Rapid effects of 17beta-estradiol on TRPV5 epithelial Ca2+ channels in rat renal cells.

Abstract

The renal distal tubules and collecting ducts play a key role in the control of electrolyte and fluid homeostasis. The discovery of highly calcium selective channels, Transient Receptor Potential Vanilloid 5 (TRPV5) of the TRP superfamily, has clarified the nature of the calcium entry channels. It has been proposed that this channel mediates the critical Ca(2+) entry step in transcellular Ca(2+) re-absorption in the kidney. The regulation of transmembrane Ca(2+) flux through TRPV5 is of particular importance for whole body calcium homeostasis.In this study, we provide evidence that the TRPV5 channel is present in rat cortical collecting duct (RCCD(2)) cells at mRNA and protein levels. We demonstrate that 17beta-estradiol (E(2)) is involved in the regulation of Ca(2+) influx in these cells via the epithelial Ca(2+) channels TRPV5. By combining whole-cell patch-clamp and Ca(2+)-imaging techniques, we have characterized the electrophysiological properties of the TRPV5 channel and showed that treatment with 20-50nM E(2) rapidly (<5min) induced a transient increase in inward whole-cell currents and intracellular Ca(2+) via TRPV5 channels. This rise was significantly prevented when cells were pre-treated with ruthenium red and completely abolished in cells treated with siRNA specifically targeting TRPV5.These data demonstrate for the first time, a novel rapid modulation of endogenously expressed TRPV5 channels by E(2) in kidney cells. Furthermore, the results suggest calcitropic effects of E(2). The results are discussed in relation to present concepts of non-genomic actions of E(2) in Ca(2+) homeostasis.

Full text

Page 1

Steroids 74 (2009) 642–649
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Rapid effects of 17?-estradiol on TRPV5 epithelial Ca2+channels in rat renal cells
Mustapha Irnatena,∗,1, Nicolas Blanchard-Guttona,1, Jeppe Praetoriusb, Brian J. Harveya
aMolecular Medicine Laboratories, Royal College of Surgeons in Ireland, Beaumont Hospital, PO Box 9063, Dublin 9, Ireland
bInstitute of Anatomy & the Water and Salt Research Center, University of Aarhus, Wilhelm Meyers Alle, Building, 1-234, DK-8000 Aarhus, Denmark
a r t i c l ei n f o
Article history:
Received 15 December 2008
Received in revised form 27 January 2009
Accepted 9 February 2009
Available online 21 February 2009
Keywords:
Estrogen
Renal cortical collecting duct
Epithelial calcium channel
TRPV5
a b s t r a c t
Therenaldistaltubulesandcollectingductsplayakeyroleinthecontrolofelectrolyteandfluidhomeosta-
sis. The discovery of highly calcium selective channels, Transient Receptor Potential Vanilloid 5 (TRPV5)
of the TRP superfamily, has clarified the nature of the calcium entry channels. It has been proposed that
this channel mediates the critical Ca2+entry step in transcellular Ca2+re-absorption in the kidney. The
regulationoftransmembraneCa2+fluxthroughTRPV5isofparticularimportanceforwholebodycalcium
homeostasis.In this study, we provide evidence that the TRPV5 channel is present in rat cortical collecting
duct (RCCD2) cells at mRNA and protein levels. We demonstrate that 17?-estradiol (E2) is involved in the
regulation of Ca2+influx in these cells via the epithelial Ca2+channels TRPV5. By combining whole-cell
patch–clamp and Ca2+-imaging techniques, we have characterized the electrophysiological properties of
the TRPV5 channel and showed that treatment with 20–50nM E2rapidly (<5min) induced a transient
increase in inward whole-cell currents and intracellular Ca2+via TRPV5 channels. This rise was signif-
icantly prevented when cells were pre-treated with ruthenium red and completely abolished in cells
treated with siRNA specifically targeting TRPV5.These data demonstrate for the first time, a novel rapid
modulation of endogenously expressed TRPV5 channels by E2in kidney cells. Furthermore, the results
suggest calcitropic effects of E2. The results are discussed in relation to present concepts of non-genomic
actions of E2in Ca2+homeostasis.
© 2009 Elsevier Inc. All rights reserved.
1. Introduction
Calcium metabolism is of crucial importance to many vital
physiological functions and the maintenance of the body Ca2+
homeostasisisessentialtolife.ThemodulationofintracellularCa2+
activity by Ca2+influx is one of the most universal signal transduc-
tionpathwaysinallcelltypes,fromsensorysignaltransduction,cell
growth, cardiovascular functions to gene expression [1]. Extracel-
lular fluid calcium levels are handled and regulated by calcitropic
hormones at three potential sites including, the gastrointestinal
tract, kidney and bone. It is well established that Ca2+is reabsorbed
in kidney by a transcellular pathway. Ca2+enters the epithelial
cell passively across the apical membrane via Ca2+-selective chan-
nelsdownanelectrochemicalgradient,diffusesthroughthecytosol
bound to intracellular proteins as calbindins and parvalbumin and
activelyextrudedatthebasolateralmembranethroughCa2+ATPase
activity and Na/Ca exchange [2].
Hormones, which are classically involved in Ca2+homeostasis,
include 1,25-dihydroxyvitamin D3, parathyroid hormone and calci-
tonin [3,4]. The idea that estrogen plays a role in Ca2+homeostasis
∗Corresponding author. Tel.: +353 85 133 4932.
E-mail address: irnatenm@yahoo.fr (M. Irnaten).
1These authors have contributed equally to this work.
hasbeenalsoestablished[5].Theinvolvementof17?-estradiol(E2)
in Ca2+homeostasis is clearly illustrated by the role of the hor-
mone in bone mineralization and the finding that E2deficiency in
postmenopausal women results in bone loss arising from a nega-
tive Ca2+balance [6,7], which is associated with a rise in plasma
and urinary Ca2+[8]. However, studies have shown that the rise
in urinary Ca2+at menopause is not due to an increased filtered
load, suggesting that E2also has a role in regulating renal Ca2+han-
dling [9]. Ca2+supplementation can also reduce bone loss in these
patients, suggesting an interaction between estrogen deficiency
and Ca2+balance [10]. However, the cellular mechanisms under-
lying E2regulation of Ca2+reabsorption is still poorly understood
in kidney.
AfamilyofCa2+-permeablecationchannelshasbeendiscovered
in the early 1990s; the Transient Receptor Potential (TRP) chan-
nels [11]. TRPV5 is a member of the TRPV subfamily [12] which is
implicated in apical calcium entry in epithelia [13], shows a consti-
tutive activity, and functions as a facilitative transporter. Although
our knowledge of TRPV5 channel physiology is still relatively lim-
ited,TRPmutationshavealreadybeendescribedthatleadtokidney
diseases [14].
Because transcellular Ca2+transport is fine-tuned to the body’s
specific requirements, regulation of the transmembrane Ca2+flux
through TRPV5 is of particular importance for whole body Ca2+
homeostasis and is tightly controlled by hormones [15].
0039-128X/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2009.02.002

Page 2

M. Irnaten et al. / Steroids 74 (2009) 642–649
643
It has been demonstrated that TRPV5 protein expression is
highly modulated by E2in kidney [16]. In addition to genomic
effects, it is well established that E2can also exert rapid non-
genomiceffectsonintracellularCa2+invariouscelltypes:including
hepatocytes, osteoblasts, enterocytes/monocytes and in distal
colon [17–21]. Furthermore, there is increasing evidence for rapid,
non-genomic, effects of E2on epithelial ion transport [22–25]. In
particular, in the kidney collecting duct M-1 cells, E2has been
showntomodulateintracellularCa2+levelsthroughacalciumentry
pathway [26]. On the basis of others and our previous studies, we
hypothesize that E2may have the ability to regulate renal Ca2+
re-absorption through rapid effects on TRPV5 channel activity.
Most electrophysiological studies to date on TRPV5 channels
have been performed on over expressing channels in heterologous
systems, CHO and HEK being the most commonly employed cell
models.Ourstudyisoneofthefewreportsofhormonalmodulation
of endogenous TRPV5 channels.
2. Experimental
2.1. Cell culture
RCCD2cell line used in this study was previously characterized
byProfN.Farman’sgroup.ImmortalizedRCCD2cellswereobtained
by infection of primary cultured CCD cells with the wild-type
simianvirus40.Ithasbeenshownthatthiscelllinehasmaintained
many of the original properties of rat CCD from which they were
derived [28]. RCCD2cells were cultured in 75cm2flasks or 35mm
glass-bottom dishes that had been coated with rat type I collagen,
as previously described [27,28]. Cells were cultured in a medium
thatcontained1:1Ham’sF-12-DMEMwith14mMNaHCO3,20mM
HEPES buffer (pH 7.4), 10U/ml penicillin–streptomycin, 2mM
glutamine, 5?g/ml insulin, 50nM dexamethasone, 5?g/ml trans-
ferrin, 30nM sodium selenite, 10nM triiodothyronine, 10ng/ml
EGF, and 2% FBS (Gibco, Paisley, UK). RCCD2 cells were maintained
in serum-free medium and in the absence of dexamethasone and
EGF overnight before treatment with oestrogen.
2.2. RT-PCR analysis
Total mRNA was isolated using TRI-REAGENT®(Molecular
Research Center, USA) according to the manufacturer’s directions.
Total RNA was reverse-transcribed into cDNA using ImProm-
IITMReverse Transcription System (Promega, USA). Primers for
TRPV5 (accession no. NM 019841) were designed using GeneFisher-
software [29], and synthesized by MWG (Germany).
The primers used for amplification of TRPV5 were [forward:
ACCACTACAGGAAGC-GTA; reverse:
PCR amplification was performed with initial heating for 2min at
94◦C, followed by 35 cycles of 1min denaturation at 94◦C, anneal-
ing for 1min at 51.5◦C and extension for 2min at 72◦C. BLASTN
search was performed on primers to confirm that the sequences
were not shared with other known genes. The PCR products were
resolved using a 1% Tris-Borate-EDTA (TBE) agarose gel and the
bands were analyzed via Gene Tools software (Syngene, UK). cDNA
bands corresponding to TRPV5 transcripts were extracted from
the agarose gel, purified using Quiaquick kit (Quiagen, UK) and
subsequently sequenced (MWG, Germany).
CCGTCAATGATGGTAAGGA].
2.3. Western blot analysis
Western blotting experiments were carried out using standard
techniquewithmodifications[30].Briefly,cellsweregentlywashed
twice with PBS and scraped into SDS sample buffer [62.5mM
Tris–HCl (pH 6.8 at 25◦C), 2% (w/v) SDS, 10% glycerol, protease
inhibitors and 2mM DTT]. Equal amounts of total protein were
resolvedon8%SDS-PAGEgelsandtransferredontopolyvinyldifluo-
ridemembranesusingthesemi-drytransfertechnique.Membranes
were blocked for 2h at room temperature in Tris buffered saline
with Tween-20 (TBST) containing 5% non-fat dry milk and incu-
bated for 1h at room temperature with rabbit-anti rat TRPV5
primary antibody diluted in blocking solution, (1/500 dilution)
(ACC-035, Alomone labs). Membranes were washed 3×10min in
TBST and incubated for 1h with HRP-Rabbit secondary antibody
(1/5000dilutioninTBSTcontaining5%driedmilk).After3×10min
wash,immuno-reactiveproteinsweredetectedusingenhancedECL
plus chemiluminescent reagent (Amersham Biosciences, UK).
Anti-?-actin monoclonal antibody (1/4000 dilution; Sigma, Ire-
land) was used as a loading control. Rat kidney cortex whole lysate
and Chinese Hamster Ovary (CHO) cells were used as positive and
negative controls, respectively.
2.4. Immunofluorescence microscopy
Four male Wistar rats were anesthetized by isoflurane inhala-
tion and kidneys were perfusion fixed with 3% formaldehyde in
phosphate buffer, pH 7.4 through the abdominal aorta. The tissues
were dehydrated in graded ethanol, incubated overnight in xylene,
and embedded in paraffin wax. 2?m thick sections were cut using
a rotary microtome (Leica, Heidelberg, Germany). All procedures
were approved by the Danish Ethics Committee. The sections were
dewaxedwithxyleneandrehydratedwithgradedethanol.Endoge-
nous peroxidase was blocked by 0.5% H2O2in absolute methanol
for 10min. The sections were boiled in 10mM Tris with 0.5mM
EGTA,pH9,for10min.Aftercooling,thesectionswerewashedwith
50mM NH4Cl in PBS for 30min followed by incubation with PBS
blocking buffer containing 1% BSA, 0.2% gelatin, and 0.05% saponin.
The sections were incubated overnight at 4◦C with primary anti-
bodies diluted in PBS supplemented with 0.1% BSA and 0.3% Triton
X-100.UponwashinPBSsupplementedwith0.1%BSA,0.2%gelatin,
and 0.05% saponin dual or triple color fluorescence labeling were
performed. The sections were incubated with fluorophore conju-
gated secondary antibodies (see below) in PBS supplemented with
BSA and Triton-X-100. After washing, sections were mounted with
a cover slip in Glycergel Antifade Medium (Dako), and inspected on
a Leica DMRS confocal microscope using an HCX PlApo 64x (1.32
NA) objective.
2.5. Antibodies
Primaryantibodieswere:mouseanti-calbindinD-28K(dilution:
1/40000) (Research Diagnostics, Flanders, NJ) and rabbit anti-
TRPV5 (dilution: 1/100) (ECAC1AP, alpha diagnostics). Fluorescent
secondary antibodies were donkey anti-rabbit Alexa488 and don-
key anti-mouse Alexa555 (dilution: 1/1000) (Invitrogen/Molecular
Probes,Eugene,OR).Topro3(dilution1/1000)wasusedasanuclear
marker (Invitrogen).
2.6. Electrophysiology
It was impossible to patch RCCD2cells after 48h plating as the
cells are flattened and whole-cell access configuration is difficult
to achieve (Basolateral membrane not accessible to the bath solu-
tion changes). We therefore detached subconfluent cells from six
wellplatesusingtrypsin–EDTA.Aftercentrifugationat1100rpmfor
3mincellswererecoveredinculturemediumatroomtemperature.
Patch–clamp experiments were performed using the whole-cell
patch–clamp configuration [31]. Isolated RCCD2cells were trans-
ferred into a 1ml superfusion chamber mounted on the stage
of an inverted microscope (Nikon) and perfused with modified
Krebs “bath” solution. Patch pipettes were prepared from capil-
lary glass (GC150 F-10, Harvard Apparatus Ltd., UK) using a puller

Page 3

644
M. Irnaten et al. / Steroids 74 (2009) 642–649
(DMZ-Universal Puller, Zeitz-Instruments, Germany) and had a
resistance of 3–5M? when filled with the pipette solution. The
reference electrode was an Ag–AgCl wire in direct contact with the
superfusion bath. The patch–clamp apparatus consisted of a CV-
203BU head stage (Axon Instruments Inc., CA, USA) connected to
an Axopatch 200B series amplifier (Axon). Seal resistance was typi-
callyinthe1–5G?range.Recordedmembranewhole-cellcurrents
wereamplifiedanddigitizedat5kHzandlowpass-filteredat1kHz.
Membrane voltage was clamped from −120mV to +100mV with
steps of 20mV. Average capacitance of the cell was approximately
14.5±2.2pF. Drug actions were measured only after steady-state
conditions were reached.
2.7. Calcium-imaging microscopy
Initially we used the same concentration of E2(20nM) in cal-
cium imaging and patch–clamp experiments, but E2did not show
any significant effect on intracellular calcium in calcium imaging.
This could be due to the fact that at this concentration (20nM) the
accessibility of E2is limited to the apical side of the cells. For Ca2+
measurements cells were not trypsinised and were plated in glass
cover slips and the basolateral membranes of the cells are stuck to
the glass cover slip limiting drug and bathing solution access. How-
ever,inpatch–clampexperiments,trypsinisationofcellsallowsthe
access of E2to the whole-cell membrane.
RCCD2cells were loaded for 45min with 5?M FURA-2/AM. All
experiments were performed in physiological solution (as above),
at room temperature (20–22◦C). Ca2+“free” experiments per-
formed in Krebs solution where calcium is omitted as previously
described by Nilius et al. [13]. Cells were mounted on an inverted
epi-fluorescence microscope (Diaphot 200, Nikon, Japan) and illu-
minatedwithaXenonlamp(Cairn,UK)filteredthroughalternating
340 and 380nm interference filters. The resultant fluorescence was
passed through a 400nm dichroic mirror, filtered at 510nm and
thencollectedusinganintensifiedCCDcamerasystem(Hammatsu,
Japan). Images were recorded for 30min, digitised and analyzed
using Openlab2 (Improvision, UK). Drug actions were measured
only after steady-state conditions were reached.
2.8. Solutions and chemicals
For both calcium imaging and whole-cell patch–clamp exper-
iments, cells were superfused with a “Krebs” solution containing
(in mM): 145 NaCl; 6 CsCl; 1 MgCl2; 10 CaCl210 HEPES and 10 glu-
cose,pH7.4withCsOH.Na+-freeconditionswereobtainedbyusing
NMDG+instead of Na+. When the extracellular CaCl2concentra-
tion was increased, extracellular NaCl was equimolarly decreased
respectively to keep the osmolarity constant. In divalent-free (DVF)
solutions, Ca2+and Mg2+were omitted from the bathing solu-
tion. The patch pipette solution contained in all experiments (in
mM): 20 CsCl; 100 Cs–aspartate; 1 MgCl2; 10 BAPTA (1,2-bis(2-
aminophenoxy)ethane-N,N,N’,N’-tetra-acetic acid); Na2ATP 4, 10
HEPES (pH 7.2). Chemicals were purchased from Sigma Chemical
Co.(Ireland).Inallexperiments,stocksolutionsofE2wereprepared
in methanol. No effect of the methanol vehicle on TRPV5 currents
was observed at the concentrations used to study E2effects.
2.9. Synthesis and transfection of siRNA for TRPV5
SiRNA sequences targeting rat TRPV5 were designed and syn-
thesized using the Silencer Pre-designed siRNA construction kit
(AmbionResearchInc.,UK).ThethreespecificratTRPV5DNAtarget
sequences of the annealed double stand siRNA that we used were
as follows: siRNA1: sense: CAUGUGGAUCAGCUUCAUtt (240–261),
antisense: AUG AAGCUGAUCCACAUGCtg; siRNA2: sense: GC
UUCAUAUGCUACAGCAGtt (252–273),antisense:CUGCUGUAG
CAUAUGAAGCt; siRNA3: sense: CGUAUAAUGUG GAAAGAACtt
(2664–2685); antisense: GUUCUUUCCACAUUAUACGtt.
with a non-silencing oligonucleotide sequence (functional non-
codingsiRNA#1obtainedfromAmbion)thatdoesnotrecognizeany
knownhomologytomammaliangenes,wasgeneratedasanegative
control.
RCCD2cells were cultured in an Opti-MEM®I reduced Serum
Medium (GIBCO, UK) for 12h prior to transfection. Sub-confluent
cells(∼50%confluence)weretransfected,betweenpassages10–25.
For each transfection, oligomer-LipofectamineTM2000 com-
plexes were prepared as follows: (i) siRNA oligomer 50 pmol was
diluted in 100?l Opti-MEM®(resulting concentration of siRNA
is 100nM) and the solution was gently mixed. (ii) Separately,
LipofectamineTM2000 (5?l) was diluted in 100?l of Opti-MEM®.
Thesolutionwasgentlymixedandincubatedfor5minatroomtem-
perature. (iii) The diluted oligomer and Lipofectamine 2000 were
gently mixed and incubated for 30min at room temperature. The
siRNA and Lipofectamine 2000 complex was added to 1ml Opti-
MEM®per well. Six-well plates were incubated at 37◦C for 6h.
thereafter; the medium was changed to a full culture medium for
48h.
For functional studies and to monitor transfection efficiencies
cells were transfected with a FAM tagged siRNA.
SiRNA,
2.10. Data analysis
The densitometry of the TRPV5 bands were normalized to the
loadingcontrol,?-actin.Densitometricanalysisofthewesternblots
were performed using GeneTools software (SYNGENE, Cambridge,
UK).Alldataarereportedasmean±SEMforaseriesoftheindicated
number of experiments. Patch–clamp data analysis was performed
using the clampfit software of the p-clamp suite version 9.2 and
Origin 7.5 (OriginLab Corp, MA, USA). Statistical analysis of the data
was obtained using a paired t-test for analysis between two groups,
a p value<0.05 was considered significant. One-way ANOVA was
used for multiple analyses of more than two groups.
3. Results
3.1. Expression and electophysiological properties of TRPV5 in
RCCD2cells
We explored the presence of TRPV5 channels in RCCD2cells by
RT-PCR. Fig. 1A shows a single band of about 550bp correspond-
ing to TRPV5 transcript in RCCD2cells. Subsequent sequencing of
the band confirmed that it corresponded to the cDNA sequence of
TRPV5. To further confirm the expression of TRPV5 in RCCD2cells,
Western blot analysis was performed and revealed a band with a
molecularsizeof∼90kDa,correspondingtoTRPV5protein(Fig.1B).
RatkidneycortexwasincludedasapositivecontrolforbothRT-PCR
and Western blot. CHO cells were included as negative control for
Western immunoblotting. To reinforce the evidence of the pres-
ence of TRPV5 in cortical collecting ducts, immunolocalization
experiments were performed. Immunofluorescence microscopy
revealed that luminal anti-TRPV5 immunoreactivity extends from
the calbindin-positive late distal convoluted tubules (DCT2) and
connecting tubules (CNT) into a subset of cells of the initial cortical
collecting ducts (iCCD, Fig. 1C). These patterns were observed in all
four rats tested. Thus, luminal anti-TRPV5 labelling was restricted
to DCT2, CNT and iCCD. Anti-TRPV5 staining was not detected in
any other renal structures.
Based on these results, we investigated the electrophysiological
properties of TRPV5 in these cells. Fig. 2A compares the whole-
cell current bathed with either external solution containing 1, 10
and 100mM Ca2+or in divalent free (DVF) solution with Na+as

Page 4

M. Irnaten et al. / Steroids 74 (2009) 642–649
645
Fig. 1. Expression of TRPV5 in RCCD2cells. (A) RT-PCR of mRNA products for TRPV5.
Lane 1: DNA ladder. Lane 2: TRPV5 transcript. Lanes 3 and 4: negative controls
with no mRNA template or reverse transcriptase, respectively. Lower panel: pos-
itive control of the expression of TRPV5 mRNA in native tissue kidney cortex. (B)
Representative blot of TRPV5 protein levels in cellular extracts from RCCD2 cells
(lane 2), rat kidney cortex (lane 1, positive control) and CHO cells (lane 3, negative
control). Anti-?-actin antibody was used as a loading control. Bands correspond-
ing to TRPV5 and ?-actin are indicated by arrows. The data are representative of
three experiments. (C) Representative image of TRPV5 and calbindin D-28K in rat
isolated CCD sections. Rat kidney sections were incubated for overnight at 4◦C with
the corresponding primary antibodies mouse anti-calbindin-D28K and rabbit anti-
TRPV5.TovisualizetheproteinscorrespondingsecondaryAlexa488/555conjugated
antibodies were used. Tropro3 was used as a nuclear marker.
the major charge carrier of the currents. Under DVF solution, cells
generated large inward currents that are completely abolished by
replacing extracellular NaCl with NMDG-Cl. Re-application of NaCl
to the bathing solution re-established the inward currents. Fig. 2B
shows the current–voltage (I–V) relationship in the presence of
increasing [Ca2+]e(in mM: 1.5, 10, 20 and 100). Extracellular Ca2+
inducesinwardcurrentwithamplitudethatdependsonthe[Ca2+]e
indicating that it is carried by Ca2+.
We examined the effects of various membrane holding poten-
tials on whole-cell currents in RCCD2cells. Fig. 3A shows typical
whole-cell current traces recorded at −120mV at different hold-
ing potentials of +20, −50, −80 and −110mV as indicated in
the graph. Whole-cell current measurements showed that current
amplitudes increased in a voltage-dependent manner upon hyper-
polarization(Fig.3B).Ataholdingpotentialof−110mV,thecurrent
measured at −120mV was larger than the currents recorded at
the same voltage when initiated from more depolarised holding
potentials (−1242±117pA at −110mV; −997±121pA at −80mV;
−312±89pA,at−50mV;−88±71pAat+20mV,n=7).Inaddition,
The channel exhibits an inwardly rectifying I–V characteristic and
a transient monovalent current upon withdrawal of external diva-
lent cations, its store dependence and pharmacology (sensitivity to
rutheniumred)madeustobelievethatthischannelisTRPV5chan-
nel. In addition, these characteristics are in agreement with those
previously reported by Clapham group [32]. These results support
the hypothesis that calcium influx may occur via TRPV5 in RCCD2
cells.
A dose–response study of the rapid effects of E2on whole-cell
Ca2+currents was performed in RCCD2cells (Fig. 4). Treatment
Fig.2. Whole-cellcurrentsare[Ca2+]edependentinRCCD2cells.Whole-cellrecord-
ings were elicited from +20mV holding potential in 20mV steps from −120mV to
+100mV. The patch pipette was filled with a standard cesium-aspartate solution
supplemented with 10mM of BAPTA. Cells were first perfused with DVF solution
then with increasing of extracellular Ca2+concentrations. (A) Whole-cell currents
were recorded in DVF conditions. The presence of Na+as the major charge carrier in
the external solution resulted in large inward current amplitudes which were com-
pletely abolished when Na+was replaced by NMDG+. Addition of 1, 10 and 100mM
of Ca2+in monovalent free external solution induced an inward transient current
that amplitude depends on the [Ca2+]e. (B) Current–voltage relationships recorded
by measuring the amplitude of currents at: 1.5mM Ca2+(?), 10mM Ca2+(?), 20mM
Ca2+(?) and 100mM Ca2+(?). Data are mean±SEM, n=7 cells obtained from seven
separate experiments.
with increasing concentration of E2(in nM; 1, 10, 20, and 50)
stimulated inward whole-cell Ca2+currents in a dose-dependent
manner. Cells generated inward currents that amplitudes increases
from −225±87pA at 1nM E2to −306±108pA at 10nM E2, −
844±122pA, at 20nM E2and to − 788±113pA at 50nM E2, n=9).
The maximum peak current amplitude obtained was at 20nM E2
therefore, we used this concentration for subsequent studies in all
patch clamp experiments.
3.2. Intracellular Ca2+rise induced by E2is sensitive to ruthenium
red in RCCD2cells
3.2.1. Calcium imaging
The effect of 20nM E2on [Ca2+]iwas examined in RCCD2cells.
As shown in Fig. 5A, E2treatment modulates cytosolic Ca2+levels.
In 35% (n=108) of the cells examined, E2induced a rapid transient
peak rise in [Ca2+]i. Vehicle controls (physiological solution alone
or with 5×10−5% methanol) had no effect on [Ca2+]i(Fig. 5A).
To test if the E2-induced increase in [Ca2+]ioriginated from
an extracellular source, we monitored the effect of E2in RCCD2
cellsbathedwith“low”(1.5mM)extracellularCa2+)solution.Under
these conditions, E2treatment did not alter [Ca2+]i, and subsequent
thapsigargin treatment triggered a rise in intracellular Ca2+indi-

Page 5

646
M. Irnaten et al. / Steroids 74 (2009) 642–649
Fig. 3. Whole-cell currents are voltage-dependent in RCCD2cells. Cells were incu-
bated in Krebs solution containing 10mM Ca2+in the perfusion medium and 10mM
BAPTA in the pipette solution. From a holding potential of +20mV the membrane
potential was changed to −50mV, −80mV and −110mV. Currents were measured
at the first voltage step of −120mV. (A) Original typical whole-cell currents traces
recorded at different holding potentials as indicated in the graph. (B) Averaged data
representing currents recorded from seven cells at different holding potentials at
the first step of −120mV. Data represent the mean±SEM of seven cells.
cating that the release of calcium ions from internal stores was still
possible but was not activated by E2. These data indicate that the
rise in (Ca2+)iinduced by E2is initiated by an influx of Ca2+into the
cell from the extracellular space and not by an initial release from
intracellular stores.
To investigate the contribution of TRPV5 in the E2-induced Ca2+
risecellswerepre-treatedwithrutheniumred,awell-knowneffec-
tive blocker of highly-Ca2+selective channels [33]. Fig. 5B shows
that E2induced a rise in Ca2+of 18.3±1.6% above basal (n=38 cells)
this rise was significantly reduced to 9.7±0.6% above basal (n=25
cells, p<0.002) when cells were pre-treated with ruthenium red
(50?M).
Interestingly, the number of cells responding to E2with a rise in
Ca2+was also significantly decreased when cells were pre-treated
Fig.4. EffectofE2onwhole-cellcurrentsinRCCD2cells.Whole-cellrecordingswere
elicited from +20mV holding potential in 20mV steps from −120mV to +100mV in
RCCD2cells. Cells were allowed to stabilize and dialyze for at about 5min before
exposure to E2. Treatment of cells with increasing concentrations of E2(in nM: 1, 10,
20,50)inducedadose-dependentcurrentamplitudesincreases.Themaximumpeak
increase was obtained at 20nM E2. The results shown are tracings of representative
cells with similar results observed in separate experiments from nine cells. Arrow
indicates time of addition of E2.
Fig. 5. 17?-estradiol induces increase in [Ca2+]iin RCCD2cells. (A) Addition of E2
(50nM) induced a rapid increase in [Ca2+]i(?) E2-induced [Ca2+]iincrease were typ-
ically rapid in onset and transient and returned to basal levels within 1–2min. No
increase in [Ca2+]ilevels were observed in response to vehicle (5×10−5% methanol)
(?), or physiological solution alone (?). The X-axis represents time after the drug
addition (the arrow represents time of E2addition). All measurements have been
normalized with the base line emission wavelength ratio being fixed at 1. As a pos-
itive control to store filling, thapsigargin (1?M) was added to the bathing solution
at the end of each experiment. (B) E2-induced Ca2+entry is sensitive to ruthe-
nium red. 50nM E2induced a significant increase over control [Ca2+]i(normalized
increase above basal=118±2; n=38 vs control 100, nt=38). Pre-incubation with
the potent TRPV5 blocker, ruthenium red (50?M), significantly inhibited the E2-
induced increases in [Ca2+]ito 110±1 (n=25). Values are presented as means±SEM
(*denotes significant differences: ***p<0.001).
with ruthenium red (19% in ruthenim red+E2versus 35% of cells
responding in E2alone, n=131).
Taken together, these data further strengthen the conclusion
that E2stimulates calcium influx via TRPV5 in RCCD2cells.
3.2.2. Patch–clamp
RCCD2cells were stimulated with E2in the presence or absence
of ruthenium red. Fig. 6 illustrates representative whole-cell cur-
rent traces in (a) control (untreated) and in E2(20nM) treated cells
in the absence (b) or presence (c) of ruthenium red (50?M). Addi-
tion of ruthenium red to the bath induces substantial decrease in
current amplitudes. Fig. 6d shows I–V relationship measurements
demonstrating that E2application stimulated a mean maximal
increase in inward whole-cell currents. E2increased whole-cell
current amplitudes from basal values in control (untreated) cells
of −222±47pA to −734±68pA at a Vp−120mV (n=10, p<0.01).
Adding ruthenium red significantly reduces the E2-activated mean
maximal increase in whole-cell currents from −734±68pA to
−255±57pA at a Vp−120mV (n=7, p<0.02), corresponding to a
reduction in current of approximately 65%.

Page 6

M. Irnaten et al. / Steroids 74 (2009) 642–649
647
Fig. 6. E2-induced inward Ca2+currents are sensitive to ruthenium red. Cells were
stimulated with 20nM E2, either in the presence or absence 50?M of ruthenium
red (RuR), or “low” (1.5mM) extracellular Ca2+conditions. The pipette solution was
supplemented with 10mM BAPTA. (a) Typical whole-cell current traces recorded in
control (untreated) cells. (b) Typical whole-cell current traces recorded in E2treated
cells (20nM E2). (c) Typical whole-cell current traces recorded showed that the
increase of Ca2+currents induced by E2treatment was prevented when cells were
pretreated for 15min with 50?M RuR. (d) Current–voltage relationships of TRPV5
channels: 20nM E2in “low” Ca2+conditions (?); Krebs supplemented with 10mM
Ca2+, 20nM E2+50?M RuR (?); 20nM E2alone (?). Data represent the mean±SEM
of seven cells (p<0.02).
The whole-cell conductance (Gc) calculated over the Vprange
−120 to −60mV was increased to 403±23pS (n=10, p<0.01) with
E2treatmentcomparedtocontroluntreatedcells(111±5pS).Expo-
sure of cells to ruthenium red (50?M) reduced the E2-induced Gc
to 119±12pS (n=7, p<0.02), which was not significantly different
from the Gc recorded in control (untreated) cells (111±5pS, n=7,
p>0.05). These data reinforce the conclusion of the contribution of
TRPV5 channels in response to E2induced whole-cell inward Ca2+
currents in RCCD2cells.
3.3. E2-induced calcium entry channels activation is suppressed
by siRNA targeting TRPV5 in RCCD2cells
As ruthenium red has been only reported as an effective but not
specific inhibitor of TRPV5 channel [33], siRNA has been employed
to specifically suppress TRPV5 expression and activity. Three dif-
ferent siRNAs (siRNA#1, siRNA#2 and siRNA#3) targeting different
sequences of TRPV5 were tested in RCCD2cells. Fig. 7A and B illus-
trate the expression of TRPV5 protein in control siRNA (Functional
non-coding siRNA #1, Ambion Research Inc., UK) and three TRPV5-
siRNAs transfected RCCD2cells. Western blot analysis revealed no
significant decrease in TRPV5 expression following transfection of
individual siRNA (Fig. 7A and B; lanes 2–4). However, combination
of co-transfections is more efficient. The most efficient silencing
expression of TRPV5 was the co-transfection with siRNA#2 and #3
(lane 7). Therefore, co-transfection of RCCD2cells with siRNA#2
and#3 has been used for functional studies and the control func-
tional non-coding siRNA#1 has been used as control-siRNA. The
possibility that the calcium influx is mediated by TRPV6 has been
tested and no significant knockdown expression of TRPV6 was
obtained when cells were transfected or co-transfected with differ-
ent siRNA targeting TRPV5 (Fig. 7A). Western blot in Fig. 7A TPRV6
Fig.7. SiRNAknockdownofratTRPV5proteinexpressioninRCCD2cells.(A)Western
blotexperimentsperformedwithtotalproteinpreparedfromRCCD2epithelialcells.
Lane 1: non-coding siRNA transfected cells (control); lane 2, 3 and 4: transfected
cells with siRNA#1; #2 and #3, respectively. Lanes 5–8: combined co-transfected
cells with siRNA#1 & #2; siRNA#1 and #3; siRNA#2 and #3; and siRNA#1, #2
and #3, respectively. Data are expressed as mean±SEM (* denotes significant
differencesbetweennon-transfected(control)cellsandcellstransfectedwithcorre-
sponding siRNA. *p<0.05; ***P<0.001). (B) Immuno-blot analysis showed that cells
co-transfected with siRNA#2, #3 are the most efficient for silencing TRPV5 protein
expressionrelativetotheircontrols.Functionalnon-codingcontrol#1(Ambion,Ltd.,
UK) has been used as siRNA control. TRPV6 has been used as a control to examine
the specificity of TRPV5 knockdown expression. Note that no significant knockdown
expression of TRPV6 in all RCCD2cells transfected with different siRNAs targeting
TRPV5. ?-actin has been used as loading control. Data are representative of three
similar experiments.
expressionseemstobedownregulatedbysomesiRNAs,particularly
siRNA 2 and 3. This is a representative Western blot, on average of
three different experiments, no statistically significant difference
has been found.
Fig. 8 shows (A) a typical I–V relationship experiment and (B)
averaged data of whole-cell Ca2+currents entering through TRPV5
obtained at the first voltage step of −120mV. The results showed
that E2induced inward whole-cell currents rise in cells trans-
fected with control functional non-coding siRNA, but E2failed in
cells co-transfected with siRNA#2/3, corresponding to E2control
of −1089±92pA (at Vp=−120mV) (n=12, p<0.005) compared to
E2responses in siRNA#2/3 cells of −141±44pA (at Vp=−120mV)
(n=6, p<0.005). Taken together, these results demonstrate that co-
transfectionwithsiRNA#2/3substantiallydecreasedTRPV5protein
expression level leading to the inhibition of TRPV5 activity and E2
activation of [Ca2+]irise involving TRPV5 channels.
4. Discussion
This study provides, for the first time, evidence of a rapid stim-
ulatory effect of 17?-estradiol on [Ca2+]iinvolving TRPV5.

Page 7

648
M. Irnaten et al. / Steroids 74 (2009) 642–649
Fig. 8. Rapid effect of E2on Ca2+currents in siRNA-TRPV5 transfected cells. Patch
clamp experiments were performed in cells either transfected with control siRNA or
co-transfected with siRNA#2/3. Cells were treated with E2(20nM) in the presence
of 10mM Ca2+in the bath solution. (A) Representative current–voltage relationship
experiment performed in cells transfected with functional non-coding siRNA (n=8)
and cells co-transfected with siRNA#2/3 (n=8). (B) Normalized data of E2induced
TRPV5 currents recorded at the first voltage step of −120mV from cells transfected
with control siRNA and siRNA#2/#3. Data represent the mean±SEM (*** indicates
significant differences between control and transfected cells, p<0.02).
Using RT-PCR and Western blot analysis we have demonstrated
that TRPV5 is present at both mRNA and protein expression levels
in RCCD2cells. We have also provided evidence for expression of
TRPV5 in native iCCD in addition to the DCT2 and CNT localization.
Usingthepatch–clamptechnique,westudiedtheelectrophysiolog-
ical properties of Ca2+-dependent channels in RCCD2cells. In the
presence of monovalent cations and Ca2+these channels initially
displaydecreaseofcurrentsasextracellularCa2+levelsarereduced,
but at very low extracellular Ca2+levels, currents then increase
beyond the amplitude observed in the presence of high extracel-
lular Ca2+levels, due to the increasing permeability to monovalent
cations including Na+, loss of selectivity in the absence of diva-
lent cations. This behaviour is thought to be related to the affinity
difference between monovalent and divalent cations in the chan-
nel pore. These Na+/Ca+selectivity features are in agreement with
those reported previously by Nilius et al. [33]. Whole-cell current
was minimal over a ±100mV range under 1.5mM Ca2+conditions
and was significantly activated by increasing [Ca2+]e. The Ca2+-
dependent whole-cell current could also be activated at any given
extracellular calcium concentration by membrane hyperpolariza-
tion.The[Ca2+]eandvoltagedependencecharacteristicsareknown
featuresofTRPV5channelsand,takentogetherwiththeRT-PCRand
Western blotting data, indicate the functional expression of TRPV5
channels in RCCD2cells.
The E2-induced whole-cell and [Ca2+]iwere increased when
cellswereexposedtoincreasing[Ca2+]inthebathsolution,indicat-
ing that Ca2+influx is initiated from the extracellular compartment
and involves Ca2+entry into the cell. Patch–clamp measurements
performed under similar conditions demonstrated that the whole-
cell conductance has been substantially decreased and remained
unaffectedbyE2treatmentwhenCa2+wasremovedfromtheextra-
cellular bathing solution. Ruthenium red is known to be the most
effective blocker of TRPV5 (IC50=111nM) [33].
In RCCD2 cells, the maximum inhibitory effect of ruthenium red
(RR) was obtained only at 50?M, 1–10?M RR have been tested
and no significantly inhibitory effect on the E2induced currents
was observed (data not shown). Pre-treatment of cells with 50?M
ruthenium red in the bathing solution significantly reduced the
number of cells responding with a rise in [Ca2+]iafter E2exposure.
Moreover, patch–clamp analysis showed a decrease in whole-cell
conductance by 70% when the cells were pre-treated with ruthe-
nium red before E2stimulation. These results implicate Ca2+entry
via possibly TRPV5 during E2response.
SiRNA knockdown was employed as an alternative approach
to specifically suppress TRPV5 channel expression and activity. To
assess the importance of TRPV5 expression in RCCD2cells, and
its contribution to E2induced rise in [Ca2+]i, specific siRNA was
transfected into RCCD2cells. Since different siRNAs targeting the
same gene are often differentially effective in silencing the expres-
sion of their target, three different siRNAs (siRNA#1, siRNA#2 and
siRNA#3) directed against different target sequences in rat TRPV5
were transfected into RCCD2cells. Combined co-transfection with
siRNA#2 and siRNA#3 showed substantial silencing of TRPV5 pro-
tein expression level in RCCD2cells. To associate the expression
silencing to functional silencing, whole-cell current measurements
were performed in TRPV5 siRNA transfected cells compared to
controlfunctionalnon-codingsiRNAtreatedcells.ThesiRNAknock-
down experiments clearly provide the evidence of the contribution
of TRPV5 in E2-induced Ca2+influx in RCCD2cells.
As the latency of onset of steroid hormone genomic responses
is approximately 30min, our results represent a rapid (within
3–6min)non-genomicresponsetoE2.Theprimarycharacteristicof
the rapid non-genomic pathway shared by all steroid hormones is
that signaling responses occur with a rapid time course of seconds
to minutes, which is incompatible with either RNA transcription
or protein synthesis. In the case of estrogen, studies have demon-
strated rapid non-genomic effects of this steroid on a variety of
target cells and tissues and in a wide variety of signaling pathways
[34].
The physiological role for non-genomic action of 17?-estradiol
in renal cortical duct cells may serve to enhance the retention and
re-absorption of Ca2+. Taken together, the results of our studies
indicate that 17?-estradiol induces a rapid intracellular calcium
response via Ca2+entry through the epithelial Ca2+channels TRPV5
in RCCD2cells. The signaling mechanisms and receptor(s) involved
inE2modulationofTRPV5channelsandtheirroleintransepithelial
Ca2+transport remain to be identified.
To date TRPV5 has not been localized to CCD while TRPV6 has.
Thus, we showed that TRPV6 is expressed in these cells. Further,
the close homology between these channels may falsely identify
one as the other despite several approaches. Functionally, it will
be hard to discriminate between both channels. To this end proof
that the siRNA and antibody is specific for TRPV5 and not TRPV6
has been preformed. To discriminate between TRPV5 and TRPV6,
Westernblotexperimentsdemonstratedthatsi-RNA-TRPV5knock-
down does not affect the expression of TRPV6 (see Fig. 7A). In
addition, based on knock-down functional experiments (i.e. in both
patch–clamp or Ca2+imaging experiments we have not seen any
significant [Ca2+]iincrease after E2stimulation in TRPV5-siRNA
transfected cells), we concluded that TRPV5 play a key role in this
Ca2+response and we considered that TRPV5 was the major Ca2+
channel involved in response to E2 stimulation in RCCD2 cells.

Page 8

M. Irnaten et al. / Steroids 74 (2009) 642–649
649
Functional regulatory mechanism of TRPV channels by estrogen
in renal collecting duct cells is of great importance for the bet-
ter understanding of transepithelial reabsorption of calcium and,
as a consequence, may reveal novel pharmacological and thera-
peutic targets for the treatment of several disorders of calcium
metabolism such as idiopathic hypercalciuria, stone disease and
postmenopausal osteoporosis.
Acknowledgements
We thank Dr N. Farman for providing the RCCD2cell line. This
research was supported by the Higher Education Authority (HEA),
Ireland, Programme for Human Genomics PRTLI Cycle 3 grant.
References
[1] Berridge MJ, Lipp P, Bootman MD. The versatility and universality of calcium
signalling. Nat Rev Mol Cell Biol 2000;1:11–21.
[2] Bronner F. Renal calcium transport: mechanisms and regulation—an overview.
Am J Physiol 1989;257:707–11.
[3] Tashjian Jr AH. Homeostasis of plasma calcium: effects of actinomycin D,
parathyroidectomy and thyrocalcitonin. Endocrinology 1965;77:375–81.
[4] Norman AW, Henry HL, Malluche HH. 24R,25-Dihydroxyvitamin D3 and 1
alpha,25-dihydroxyvitamin D3are both indispensable for calcium and phos-
phorus homeostasis. Life Sci 1980;27:229–37.
[5] Prince RL. Counterpoint: estrogen effects on calcitropic hormones and calcium
homeostasis. Endocr Rev 1994;15:301–9.
[6] Young MM, Jasani C, Smith DA, Nordin BE. Some effects of ethinyl oestradiol on
calcium and phosphorus metabolism in osteoporosis. Clin Sci 1968;34:411–7.
[7] Young MM, Nordin BE. The effect of the natural and artificial menopause on
bone density and fracture. Proc R Soc Med 1969;62:242.
[8] Nordin BE, Horsman A, Marshall DH, Simpson M, Waterhouse GM. Calcium
requirement and calcium therapy. Clin Orthop Relat Res 1979;140:216–39.
[9] Adami S, Gatti D, Bertoldo F, Rossini M, Fratta-Pasini A, Zamberlan N, et al. The
effects of menopause and estrogen replacement therapy on the renal handling
of calcium. Osteoporos Int 1992;2:180–5.
[10] Prince RL, Smith M, Dick IM, Price RI, Webb PG, Henderson NK, et al. Pre-
vention of postmenopausal osteoporosis. A comparative study of exercise,
calcium supplementation, and hormone-replacement therapy. N Engl J Med
1991;325:1189–95.
[11] Montell C, Rubin GM. Molecular characterization of the Drosophila trp locus:
a putative integral membrane protein required for phototransduction. Neuron
1989;2:1313–23.
[12] Hoenharderop JG, van Der Kemp AW, Hartog A, van de Graaf SF, van Os
CH, Willems PH, et al. Molecular identification of the apical Ca2+chan-
nel in 1,25-dihydroxyvitamin D3-responsive epithelia. J Biol Chem 1999;274:
8375–8.
[13] Nilius B, Vennekens R, Prenen J, Hoenderop JG, Droogmans G, Bindels RJ. The
single pore residue Asp542 determines Ca2+permeation and Mg2+block of the
epithelial Ca2+channel. J Biol Chem 2001;276:1020–5.
[14] MontellC.Physiology,phylogeny,andfunctionsoftheTRPsuperfamilyofcation
channels. Sci STKE 2001;90:RE1.
[15] Berridge MJ. Elementary and global aspects of calcium signalling. J Physiol
1997;499:291–306.
[16] van Abel M, Hoenderop JG, van der Kemp AW, van Leeuwen JP, Bindels
RJ. Regulation of the epithelial Ca2+channels in small intestine as stud-
ied by quantitative mRNA detection. Am J Physiol Gastrointest Liver Physiol
2003;285:78–85.
[17] Sanchez-BuenoA,SanchoMJ,CobboldPH.Progesteroneandoestradiolincrease
cytosolic Ca2+in single rat hepatocytes. Biochem J 1991;280:273–6.
[18] Lieberherr M, Grosse B, Kachkache M, Balsan S. Cell signaling and estrogens in
female rat osteoblasts: a possible involvement of unconventional nonnuclear
receptors. J Bone Miner Res 1993;8:1365–76.
[19] Picotto G, Massheimer V, Boland R. Acute stimulation of intestinal cell calcium
influx induced by 17 beta-estradiol via the cAMP messenger system. Mol Cell
Endocrinol 1996;119:129–34.
[20] DoolanCM,HarveyBJ.ModulationofcytosolicproteinkinaseCandcalciumion
activity by steroid hormones in rat distal colon. J Biol Chem 1996;271:8763–7.
[21] Stefano GB, Prevot V, Beauvillain JC, Fimiani C, Welters I, Cadet P, et al. Estradiol
coupling to human monocyte nitric oxide release is dependent on intracellu-
lar calcium transients: evidence for an estrogen surface receptor. J Immunol
1999;163:3758–63.
[22] MacNamara B, Winter DC, Cuffe J, O’Sullivan G, Harvey BJ. Mechanism of
oestrogen-induced salt and water retention. Surg Forum 1995;81:560–2.
[23] MacNamara B, Winter DC, Cuffe J, O’Sullivan G, Geibel J, Harvey BJ. Real time
imaging of the rapid nongenomic oestrogen effects on cellular ion transport.
Surg Forum 1997;83:522–4.
[24] Condliffe SB, Doolan CM, Harvey BJ. 17b-Oestradiol acutely regulates Cl−secre-
tion in rat distal colonic epithelium. J Physiol 2001;530:47–54.
[25] HarveyBJ,AlzamoraR,HealyV,RenardC,DoolanCM.Rapidresponsestosteroid
hormones: from frog skin to human colon. A homage to Hans Ussing. Biochim
Biophys Acta 2002;1566:116–28.
[26] HarveyBJ,HigginsM.NongenomiceffectsofaldosteroneonCa2+inM-1cortical
collecting duct cells. Kidney Int 2000;57:1395–403.
[27] Blot-Chabaud M, Laplace M, Cluzeaud F, Capurro C, Cassingéna R, Vandewalle
A, et al. Characteristics of a rat cortical collecting duct cell line that maintains
high transepithelial resistance. Kidney Int 1996;50:367–76.
[28] Djelidi S, Beggah A, Courtois-Coutry N, Jay M, Cluzeau F, Viengchareun S, et
al. Basolateral translocation by vasopressin of the aldosterone-induced pool of
latent Na-K-ATPases is accompanied by a1-subunit dephosphorylation: study
in a new aldosterone-sensitive rat cortical collecting duct cell line. J Am Soc
Nephrol 2001;12:1805–18.
[29] Giegerich R, Meyer F, Schleiermacher C. GeneFisher-software support for the
detection of postulated genes. Proc Int Conf Intell Syst Mol Biol 1996;4:68–77.
[30] Burnette WN. Western blotting: electrophoretic transfer of proteins from
sodium dodecyl sulphate–polyacrylamide gels to unmodified nitrocellulose
and radiographic detection with antibody and radioiodinated protein A. Anal
Biochem 1981;112:195–203.
[31] Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch–clamp
techniques for high-resolution current recording from cells and cell-free mem-
brane patches. Pflügers Archiv 1981;391:85–100.
[32] David E, Clapham. Sorting out MIC, TRP, and CRAC Ion Channels. J Gen Physiol
2002;120:217–20.
[33] Nilius B, Prenen J, Vennekens R, Hoenderop JG, Bindels RJ, Droogmans G. Phar-
macological modulation of monovalent cation currents through the epithelial
Ca2+channel ECaC1. Br J Pharmacol 2001;134:453–62.
[34] Falkenstein E, Tillmann HC, Christ M, Feuring M, Wehling M. Multiple actions
of steroid hormones—a focus on rapid, non-genomic effects. Pharmacol Rev
2000;52(4):513–56.