Basal Calcium Entry in Retinal Pigment Epithelial Cells
Is Mediated by TRPC Channels
Sönke Wimmers1 and Olaf Strauss1,2
PURPOSE. Ca2⫹ is a major regulator of cell function. In the
retinal pigment epithelium (RPE), intracellular free Ca2⫹ concentration ([Ca2⫹]i) is essential for the maintenance of normal
retinal function. Therefore, accurate control of [Ca2⫹]i is vital
in these cells. Because Ca2⫹ is permanently extruded from the
cytosol, RPE cells need a basal Ca2⫹ entry pathway that counteracts this Ca2⫹ efflux. The purpose of this study was to
identify the molecular basis of basal Ca2⫹ entry into the RPE.
METHODS. [Ca2⫹]i was measured using Fura-2–loaded ARPE-19
cells. The expression pattern of TRPC channels was investigated by RT-PCR with RNA extracted from ARPE-19 cells and
freshly isolated RPE cells from human donor eyes.
RESULTS. In most cells, basal [Ca2⫹]i is highly controlled by cell
membranes that are only slightly permeable to Ca2⫹ and by the
activity of Ca2⫹ pumps and transporters. The authors show
here that RPE cells have a basal Ca2⫹ conductance that is dose
dependently blocked by La3⫹. Basal [Ca2⫹]i was also strongly
reduced by the TRP channel blockers Gd3⫹, Ni2⫹, 2-APB, and
SKF96365 and was insensitive to blockers of other Ca2⫹ channels. In confirmation of this pharmacologic profile, RPE cells
expressed TRPC1 and TRPC4 channels, as shown by RT-PCR
experiments.
CONCLUSIONS. Ca2⫹ is needed for several permanently occurring
regulatory processes in RPE cells. The Ca2⫹ influx pathway
identified in this study is essential to define a resting basal
[Ca2⫹]i. This resting [Ca2⫹]i may contribute, for example, to
basal cytokine secretion essential for the maintenance of normal retinal function. (Invest Ophthalmol Vis Sci. 2007;48:
5767–5772) DOI:10.1167/iovs.07-0412
M
any cellular processes are controlled by the intracellular
free Ca2⫹ concentration ([Ca2⫹]i).1,2 These include excitation, secretion, cell differentiation, gene expression, endocytosis, and apoptosis. Altered Ca2⫹ homeostasis may lead to
physical impairment, as seen in genetic diseases associated
with Ca2⫹ transporters and channels.3 Generally, two pathways for the elevation of [Ca2⫹]i exist: Ca2⫹ may enter through
plasma membrane Ca2⫹ channels or Ca2⫹ may be released
from intracellular Ca2⫹ stores by the activation of ryanodine or
inositol trisphosphate receptors. For each case, the cells use a
From the 1Experimentelle Ophthalmologie, Klinik und Poliklinik
für Augenheilkunde, Universitätsklinkikum Hamburg-Eppendorf, Hamburg, Germany; and the 2Experimentelle Ophthalmologie, Klinik und
Poliklinik für Augenheilkunde, Klinikum der Universität Regensburg,
Regensburg, Germany.
Supported by the Deutsche Forschungsgemeinschaft DFG Grants
STR 480/8-2 and STR 480/9-1.
Submitted for publication April 5, 2007; revised July 9 and August
28, 2007; accepted October 19, 2007.
Disclosure: S. Wimmers, None; O. Strauss, None
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Corresponding author: Sönke Wimmers, Experimentelle Ophthalmologie, Klinik und Poliklinik für Augenheilkunde, Universitätsklinkikum Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany; wimmers@uke.uni-hamburg.de.
Investigative Ophthalmology & Visual Science, December 2007, Vol. 48, No. 12
Copyright © Association for Research in Vision and Ophthalmology
variety of Ca2⫹-transporting proteins.2 The Ca2⫹ extrusion is
mediated by Ca2⫹ exchangers and pumps. In resting cells in
which the [Ca2⫹]i is approximately 100 nM, the Ca2⫹ influx,
efflux, and intracellular Ca2⫹ buffering are in equilibrium.
The retinal pigment epithelium (RPE) is located between
the neural retina and the choroidal vasculature.4,5 With its tight
junctions, it forms part of the blood-retina barrier and is important for the maintenance of retinal function. Its pigment
absorbs excess light, it reisomerizes all-trans retinal to 11-cis
retinal and delivers it back to the photoreceptors, it controls
the transport of metabolites, nutrients, ions, and water between the subretinal space and the choroidal vessels, and it
phagocytes shed outer segments of photoreceptors. As a target
and source of a variety of growth factors, the RPE maintains
retinal integrity and is part of the immune privilege of the
subretinal space.4 – 6 Many of these tasks are influenced by
changes in the [Ca2⫹]i.7 Therefore, accurate control of [Ca2⫹]i
in the RPE is vital.
In the RPE, it has been shown that Ca2⫹ influx through the
voltage-operated L-type Ca2⫹ channel ␣1D plays a role in the
control of growth factor secretion.8 Additionally, a specific
block of L-type channels led to a reduced light peak in electroretinograms in mice and rats, indicating that these channels
are involved in light-induced responses of the RPE.9,10 Furthermore, purinergic stimulation of RPE cells leads to an increase in
[Ca2⫹]i, which seems to be at least partially mediated by
ionotropic purinergic receptors (P2X).11 This ATP-induced
[Ca2⫹]i increase leads to increased transepithelial Cl⫺ and
water transport.
Both L-type and purinergic receptor channels open only in
response to specific stimulation, such as depolarization (␣1D)
or binding of ATP (P2X). Ca2⫹ is permanently extruded from
RPE cells through constitutively active plasma-membrane Ca2⫹
ATPases and Na⫹/Ca2⫹ exchangers.12,13 Therefore, unstimulated RPE cells seem to have a Ca2⫹ leak pathway for Ca2⫹
influx that stabilizes the basal [Ca2⫹]i of 100 nM. In another
study we have already demonstrated that this Ca2⫹ influx is not
driven by a reverse mode of the Na⫹/Ca2⫹ exchanger.14 Here
we show in addition that it is not driven by the already identified voltage-operated Ca2⫹ channels or the ionotropic purinergic receptor-operated channels. Instead, we show by pharmacologic reduction of the basal [Ca2⫹]i that this background
Ca2⫹ influx is carried out by a member of the transient receptor potential (TRP) channels.
MATERIALS
AND
METHODS
Cell Culture
The human retinal pigment epithelial cell line ARPE-19 was cultured in
Dulbecco modified eagle medium/F-12 nutrient mixture (D-MEM/F-12)
containing 10% fetal bovine serum, insulin-transferrin-sodium (Roche,
Basel, Switzerland), nonessential amino acids, and penicillin/streptomycin at 37°C in a humidified ambient atmosphere containing 5% CO2.
They were passaged twice per week. For Fura-2 measurements, they
were seeded onto coverslips and cultured to confluence.
For primary cultures of human RPE cells, the anterior part of human
donor eyes, including vitreous and retina, were removed. The RPE and
choroidea were carefully separated from the sclera, washed with PBS,
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IOVS, December 2007, Vol. 48, No. 12
TABLE 1. Oligonucleotides Used to Amplify Transcripts of TRPC1–7
Gene
Accession No.
TRPC1
NM_003304
TRPC3
NM_003305
TRPC4
NM_016179
TRPC5
NM_012471
TRPC6
NM_004621
TRPC7
NM_020389
Sequence
Forward: 5⬘-TGCTTACCAAACTGCTGGTG-3⬘
Reverse: 5⬘-AACTGTTTTGCCGTTTGACC-3⬘
Forward: 5⬘-GACTTTGGGATGCTGTCCAT-3⬘
Reverse: 5⬘-ATTTGCAGGGAGGATGTACG-3⬘
Forward: 5⬘-TACTCCCTTCAATGTCATCC-3⬘
Reverse: 5⬘-TTCACCAGGTTCCTCATAAC-3⬘
Forward: 5⬘-CAACTGTCGTGGAATGGATG-3⬘
Reverse: 5⬘-AGTGCTTCCGCAATCAGAGT-3⬘
Forward: 5⬘-GGCTCTCATTTACTGGTTTG-3⬘
Reverse: 5⬘-GTGCTGGTTTCATTAGGAAG-3⬘
Forward: 5⬘-GCCCACCAGATACCAGAAAA-3⬘
Reverse: 5⬘-CACCCTCAGGTGGTCTTTGT-3⬘
Length
(bp)
Annealing Temperature
(°C)
243
59
264
59
183
54
244
59
171
54
241
56
and incubated overnight with collagenase IA/IV (0.5 mg/mL each) in
serum-free culture medium. The dissociated cells were collected by
centrifugation (50g, 5 minutes) and cultured on coverslips in the same
medium as the ARPE-19 cells. These still pigmented RPE cells were
used for Ca2⫹ measurements after they reached confluence. Human
material was used in accordance with the tenets of the Declaration of
Helsinki.
transcriptase (Invitrogen). For control PCR reactions, human total
brain RNA (Stratagene, La Jolla, CA) was reverse transcribed under the
same conditions. PCR experiments were performed with 1 ␮L cDNA in
50-␮L PCR reaction mixtures with Taq DNA polymerase (Stratagene)
and 1.5 pmol of sense and antisense oligonucleotides specific to the
various TRPC channel subunits (Table 1). The identity of the amplification product was confirmed by sequencing.
Measurement of Intracellular Free
Ca2ⴙ Concentrations
Data Analysis
ARPE-19 cells grown on coverslips to confluence were washed with
Ringer solution (130 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2,
5 mM glucose, 10 mM HEPES, pH 7.3, with NaOH) and loaded with
Fura-2 AM ester (Fluka, Buchs, Switzerland) for 40 minutes in the dark
at room temperature in Ringer solution containing 10 ␮M Fura-2 AM.
Cells were washed and incubated for at least 30 minutes with Ringer
solution. The coverslips were placed into a bath chamber perfused
constantly with Ringer solution and mounted onto an inverted microscope (Axiovert 35; Carl Zeiss, Oberkochen, Germany) equipped with
a 40⫻ objective (Fluar; Carl Zeiss). To exclude the possible stimulation
of mechanosensitive channels by the perfusion system, we stopped the
perfusion and found no change in [Ca2⫹]i. We performed ratiometric
measurement Fura-2 fluorescence at 5-second intervals using a highspeed polychromator system (VisiChrome; Visitron Systems, Puchheim, Germany) altering the wavelength of excitation light between
340 and 380 nm. Emitted light was filtered with a 510-nm filter and was
detected by a cooled charged-coupled device camera (CoolSNAP;
Roper Scientific GmbH, Ottobrunn, Germany). Data were collected
(MetaFluor software; Universal Imaging) and analyzed (MetaAnalysis
software; Universal Imaging). Intracellular free Ca2⫹ ([Ca2⫹]i) was
calculated from the Fura-2 fluorescence ratio (F340/F380).15 Mean fluorescence intensities after excitation with 340 nm were between 153
and 197 arbitrary units, indicating equal loading of the cells throughout
all experiments.
Results were presented as mean ⫾ SEM. Statistical significance was
tested using one-way analysis of variance (P ⬍ 0.05; statistical significance; P ⬍ 0.01, strong statistical significance; P ⬍ 0.001, very strong
statistical significance).
RESULTS
Blocking of the plasma membrane Ca2⫹ ATPase in RPE cells by
the application of 2 mM orthovanadate led to a pronounced
increase in [Ca2⫹]i. The [Ca2⫹]i increased to 216.78% ⫾
10.46% of the basal value (n ⫽ 4; Fig. 1). Given that plasma
membrane Ca2⫹ ATPases are responsible for Ca2⫹ efflux to
maintain the low [Ca2⫹]i, this increase must have been caused
by a permanent Ca2⫹ influx through a sustained membrane
conductance for Ca2⫹.
It has already been shown that RPE cells have some types of
Ca2⫹-conducting ion channels, such as voltage-operated L-type
Ca2⫹ channels and purinergic receptors. Therefore, we first
tested the influence of blockers of these currents on the resting
[Ca2⫹]i. As shown in Figure 2, neither 10 ␮M nifedipine (a
blocker of L-type Ca2⫹ channels, n ⫽ 9) nor 50 ␮M PPADS (a
blocker of P2X channels, n ⫽ 7) had any effect on [Ca2⫹]i,
suggesting that there should be background Ca2⫹ channels
RNA Isolation and RT-PCR
Human RPE was obtained from organ donors within 24 hours of death.
After the cornea was removed for transplantation, the eyes were
subjected for RPE preparation. The anterior parts of the eyes, including
the vitreous and the retina, were removed. The posterior part was
rinsed with ice-cold PBS (without Ca2⫹ and Mg2⫹) to wash away
residual material from the neural retina. With the use of fine forceps,
the RPE was gently brushed away. RPE cells were collected and lysed
in lysis buffer (RNeasy Mini Kit; Qiagen, Valencia, CA). Total RNA from
ARPE-19 cells was prepared from confluent cultures grown in a 25-cm2
culture flask. RNA was isolated (RNeasy Mini Kit; Qiagen) according to
manufacturer’s instructions. RNA (1 ␮g) was reverse transcribed at
37°C for 1 hour in the following reaction mixture: 1 ␮g oligo dT primer
(Invitrogen, Carlsbad, CA), 1 mM of each dNTP, 20 U RNAguard
(Amersham Biosciences, Freiburg, Germany), and 20 U M-MLV reverse
FIGURE 1. Inhibition of plasma membrane ATPases by 2 mM Na3VO4
led to increased [Ca2⫹]i in RPE cells. (a) [Ca2⫹]i changes after application of 2 mM Na3VO4 calculated from Fura-2 measurements. (b)
Mean [Ca2⫹]i before (control) and maximal [Ca2⫹]i during application
of Na3VO4 (n ⫽ 4).
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Basal Calcium Entry in RPE Mediated by TRPC Channels
5769
TRPC1 and TRPC4 were expressed in the RPE. TRPC7 was also
expressed in freshly isolated cells (Fig. 7).
DISCUSSION
FIGURE 2. Basal [Ca2⫹]i is independent from the activity of voltageoperated Ca2⫹ channels and ionotropic purinergic receptors. (a)
[Ca2⫹]i changes during application of nifedipine (10 ␮M) or PPADS (50
␮M) calculated from Fura-2 measurements. (b) Mean [Ca2⫹]i before
(black bars) and during (gray bars) application of the drugs (5 ␮M
nifedipine, n ⫽ 9; 50 ␮M PPADS, n ⫽ 7) did not differ from each other.
distinctive from voltage-operated Ca2⫹ channels and P2X channels expressed in these cells.
Because lanthanides are known to inhibit Ca2⫹ influx, we
tested the effect of La3⫹ and Gd3⫹ on basal [Ca2⫹]i in RPE cells.
Both ions reduced [Ca2⫹]i significantly; 100 ␮M La3⫹ led to a
reduction to 25.26% ⫾ 7.21% (n ⫽ 10), and 100 ␮M Gd3⫹ to
14.57 ⫾ 2.67% (n ⫽ 5) of the resting [Ca2⫹]i (Fig. 3). Although
La3⫹ is an unspecific blocker of different Ca2⫹ channels, the
sensitivity of the individual Ca2⫹ channel types to La3⫹ is very
different. Therefore, we investigated the concentration dependence of the La3⫹ effect on the basal [Ca2⫹]i. At the lowest
concentration of 0.2 ␮M, La3⫹ already reduced basal [Ca2⫹]i to
39.7% ⫾ 11.72% (n ⫽ 7; Fig. 4). Ni2⫹ is also known to inhibit
Ca2⫹ influx through different Ca2⫹ channels. Application of 2
mM Ni2⫹ reduced the [Ca2⫹]i to 33.08% ⫾ 8.95% (n ⫽ 6).
Lanthanides and Ni2⫹ are relatively unspecific inhibitors
that affect various ion channels and transporters, but at low
concentrations they are known to be efficient blockers of some
members of the TRP channel family. In addition, we used
blockers influencing a more narrow number of channels, including TRPC channels. The application of 2-APB and SKF
96365 to RPE cells resulted in a decrease of [Ca2⫹]i, comparable to that seen with La3⫹ or Gd3⫹. 2-APB (75 ␮M, n ⫽ 8) and
SKF 96365 (50 ␮M, n ⫽ 8) led to a decrease of [Ca2⫹]i, to
23.55% ⫾ 5.83% and 63.78% ⫾ 8.85%, respectively (Fig. 5).
This reduction of basal [Ca2⫹]i was qualitatively reproduced
with primary cultures of RPE cells from human donor eyes (Fig.
6). The basal [Ca2⫹]i was reduced in these cells by the application of 75 ␮M 2-APB and 50 ␮M SKF 96365 to 55.7% ⫾ 9.38%
and 67.36% ⫾ 4.63%, respectively.
The pharmacologic profile of blockage of basal Ca2⫹ entry
observed here suggested that it was driven by members of the
TRPC subfamily. RT-PCR with RNA from the RPE cell line
ARPE-19 and from freshly isolated RPE cells showed that
In this study, we showed for the first time that RPE cells have
large basal membrane permeability for Ca2⫹, as indicated by an
increase in [Ca2⫹]i after inhibition of the plasma membrane
Ca2⫹ ATPase. This basal Ca2⫹ conductance was inhibited by
blockers with the same pharmacologic profiles as TRPC channels. We were able to exclude the contribution of voltageoperated Ca2⫹ channels and purinergic receptors to basal Ca2⫹
entry. By RT-PCR we confirmed the expression of TRPC channels in the RPE.
The RPE is a target for and a source of various cytokines
whose intracellular signaling cascades are coupled to [Ca2⫹]i.
Furthermore, [Ca2⫹]i changes are involved in many other RPE
functions, such as photoreceptor outer segment phagocytosis,
transcellular fluid and ion transport, cell differentiation, and
the control of gene expression.5 Nevertheless, until now, only
voltage-operated L-type Ca2⫹ channel ␣1D expression has
been shown in RPE cells. These channels are involved in
growth factor signaling in the RPE.8,16 In addition to these
studies about voltage-operated channels, only one other study
based on pharmacologic evidence of purinergic receptors suggests that ionotropic purinergic receptors (P2X) are involved
in the regulation of fluid and ion transport across the RPE.11 In
addition, it has been reported that NMDA receptors are expressed in the RPE,17–21 though it seems unlikely that these
channels may contribute to resting [Ca2⫹]i because they need
glutamate for stimulation.
FIGURE 3. Basal [Ca2⫹]i is significantly reduced by the application of
different nonspecific ion channel blocking ions. (a) [Ca2⫹]i changes in
the presence of La3⫹ (100 ␮M), Gd3⫹ (100 ␮M), or Ni2⫹ (2 mM)
calculated from Fura-2 measurements. (b) Mean [Ca2⫹]i before (black
bars) and during (gray bars) application of La3⫹ (n ⫽ 10), Gd3⫹ (n ⫽
5), and Ni2⫹ (n ⫽ 6). All three ions reduced the [Ca2⫹]i significantly.
Data were normalized to the mean values before application of the
respective drug. *P ⬍ 0.05. ***P ⬍ 0.001.
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Wimmers and Strauss
FIGURE 4. Concentration-response curve for the influence of La3⫹ on
basal [Ca2⫹]i. (a) Stepwise reduction of [Ca2⫹]i after application of
increasing concentrations of La3⫹ calculated from Fura-2 measurements. (b) Influence of increasing concentrations La3⫹ on [Ca2⫹]i.
Compared are the steady state values with the different La3⫹ concentrations. Mean ⫾ SEM [Ca2⫹]i normalized to the concentration before
La3⫹ application (n ⫽ 2–15). Note that the x-axis is interrupted between 15 and 45 ␮M La3⫹.
Thus, considering the multitude of different [Ca2⫹]i-regulated RPE cell functions, additional Ca2⫹ channels are likely to
be expressed in the RPE. TRP channels are good candidates
because they are coupled to a variety of intracellular signaling
pathways. RPE cells use energy to continuously extrude Ca2⫹
effectively from the intracellular space through a plasma-membrane Ca2⫹ ATPase and a Na⫹/Ca2⫹ exchanger. Because some
of the Ca2⫹-induced processes, such as basal VEGF secretion,
must persist without stimulation, RPE cells need an equally
effective Ca2⫹ entry pathway. To our knowledge, such a leakage Ca2⫹ pathway is only described for vascular smooth muscle cells,22–24 osteoblastlike cells,25 and chromaffin cells.26
Our findings that La3⫹, Gd3⫹, Ni2⫹, 2-APB, and SKF 96365
inhibited this nonstimulated basal Ca2⫹ entry indicated that
TRP channels are the molecular correlate of this Ca2⫹ leak.
Although none of these blockers is specific for a particular
class of TRP channels, conclusions concerning the molecular
identity of the channels can be drawn from the combination of
their effects. TRPV channels are either insensitive to or activated by 2-APB.27 Accordingly, these channels can be excluded
from their possible role in basal Ca2⫹ entry in RPE cells. TRPM
channels can be excluded because of their insensitivity to
Ni2⫹. Thus far, it has been found that TRPM channels are
highly permeable to Ni2⫹.28,29 Therefore, we concentrated on
the TRPC channels.
Our RT-PCR experiments revealed that TRPC1 and TRPC4
are expressed in ARPE-19 and cultured RPE cells from human
donor eyes. In the latter, we also found TRPC7 to be expressed. La3⫹ and Gd3⫹ block a variety of TRP channels.30
They also inhibit a variety of other ion channels and transporters.31–35 All non-TRP channels, however, have a much lower
La3⫹ sensitivity than the leak Ca2⫹ channel investigated in this
IOVS, December 2007, Vol. 48, No. 12
FIGURE 5. Influence of 2-APB and SKF 96365 on basal [Ca2⫹]i in RPE
cells. (a) [Ca2⫹]i changes after application of 2-APB (75 ␮M) or SKF
96365 (50 ␮M) calculated from Fura-2 measurements. (b) [Ca2⫹]i
before (black bars) and during (gray bars) application of the TRP
channel blockers 2-APB (n ⫽ 8) and SKF 96365 (n ⫽ 8). Both molecules reduced [Ca2⫹]i significantly. *P ⬍ 0.05. ***P ⬍ 0.001.
study. Voltage-operated Ca2⫹ channels had an IC50 of 1.1
mM.32 Most TRP channels have much higher La3⫹ sensitivity
than voltage-operated Ca2⫹ channels. Nevertheless, their sensitivity is much higher (IC50, 3–100 ␮M) than the IC50 for La3⫹
block of basal Ca2⫹ entry in RPE cells.36 –39
Some study results conflict with ours. In these studies,
TRPC4-mediated currents were potentiated rather than
blocked, as in our study, by La3⫹ in micromolar concentrations.40 – 42 In other studies, TRPC4 channels were blocked by
La3⫹.43– 45 Comparative studies of wild-type and TRPC4 knockout mice revealed very high La3⫹ sensitivity of these native
TRPC4 channels in the nanomolar range.43,46 This discrepancy
might be explained by the fact that the potentiation of these
currents could only be observed in the heterologous expression system, whereas studies indicating high La3⫹ sensitivity of
FIGURE 6. Influence of 2-APB and SKF96365 on basal [Ca2⫹]i in primary cultures of RPE cells. (a) [Ca2⫹]i changes after the addition of
SKF96365 (50 ␮M) calculated from Fura-2 measurements. (b) Mean
[Ca2⫹]i before (black bars) and during (gray bars) application of
SKF96365 (n ⫽ 5) and 2-APB (n ⫽ 5). Both molecules reduced [Ca2⫹]i
significantly in all five independent experiments. *P ⬍ 0.05.
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Basal Calcium Entry in RPE Mediated by TRPC Channels
5771
FIGURE 7. Expression profile of TRPC
channels in ARPE-19 cells compared
with the expression pattern in freshly
isolated RPE cells from human donor
eyes.
TRPC4 were conducted with endogenously expressed channels in native cells.
TRPC4 has been shown to coassemble with TRPC1 to form
heteromultimeric channels.47 These heteromultimeric channels might be dominated by the high La3⫹ sensitivity of native
TRPC4 channels.
In another single study on TRP channels in the RPE, it was
found that only TRPC1 is expressed in ARPE-19 cells.48 TRPC4
is known to be differentially spliced.41,49,50 Because we amplified different parts of the gene, our results may be attributed to
a splice variant that Bollimuntha et al.48 could not detect with
their oligonucleotides. In addition, we confirmed the expression of TRPC1 and TRPC4 in native RPE cells. We also found
TRPC7 to be expressed in native RPE cells. It has been thought
that heteromultimers could only be formed among TRPC1,
TRPC4, and TRPC5 and among TRPC3, TRPC6, and TRPC7.47
Nevertheless, some studies show that heteromultimers can also
be formed between these groups.51,52 Hence, the additional
expressed TRPC7 might be a part of a heteromultimeric channel carrying the basal influx into native RPE cells.
Ca2⫹ is one of the fundamental intracellular signaling molecules. TRPC channels mediating the leak Ca2⫹ entry to the
RPE may be involved in basal cellular processes controlled by
[Ca2⫹]i, such as basal secretion of cytokines. Additionally,
TRPC channels are coupled to intracellular signaling pathways
that are activated by G protein– coupled receptors. On stimulation, these channels may contribute not only to the adjustment of resting [Ca2⫹]i but also to that of elevated [Ca2⫹]i.53
Because TRPC channels are nonselective cation channels, the
TRPC channels identified in this study may also contribute to
the resting membrane potential in RPE cells. Human RPE cells
have a resting membrane potential of approximately ⫺45
mV.54,55 Given that they have high K⫹ permeability,54 the
observed resting potential requires an additional Na⫹-permeable current that shifts the resting potential to values positive
to the equilibrium potential of K⫹. TRPC channels are known
to set the membrane potential and the basal Ca2⫹ entry56 in
other cell types. They may also do so in the RPE.
Acknowledgments
The authors thank Stefanie Schlichting for excellent technical assistance.
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