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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
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: capacitative Ca2+ entry • ICRAC • signal transduction • endothelial cells
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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
[
-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
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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 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
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 pseudogene.
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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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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.
|
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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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.
|
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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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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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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 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.
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ACKNOWLEDGMENTS |
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Received for publication February 14, 2001. Accepted for publication April 17, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This article has been cited by other articles:
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  M. Trebak STIM1/Orai1, ICRAC, and Endothelial SOC Circ. Res., May 8, 2009; 104(9): e56 - e57. [Full Text] [PDF]
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 P. C. Sundivakkam, A. M. Kwiatek, T. T. Sharma, R. D. Minshall, A. B. Malik, and C. Tiruppathi Caveolin-1 scaffold domain interacts with TRPC1 and IP3R3 to regulate Ca2+ store release-induced Ca2+ entry in endothelial cells Am J Physiol Cell Physiol, March 1, 2009; 296(3): C403 - C413. [Abstract] [Full Text] [PDF]
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I. F. Abdullaev, J. M. Bisaillon, M. Potier, J. C. Gonzalez, R. K. Motiani, and M. Trebak Stim1 and Orai1 Mediate CRAC Currents and Store-Operated Calcium Entry Important for Endothelial Cell Proliferation Circ. Res., November 21, 2008; 103(11): 1289 - 1299. [Abstract] [Full Text] [PDF]
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 K. T. Cheng, X. Liu, H. L. Ong, and I. S. Ambudkar Functional Requirement for Orai1 in Store-operated TRPC1-STIM1 Channels J. Biol. Chem., May 9, 2008; 283(19): 12935 - 12940. [Abstract] [Full Text] [PDF]
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 H.-T. Ma, Z. Peng, T. Hiragun, S. Iwaki, A. M. Gilfillan, and M. A. Beaven Canonical Transient Receptor Potential 5 Channel in Conjunction with Orai1 and STIM1 Allows Sr2+ Entry, Optimal Influx of Ca2+, and Degranulation in a Rat Mast Cell Line J. Immunol., February 15, 2008; 180(4): 2233 - 2239. [Abstract] [Full Text] [PDF]
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S. Wu, H. Chen, M. F. Alexeyev, J. A. C. King, T. M. Moore, T. Stevens, and R. D. Balczon Microtubule Motors Regulate ISOC Activation Necessary to Increase Endothelial Cell Permeability J. Biol. Chem., November 30, 2007; 282(48): 34801 - 34808. [Abstract] [Full Text] [PDF]
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 S. Tatur, N. Groulx, S. N. Orlov, and R. Grygorczyk Ca2+-dependent ATP release from A549 cells involves synergistic autocrine stimulation by coreleased uridine nucleotides J. Physiol., October 15, 2007; 584(2): 419 - 435. [Abstract] [Full Text] [PDF]
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 R. I. Clyman, N. Waleh, H. Kajino, C. Roman, and F. Mauray Calcium-dependent and calcium-sensitizing pathways in the mature and immature ductus arteriosus Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2007; 293(4): R1650 - R1656. [Abstract] [Full Text] [PDF]
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A. P. Albert, S. N. Saleh, C. M. Peppiatt-Wildman, and W. A. Large Multiple activation mechanisms of store-operated TRPC channels in smooth muscle cells J. Physiol., August 15, 2007; 583(1): 25 - 36. [Abstract] [Full Text] [PDF]
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I. Singh, N. Knezevic, G. U. Ahmmed, V. Kini, A. B. Malik, and D. Mehta G{alpha}q-TRPC6-mediated Ca2+ Entry Induces RhoA Activation and Resultant Endothelial Cell Shape Change in Response to Thrombin J. Biol. Chem., March 16, 2007; 282(11): 7833 - 7843. [Abstract] [Full Text] [PDF]
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D. F. Alvarez, J. A. King, D. Weber, E. Addison, W. Liedtke, and M. I. Townsley Transient Receptor Potential Vanilloid 4-Mediated Disruption of the Alveolar Septal Barrier: A Novel Mechanism of Acute Lung Injury Circ. Res., October 27, 2006; 99(9): 988 - 995. [Abstract] [Full Text] [PDF]
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 A. M. Kwiatek, R. D. Minshall, D. R. Cool, R. A. Skidgel, A. B. Malik, and C. Tiruppathi Caveolin-1 Regulates Store-Operated Ca2+ Influx by Binding of Its Scaffolding Domain to Transient Receptor Potential Channel-1 in Endothelial Cells Mol. Pharmacol., October 1, 2006; 70(4): 1174 - 1183. [Abstract] [Full Text] [PDF]
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J. Wang, L. Weigand, W. Lu, J.T. Sylvester, G. L. Semenza, and L. A. Shimoda Hypoxia Inducible Factor 1 Mediates Hypoxia-Induced TRPC Expression and Elevated Intracellular Ca2+ in Pulmonary Arterial Smooth Muscle Cells Circ. Res., June 23, 2006; 98(12): 1528 - 1537. [Abstract] [Full Text] [PDF]
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 D. Mehta and A. B. Malik Signaling Mechanisms Regulating Endothelial Permeability Physiol Rev, January 1, 2006; 86(1): 279 - 367. [Abstract] [Full Text] [PDF]
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D. L. Cioffi, S. Wu, M. Alexeyev, S. R. Goodman, M. X. Zhu, and T. Stevens Activation of the Endothelial Store-Operated ISOC Ca2+ Channel Requires Interaction of Protein 4.1 With TRPC4 Circ. Res., November 25, 2005; 97(11): 1164 - 1172. [Abstract] [Full Text] [PDF]
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 D. F. Alvarez, J. A. King, and M. I. Townsley Resistance to Store Depletion-induced Endothelial Injury in Rat Lung after Chronic Heart Failure Am. J. Respir. Crit. Care Med., November 1, 2005; 172(9): 1153 - 1160. [Abstract] [Full Text] [PDF]
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X. Yao and C. J. Garland Recent Developments in Vascular Endothelial Cell Transient Receptor Potential Channels Circ. Res., October 28, 2005; 97(9): 853 - 863. [Abstract] [Full Text] [PDF]
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T. K. Zagranichnaya, X. Wu, and M. L. Villereal Endogenous TRPC1, TRPC3, and TRPC7 Proteins Combine to Form Native Store-operated Channels in HEK-293 Cells J. Biol. Chem., August 19, 2005; 280(33): 29559 - 29569. [Abstract] [Full Text] [PDF]
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M. Freichel, R. Vennekens, J. Olausson, S. Stolz, S. E Philipp, P. Weissgerber, and V. Flockerzi Functional role of TRPC proteins in native systems: implications from knockout and knock-down studies J. Physiol., August 15, 2005; 567(1): 59 - 66. [Abstract] [Full Text] [PDF]
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S. Wu, E. A. Cioffi, D. Alvarez, S. L. Sayner, H. Chen, D. L. Cioffi, J. King, J. R. Creighton, M. Townsley, S. R. Goodman, et al. Essential Role of a Ca2+-Selective, Store-Operated Current (ISOC) in Endothelial Cell Permeability: Determinants of the Vascular Leak Site Circ. Res., April 29, 2005; 96(8): 856 - 863. [Abstract] [Full Text] [PDF]
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A. B. Parekh and J. W. Putney Jr. Store-Operated Calcium Channels Physiol Rev, April 1, 2005; 85(2): 757 - 810. [Abstract] [Full Text] [PDF]
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S. Zhang, J. X.-J. Yuan, K. E. Barrett, and H. Dong Role of Na+/Ca2+ exchange in regulating cytosolic Ca2+ in cultured human pulmonary artery smooth muscle cells Am J Physiol Cell Physiol, February 1, 2005; 288(2): C245 - C252. [Abstract] [Full Text] [PDF]
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 N. Kunichika, Y. Yu, C. V. Remillard, O. Platoshyn, S. Zhang, and J. X.-J. Yuan Overexpression of TRPC1 enhances pulmonary vasoconstriction induced by capacitative Ca2+ entry Am J Physiol Lung Cell Mol Physiol, November 1, 2004; 287(5): L962 - L969. [Abstract] [Full Text] [PDF]
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S. Zhang, C. V. Remillard, I. Fantozzi, and J. X.-J. Yuan ATP-induced mitogenesis is mediated by cyclic AMP response element-binding protein-enhanced TRPC4 expression and activity in human pulmonary artery smooth muscle cells Am J Physiol Cell Physiol, November 1, 2004; 287(5): C1192 - C1201. [Abstract] [Full Text] [PDF]
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 Y. Yu, I. Fantozzi, C. V. Remillard, J. W. Landsberg, N. Kunichika, O. Platoshyn, D. D. Tigno, P. A. Thistlethwaite, L. J. Rubin, and J. X.-J. Yuan Enhanced expression of transient receptor potential channels in idiopathic pulmonary arterial hypertension PNAS, September 21, 2004; 101(38): 13861 - 13866. [Abstract] [Full Text] [PDF]
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M.-J. Lin, G. P.H. Leung, W.-M. Zhang, X.-R. Yang, K.-P. Yip, C.-M. Tse, and J. S.K. Sham Chronic Hypoxia-Induced Upregulation of Store-Operated and Receptor-Operated Ca2+ Channels in Pulmonary Arterial Smooth Muscle Cells: A Novel Mechanism of Hypoxic Pulmonary Hypertension Circ. Res., September 3, 2004; 95(5): 496 - 505. [Abstract] [Full Text] [PDF]
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X. Wang, J. L. Pluznick, P. Wei, B. J. Padanilam, and S. C. Sansom TRPC4 forms store-operated Ca2+ channels in mouse mesangial cells Am J Physiol Cell Physiol, August 1, 2004; 287(2): C357 - C364. [Abstract] [Full Text] [PDF]
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G. U. Ahmmed, D. Mehta, S. Vogel, M. Holinstat, B. C. Paria, C. Tiruppathi, and A. B. Malik Protein Kinase C{alpha} Phosphorylates the TRPC1 Channel and Regulates Store-operated Ca2+ Entry in Endothelial Cells J. Biol. Chem., May 14, 2004; 279(20): 20941 - 20949. [Abstract] [Full Text] [PDF]
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J. Kawasaki, G. E. Davis, and M. J. Davis Regulation of Ca2+-dependent K+ Current by {alpha}v{beta}3 Integrin Engagement in Vascular Endothelium J. Biol. Chem., March 26, 2004; 279(13): 12959 - 12966. [Abstract] [Full Text] [PDF]
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D. F. Alvarez, E.-A. B. Gjerde, and M. I. Townsley Role of EETs in regulation of endothelial permeability in rat lung Am J Physiol Lung Cell Mol Physiol, February 1, 2004; 286(2): L445 - L451. [Abstract] [Full Text] [PDF]
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R. Ma, D. Rundle, J. Jacks, M. Koch, T. Downs, and L. Tsiokas Inhibitor of Myogenic Family, a Novel Suppressor of Store-operated Currents through an Interaction with TRPC1 J. Biol. Chem., December 26, 2003; 278(52): 52763 - 52772. [Abstract] [Full Text] [PDF]
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D. Mehta, G. U. Ahmmed, B. C. Paria, M. Holinstat, T. Voyno-Yasenetskaya, C. Tiruppathi, R. D. Minshall, and A. B. Malik RhoA Interaction with Inositol 1,4,5-Trisphosphate Receptor and Transient Receptor Potential Channel-1 Regulates Ca2+ Entry: ROLE IN SIGNALING INCREASED ENDOTHELIAL PERMEABILITY J. Biol. Chem., August 29, 2003; 278(35): 33492 - 33500. [Abstract] [Full Text] [PDF]
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S.-c. W. Brazer, B. B. Singh, X. Liu, W. Swaim, and I. S. Ambudkar Caveolin-1 Contributes to Assembly of Store-operated Ca2+ Influx Channels by Regulating Plasma Membrane Localization of TRPC1 J. Biol. Chem., July 11, 2003; 278(29): 27208 - 27215. [Abstract] [Full Text] [PDF]
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V Sydorenko, Y Shuba, S Thebault, M Roudbaraki, G Lepage, N Prevarskaya, and R Skryma Receptor-coupled, DAG-gated Ca2+-permeable cationic channels in LNCaP human prostate cancer epithelial cells J. Physiol., May 1, 2003; 548(3): 823 - 836. [Abstract] [Full Text] [PDF]
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X. Liu, B. B. Singh, and I. S. Ambudkar TRPC1 Is Required for Functional Store-operated Ca2+ Channels. ROLE OF ACIDIC AMINO ACID RESIDUES IN THE S5-S6 REGION J. Biol. Chem., March 21, 2003; 278(13): 11337 - 11343. [Abstract] [Full Text] [PDF]
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Y. Yu, M. Sweeney, S. Zhang, O. Platoshyn, J. Landsberg, A. Rothman, and J. X.-J. Yuan PDGF stimulates pulmonary vascular smooth muscle cell proliferation by upregulating TRPC6 expression Am J Physiol Cell Physiol, February 1, 2003; 284(2): C316 - C330. [Abstract] [Full Text] [PDF]
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M. Goel, W. G. Sinkins, and W. P. Schilling Selective Association of TRPC Channel Subunits in Rat Brain Synaptosomes J. Biol. Chem., December 6, 2002; 277(50): 48303 - 48310. [Abstract] [Full Text] [PDF]
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R. S. Ostrom, X. Liu, B. P. Head, C. Gregorian, T. M. Seasholtz, and P. A. Insel Localization of Adenylyl Cyclase Isoforms and G Protein-Coupled Receptors in Vascular Smooth Muscle Cells: Expression in Caveolin-Rich and Noncaveolin Domains Mol. Pharmacol., November 1, 2002; 62(5): 983 - 992. [Abstract] [Full Text] [PDF]
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 S. R. Hassock, M. X. Zhu, C. Trost, V. Flockerzi, and K. S. Authi Expression and role of TRPC proteins in human platelets: evidence that TRPC6 forms the store-independent calcium entry channel Blood, September 26, 2002; 100(8): 2801 - 2811. [Abstract] [Full Text] [PDF]
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R Flemming, A Cheong, A M Dedman, and D J Beech Discrete store-operated calcium influx into an intracellular compartment in rabbit arteriolar smooth muscle J. Physiol., September 1, 2002; 543(2): 455 - 464. [Abstract] [Full Text] [PDF]
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C. Tiruppathi, M. Freichel, S. M. Vogel, B. C. Paria, D. Mehta, V. Flockerzi, and A. B. Malik Impairment of Store-Operated Ca2+ Entry in TRPC4-/- Mice Interferes With Increase in Lung Microvascular Permeability Circ. Res., July 12, 2002; 91(1): 70 - 76. [Abstract] [Full Text] [PDF]
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M. Sweeney, Y. Yu, O. Platoshyn, S. Zhang, S. S. McDaniel, and J. X.-J. Yuan Inhibition of endogenous TRP1 decreases capacitative Ca2+ entry and attenuates pulmonary artery smooth muscle cell proliferation Am J Physiol Lung Cell Mol Physiol, July 1, 2002; 283(1): L144 - L155. [Abstract] [Full Text] [PDF]
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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.