced in HTRP1AS, HTRP3AS, and HTRP4AS cells in which antisense cDNA constructs were stably expressed.
Level of Expression of Trp Proteins in HTRP1AS, HTRP3AS, and HTRP4AS Cells-To confirm that antisense expression was reducing the protein levels as well as the mRNA levels of the various endogenous Trps, we performed Western blot analysis on all of the cells utilized. Although we were ultimately successful in observing endogenous Trp proteins on Western blots of HEK-293 cells utilizing the Alomone anti-Trp antibodies, this occurred only after an extensive characterization procedure for the anti-Trp antibodies (see "Experimental Procedures"). Initial experiments gave high background, multiband blots where the differences in the appropriate molecular weight band was barely discernible between lanes for control and Trp-overexpressing cells. However, after optimization of the  D). The SOCE or CChstimulated entry was assessed by the readdition of BaCl 2 (Ba 2ϩ ; 5 mM) in Ca 2ϩfree medium following store depletion with either thapsigargin or CCh. A, the SOCE induced by thapsigargin in control HEK-293 cells; B, the inhibitory effect of 2-APB (100 M) on SOCE. C, the CChstimulated Ba 2ϩ entry in control HEK-293 cells; D, the CCh-stimulated Ba 2ϩ entry in 2-APB-treated cells. 2-APB (100 M) was added at 3 min prior to carbachol addition. To monitor OAG effects (E and F), SrCl 2 (Sr 2ϩ ; 5 mM) was added in Ca 2ϩfree medium. Stimulated Sr 2ϩ entry was initiated by the addition of OAG (100 M) into the chamber immediately after solution flow was stopped. 2-APB was added 3 min before OAG stimulation. Shown is the OAG-stimulated Sr 2ϩ entry in control cells (E) and in cells treated with 100 M 2-APB (F). Each trace represents one coverslip (ϳ800 cells). Three coverslips (n ϭ 3) were done for each condition, and similar results were observed. The basal [Ca 2ϩ ] i was ϳ50 nM, and the peak of Ca 2ϩ following thapsigargin stimulation was ϳ350 nM. The peak of Ca 2ϩ following CCh stimulation was ϳ850 nM.
techniques, we were able to see single, clear, strong bands at the appropriate molecular weight for endogenous HTRP1, HTRP3, and HTRP4. The Western blot data in Fig. 3 demonstrate that HTRP1, HTRP3, and HTRP4 are endogenously expressed in HEK-293 cells and that the expression of antisense constructs specifically reduces individual HTRP protein levels. As seen in Fig. 3C, the introduction of HTRP1 antisense results in a reduction of HTRP1 protein to a value that is 36 Ϯ 2.8% of the control protein level (significantly different from control value, p Ͻ 0.001, n ϭ 3), while having no effect on either HTRP3 or HTRP4 protein levels. The expression of antisense to HTRP3 results in a reduction of HTRP3 protein to a value that is 43.0 Ϯ 6.0% of the control protein level (significantly different from control value, p Ͻ 0.005, n ϭ 4), while having no effect on HTRP1 and HTRP4 protein levels (Fig. 3B). Finally, the expression of HTRP4 antisense results in a reduction of HTRP4 protein to a value that is 31 Ϯ 14% of the control protein level (significantly different from control value, p Ͻ 0.02, n ϭ 3), while having no effect on HTRP1 and HTRP3 proteins levels (Fig. 3A). Therefore, the antisense constructs demonstrate the specificity of action required to analyze the involvement of individual Trp isoforms in various endogenous Ca 2ϩ entry pathways.
Role of HTRP4 in Thapsigargin-stimulated, CCh-stimulated, and OAG-stimulated Ca 2ϩ Entry-To investigate the functional importance of HTRP4 in regulating Ca 2ϩ influx, we examined entry in HTRP4AS cells, compared with HTRP4S cells, in response to thapsigargin, carbachol, and OAG. We monitored either Ba 2ϩ or Sr 2ϩ entry prior to stimulation and then again after stimulation. We summarized these results in Fig. 4. In the left panels, we show traces from two representative coverslips (HTRP4AS versus HTRP4S), each with the response averaged over ϳ800 cells in the microscope field. In the study of thapsigargin-or carbachol-induced Ca 2ϩ entry pathways, we measured levels of Ba 2ϩ (or Sr 2ϩ ) before (basal leak) and after (total influx) stimulation. We then determined the stimulated influx by subtracting the basal leak from the total influx. In the study of the OAG-induced entry pathway, we simply measured the slope of Sr 2ϩ entry in the presence of OAG in HTRP4AS versus HTRP4S cells, since no basal Sr 2ϩ entry was observed. In the right panel, we show the mean values (with error bars showing the S.E.) of the stimulated Ba 2ϩ or Sr 2ϩ uptakes plotted as percentages of its control. In this case, we determined the mean value of final Ba 2ϩ (or Sr 2ϩ ) influx for a series of coverslips for each cell type, and we determine whether the difference between HTRP4AS and HTRP4S cells was statistically significant by Student's t test. Fig. 4A shows both the basal Ba 2ϩ leak (before store depletion) and the total Ba 2ϩ influx following store depletion with thapsigargin (1 M). When one compares the responses in HTRP4AS and HTRP4S cell lines, there is little difference in the initial transient Ca 2ϩ peak (Fig. 4, A and C), suggesting that the suppression of HTRP4 expression does not significantly alter the size of the internal Ca 2ϩ stores. Furthermore, there is no difference in thapsigargin-stimulated Ba 2ϩ entry (slope: HTRP4S, 0.69 Ϯ 0.07, n ϭ 15; HTRP4AS, 0.67 Ϯ 0.07, n ϭ 15; p Ͼ 0.8), suggesting that HTRP4 plays no major role in regulation of SOCE (Fig. 4B).
We also used Sr 2ϩ to monitor SOCE. Interestingly, Sr 2ϩ leak was undetectable in both HTRP4AS and HTRP4S cells (Fig.  4C). Furthermore, the Sr 2ϩ influx following store depletion was dramatically enhanced, independent of cell lines (Fig. 4C). As shown in Fig. 4D, there was no difference in Sr 2ϩ entry in HTRP4AS compared with HTRP4S cells (slope: HTRP4S, 0.62 Ϯ 0.02, n ϭ 12; HTRP4AS, 0.64 Ϯ 0.02, n ϭ 12; p Ͼ 0.50). The Ba 2ϩ and Sr 2ϩ data both agree that HTRP4 proteins are not involved in the Ca 2ϩ entry pathway activated by calcium store depletion with thapsigargin.
To evaluate whether HTRP4 plays a role in receptor-stimulated Ca 2ϩ entry, we measured Ba 2ϩ influx before and after carbachol (100 M) stimulation of HTRP4AS cells, using HTRP4S cells as a control. As shown in Fig. 4E, in the absence of Ca 2ϩ , carbachol induced a large, transient increase in [Ca 2ϩ ] i followed by a decline to basal levels. Stable transfection of antisense constructs for HTRP4 had no measurable effect on the size of the CCh-stimulated Ca 2ϩ release; however, it did cause a dramatic decrease in final Ba 2ϩ influx (Fig. 4E). As shown in Fig. 4F, the expression of HTRP4 antisense resulted in a 35% inhibition of Ba 2ϩ entry, which was a statistically significant reduction (slope: HTRP4S, 1.19 Ϯ 0.15, n ϭ 36; HTRP4AS, 0.77 Ϯ 0.10, n ϭ 36; p ϭ 0.03).
In summary, HTRP4 does not seem to play a role in regulation of SOCE. However, it does play a role in carbachol-, and OAG-induced Ca 2ϩ entry. Stably  Transfected with HTRP1AS, HTRP3AS, and HTRP4AS-In a previous study, we showed that stable expression of HTRP3 antisense in HEK-293 cells resulted in a 32% inhibition of  SOCE. Transient transfection of an HTRP1 antisense construct in HTRP3AS cells led to a higher level of inhibition (55%) of store-operated Ca 2ϩ entry (32). To evaluate whether HTRP1 or HTRP3 also play a role in mediating OAG-induced Ca 2ϩ signaling, we measured Sr 2ϩ influx induced by OAG (100 M) in HEK-293 cells expressing antisense cDNA constructs for HTRP1 (HTRP1AS) or HTRP3 (HTRP3AS). HTRP1S or HTRP3S cells were used as the corresponding controls. Sr 2ϩ leak was undetectable in any of these experiments. Sr 2ϩ influx induced by OAG was dramatically reduced in HTRP3AS compared with HTRP3S. The magnitude of the effect was a 52% reduction, which is statistically significant ( The data suggest that HTRP3 and HTRP4, but not HTRP1, play an important role in OAG-induced Sr 2ϩ entry (Fig. 5).

5Ј-TTT GTC TTC ATG ATT TGC TAT CA-3Ј
The Role of HTRP3 and HTRP4 in Ca 2ϩ Entry-dependent, CCh-induced Ca 2ϩ Oscillations-Two recent studies (42,43) indicate that CCh, at low doses, can initiate long lasting Ca 2ϩ oscillations in HEK-293 cells. Sustained oscillations of Ca 2ϩ in response to CCh (sustained for at least 1 h) are strictly dependent on the presence of extracellular Ca 2ϩ . While initial theories suggested that the Ca 2ϩ entry required for sustained Ca 2ϩ oscillations was mediated via store-operated channels, very recent data suggest that the Ca 2ϩ entry is via non-store-operated channels (42,44). Since we could reduce Ca 2ϩ entry via either store-operated or non-store-operated pathways by expressing either HTRP3 antisense or HTRP4 antisense, we investigated what effect the expressions of these antisense constructs have on cell Ca 2ϩ oscillations.
Since we observed above that HTRP4 does not play a role in SOCE but does play a role in CCh-stimulated Ca 2ϩ entry, it seemed possible that HTRP4 is involved in mediating the Ca 2ϩ entry required to support the sustained Ca 2ϩ oscillations in CCh. To test this hypothesis, we monitored CCh-induced Ca 2ϩ oscillations in individual cells for both the HTRP4S and HTRP4AS populations of cells. The data in Fig. 6 (left panel) RNA was extracted, treated with DNase, and reverse transcribed with oligo(dT) [12][13][14][15][16][17][18] primers and Superscript TM II reverse transcriptase (Invitrogen). The first strand cDNA was then amplified with PCR using the forward and outer reverse primers that are specific for each Trp isoform (Table I) to amplify the various Trp isoform cDNA. ␤-Actin was amplified as a positive control to assure that changes observed were not due to different levels of starting material (first two lanes). For a negative control, reactions were performed without reverse transcriptase (No RT; last two lanes). The results shown are representative of at least three independent experiments. FIG. 3. Western blot analysis of HTRP1, HTRP3, and HTRP4 protein levels in control and antisense-expressing cells. Total cell lysates from HTRP4S, HTRP4AS, HTRP3S, HTRP3AS, HTRP1S, and HTRP1AS cells were obtained using procedures described under "Experimental Procedures." Total protein (200 or 400 g) was separated by 7.5% SDS-PAGE and transferred to Immobilon membrane. Polyclonal anti-human Trp4, Trp3, or Trp1 antibody and horseradish peroxidase-labeled goat anti-rabbit immunoglobulin were used as primary and secondary antibodies, respectively. The signals were detected by ECL using standard protocols. The results shown are representative of at least three independent experiments. fewer repetitive Ca 2ϩ spikes in HTRP4AS cells in comparison with HTRP4S cells. In Fig. 7, we show the statistical analysis of the responses recorded from over 300 individual cells of each type. It is clear that many more HTRP4S cells than HTRP4AS cells undergo in excess of three Ca 2ϩ oscillations, the number of damped oscillations seen in normal HEK-293 cells in a Ca 2ϩfree medium (data not shown). However, it is also clear that the amplitude of the first spike is similar in the two cell lines, indicating that the difference is not in the initial intracellular Ca 2ϩ pool content but rather in the inability of the HTRP4AS cells to continue to refill the internal Ca 2ϩ stores after the initial depletion.
To determine whether the effects observed with HTRP4 antisense were specific and therefore supported a role for FIG. 4. Role of HTRP4 in thapsigargin-, CCh-, and OAG-stimulated Ba 2؉ or Sr 2؉ entry. HTRP4 antisense cDNA constructs were stably transfected into HEK-293 cells, generating a HTRP4AS cell line. Stable transfection of HTRP4 sense cDNA constructs (ϳ300 nucleotides) into HEK-293 cells (i.e. HTRP4S cells) was used as a control. The left panel shows the time course of representative experiments during which levels of Ba 2ϩ (or Sr 2ϩ ) before (basal leak) and after (total influx) agonist stimulation were measured. SOCE was determined by subtracting the basal leak from the total influx following store depletion with 1 M thapsigargin (A and C). CCh-stimulated entry was determined by subtracting basal Ba 2ϩ leak from total Ba 2ϩ influx following stimulation with 100 M carbachol (E). OAG-stimulated entry was monitored by measuring Sr 2ϩ entry following stimulation with 100 M OAG (G). Each trace represents a single coverslip with the response averaged over ϳ800 HTRP4S cells (thick line) or HTRP4AS cells (thin line). The right panel shows the statistical analysis of the data, with the mean value of the leak-subtracted Ba 2ϩ (or Sr 2ϩ ) entry (for n coverslips) plotted as a percentage of control. Statistical differences between values in HTRP4AS cells and HTRP4S cells were analyzed by Student's t test. HTRP4 in maintaining the ongoing Ca 2ϩ oscillations, we determined whether the expression of an HTRP3 antisense construct, which reduces store-operated Ca 2ϩ entry in HEK-293 cells (32), would affect the Ca 2ϩ oscillations induced by low doses of CCh. The data in Fig. 8 (left panel), show 10 representative single cell responses recorded from one HTRP3S coverslip. Cells were stimulated with 15 M CCh in a medium containing 1.8 mM Ca 2ϩ . These cells exhibited repetitive peaks over the 10-min time period shown. In Fig. 8 (right panel), we show 10 representative individual cell responses from one HTRP3AS coverslip. Note that there are no obvious differences in the number of Ca 2ϩ spikes in HTRP3AS cells compared with HTRP3S cells. In Fig. 9, we show the statistical analysis of the responses recorded from over 300 individual cells of each type. It is clear that HTRP3S and HTRP3AS cells undergo a similar number of Ca 2ϩ oscillations, arguing that reduction of store-operated Ca 2ϩ entry has no effect on the sustained Ca 2ϩ oscillations.
The Role of HTRP3 and HTRP4 in Arachidonic Acidstimulated Ca 2ϩ Entry-Since a recent report suggested that the Ca 2ϩ entry pathway required for sustained Ca 2ϩ oscillations is an arachidonic acid-activated channel (43), we sought to determine whether expression of HTRP4 antisense had any impact on the arachidonic acid-stimulated Ca 2ϩ entry. The data in Fig. 10A are representative traces that show that the addition of arachidonic acid to previously unstimulated HTRP4S cells produces a dramatic rise in Ca 2ϩ levels, while a similar addition to HTRP4AS cells produced much less of a response. The data in Fig. 10B summarize our results and demonstrate that expression of HTRP4 antisense results in a 75% reduction in the level of arachidonic acid-stimulated Ca 2ϩ entry (slope: HTRP4S, 0.21 Ϯ 0.03, n ϭ 19; HTRP4AS, 0.05 Ϯ 0.01, n ϭ 20; p Ͻ 0.0005). To determine whether this was a specific effect of HTRP4 antisense, we expressed HTRP3 antisense and determined the effect on arachidonic acid stimulation of Ca 2ϩ entry. As seen in Fig. 10, C and D, there is no significant effect of expressing HTRP3 antisense

DISCUSSION
Ever since the Trp mutation in the Drosophila phototransduction cascade was identified (8), an investigation into the mechanisms by which this light-sensitive ion channel is activated has ensued. There is good agreement that Trps are activated by G-protein-coupled receptors, which induce phospholipase C-mediated phosphoinositide breakdown. However, the phototransduction pathways that finally activate Drosophila Trp, as well as Trpl, remain highly controversial (13). The same is true for the mammalian Trp isoforms. For nearly all of the functionally expressed mammalian Trps (short isoforms), there is at least one report concerning a store-operated mechanism of activation (14 -16, 24, 28 -30, 32-35). On the other hand, there is evidence for the involvement of store-independent pathways in the regulation of Trp4 (26), Trp3 (20,31,45), and Trp1 (27). The reasons for differences in the functional properties of Trps reported by individual laboratories remain unclear.
While most of the early studies on Trp function were done by overexpressing exogenous Trps, our approach has been to investigate the role of the endogenous Trp isoforms by using antisense techniques to suppress the expression of various Trp proteins. In a previous publication (32), we showed RT-PCR evidence for the endogenous expression of HTRP1, HTRP3, HTRP4, and HTRP6 in our population of HEK-293 cells, a finding that agreed qualitatively with a previous publication showing RT-PCR evidence for the expression of these four Trp isoforms in HEK-293 cells (46). Although we did provide Northern blot evidence for endogenous expression of HTRP3 (32) and another publication provided Western blot evidence for endogenous expression of HTRP1 protein (47) in HEK-293 cells, there is not extensive data in the literature to confirm the RT-PCR evidence of endogenous expression of Trp isoforms in HEK-293 cells. Thus, our Western blot data in Fig. 3 represents important evidence for the endogenous expression of HTRP1, HTRP3, and HTRP4 proteins in HEK-293 cells. However, based on our RT-PCR and Western blot data, it would appear that HTRP1 and HTRP3 proteins are more abundant than HTRP4 proteins. In the PCR analysis, cDNA fragments for HTRP1 and HTRP3 were visible after 30 PCR cycles, whereas the cDNA fragment for HTRP4 was visible only after 35 cycles, suggesting that less mRNA for HTRP4 is present. Likewise, for the Western blot experiments, we loaded the gels with twice as much protein and exposed the films for much longer time periods for the HTRP4 blots than for the HTRP1 and HTRP3 blots.
Contrary to a number of earlier published results on exogenously expressed Trp4 (16,(22)(23)(24), we were not able to detect a role for endogenous HTRP4 in channel activation by store depletion with thapsigargin (Fig. 4, A-D). Also, our results run counter to those described in a paper that was published during the preparation of this manuscript, describing studies on vascular endothelial cells from Trp4 knockout mice (48). In that report, a reduction of store-operated Ca 2ϩ entry was observed in the absence of murine Trp4. On the other hand, our results, using the antisense approach, agree with at least part of the conclusions of two recent overexpression studies, namely that murine Trp4 (25) and HTRP4 (26) do not contribute to storeoperated channel activity. In addition, a paper that became available in electronic form during the revision our manuscript argues that neither the ␣ nor ␤ splice variant of human Trp4 is regulated by depletion of internal calcium stores (49).
Although we could see no evidence for HTRP4 involvement in capacitative calcium entry, we were able to detect a significant effect of HTRP4 antisense expression on channel activation by the muscarinic receptor. It is likely that endogenous HTRP4 functions as a component of receptor-mediated calcium entry channels (Fig. 4, E and F). Thus, our results agree with overexpression studies showing that murine Trp4 is regulated by agonist stimulation (25), but they disagree with the report that overexpressed HTRP4 is constitutively activated and not regulated by agonists (26). In terms of cation selectivity, we see a nonselective permeability for Ba 2ϩ and Sr 2ϩ (Fig. 4, A-D). This finding agrees with results from human Trp4 (26) and mouse Trp4 (25) but not bovine Trp4 (22). Subtle differences between HTRP4 and Trp4 from other species may affect, at least partially, its contribution to channel properties.
We also observed that HTRP4 was involved in the pathway responsible for OAG stimulation of Sr 2ϩ entry (Fig. 4, G and H). Since phospholipase C activation by carbachol should produce an elevation of diacylglycerol, one might expect the Ca 2ϩ entry pathway activated by OAG to be the same pathway that is activated following CCh stimulation. Consistent with this hypothesis are the findings that both the OAG-induced and the CCh-stimulated Ca 2ϩ entry pathways are at least partially dependent on the presence of HTRP4. We observed that expression of HTRP4 antisense can reduce the CCh-stimulated Ca 2ϩ entry by 35% and can reduce the OAG-stimulated Ca 2ϩ entry pathway by 46%. However, based on the effects of 2-APB on these two pathways, it seems unlikely that CCh stimulates the OAG-inducible pathway in HEK-293 cells. We see that most of the CCh-stimulated entry pathway(s) is inhibited by 100 M 2-APB, while none of the OAG-induced entry pathway is inhib- ited by this compound. Clearly, 2-APB can block the capacitative portion of the CCh-stimulated entry, based on the ability of 2-APB to block the thapsigargin-stimulated Ba 2ϩ entry (Fig. 1). However, it is also clear that the noncapacitative component of Ca 2ϩ entry stimulated by CCh is sensitive both to HTRP4 expression levels and to the presence of 2-APB. These data strongly suggest that the physiological levels of diacylglycerol produced by CCh stimulation of cells is not sufficient to activate the Ca 2ϩ entry pathway activated by pharmacological levels of OAG. While one could certainly argue that high concentrations of diacylglycerol near the membrane could be produced following the activation of phospholipase C, the ability of 2-APB to block most of the CCh-stimulated Ca 2ϩ entry, while having no effect on the OAG-stimulated entry pathway, indicates that this is not the case.
Our data demonstrating that Ba 2ϩ entry due to store depletion with thapsigargin is inhibited by 2-APB ( Fig. 1) is, on the surface, consistent with the notion that 2-APB blocks SOCE via its interaction with the InsP 3 receptor (40). Original reports showed that 2-APB inhibits agonist-induced Ca 2ϩ release in platelets, neutrophils, and aorta, probably via inhibition of InsP 3 receptors, type 1 or type 3. While it does not affect InsP 3 binding to its receptor, it does partly inhibit Ca 2ϩ uptake into the store, like thapsigargin, at least at high doses (100 M) (37). In recent studies, it was reported that 2-APB completely blocks Ca 2ϩ entry induced by different store-depleting agents, and the results were observed in several cell lines, including HEK-293 cells (40,42). 2-APB was found to block Ca 2ϩ entry mediated by overexpressed HTRP3 channels if the channels are activated through phospholipase C-coupled receptors but not by diacylglycerol. The results were interpreted to argue for a direct interaction between the InsP 3 receptor and HTRP3 (40). In our 2-APB studies, we found that there is a striking difference between the short and long term effects of 2-APB on InsP 3 signaling. While a 3-min incubation with 2-APB had essentially no effect on the CCh-induced Ca 2ϩ release (Fig. 1D), longer incubation times dramatically reduced the CCh-stimulated release of store Ca 2ϩ (data not shown). Surprisingly, the 3-min exposure to 2-APB, which had no measurable effect on CCh-induced Ca 2ϩ release, dramatically inhibited CCh-induced Ba 2ϩ entry (Fig. 1D). Therefore, the inhibitory effect of 2-APB on SOCE is not correlated with the ability to reduce the initial depletion of the Ca 2ϩ stores. This suggests that 2-APB may be directly blocking plasma membrane Ca 2ϩ channels, a finding that is consistent with recent observations that 2-APB blocks thapsigargin-induced Ca 2ϩ entry, even in cells lacking functional InsP 3 receptors (38,39).
Cyclical processes are part of the essence of life, and oscillations in cell calcium are one example of such cyclical processes, one that seems to be fundamental to the growth and differentiation of many cell types. Cytosolic calcium oscillations can be induced by a variety of agonists. Although the underlying mechanisms remain unclear, several models have been proposed, with InsP 3 being the center of attention (4, 50 -52). In the current study, we set up to record oscillations induced by carbachol (15 M) in fura-2-loaded HEK-293 cells. We observed that the addition of CCh in the continued presence of external Ca 2ϩ led to a sustained oscillatory pattern that is truncated shortly after the removal of extracellular Ca 2ϩ (data not shown). We set out to investigate the role of HTRP4 in mediating the Ca 2ϩ entry that is necessary for this sustained oscillatory pattern. We found that in cells expressing an antisense construct for HTRP4 (Fig. 6, right panel), the oscillations induced by CCh were more similar to the damped oscillations seen in nominally Ca 2ϩ -free medium than those seen in the presence of Ca 2ϩ (Fig. 6, left panel). In Fig. 7, we plot the number of cells versus the number of oscillations that occur in a cell over a 10-min period. It is clear that most of the cells expressing HTRP4 antisense are clustered in the leftmost portion of the bar graph, indicating that they are unable to sustain oscillations over a long period of time. This appears to be a specific effect of the HTRP4 antisense, since the expression of the HTRP3 antisense construct had no effect on Ca 2ϩ oscillations (Figs. 8 and 9). The determination that HTRP4 is involved in regulating agonist-induced Ca 2ϩ oscillations is a very important one. Although much of the work in the Ca 2ϩ signaling field focuses on the plateau phase of the Ca 2ϩ response following high dose agonist addition, it is likely that under physiological conditions, cells see agonist at much lower doses. Thus, the Ca 2ϩ oscillations seen in response to 15 M CCh are likely to be physiologically more important than the plateau response seen at 100 M CCh. Thus, the determination that HTRP4 is involved in supporting Ca 2ϩ oscillations is an extremely important one.
While recent publications from the Shuttleworth laboratory have suggested that the Ca 2ϩ entry pathway required for sustained Ca 2ϩ oscillations is regulated by arachidonic acid, this hypothesis is based on the use of pharmacological inhibitors (43), which can always raise questions of drug specificity. When we observed that expression of HTRP4 antisense produced a block of the CCh-induced Ca 2ϩ oscillations, we recognized that this provided an opportunity to test the arachidonic acid hypothesis utilizing a molecular approach. We first determined that the addition of arachidonic acid to control cells (HTRP4S cells) produced a significant level of Ca 2ϩ entry (Fig. 10A). While the dose of arachidonic acid used in Fig. 10 is somewhat higher than those used to define the arachidonic acid-regulated channel I(ARC) (53), we observed that the exact dose for activating Ca 2ϩ entry depended on the batch of arachidonic acid received from the supplier. With some batches, we did see activation with doses in the range of 5-10 M, while with other batches, it took 30 -40 M to stimulate robust levels of Ca 2ϩ entry. The variability is probably due to the known instability of arachidonic acid in solution. However, no matter which batch was used, we would first define a window of doses to use by titrating the amount that would stimulate Ca 2ϩ entry without giving a rise in Ca 2ϩ concentration when added to cells in a Ca 2ϩ -free environment.
We next determined whether expression of HTRP4 antisense would block the arachidonic acid-stimulated Ca 2ϩ entry pathway. The data in Fig. 10, A and B, illustrates that HTRP4AS expression blocks ϳ75% of the arachidonic acid-stimulated Ca 2ϩ entry. This appears to be a specific effect of HTRP4 antisense, since the expression of HTRP3 antisense has no significant effect on the arachidonic acid-stimulated Ca 2ϩ entry (Fig. 10, C and D). Therefore, this finding provides the first evidence for the involvement of one of the Trp proteins in the arachidonic acid-regulated Ca 2ϩ channel. It also supports the hypothesis that the arachidonic acid-regulated pathway is the one that provides the Ca 2ϩ influx necessary for the maintenance of sustained Ca 2ϩ oscillations.
Among all mammalian Trp homologs, HTRP3 has been the most extensively studied. Early studies showed that expression of full-length cDNA encoding HTRP3 increased SOCE (14), and expression of a partial cDNA fragment of HTRP3 in the antisense orientation significantly reduced SOCE (32), following store depletion with thapsigargin. Additional studies suggested that HTRP3 might also be activated by a conformational coupling mechanism by interaction with InsP 3 receptor (30,40,54). Other recent studies show that overexpressed HTRP3 can be activated by application of OAG (26,45,55). For comparison, human Trp1 has been shown to be a nonselective cation chan-nel (15), which, when expressed in mammalian cells, is activated by store depletion by thapsigargin as well as inositol 1,4,5-trisphosphate (15). Our recent studies indicated that expression of HTRP1 antisense in combination with HTRP3 antisense produces a further reduction in thapsigargin-stimulated Ca 2ϩ entry in addition to that seen with expression of HTRP3 alone (32). When HTRP1 is expressed in Sf9 insect cells, it may (15) or may not (27) be sensitive to depletion of internal Ca 2ϩ stores. In addition, overexpression of HTRP1 leads to channels that are sensitive to stimulation by carbachol (14) but insensitive to OAG (45,56). The conclusion from the present study is that HTRP3 proteins as well as HTRP4 proteins play a role in forming the endogenous channels, which are activated by OAG. In contrast, endogenous HTRP1 proteins appear to play no role in the activation of channels by OAG. As shown in Fig. 5, in HEK-293 cells expressing antisense cDNA constructs for HTRP3 or HTRP4, Sr 2ϩ entry induced by OAG was dramatically reduced. In contrast, Sr 2ϩ entry induced by OAG was unaffected in cells expressing antisense cDNA for HTRP1. This provides useful evidence that OAG is indeed acting on the HTRP3 and HTRP4 but not HTRP1 channels.
We should point out that these results differ from earlier results, in that OAG stimulates Sr 2ϩ entry via endogenous channels in our HEK-293 cells, while in other studies OAG had no effect on parental HEK-293 cells but did stimulate Sr 2ϩ entry in cells overexpressing HTRP3 (26,40). This difference in results is probably due to the variation of characteristics of cultured lines grown in different laboratories. It is widely known for cell lines such as PC12 cells that there can be dramatic differences in cultures carried for a number of years in different laboratories. Different laboratories utilize serum from different sources, and other more subtle differences in tissue culture procedure also exist. Since our previous work demonstrates dramatic clone-to-clone variation in levels of SOCE within our HEK-293 cell population, it is not difficult to imagine that culture conditions in one laboratory might offer a competitive advantage for growth of some clones over others, thereby leading to high endogenous levels of SOCE in HEK-293 populations in one laboratory versus low endogenous levels of SOCE in HEK-293 populations in another laboratory. Thus, while we see substantial SOCE via endogenous channels, some laboratories see very little endogenous SOCE. The cell lines with the low endogenous SOCE have been popular for electrophysiological studies of the overexpression of Trp proteins, since there is a low basal current, and an increment in current due to expression of Trp can be easily observed. One possible drawback to the use of cell lines with low endogenous currents might exist if the transfected Trp needs to form heterotetramers with another Trp isoform to show the correct channel characteristics. Cell lines having vanishingly small endogenous currents may not express the full complement of endogenous Trps needed to form the true physiological configuration of the channel. Thus, the characteristics of the channel formed by overexpressed Trp may be misleading in terms of the true role of that Trp protein. On the other hand, the HEK-293 cell line that we use is perfect for our approach. It has substantial levels of both SOCE and receptor-activated Ca 2ϩ entry so that we can study the role of endogenous Trps in either Ca 2ϩ entry pathway.
One extremely important question in the Trp field is why there are so many conflicting reports about the role of various Trp isoforms in mediating either store-operated or agoniststimulated Ca 2ϩ entry pathways. One possible reason is the use of different cellular expression systems with the possibility of differing genetic backgrounds in terms of the types of endogenous Trp subunits available for combination with the overex-pressed Trp isoform. It is also possible that overexpression of Trps leads to the study of homotetramers that may have very different properties from endogenous channels that may normally be assembled from heterogeneous subunits. In addition to these problems, we published a recent report that cautions about the study of low numbers of stable clones expressing Trps (36). That paper estimates the probability that clone-to-clone variation in the population of HEK-293 cells contributes to the misinterpretation of data in clones overexpressing Trp isoforms. In light of these potential problems, we decided that a strategy that reduces the level of endogenous Trps would be a more desirable way to study the functional role of individual Trp isoforms.
In summary, our results define some interesting similarities and differences in endogenous Ca 2ϩ entry pathways activated by thapsigargin, CCh, or OAG in HEK-293 cells. The endogenous Ca 2ϩ entry pathways activated by store depletion or by carbachol can be inhibited by 2-APB, while the endogenous Ca 2ϩ entry pathway stimulated by OAG is not inhibited by 2-APB. The Ca 2ϩ entry pathway activated by store depletion does not require HTRP4 as a channel subunit. On the other hand, the Ca 2ϩ entry pathways activated by either carbachol or OAG do require HTRP4 expression. The CCh-and OAG-stimulated, HTRP4-dependent Ca 2ϩ entry pathways appear not to be one and the same, since 2-APB inhibits most of the CChstimulated pathway but none of the OAG-stimulated pathway. HTRP3 proteins appear to be more widely used than HTRP4 proteins, since they are required in both the store-operated channels and the OAG-stimulated channels. In contrast, HTRP1 appears to be a functional subunit of endogenous storeoperated channels but not OAG-stimulated channels. And of greater physiological importance, HTRP4 plays a role in both the Ca 2ϩ entry pathway activated by arachidonic acid and the Ca 2ϩ entry pathway required to sustain Ca 2ϩ oscillations induced by low doses of CCh.