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(The FASEB Journal. 2001;15:1727-1738.)
© 2001 FASEB

Contribution of endogenously expressed Trp1 to a Ca2+-selective, store-operated Ca2+ entry pathway

GEORGE H. BROUGH, SONGWEI WU, DONNA CIOFFI, TIMOTHY M. MOORE, MING LI, NICHOLAS DEAN* and TROY STEVENS1

Department of Pharmacology, University of South Alabama, College of Medicine, Mobile, Alabama 36688, USA; and
* ISIS Pharmaceuticals, Carlsbad, California 92008, USA

1Correspondence: Department of Pharmacology, University of South Alabama College of Medicine, 307 N. University Blvd., MSB 3360, Mobile, AL 36688, USA. E-mail: tstevens{at}jaguar1.usouthal.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Heterologous expression of the transient receptor potential-1 gene product (Trp1) encodes for a Ca2+ entry pathway, though it is unclear whether endogenous Trp1 contributes to a selective store-operated Ca2+ entry current. We examined the role of Trp1 in regulating both store-operated Ca2+ entry and a store-operated Ca2+ entry current, ISOC, in A549 and endothelial cells. Twenty different ‘chimeric’ 2'-O-(2-methoxy)ethylphosphothioate antisense oligonucleotides were transfected separately using cationic lipids and screened for their ability to inhibit Trp1 mRNA. Two hypersensitive regions were identified, one at the 5' end of the coding region and the second in the 3' untranslated region beginning six nucleotides downstream of the stop codon. Antisense oligonucleotides stably decreased Trp1 at concentrations ranging from 10 to 300 nM, for up to 72 h. Thapsigargin increased global cytosolic Ca2+ and activated a ISOC, which was small (-35 pA @ -80 mV), reversed near +40 mV, inhibited by 50 µM La3+, and exhibited anomalous mole fraction dependence. Inhibition of Trp1 reduced the global cytosolic Ca2+ response to thapsigargin by 25% and similarly reduced ISOC by 50%. These data collectively support a role for endogenously expressed Trp1 in regulating a Ca2+-selective current activated upon Ca2+ store depletion.—Brough, G. H., Wu, S., Cioffi, D., Moore, T. M., Li, M., Dean, N., Stevens, T. Contribution of endogenously expressed Trp1 to a Ca2+-selective, store-operated Ca2+ entry pathway.


Key Words: capacitative Ca2+ entry • ICRAC • signal transduction • endothelial cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ACTIVATION OF CA2+ entry across the cell membrane is an important inflammatory stimulus in nonexcitable cells. This stimulus produces degranulation in mast cells (1 2 3) and activates cell-mediated immunity in T lymphocytes (4 5 6 7 8) . In endothelial cells, Ca2+ entry across the cell membrane initiates a retraction needed to deliver plasma-rich exudate to inflamed tissue (9 10 11 12 13 14 15) and for transmigration of circulating leukocytes (16 17 18 19) . These cell types all share a common mode of Ca2+ entry: this Ca2+ entry process is activated after the depletion of Ca2+ stored in the endoplasmic reticulum, a so-called store-operated Ca2+ entry pathway (20 21 22 23 24 25) . However, several apparent store-operated Ca2+ entry pathways exist, each possessing distinctive electrophysiological characteristics and each making unique contributions to the global rise in cytosolic Ca2+ ([Ca2+]i) after Ca2+ store depletion. Indeed, mast cells and lymphocytes express a highly Ca2+-selective current, called a Ca2+ release-activated current or ICRAC (1 , 4) . The endothelial cell current may or may not exhibit less Ca2+ selectively (26 27 28) and is generally referred to as a store-operated Ca2+ current or ISOC.

The molecular identity of ion channels that mediate store-operated Ca2+ entry is still poorly understood, though electrophysiologic studies suggest the presence of multiple, different channels. Discovery that the transient receptor potential (trp) gene product in Drosophila melanogaster encoded a retinal store-operated Ca2+ entry channel provided impetus for cloning a whole family of related mammalian homologues (22 , 29) . These products belong to a six-transmembrane spanning domain family of cation channels that include the Drosophila Trp and seven mammalian Trp homologues (Trp1-Trp7). Predicted topography suggests the functional channel is a tetramer, wherein the pore is formed by cohesion of membrane-delimited domains bridging the S5 and S6 membrane spanning regions from four different monomers (29) . Thus, multiple combinations of Trp proteins could result in unique channel characteristics. Trp1, 4, and 5 appear to be activated upon Ca2+ store depletion, though not all mammalian Trp channels are store operated (22) . Most available data are derived from the heterologous expression of individual gene products, where expressed proteins may form channels with properties that do not resemble their endogenous phenotype. Indeed, coexpression of Trp1 with Trp3 produced a channel with biophysical and regulatory properties that were different from the expression of either Trp1 or Trp3 alone (30) . There is therefore a need to examine the contribution that individual Trp proteins make to endogenous modes of Ca2+ entry.

Although Trp1 contributes to formation of a Ca2+ entry pathway, the nature of this Trp1-containing channel is not clear. Overexpression of Trp1 likely forms a nonselective cation channel that enhances store-operated Ca2+ entry, particularly in recalcification experiments (29 , 31 , 32) . However, there have been limited attempts to examine the contribution of Trp1 to its endogenously formed channel. Recent findings indicate that inhibition of Trp1 expression in salivary gland cells (33) and of Trp1 and Trp3 in HEK-293 cells (34) reduces the global [Ca2+]i response to activation of store-operated Ca2+ entry, suggesting that Trp1 may comprise a functional subunit of certain store-operated Ca2+ entry pathways. Neither of these studies specifically examined the electrophysiological nature of Trp1-containing channels. Indeed, the role of Trp1 in ISOC is controversial; expression of Trp1 augmented the whole-cell Ca2+ current in CHO cells (31) , but did not function as a store-operated Ca2+ entry channel when expressed in the baculovirus/Sf9 cell system (35) . Our present studies therefore used an antisense oligonucleotide approach to inhibit Trp1 mRNA to examine the specific role of Trp1 in an ISOC identified in two lung-derived nonexcitable cell types (A549 and endothelial cells) responsive to inflammatory stimuli.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
A549 (lung epithelial carcinoma cell line) cells were kindly provided by Dr. Richard Honkanen (University of South Alabama). The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco BRL, Rockville, MD) containing 1 g glucose per liter and 10% fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT) with 100 units/ml penicillin and 0.1 mg/ml streptomycin (Sigma, St. Louis, MO). Cells were routinely passaged at 90–95% confluence.

Human pulmonary artery endothelial cells were purchased from Clonetics, Inc. (San Diego, CA). Cells were maintained in 1:1 DMEM/Ham’s F-12 with 10% FBS. Cells were verified as endothelial by positive factor VIII staining and by the uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled, acetylated low density lipoprotein. Cells were cultured after standard procedures, as described elsewhere (14 , 15 , 36) .

Molecular biology
Standard techniques for RT-PCR subcloning were generally followed. Total RNA was extracted with RNA Stat-60 (Tel-Test ‘B’, Friendswood, TX) from A549 cells and human pulmonary artery endothelial cells were grown to 100% confluence (~107 cells) on 75 cm2 tissue culture flasks. First-strand synthesis was performed with SuperScript II reverse transcriptase (200 units) and oligo(dT)12–18 primer (Life Technologies, Rockville, MD) on ~ 1 µg of DNase I-treated total RNA. PCR was conducted with the following sets of primers for Trp1/Trpß: set A, 5'-TCG CCG AAC GAG GTG ATG G-3' (sense) and 5'-GTT ATG GTA ACA GCA TTT CTC C-3' (antisense); set B, 5'-GTG CTT GGG AGA AAT GCT G-3' (sense) and 5'-GGG GCT TGG GTA GAG ATA C-3' (antisense); PCR products were ligated into TA cloning vector pCR2.1 (Invitrogen, San Diego, CA) and transformed into chemically competent Escherichia coli. Plasmids were isolated by the QIAprep® spin prep system (Qiagen, Valencia, CA) and submitted to The Biopolymer Laboratory at The University of South Alabama for automated fluorescence sequence analysis (Applied Biosystems 373XL DNA sequencer). Sequence accuracy was confirmed by sequencing both strands with universal primers. Nucleotide and amino acid alignments were achieved with BLAST (NCBI) and DNASIS v2.0 (Hitachi Software) programs.

Oligonucleotide synthesis
Synthesis and purification of chimeric deoxyphosphorothioate/2'-O-methoxyethyl base oligonucleotides were performed using an Applied Biosystems 380B automated DNA synthesizer, as described by ISIS Pharmaceuticals (37) .

Cell treatment with antisense oligonucleotides
A549 cells were seeded at 1.2–1.5 x 105 cells per 60 mm dish until 80% confluence was reached (~3 days). At that time, 1 ml of DMEM containing ISIS antisense oligonucleotide (10–300 nM final concentration) and 15 µg/ml Lipofectin® (DOTMA/DOPE) (Gibco BRL, Rockville, MD) complexes were added to the cells for 4 h in humidified air at 37°C with 5% CO2. The complexes were washed off and cells were allowed to recover in full-growth media (DMEM containing 10% FBS with penicillin/streptomycin antibiotics).

Microinjection of antisense oligonucleotides
In specified experiments, antisense oligonucleotides were microinjected into endothelial cells as we have described (38 , 39) . For these studies, human endothelial cells were seeded onto 25 mm circle glass coverslips etched with identifying marks. Cells were grown for 24 to 48 h in DMEM supplemented with 10% FBS and penicillin/streptomycin in humidified air at 37°C with 5% CO2. Microinjection was performed using an Olympus IX70 inverted microscope with Transjector 5246, Micromanipulator 5179, and Sterile Femtotips II (time setting 0.2 s, injection pressure 7.0 psi, compensation pressure 0.6 psi). After injections, cells were incubated for periods of 24, 48, or 72 h. Injected cells were subsequently identified by their location and [Ca2+]i measurements were performed.

Measurement of Trp1 mRNA levels
A standard Northern blotting procedure with modifications to optimize detection of low abundance messages was used. After the specified treatment period (24 to 72 h) with antisense oligonucleotides/Lipofectin® complexes, total RNA was extracted from A549 cells lysed with RNA Stat-60TM reagent (Tel-Test ‘B’ Inc., Friendswood, TX). Twenty micrograms of each RNA sample was resolved on a 1% agarose-formaldehyde gel. RNA transfer to nylon membranes (BrightStar-PlusTM, Ambion, Austin, TX) was accomplished by upward capillary transfer technique for 1 h and cross-linked with 125 mJ UV light (GeneLinker, Bio-Rad, Hercules, CA). An ssDNA probe (Tst-47, 226 bases) to trp1 mRNA was radiolabeled with [{alpha}-32P]dCTP along with a single-stranded probe for the RNA molecular size marker (Millennium MarkerTM, Ambion) by linear PCR with the respective antisense primers. The blots were hybridized with 1–3 x 106 cpm/ml of Trp1 probe and 20,000 cpm/ml of size marker probe in UltraHybTM (Ambion) solution in roller tubes (Techne, Cambridge, UK). Hybridization was performed at 43°C and overnight (16–20 h). The next day, blots were washed twice with 2x SSC/0.1% SDS for 10 min and twice with 0.2x SSC/0.1% SDS for 15 min. The blots were placed on Kodak X-OMAT film with an intensifying screen for up to 5 days before development. Blots were reprobed with 32P-labeled glycerol-3-phosphate dehydrogenase to confirm equal loading of RNA samples.

Intracellular free calcium measurements
A549 cells were seeded onto 25 mm circle microscope glass coverslips (Fisher Scientific, Pittsburgh, PA) and grown to confluence. Cytosolic Ca2+ ([Ca2+]i) was estimated with the Ca2+-sensitive fluorophore Fura-2/acetoxymethylester (Molecular Probes, Eugene, OR). Coverslip-attached A549 cells were transferred into HEPES-buffered physiological salt solution (in mM: 107 NaCl, 6 KCl, 1.2 MgSO4, 1.2 KH2PO4 2 CaCl2, 11.5 D-glucose, 25 HEPES, pH 7.35 adjusted with NaOH; HPSS) and loaded with 2 µM Fura-2/acetoxymethylester for 20 min at room temperature in the dark. After de-esterification in fresh-loading HPSS for an additional 20 min at room temperature, coverslip-attached cells were mounted in an Attofluor® cell chamber. The fluorescence of Fura-2-loaded cells was then monitored with an Olympus IX70 inverted microscope at x 400 with a xenon arc lamp photomultiplier system (Photon Technology International Ltd., Monmouth Junction, NJ), and data were acquired and analyzed with PTI Felix software. Epifluorescence (signal averaged) was measured from three to five A549 cells in a confluent monolayer and the changes in [Ca2+]i were expressed as the fluorescence ratio of the Ca2+-bound (340 nm) to Ca2+-unbound (380 nm) excitation wavelengths emitted at 510 nm.

Patch-clamp electrophysiology
Conventional whole-cell voltage-clamp configuration was performed to measure transmembrane currents in single A549 cells by the standard giga seal patch-clamp technique. Confluent A549 cells were trypsin dispersed, seeded onto 35 mm plastic culture dishes, and allowed to reattach 4 to 6 h before patch-clamp experiments were performed. Patch-clamp recordings were obtained using single (electrically isolated) A549 cells. Recording pipettes were heat polished to produce a tip resistance in the range of 3 to 5 M{Omega} in our internal solution. The pipette solution contained (in mM) 130 N-methyl-D-glucamine, 10 HEPES, 1.15 EGTA, 1 Ca(OH)2, 2 Mg2+-ATP, 1 NPA, 0.1 NPPB ([Ca2+] was estimated as 100 nM) (pH 7.2 adjusted with methane sulfonic acid). The external (bath) solution contained (in mM) 120 aspartic acid, 5 Ca(OH)2, 5 CaCl2, 10 HEPES, 0.5 3,4-diaminopyridine (pH 7.4 adjusted with tetraethylammonium hydroxide). Both solutions were adjusted to 290–300 mosM with sucrose. Currents were recorded with a computer-controlled EPC9 patch-clamp amplifier (HEKA; Lambrecht, Germany). Cell capacitance and series resistance were calculated with the software supported internal routines of the EPC9 and compensated before each experiment. Voltage pulses were applied from -100 to +60 mV in 20 mV increments after the whole-cell configuration was achieved, with 200 ms duration during each voltage step and 2 s interval between steps. The holding potential between each step was 0 mV. Data acquisition and analysis were performed with Pulse/PulseFit software (HEKA) and filtered at 2.9 kHz.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Trp1 in A549 cells
Expression of Trp proteins has not been established in A549 cells. Therefore, initial experiments used RT-PCR cloning to examine whether Trp1 and its splice variant, Trp1ß, are expressed in both A549 and human pulmonary artery endothelial cells. Sequence specific primers were generated against discrete regions encoding for portions of the amino-terminal cytosolic loop of Trp1. As indicated in Fig. 1A , primer set A targeted an upstream nucleotide region whereas primer set B spanned the 102 nucleotide deletion present in Trp1ß. Primer set A generated a RT-PCR product of expected size in both A549 and endothelial cells which sequence analysis verified was Trp1 (100% nucleotide identity) (Fig. 1B , 1C ). Primer set B generated two products of predicted size for Trp1 and Trp1ß (Fig. 1D , 1E ) that, when sequenced, verified expression of both the full-length and splice variant products. Additional primer sets were used to test for homology to Trp1 in transmembrane spanning and carboxy-terminal regions (data not shown). In each case, single products of expected sizes were obtained and shown to be Trp1 by sequence analysis. Using this RT-PCR approach, we also identified a product that encoded an apparent pseudogene with high homology to the Trp1{delta} reported in mouse (data not shown) (40) . These data collectively demonstrate that A549 and endothelial cells express Trp1, its splice variant Trp1ß, and the Trp1{delta} pseudogene.



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  		Figure 1. A549 and pulmonary artery endothelial cells express Trp1 and its splice variant, Trp1ß. A) Model of Trp1 (modified from ref 29 ) illustrating discrete sites targeted by RT-PCR primer sets. Forward and reverse primers are indicated and were directed against nucleotides in the amino-terminal coding sequence. Primer set B spanned the region demarcating the Trp1 splice variant, Trp1ß. B) Primer set A generated a product of expected size in both cell types. C) Cloning and nucleotide alignment revealed 100% identity between the cloned products and human Trp1. Nucleotide sequence of Trp1 is depicted and compared with the currently cloned partial product. D) Primer set B, which spans the splice site in Trp1ß, generated two products. E) Cloning and nucleotide alignment of these two products revealed 100% homology between the cloned products and Trp1 and Trp1ß, respectively. Nucleotide numbers of Trp1 are depicted and compared with the currently cloned partial product. The spliced region, deleted in Trp1ß, is depicted in red.

Inhibition of Trp1 translation
To examine the role of endogenous Trp1 in store-operated Ca2+ entry, we used a gene walking approach with 20 different chimeric 2'-O-(2-methoxy)ethylphosphothioate antisense oligonucleotides (Table 1 ) that contained 10 central phosphorothioate oligodeoxy residues (‘oligodeoxy gap’) flanked by five 2'-O-(2-methoxy) residues on the 3' and 5' ends, similar to earlier descriptions for other mRNA targets (Fig. 2 ) (41 , 42) . These methoxy, ethoxy modifications are used to impart greater hybridizing affinity of oligonucleotides for their target and to improve nuclease resistance. Test oligonucleotides were designed to span the start codon and extend through the coding region and into the 3'-untranslated region of human Trp1 mRNA.


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  		Table 1. Antisense oligonucleotides targeting Trp1



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  		Figure 2. Target regions in Trp1 mRNA. Trp1 gene start (green) and stop (red) codons are shown. Sites targeted by ISIS antisense probes that stretch across the start codon, through the coding region, and in the 3'-untranslated region are underlined and numbered. Two effective antisense probes, ISIS 17193 and ISIS 17206, are shown in red. The probe generated for Northern blot analyses is illustrated in blue. Note this probe detects both Trp1 and Trp1ß mRNA.

Since phosphorothioate oligonucleotides act principally through RNase H-mediated cleavage of mRNA (43) , antisense inhibition of the Trp1 message was determined by Northern blot analysis using a cDNA probe that hybridized upstream of the stop codon (Fig. 2) . This probe was designed to detect both Trp1 and Trp1ß mRNA. Cationic lipids (DOTMA/DOPE; Lipofectin®) were used to facilitate uptake of antisense oligonucleotides into A549 cells. Figure 3 illustrates hybridization of the cDNA probe for Trp1 with an ~4.5 kb pair transcript, similar to previous reports for Trp1 (44 , 45) . Incubation of A549 cells with cationic lipids did not influence mRNA levels. However, two ISIS oligonucleotides, 17193 and 17206, produced dose-dependent decreases in Trp1 mRNA. Indeed, of the 20 oligonucleotides screened, ISIS 17193 and ISIS 17206 reproducibly generated the greatest level of inhibition (data not shown) suggesting these oligonucleotides target hypersensitive sites in the Trp1 coding and 3'-untranslated regions, respectively. Thus, ISIS 17193 and ISIS 17206 were selected on the basis of greater efficacy for further evaluation.



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  		Figure 3. Transfection of A549 cells with antisense oligonucleotides inhibit Trp1 mRNA. Northern blot of Trp1 revealed a single ~4.5 kb transcript that was not altered by treatment with cationic lipids. Transfection of cells with ISIS 17193 and ISIS 17206, however, produced dose-dependent decreases in Trp1 mRNA. M refers to the RNA molecular weight ladder; Con refers to control experiments, without treatment; L denotes Lipofectin®. Data reflect results obtained from five separate experiments.

Sequence specificity of antisense oligonucleotides for Trp1 mRNA was examined using a series of ‘mismatch’ control analogs, which contain the same base composition as ISIS 17193 and ISIS 17206 but are comprised of scrambled and noncomplementary sequences to Trp1 (Table 1) . These studies were conducted over a 72 h time course (Fig. 4 ). Consistent with the findings in Fig. 3 , ISIS 17193 inhibited Trp1 mRNA for 72 h. Mismatch controls were without effect until at 72 h, when mismatch probes slightly reduced the Trp1 message. Similarly, ISIS 17206 inhibited Trp1 mRNA for 72 h, but in this case the mismatch controls were without effect over the entire time course. These data support the specific and stable inhibition of Trp1 mRNA by antisense probes.



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  		Figure 4. The effect of ISIS 17193 and ISIS 17206 is selective. A549 cells were transfected with 300 nM of either the active probes (A, ISIS 17193; B, ISIS 17206) or mismatch controls (see Table 1 for details). Two different lots of ISIS 17193 and ISIS 17206 were tested, labeled a and b, respectively. Experiments were conducted over 24–72 h to determine whether antisense oligonucleotides produced a stable decrease in Trp1 message. Whereas the active probes inhibited Trp1 mRNA over 72 h, mismatch controls were without effect. Con refers to control experiments without treatment; L denotes Lipofectin®. Data reflect the results obtained from five separate experiments.

Trp1 and store-operated Ca2+ entry
We next examined the role of Trp1 in store-operated Ca2+ entry, using the endoplasmic reticulum Ca2+ ATPase inhibitor thapsigargin. Application of thapsigargin produced a slowly developing increase in [Ca2+]i that peaked near 600 nM, followed by a sustained plateau near 300 nM. The thapsigargin response was associated with two clearly discernible increases in [Ca2+]i, the first due to Ca2+ release from intracellular stores and the second due exclusively to Ca2+ entry across the cell membrane (Fig. 5A ). In typical [Ca2+]i responses to thapsigargin in other cell types (14 , 15 , 36) , Ca2+ release and entry phases are not clearly resolved when physiological concentrations of extracellular Ca2+ are present. Individual 340 and 380 wavelengths are shown in Fig. 5A to show that they move in opposition, demonstrating that the lag between Ca2+ release and Ca2+ entry is not an artifact of fura fluorescence.



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  		Figure 5. Inhibition of Trp1 reduced the [Ca2+]i response to thapsigargin. A549 cells were transfected with ISIS 17193 and the [Ca2+]i response to thapsigargin (1 µM) was examined 24 (data not shown), 48, and 72 h later. A) The typical [Ca2+]i response to thapsigargin clearly illustrates an initial Ca2+ release (first peak) and subsequent Ca2+ entry component. The Ca2+ release component is not reduced when experiments are conducted in nominal extracellular Ca2+ (data not shown). Individual wavelengths demonstrate that the delay in Ca2+ entry is not due to an artifact of fura fluorescence. B) The decrease in Trp1 observed at both 48 and 72 h caused an attenuation of the global [Ca2+]i response to thapsigargin. Responses represent the average of four to six cells per experiment.

We examined the role of Trp1 on the thapsigargin-induced rise in global [Ca2+]i. Twenty-four hours after A549 cells were treated with ISIS 17193 to inhibit Trp1 mRNA, the [Ca2+]i response was not different from the control response (data not shown), suggesting Trp1 protein turnover may be slow. However, both 48 and 72 h treatment with ISIS 17193 resulted in a 25% decrease in the [Ca2+]i response to thapsigargin (Fig. 5B ). We confirmed the role of Trp1 in store-operated Ca2+ entry in endothelial cells, first microinjecting the antisense oligonucleotides into the cytosol and measuring the [Ca2+]i response 24, 48, and 72 h later. Again, the [Ca2+]i response to thapsigargin was not different from control responses at 24 h (data not shown), but was decreased after 48 and 72 h exposure to antisense oligonucleotide inhibition of Trp1 mRNA (Fig. 6 ). These data therefore support a central role for Trp1 as an endogenous component of store-operated Ca2+ entry channels.



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  		Figure 6. Inhibition of Trp1 decreases the endothelial cell response to thapsigargin. Pulmonary artery endothelial cells were microinjected with ISIS 17193 and [Ca2+]i responses to thapsigargin (1 µM) evaluated 24 and 48 h later. Antisense inhibition of Trp1 did not effect the response 24 h after injection (data not shown), though at the 48 h time point [Ca2+]i responses were diminished. n = 5 experiments per group; responses represent an average of four to six cells per experiment. *Significantly different from the control response.

Trp1 and ISOC
Heterologous expression of Trp1ß encoded for a nonselective channel in CHO cells (31) , although this channel was not store operated when expressed in the baculovirus/Sf9 system (35) . Similarly, expression of Trp1 encoded for a nonselective channel that increased whole-cell currents or increased global [Ca2+]i responses (32) . The contribution of endogenous Trp1 to ISOC, however, is poorly resolved, especially considering that cells express multiple Trp proteins likely to form heteromultimers of various combinations (29) . Our next studies therefore examined the contribution of Trp1 to an endogenous ISOC in A549 cells. Figure 7 illustrates a typical whole-cell Ca2+ current activated on internal dialysis with thapsigargin through the patch pipette. The Ca2+ current was small (~ -35 pA at -80 mV), inwardly rectifying, and had a positive reversal potential near +40 mV. Indeed, this current resembled that seen in endothelial cells similarly stimulated with internal dialysis of thapsigargin (38 , 46) . Unlike other cell types, however, 100% of the A549 cells studied possessed a thapsigargin-activated current. Perfusion with buffer containing 50 µM La3+ abolished the thapsigargin-evoked current and left-shifted the reversal potential to 0 mV, consistent with the inhibition of a Ca2+-selective current.



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  		Figure 7. Thapsigargin activates a Ca2+-selective ISOC in A549 cells. Current-voltage relationships were recorded 3–5 min after a whole-cell configuration was established in single A549 cells. Pulses of 200 ms duration were applied every 3 s from [minus100 to +100 mV in 20 mV steps; the holding potential was 0 mV. Thapsigargin (1 µM) was included in the patch pipette to activate ISOC. A) Dialysis of thapsigargin through the patch pipette activated a small inward current that reversed near +40 mV and was inwardly rectifying. Perfusion of 50 µM La3+ reversed this current within 2–3 min of its application; the remaining current was left-shifted to reverse at 0 mV. B) Replacement of extracellular Ca2+ with Na+ revealed that thapsigargin activated a larger Na+ current that also reversed near +40 mV and was inwardly rectifying. Replenishment of Ca2+ to the extracellular bath reduced this Na+ current at low concentrations (<=1 µM) and promoted a Ca2+ current at higher concentrations (>=100 µM) (C, D). These data are consistent with an anomalous mole fraction behavior of Ca2+-selective currents like voltage gated Ca2+ channels and ICRAC.

Several store-operated currents have been identified and differ significantly as to their ion selectivity. The original characterization of a store-operated current in mast cells and lymphocytes determined that the ICRAC was highly Ca2+ selective (1 , 4) . Subsequent findings indicated that thapsigargin activates multiple channels (22 , 47) ; of these many channels, some cation-permeable channels are nonselective (48) . One feature of all store-operated cation channels is they readily conduct monovalent cations in the absence of extracellular Ca2+ (7) . However, addition of Ca2+ or other divalent cations reduces the monovalent cation current through an anomalous mole fraction effect in Ca2+-selective channels, like ICRAC (2) . We therefore examined whether the thapsigargin-activated current in A549 cells conducts Na+ in the absence of divalent cations and whether readdition of Ca2+ decreases Na+ conductance through the channel’s anomalous mole fraction dependence. Figure 7B demonstrates that thapsigargin activates an Na+ current larger than the typical Ca2+ current. Low concentrations of Ca2+ inhibited the Na+ current, but a Ca2+-dependent current was observed at higher concentrations. These findings are consistent with ion permeation through a Ca2+-selective channel, similar to voltage-dependent Ca2+ channels and Ca2+-selective, store-operated channels (2 , 7 , 49 , 50) .

To examine the contribution of Trp1 to ISOC, cells were treated with antisense oligonucleotides for 48 or 72 h and the response to thapsigargin was assessed in a whole-cell, voltage-clamp configuration. Neither Lipofectin® nor mismatch controls reduced ISOC, as seen in Fig. 8A , B , respectively. ISIS 17206 (48 h) and ISIS 17193 (72 h) both reduced the thapsigargin-activated current by ~50%, from -35 pA to -18 pA at -[80 mV, though such inhibition of Trp1 did not cause a left shift in the reversal potential (Figs. 8C , D ). These data provide evidence that endogenous Trp1 contributes to a ISOC and, in considering these findings with Fig. 7 , they suggest that the Trp1-containing channel possesses Ca2+ selectivity.



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  		Figure 8. Inhibition of Trp1 reduces the Ca2+-selective ISOC in A549 cells. Current-voltage relationships were recorded 3–5 min after a whole-cell configuration was established in single A549 cells. Pulses of 200 ms duration were applied every 3 s from -100 to +60 mV in 20 mV steps; the holding potential was 0 mV. Thapsigargin (1 µM) was included in the patch pipette to activate ISOC. This current was not altered by either Lipofectin® (A) or transfection with a mismatch control oligonucleotide (B, ISIS 122207). However, 48 and 72 h after transfection with ISIS 17206 (C) and ISIS 17193 (D), respectively, the thapsigargin-activated ISOC was decreased by ~50%.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Ca2+ signaling is a key inflammatory stimulus that activates mast cells, T lymphocytes, and endothelial cells (3 , 6 , 13) . Inflammatory Ca2+ signals promote proliferation and motility in A549 cells (human lung carcinoma cell line), though Ca2+ entry pathways in these cells have not been rigorously studied. Indeed, insight into mechanisms underlying the activation of store-operated Ca2+ entry pathways is still limited, largely because until recently putative molecular candidates for Ca2+ entry channels were lacking. Identification of Trp1, a mammalian homologue of Drosophila melanogaster Trp, provided such a molecular candidate (44 , 45) . Seven related mammalian Trp homologues have now been cloned and likely recombine to form cell-specific heterotetrameric channels (29) . To overcome problems associated with examining the contribution of individual subunits to the function of a multimeric channel, we developed antisense oligonucleotides (ISIS 17193 and ISIS 17206) that potently inhibit Trp1 expression without influencing other components of the endogenous channel. This approach allowed examination of the specific effect of Trp1 on store-operated Ca2+ entry activated by passive Ca2+ store depletion using thapsigargin. Data support a central role for Trp1 in the global [Ca2+]i rise evoked upon Ca2+ store depletion and, for the first time, provide direct evidence that endogenous Trp1 contributes to a Ca2+-selective ISOC.

The initial cloning of human Trp1 was followed shortly by evidence that it and its splice variant Trp1ß encoded for a Ca2+ entry pathway (31) . However, heterologous expression of Trp1 did not increase the global [Ca2+]i response to receptor Gq-coupled agonists and only slightly increased the [Ca2+]i response in recalcification experiments, particularly compared with cells expressing Trp3 (32) . Recently, a role for Trp1 in store-operated Ca2+ entry has been more clearly resolved using salivary gland cells (33) . In these cells, overexpression of Trp1 increased the global [Ca2+]i response to thapsigargin and antisense inhibition of Trp1 reduced endogenous [Ca2+]i flux. Thus, in certain cell types Trp1 may play a prominent role in store-operated Ca2+ entry pathways. Attempts to characterize the electrophysiological properties of Trp1 in order to ascertain whether it contributes to a Ca2+-selective or nonselective current are inconclusive. Overexpression of Trp1ß in CHO cells resulted in the appearance of a nonselective cation current upon Ca2+ store depletion (31) , though in a baculovirus system Trp1 was not store operated (35) ; in particular, neither inositol 1,4,5-trisphosphate nor thapsigargin activated a cation current. Such disparate results may reflect a functional difference between Trp1 and Trp1ß. Alternatively, these incongruent findings may arise when the overexpressed subunits form channels distinct from their endogenous phenotype.

We therefore sought to determine whether Trp1 is a component of endogenous store-operated Ca2+ entry channels in A549 and endothelial cells. Both A549 and endothelial cells expressed Trp1 and its splice variant Trp1ß. Northern blot analysis revealed that the Trp1 message appeared similar in size to previous reports of Trp1 isolated from various organs, including lung (44 , 45) . Antisense probes identified discrete regions of Trp1 mRNA that were hypersensitive to inhibition and efficiently decreased Trp1 for 48 and 72 h. Two separate findings support the specific role for Trp1 in regulating store-operated Ca2+ entry. First, the [Ca2+]i response to thapsigargin was not reduced by either mismatch controls or antisense treatments, which did not decrease Trp1. Second, the [Ca2+]i response to thapsigargin was attenuated when Trp1 was decreased (e.g., 48 and 72 h time points). Clearly, however, antisense treatment affected only a fraction of the global [Ca2+]i response to thapsigargin (25%), bringing into question characteristics of the Trp1-containing channel.

To address this issue, we performed whole-cell patch-clamp experiments designed to isolate ISOC. Previous work from our laboratory (38 , 39 , 51) and others (27 , 28 , 46 , 52) found that this ISOC is small, inwardly rectifying, reverses near +40 mV, and is inhibited by La3+. Moreover, the current requires intracellular ATP, an intact cytoskeleton, and myosin light chain kinase activity. Absolute Ca2+ selectivity has not been established, particularly in direct comparison to ICRAC reported in mast cells and lymphocytes. Highly Ca2+-selective channels possess an anomalous mole fraction effect in which they conduct monovalent cations in the absence of Ca2+ (2) and Ca2+ replenishment blocks monovalent cation flux. Our present findings indicate the ISOC in A549 cells possesses an anomalous mole fraction effect since Ca2+ blocks Na+ conductance, suggesting that store depletion activates a Ca2+-selective entry pathway. Thus, inhibition of this current by ISIS 17193 and ISIS 17206 demonstrates that Trp1 forms an essential component of a Ca2+-selective, store-operated pathway. ISOC was not abolished by antisense treatments, however, and the reversal potential did not shift to the left; thus, Trp1 must not be essential for channel activation and Ca2+ selectivity apparently is not altered by the decrease in Trp1 expression. We interpret these findings to suggest that Trp1 alone does not comprise the endogenous ISOC channel, unless the residual Trp1 protein (e.g., that not inhibited by antisense treatments) is sufficiently high to account for the remaining current. Both A549 and endothelial cells express other Trp proteins, particularly Trp4; indeed, Trp4 is more abundantly expressed than is Trp1 (T. Stevens, unpublished observations). Work by Flockerzi and colleagues has illustrated that Trp4 encodes for a Ca2+-selective channel (53 54 55) . Though speculative, Trp1 and Trp4 may form an endogenous channel in which gating and Ca2+ selectivity are determined by the presence of Trp4. Future studies will be required to assess whether Trp1/Trp4 heteromultimers form the endogenous A549 and endothelial cell ISOC channel and whether the current remaining after Trp1 inhibition is due to the presence of Trp4.

In summary, our findings support a role for Trp1 in an endogenous store-operated and selective Ca2+ entry pathway. This conclusion was made possible through antisense inhibition of Trp1 mRNA translation at two discrete hypersensitive sites, including one in the coding region and one in the 3'-untranslated region, that allowed for the selective inhibition of endogenous Trp1. The physiological significance of these observations remain speculative. However, earlier studies have established a central role for store-operated Ca2+ entry in the response to inflammation. Thus, it is likely that Trp1 makes an important contribution to the cell’s response to inflammation.


   ACKNOWLEDGMENTS
 
The authors wish to thank Ms. Judy Creighton and Tray Weathington for their excellent assistance in cell culture experiments. This work was supported by HL56056 and HL60024 (to T.S.), and DK50151 (to M.L.). S.W. is an American Heart Association, Southeastern Consortium Fellow.

Received for publication February 14, 2001. Accepted for publication April 17, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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