H

GYa

b

c

a

ARR1AA

KTZCC

1

C[ebTtaTTwTtttTn

U

m

0d

Cell Calcium 49 (2011) 43–55

Contents lists available at ScienceDirect

Cell Calcium

journa l homepage: www.e lsev ier .com/ locate /ceca

eavy metal cations permeate the TRPV6 epithelial cation channel

ergely Kovacsa,c,∗, Tamas Dankob, Marc J. Bergerona,c, Bernadett Balazsb,oshiro Suzukia, Akos Zsemberyb, Matthias A. Hedigera,c,∗

Institute of Biochemistry and Molecular Medicine, University of Bern, 3012 Bern, SwitzerlandInstitute of Human Physiology and Clinical Experimental Research, Semmelweis University, 1094 Budapest, HungarySwiss National Centre of Competence in Research, NCCR TransCure, University of Bern, Switzerland

r t i c l e i n f o

rticle history:eceived 16 July 2010eceived in revised form0 November 2010ccepted 15 November 2010vailable online 13 December 2010

eywords:

a b s t r a c t

TRPV6 belongs to the vanilloid family of the transient receptor potential channel (TRP) superfamily. Thiscalcium-selective channel is highly expressed in the duodenum and the placenta, being responsible forcalcium absorption in the body and fetus. Previous observations have suggested that TRPV6 is not onlypermeable to calcium but also to other divalent cations in epithelial tissues. In this study, we testedwhether TRPV6 is indeed also permeable to cations such as zinc and cadmium. We found that the basalintracellular calcium concentration was higher in HEK293 cells transfected with hTRPV6 than in non-transfected cells, and that this difference almost disappeared in nominally calcium-free solution. Live cell

RPV6incadmiumalcium channel

imaging experiments with Fura-2 and NewPort Green DCF showed that overexpression of human TRPV6increased the permeability for Ca2+, Ba2+, Sr2+, Mn2+, Zn2+, Cd2+, and interestingly also for La3+ and Gd3+.These results were confirmed using the patch clamp technique. 45Ca uptake experiments showed thatcadmium, lanthanum and gadolinium were also highly efficient inhibitors of TRPV6-mediated calciuminflux at higher micromolar concentrations. Our results suggest that TRPV6 is not only involved in calcium

ransps.

transport but also in the ttoxicological implication

. Introduction

The transient receptor potential channel TRPV6 also known asaT1, was first cloned from rat duodenum by Peng et al. in 19991]. The TRPV6 gene is located on chromosome 7 in humans. Thencoded protein comprises 725 amino acids, has six transmem-rane domains, and intracellular N- and C-termini like all otherRPV family members. There are three ankyrin repeats on the N-erminal part of the protein, whereas a calmodulin-binding sitend a PKC phosphorylation site reside on the C-terminal part.he putative pore region is localized between TM5 and TM6.RPV6 shares relatively high sequence homology (75% identity)ith TRPV5 but significantly less with the other members of the

RPV family (30–35%) [2]. The �-glucuronidase Klotho enhanceshe activity of TRPV6 via alteration of the N-glycosylation site on

he first extracellular loop [3]. TRPV6 also shows greater func-ional similarity to TRPV5 than to other TRPV channels. UnlikeRPV1–4, TRPV5 and TRPV6 are highly calcium-selective chan-els, and can form homo- or heteromultimer channels. They are

∗ Corresponding authors at: Institute of Biochemistry and Molecular Medicine,niversity of Bern, 3012 Bern, Switzerland.

E-mail addresses: gergely.kovacs@mci.unibe.ch (G. Kovacs),atthias.hediger@mci.unibe.ch (M.A. Hediger).

143-4160/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.oi:10.1016/j.ceca.2010.11.007

ort of other divalent cations, including heavy metal ions, which may have

© 2010 Elsevier Ltd. All rights reserved.

not sensitive to capsaicin, heat or osmotic stress. Their pivotalrole is in calcium (re)absorption in renal and intestinal epithe-lia.

TRPV6 is mainly expressed in absorptive and secretory epithe-lia, where it is involved in transplacental calcium transport andin duodenal calcium absorption. The role of placental and duode-nal TRPV6 in the calcium homeostasis of the human body (fetusand adult) has been widely investigated. In the placenta, wherethe expression of TRPV6 is the highest, TRPV6 is a major pathwaythrough which calcium is supplied to the fetus. There it is expressedin the syncytiotrophoblasts [4]. In the gastrointestinal tract, TRPV6is expressed in the duodenum, proximal jejunum and in the colon[1,5]. In addition to the placenta and gut, TRPV6 is also expressedin the pancreas, salivary gland, mammary gland and prostate butits function is still unclear in these organs.

Our group showed that the serum and amniotic calcium con-centrations as well as the growth rate of murine TRPV6-knockoutfetuses were significantly lower [6], suggesting that TRPV6 is essen-tial for calcium supplementation and optimal development of thefetus. Moreover, the implication of TRVP6 and several other placen-

tal calcium transporters in pre-eclampsia was recently reported [7].Under calcium-restricted conditions, a lack of TRPV6 in adult ani-mals exerts a significant effect on calcium homeostasis, suggestingthat duodenal TRPV6 is a major calcium absorptive mechanism inthe gut [8,9].

4 Calciu

aonRCcis[

tcitr[tvisst

To

2

2H

1wcs

cpc85tab

c2mmAcc

2

cPw11iq

4 G. Kovacs et al. / Cell

There have been several studies that investigated the perme-bility of TRPV6; however none of them looked at the permeabilityf human TRPV6 in mammalian expression systems, or of endoge-ous TRPV6 to different trace elements and toxic heavy metals.at TRPV6 expressed in Xenopus oocytes was reported to transporta2+, Ba2+, Sr2+ but not Mg2+ or any of the other di- or trivalentations examined [1]. When human TRPV6 (hTRPV6) was studiedn the same system, similar results were obtained [10]. Barium andtrontium also evoked a current in mammalian expression systems11].

Magnesium was not found to be transported by TRPV6, evenhough intracellular magnesium is a key player in determining thealcium selectivity of TRPV6 [1,12]. There are a few studies report-ng that the ancestral form of mammalian TRPV5 and 6 in fish alsoransports zinc. The FrECaC (epithelial calcium channel from Fuguubripes) was found to transport Zn2+ and to a smaller extent Fe2+

13]. In freshwater rainbow trout gill, calcium and zinc were showno use the same apical entry pathway, which had a much higher Km

alue for calcium than for zinc [14]. In addition, 1�,25-(OH)2D3ncreased zinc uptake as well as OmECaC and OmSLC39A1 expres-ion [15]. In rat or pig brush border membrane vesicles from themall intestine, zinc, cadmium and calcium were reported to usehe same channel-like pathway to enter the cells [3,16,17].

In this study, we provide evidence for the first time that humanRPV6 is indeed permeable to zinc and cadmium, as well as to somether divalent and trivalent cations.

. Methods

.1. Cell culture and transfection of pEYFP-C1-TRPV6 intoEK293 cells

HEK293 and MCF-7 cells were cultured in DMEM and RPMI640 cell culture media respectively. Media were supplementedith 10% FBS, 10 mM HEPES, 1 mM sodium pyruvate, and 1% peni-

illin/streptomycin at 37 ◦C in a cell culture incubator. Cells wereubcultivated when confluency reached 90%.

For ion imaging experiments, cells were plated at 300,000ells/well density onto No. 00 coverslips coated with 100 �g/mloly-d-lysine in 35 mm dishes. For 45calcium uptake, 400,000ells were placed into poly-d-lysine-coated 35 mm dishes. After–16 h, cells were transfected with 2 �g pEYFP-C1-TRPV6 using�l Lipofectamine 2000 per dish, as described in the manufac-

urer’s protocol. After 4 h, transfection medium was replaced withntibiotic-free medium. Transfection efficiency was estimated toe 60–70% using fluorescence microscopy.

To establish stable pTagRFP-C1-hTRPV6 expressing HEK 293ells, first we plated and transfected cells as described above. After4 h of transfection when cells started to express our construct,edium was exchanged with normal growth medium supple-ented with 500 �g/ml G418. Medium was replaced every day.fter several days, when most of the non-transfected cells died,ells were plated into 96-well plates at 1 cell/well density. Stablelones were selected by fluorescence microscopy.

.2. Cell biotinylation and Western blotting

All steps of cell surface biotinylation were carried out in a 4 ◦Cold room using ice-cold solutions. First, cells were rinsed once withBS–Ca–Mg (PBS containing 0.1 mM CaCl2 and 1 mM MgCl2). After-

ards, surface proteins were biotinylated by incubating cells with

.5 mg/ml sulfo-NHS-LC-biotin in 10 mM triethanolamine (pH 7.4),mM MgCl2, 2 mM CaCl2, and 150 mM NaCl for 90 min with hor-

zontal shaking at 4 ◦C. After labeling, samples were washed withuenching buffer (PBS containing 1 mM MgCl2, 0.1 mM CaCl2, and

m 49 (2011) 43–55

100 mM glycine) for 20 min at 4 ◦C, and then rinsed three timeswith PBS. Cells were finally lysed in lysis buffer for 30 min accord-ing to Ref. [18] and lysates were cleared by centrifugation. Proteinconcentrations were determined by DC Protein Assay. Portion ofcell lysates of equivalent amounts of protein were equilibratedovernight with streptavidin agarose beads at 4 ◦C. Beads werewashed sequentially with solutions A [50 mM Tris·HCl (pH 7.4),100 mM NaCl, and 5 mM EDTA] three times, B [50 mM Tris·HCl (pH7.4) and 500 mM NaCl] twice, and C (50 mM Tris·HCl, pH 7.4) once.Biotinylated surface proteins were then released by heating to 95 ◦Cwith 4× Lämmli buffer. Proteins from the total lysate or intracel-lular fraction were also heated to 95 ◦C for 5 min with 4× Laemmlibuffer, after adjusting the protein concentration to 1 mg/ml.

When different lysis buffers were tested, cells were washedthree times with ice-cold PBS–Ca–Mg followed by lysis in the differ-ent buffers for 30 min in a 4 ◦C cold room. Samples were spun downand supernatants were saved. Protein concentration was measuredand adjusted to 1 mg/ml. Samples were diluted with 4× Laemmlibuffer and boiled at 95 ◦C for 5 min.

Samples were run on a 6% SDS gel with 15 �g protein loadedinto each lane. Using the semi-dry transfer method, samples weretransferred onto a PVDF membrane in Dunn’s buffer. Membraneswere blocked overnight with PBS containing 5% milk, 0.5% BSAand 0.02% NaN3. Afterwards, samples were incubated in blockingbuffer containing the primary antibody (1:2000 for chicken anti-GFP (Abcam ab13970) or 1:200 for goat anti-rabbit TRPV6 (BiomolBML-SA567)) at room temperature for 1.5 h followed by threewashes with PBS. HRP-conjugated donkey anti-chicken (1:8000)and HRP-conjugated goat anti-rabbit (1:20,000) antibodies wereused as secondary antibodies. After three consecutive washes withPBS, the enhanced chemiluminescence (ECL) method was used fordetection.

2.3. Deglycosylation experiments

In these experiments, 50 �l of biotinylated, cell surface pro-teins were first denatured in 5 �l of 10× denaturing buffer (2% SDSand 10% 2-mercapto-ethanol) at 99 ◦C for 10 min. They were thendigested for 3 h at 37 ◦C with 4 �l PNGase F or 10 �l Endo H enzymein 50 �l of 2× digestion buffer (100 mM Na2HPO4 and 2% NP40, pHadjusted to 7.5 for PNGaseF, and to 5.5 for EndoH). The digestionswere terminated by the addition of 100 �l 2× protein sample buffer.Samples were run, transferred and developed as described above.

2.4. Confocal microscopy

HEK293 cells transfected with pEYFP-C1-hTRPV6 were fixedwith 4% paraformaldehyde at 37 ◦C for 15 min after rinsing thecells thoroughly with PBS. After three washes with PBS, nuclei werecounterstained with Hoechst33342 (1:1000) at room temperaturefor 5 min. After three final washes with PBS, cells were mounted,and imaged using a Nikon C1 confocal laser scanning microscopysystem.

2.5. Live cell ion imaging

Cells were imaged after 48–72 h. Non-transfected and trans-fected cells on the same piece of coverslip were loaded with either5 �g/ml Fura-2 AM (stock solution 5 �g/�l) for one hour, 6.8 �MMag-Fura-2 for 10 min or 5 �M NewPort Green DCF (stock solution5 mM) for 45 min at 37 ◦C in serum-free medium in a cell culture

incubator. Fluorescence dyes were dissolved in DMSO containing20% Pluronic-F127. Additionally, the loading medium contained1 mM probenecid to prevent dye leakage. Thereafter, cells wereplaced into modified Krebs–Ringer HEPES (KRH) (150 mM NaCl,4.8 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mMd-glucose, and 10 mM

Calciu

HtHeteFFtGow

Tstmd

2fl

2aw1FS4cKww

2

cmtmoiasdtdcbabwSfDwi[r1tpcn

G. Kovacs et al. / Cell

EPES, pH 7.4) for 20 min in order to achieve full de-esterification ofhe fluorescence dye. For Ca2+, Ba2+, Sr2+, La3+, Gd3+, Zn2+, Cd2+, andg2+ experiments, Fura-2 as well as Mag-Fura-2 were alternatelyxcited at 340 nm and 380 nm, and the F340/F380 ratio was moni-ored. In all other experiments where Fura-2 was applied, 359 nmxcitation wavelength (isobestic point) was used, and quenching ofura-2 intensity was recorded. The initial rate of the increase of theura-2 ratio or of the decrease of Fura-2 intensity at 359 nm wasaken as the measure of the influx of the particular cation. NewPortreen DCF was excited at 500 nm, and the initial increase of the flu-rescence intensity in response to the addition of different cationsas measured.

Fluorescence measurements were performed on a Nikon EclipseiU inverted microscope equipped with a polychrome V+ lightource. Cells were visualized with a Nikon 40× S Fluor objec-ive. Images were taken with a Hamamatsu Orca-EG cooled,

onochrome CCD camera. Image acquisition and analysis wereone with SimplePCI 6.2 from CImaging.

.6. Fluorescence ion measurements using Flexstation IIuorescence microplate reader

Non-transfected and stable clones of hTRPV6-expressing HEK93 cells were plated on poly-d-lysine-coated, black 96-well platest cell density of 40,000 cells/well. After 24 h, growth mediumas aspirated and cells were loaded with Calcium 3 FLIPR dye in

00 �l Krebs–Ringer buffer for 1 h at 37 ◦C. Plates were placed intolexstation II, fluorescence microplate reader (Molecular Devices,unnyvale, CA, USA). Fluorescence intensities were measured at85 nm excitation and 525 nm emission wavelengths and 515 nmut-off filter was used as well. After the first 100 s, 50 �l ofrebs–Ringer buffer containing various concentrations of cadmiumere added to the wells and changes in fluorescence intensitiesere monitored. Experiments were performed at 37 ◦C.

.7. Patch clamp experiments

Voltage clamp recordings were performed in the whole-cellonfiguration [19] using an Axopatch 200B amplifier (Axon Instru-ents). Micropipettes were pulled from borosilicate glass capillary

ubes (Harvard Apparatus) using a P-97 Flaming-Brown typeicropipette puller (Sutter Instrument), and had a tip resistance

f 3–6 M� when filled with pipette solution. Patch pipette fill-ng solution contained (in mM): 140 NMDG, 1 MgCl2, 20 EGTAnd 10 HEPES, adjusted to pH 7.2 with HCl. Normal extracellularolution contained (in mM): 147 NaCl, 3 KCl, 1 CaCl2, 2 MgCl2, 10-glucose, 10 HEPES, adjusted to pH 7.4 with NaOH. Control solu-ion with no divalent cations contained (in mM): 147 NMDG, 15-glucose, 10 HEPES, adjusted to pH 7.4 with HCl. After whole-cellonfiguration was obtained in normal solution, cells were initiallyathed in control solution without divalent cations for 5 min tollow proper dialysis of the cell interior. During recordings, theath solution was changed to a control solution supplementedith 2 mM test divalent cation (XCl2; X = Ca2+, Cd2+, Zn2+, Co2+).

olutions were delivered by continuous perfusion with a gravity-ed delivery system. TRPV6-expressing cells were selected using aiaphot 300 inverted patch clamp microscope (Nikon) equippedith an epifluorescent attachment (Elektro-Optika). Electrophys-

ological measurements were performed as described previously20], with minor modifications. Voltage clamp recordings were car-ied out using ramp commands (−110 mV to +90 mV in 200 ms,

mV/ms) applied every 5 s, starting 40–60 s prior to the introduc-

ion of any test cations (background current detection). The holdingotential was −20 mV between ramps. Capacitative currents wereompensated with analog compensation. Linear leak currents wereot compensated. Series resistance was accepted if lower than five

m 49 (2011) 43–55 45

times the pipette tip resistance. Currents were filtered at 1 kHz(four-pole Bessel filter) and digitized at 5 kHz. Command protocols,data acquisition and analysis were controlled by pClamp 6.03 soft-ware (Axon Instruments). Membrane potentials were corrected forliquid junction potential if greater than 2 mV values were detected.All experiments were performed at room temperature.

2.8. 45Ca uptake assay

Radioactive uptake assays were performed 48 h after trans-fection. Cells were washed twice with nominally calcium-free,modified KRH solution. Afterwards, cells were exposed for a settime to 2 �Ci 45Ca in KRH solution containing 100 �M cold cal-cium with/without the other cations under investigation. Calciumuptake was stopped by rinsing the cells with ice-cold KRH-solution.Cells were lysed in 1 ml 1 N NaOH overnight. The samples weremixed with scintillation liquid, and measured in a liquid scintilla-tion counter (Packard Tri-Carb 2100 TR). Protein concentration wasdetermined by DC Protein Assay.

2.9. Materials

HEK293 cells were obtained from American Type Culture Col-lection (ATCC). Lipofectamine 2000, Fura-2 AM, NewPort GreenDCF, Mag-Fura-2 AM, Pluronic Acid F-127, cell culture medium,fetal bovine serum, cell culture supplements and antibiotics werefrom Invitrogen. The selectivity profile of Newport Green DCF isavailable in the section 19.7 (Fluorescent Indicators for Zn2+ andOther Metal Ions) of The Handbook from Molecular Probes. DCProtein Assay was purchased from Bio-Rad Laboratories (Reinach,Switzerland). pEYFP-C1-TRPV6 was a generous gift from Prof.Christoph Romanin (Institute for Biophysics, Johannes Kepler Uni-versity, Linz, Austria). Calcium 3 assay kit was purchased fromMolecular Devices (Sunnyvale, CA, USA). All other materials wereobtained from Sigma–Aldrich Chemie GmbH (Buchs, Switzerland).

2.10. Statistics

Results are presented as mean ± SEM. Data were compared withthe rank sum t-test. P < 0.05 was considered significant. Non-linearcurve fitting was performed using the Sigma-Plot 11.0 program.

3. Results

3.1. Overexpression of hTRPV6 in HEK293 cells

EYFP-hTRPV6 protein expressed in HEK293 cells was initiallyisolated using three different lysis buffers, as described in refer-ences [5,18], and by PepTag assay for non-radioactive detection ofPKC (Promega). After probing the blots with anti-GFP (Fig. 1A upperpanel) or anti-TRPV6 (Fig. 1A lower panel), we determined that lysisbuffers 2 and 3 were the best for the isolation. We observed anupper ∼105 kDa band and a lower ∼90 kDa band. Surface biotiny-lation experiments revealed that both forms of TRPV6 are present atthe plasma membrane with the higher expression of the lower band(Fig. 1B) in our overexpression system. Pretreatment with PNGaseF which cleaves all types of N-linked oligosaccharides resultedin a complete disappearance of the upper band and lower bandsand appearance of a new more intense band ∼85 kDa (Fig. 1B). Inresponse to Endo H treatment the upper band was resistant, but

the mass of the lower band decreased (Fig. 1B). These data sug-gest that the upper band is the fully glycosylated form of TRPV6whereas the lower band represents the immature form of TRPV6. Arepresentative immunofluorescence image shows the punctuatedexpression pattern of EYFP-TRPV6 overexpressed in HEK293 cells

46 G. Kovacs et al. / Cell Calcium 49 (2011) 43–55

Fig. 1. Expression of pEYFP-C1-hTRPV6 plasmid in HEK293 cells.P g theB EYFP-a uores( olor in

(i

3p

iH3bccdai

FRi

anel A: Western blot analysis using anti-GFP (top) or anti-TRPV6 (bottom) showin: Deglycosylation experiments with PNGase F and Endo H on surface biotinylatedn immature (lower band) form at the plasma membrane. Panel C: Representative flgreen), nucleus (blue). Scale bar is 50 �m. (For interpretation of the references to c

Fig. 1C). Delineation of the plasma membrane could be observedn several cells.

.2. Determination of hTRPV6 di- and trivalent cationermeability with Fura-2 imaging

When intracellular calcium was measured with Fura-2, basalntracellular calcium was increased in EYFP-hTRPV6-transfectedEK cells compared to non-transfected cells (340/380 ratios:.227 ± 0.036 (cell number = 682) vs. 1.435 ± 0.01 (cell num-er = 421), Fig. 2A). When only EYFP was transfected into the

ells, no difference was observed (Fig. 2B). Removal of extra-ellular calcium almost completely abolished this significantifference (340/380 ratios: 1.1317 ± 0.005 vs. 1.269 ± 0.004). Re-dministration of 1 mM Ca2+ evoked a much larger increase ofntracellular calcium in EYFP-hTRPV6-transfected compared to

ig. 2. The expressed EYFP-hTRPV6 is fully functional as a calcium channel in HEK293 ceepresentative Fura-2 tracings show that in EYFP-hTRPV6- (Panel A) but not EYFP-expres

nitial calcium entry after re-administration of extracellular calcium are significantly incr

expression of EYFP-hTRPV6 protein extracted in three different lysis buffers. PanelhTRPV6 shows that TRPV6 is expressed in a fully glycosylated (upper band) and incence confocal image of HEK293 cells transfected with EYFP-TRPV6. EYFP-hTRPV6this figure legend, the reader is referred to the web version of the article.)

non-transfected cells (Figs. 2A and 3A). In EYFP-transfected cells,the 1 mM Ca2+-induced change was the same in both transfectedand non-transfected cells (Fig. 2B).

Next, we tested whether TRPV6 is permeable to other di- andtrivalent cations. In the case of Ba2+, Sr2+, Zn2+, Cd2+, Hg2+ La3+,and Gd3+, the initial rate of increase of the Fura-2 ratio was mea-sured, whereas for Co2+, Ni2+ and Mn2+, it was the initial rate ofdecrease of fluorescence intensity at 359 nm. As shown by othergroups, both barium and strontium can enter the cells throughTRPV6 (Fig. 3A). The cells’ permeability to zinc and cadmium butnot to mercury also greatly increased when TRPV6 was trans-

fected into the cells (Fig. 3B). The rate of increase of the Fura-2ratio in response to lanthanum and gadolinium was also faster inEYFP-hTRPV6-expressing cells (Fig. 3D). The quenching of Fura-2intensity at 359 nm (calcium-insensitive) wavelength was signifi-cantly enhanced in response to manganese, very slightly to cobalt,

lls.sing cells (Panel B) both the basal intracellular calcium concentration and the basaleased.

G. Kovacs et al. / Cell Calcium 49 (2011) 43–55 47

F lent cB flux oz lanthr

bbTttbts

3i

Gecevisiiol(flh

D

ig. 3. EYFP-hTRPV6 significantly increases the permeability of certain di- and trivaar graphs show the summarized data of Fura-2 calcium imaging experiments. Ininc and cadmium from group IIB (Panel B); some transitional metals (Panel C), andepresents at least six separate experiments. *P < 0.05.

ut not at all to nickel (Fig. 3C). We also found a correlation betweenasal calcium level and the rate of entry of the cation permeating viaRPV6 in EYFP-hTRPV6-expressing HEK293 cells (Fig. 4), suggestinghat the more TRPV6 protein expressed at the plasma membrane,he higher the basal intracellular calcium entry and the entry of theasal calcium or other permeable cations. This indirectly indicateshat calcium or other divalent cations release from the intracellulartores is not affected.

.3. Assessment of TRPV6 divalent cation channeling with ionmaging using other fluorescence dyes

To verify the results obtained with Fura-2, we used NewPortreen DCF, a calcium-insensitive fluorescence dye in the nextxperiments. For these measurements, we transfected HEK293ells with a pTagRFP-C1-hTRPV6 construct to avoid the interfer-nce of EYFP and Newport Green DCF fluorescence due to theirery similar spectra. Transfection of RFP-hTRPV6 protein resultedn a punctuated distributional pattern, analogous to the pEYFP con-truct (see Fig. 1C), whereas RFP alone distributed homogenouslyn the cells (Fig. 5A and C). As shown in the representative trac-ngs, Newport Green DCF was indeed insensitive to calcium underur experimental conditions because removal of 1 mM extracel-ular calcium did not induce any change in fluorescence intensity

Fig. 5B and D). Furthermore, there was no difference in baselineuorescence intensity between non-transfected cells and RFP-TRPV6-expressing cells (Fig. 5D).

When we tested cadmium entry, to which ion Newport GreenCF is the most sensitive, we found that the increase of fluores-

ations in HEK293 cells.f calcium, barium, and strontium from the alkaline earth metals group (Panel A);anides (Panel D) are remarkably increased in hTRPV6-expressing cells. Each group

cence intensity in response to 1 mM cadmium was several ordersof magnitude larger in RFP-hTRPV6-transfected cells than in non-transfected cells (Fig. 5B). The transfection of RFP alone did notincrease cadmium influx (Fig. 5D). The increase of fluorescenceintensity evoked by zinc (Fig. 6A) and cadmium (Fig. 6B) was sig-nificantly larger in hTRPV6-transfected cells than non-transfectedcells. There was no difference in mercury and nickel entry (data notshown).

We next tested whether Zn2+ or Cd2+ at micromolar level arepermeable through hTRPV6. Our attempt failed to detect any influxof zinc at micromolar level in HEK293 cells that were transientlywith hTRPV6 (Fig. 7A and C), but we could observe cadmiuminflux through hTRPV6 (Fig. 7B and D). Furthermore, we could alsodemonstrate cadmium influx in stably transfected HEK293 cells(Fig. 8A and B).

We also examined the effect of calcium on zinc and cadmiuminflux through hTRPV6. Our results showed that 1 mM calciumsignificantly blocked the entry of zinc (1 mM) whereas it wascompletely ineffective on the influx of cadmium (1 mM) throughhTRPV6 (Fig. 9A and B, respectively).

3.4. Measurement of hTRPV6 permeability properties with thepatch clamp technique

To further confirm the previously obtained results withzinc, cadmium and cobalt, we used the patch clamp tech-nique in whole-cell configuration. Divalent cation influx wasdetected by monitoring inward currents at -80 mV duringvoltage ramps. Initially, both EYFP-hTRPV6-transfected and non-

48 G. Kovacs et al. / Cell Calcium 49 (2011) 43–55

F in di-B PV6 cc embd s, whi

tstrsa

wmblpttptrri

ig. 4. Correlation between basal intracellular calcium and basal influx rate of certaasal intracellular calcium concentration is dependent on the number of active hTRations channeled by TRPV6 is also determined by TRPV6 channels at the plasma mi- or trivalent cations showed a strong correlation in EYFP-hTRPV6-expressing cell

ransfected HEK293 cells were perfused with an extracellularolution lacking charge carriers (see Section 2). Supplementa-ion of the solution with 2 mM Ca2+ evoked large, inwardlyectifying currents in EYFP-hTRPV6-transfected cells, while nouch current was detected in non-transfected cells (Fig. 10And B).

Next, we found that 2 mM Zn2+ produced a transient currentith peak amplitude of 20–100 pA, suggesting that Zn2+ also per-eates TRPV6 (Fig. 10C). The decay of this transient current may

e due to inhibition of TRPV6 by Zn2+ itself (see Section 4). Fol-owing the transient increase in inward currents, the continuousresence of external Zn2+ reduced current densities below con-rol levels (Fig. 10C). This latter phenomenon is most likely due tohe inhibition of basal Cl− conductance by Zn2+ [21]. Cd2+ (2 mM)

roduced sustained inward currents similar to those observed inhe presence of Ca2+ (Fig. 10D). Additionally, the Cd2+-evoked cur-ent did not decay as fast as the Ca2+-induced current but ratheremained at a relatively high level. Unlike the other divalent cationsnvestigated, the transition metal Co2+ (2 mM) did not evoke an

and trivalent cations in EYFP-hTRPV6-expressing cells.hannels expressed at the plasma membrane. The basal influx rate of di- or trivalentrane. As expected, both intracellular calcium concentration and the influx of thesech further supports our previous findings that TRPV6 is permeable to these cations.

increase in inward currents (Fig. 10E). Furthermore, we also foundthat Zn2+ not only permeated but also modulated hTRPV6 chan-nel activity in a biphasic manner. Inward currents evoked by 2 mMCa2+were efficiently blocked by 2 mM Zn2+ whereas in the pres-ence of 2 �M zinc an increase in peak inward currents was observed(Fig. 10F).

3.5. Zinc and cadmium influx via hTRPV6 in MCF-7 human breastcancer cells

To further confirm that the ability of hTRPV6 to conduct zincand cadmium is not cell type-dependent, we performed ion imag-ing experiments using Fura-2 in MCF-7 human breast cancercells that express very low amount of endogenous TRPV6 as

we showed previously [22]. We found that basal calcium con-centration was significantly higher in MCF-7 cells transientlytransfected with pEYFP-C1-hTRPV6 compared to non-transfectedcells (2.378 ± 0.055, n = 137 vs. 1.320 ± 0.013, n = 160, P < 0.001).This difference almost completely vanished in nominally calcium

G. Kovacs et al. / Cell Calcium 49 (2011) 43–55 49

FI cellsn cellul( e cells

fZnc(

FAt

ig. 5. Expression of pTagRFP-C1-hTRPV6 in HEK293 cells.mages show the expression of RFP-hTRPV6 (Panel A) and RFP (Panel C) in HEK293ot sensitive to changes in intracellular calcium, as there was no change when extraPanel B) but not RFP alone (Panel D) significantly increased cadmium entry into th

ree buffer (1.355 ± 0.015 vs. 1.210 ± 0.007, P < 0.001). When 1 mMn2+ or 250 �M Cd2+ was added to the extracellular milieu a sig-ificant higher increase in Fura-2 ratio was observed in MCF-7ells that overexpressed hTRPV6 compared to non-transfected cellsFig. 11).

ig. 6. Confirmation of increased zinc and cadmium permeabilities in EYFP-TRPV6-expres shown in the vertical point plots, non-transfected HEK293 cells are only slightly permea

he influx of zinc (Panel A) and cadmium (Panel B) in HEK293 cells. Each group contains a

. Representative NewPort Green DCF tracings show that this fluorescence dye wasar calcium was removed (Panels B and D, first arrows). Transfection of RFP-hTRPV6under control conditions.

45

3.6. Modulation of Ca uptake via hTRPV6 by several di- andtrivalent cations

We first established the optimal duration of 45Ca uptake exper-iments in the cells. We found that radioactive calcium uptake

ssing HEK293 cells using the calcium-insensitive Newport Green DCF dye.ble to zinc or cadmium. However, overexpression of hTRPV6 significantly enhancedt least six separate experiments.

50 G. Kovacs et al. / Cell Calcium 49 (2011) 43–55

F ura-2A ag Fun t at 10i

i(riivewhvhm

FA(

ig. 7. Representative tracings showing the effect of zinc and cadmium on mag Fdministration of 1 mM, but neither 1 nor 10 �M zinc induced large increase of mon-transfected cells (Panels A and C). In contrast, cadmium already evoked an effec

n HEK 293 cells transfected with hTRPV6 (Panels B and D)

ncreased with time up to 10 min, whereupon the uptake plateauedFig. 12). A two-minute incubation time was selected because theelative difference between the two groups was the largest in thencreasing part of the curve at this time point. We found that bar-um, cobalt, and mercury did not significantly change 45Ca uptakeia TRPV6 (less than 25%) (Fig. 13A–C). Mercury at a higher dosexerts unspecific effects on cellular calcium homeostasis, which

ould explain its activating effect at 100 �M (Fig. 13B). Strontiumad a slight activating effect (Fig. 13A). Biphasic effects (i.e., acti-ation at low concentration (nanomolar level) and inhibition atigh concentration (micromolar level)) were observed with zinc,anganese, and lanthanum (Fig. 13B–D). Remarkably, gadolin-

ig. 8. Cadmium at low micromolar concentrations is channeled by hTRPV6 in stable TRPverage traces show the effect of various concentrations of cadmium on FLIPR Calcium 3

Panel A) and in non-transfected HEK 293 cells (Panel B). Each panel presents the average

fluorescence in pEYFP-C1-hTRPV6 expressing and non-transfected HEK 293 cells.ra-2 ratio that was significantly robuster in hTRPV6 expressing cells compared to�M (Panel B). The observed response to 10 �M and 1 mM cadmium was also larger

ium, cadmium, and nickel inhibited TRPV6 with no biphasic effect(Fig. 13B–D). The most efficient inhibitors were found to be gadolin-ium, lanthanum, and cadmium.

4. Discussion

TRPV6 is known to be the most highly calcium-selective mem-

ber of the vanilloid transient receptor potential channel family.Although many studies have been conducted to investigate therole of TRPV6 in fetal and adult calcium homeostasis, there is onlyscant information available about TRPV6 as a potential transporterof other di- and trivalent cations. This might be a consequence of

V6-expressing HEK293 clones.fluorescence intensity in HEK 293 cells that were stably transfected with hTRPV6trace derived from 6 individual traces in each condition.

G. Kovacs et al. / Cell Calcium 49 (2011) 43–55 51

F V6. Rep iminisc

tdwspa[rmanigd

Hpwtbmitetd4

igdtpos

ItazAfiiaapda

ig. 9. Effect of extracellular calcium on zinc and cadmium influx mediated by hTRPresence of 1 mM extracellular calcium zinc influx through hTRPV6 is significantly dalcium (Panel B).

he fact that in the first publication characterizing TRPV6 in rat duo-enum in an oocyte expression system, we concluded that TRPV6as permeable only to Ca2+, Ba2+, and Sr2+ [1]. However, there are

everal findings that suggest that mammalian TRPV6 can trans-ort other cations such as zinc, cadmium, etc. Firstly, the commonncestor of TRPV5 and 6 in fish was found to be permeable to zinc13,15]. Secondly, expression of a multidivalent cation channel inat and pig small intestine that transports zinc, calcium, barium, andanganese was also reported [16,17]. Thirdly, increased intestinal

bsorption of cadmium and zinc, as well as increased TRPV6 butot divalent metal iontransporter, DMT1 expression, was observed

n mice kept on a calcium-deficient diet [23,24]. These data sug-est that TRPV6 is permeable not only to calcium but also to otherivalent cations in the duodenum, as well as the placenta.

When we tested the expression of our tagged TRPV6 protein inEK293 cells, we found that TRPV6 protein was expressed at thelasma membrane 48 h after transfection. No endogenous TRPV6as detected in non-transfected cells. We could also confirm that

he TRPV6 expressed at the plasma membrane was functional, asasal intracellular calcium concentration and basal calcium entryeasured with Fura-2 was significantly higher (70-fold difference)

n transfected cells. The 45Ca uptake experiments also showed thatransfected cells had higher calcium permeability but the calciumntry was only twice that of non-transfected cells. This showshat TRPV6 was continuously open under our experimental con-itions. The difference between the Fura-2 measurement and the5Ca uptake assay in terms of TRPV6 activity is probably due to anncrease in calcium buffering capacity by Fura-2, as previously sug-ested [25]. Fura-2 is sensitive not just to calcium but also to otherivalent and trivalent cations. First, we were able to demonstratehat indeed barium and strontium are transported by TRPV6, asreviously reported [26]. Furthermore, these two divalent cationsnly slightly inhibited calcium transport via TRPV6, again as alreadyhown [1,10].

Next, we wanted to test whether elements of periodic groupIB (zinc, cadmium, mercury) could permeate TRPV6, because ofhe importance of trace metals for the human body. Firstly, zinc isn important component of the catalytic site of several metalloen-ymes such as alkaline phosphatase and alcohol dehydrogenase.lso, it is a crucial structural component of hundreds of “zincnger”-containing proteins. An alteration in zinc metabolism can

nduce certain diseases. For example, acrodermatitis enteropathica,

n autosomal recessive disorder causing zinc deficiency, developss a result of a mutation in the gene encoding the zinc transporterrotein ZIP4. Acute zinc toxicity is manifested by nausea, vomiting,iarrhea, headache, and abdominal cramps. Secondly, cadmiumnd mercury are highly toxic to the human body. Mercury binds

presentative tracings of Newport Green DCF fluorescence intensity show that in thehed (Panel A). The TRPV6-mediated cadmium entry was unaffected by extracellular

to the thiol group of the amino acid cysteine and thereby altersthe function of many proteins. Since TRPV6 resides in the duodenaland placental epithelia, it could be important for zinc homeostasisand/or cadmium, mercury toxicity.

Our results with Fura-2 showed that both zinc and cadmiumbut not mercury were transported by TRPV6 overexpressed inHEK293 cells. We further confirmed these findings with thecalcium-insensitive dye, NewPort Green DFC, and the patch clamptechnique. Previously we have shown that overexpression ofhTRPV6 in MCF-7 human breast cancer cells increased basal cal-cium levels as well as basal calcium and barium influx [22]. Inour present study, we found augmentations of zinc and cadmiumpermeabilities by the overexpressing hTRPV6 in these cells.

We could also demonstrate TRPV6-dependent cadmium butnot zinc transport when these cations were administered at lowmicromolar concentration. The negative results with zinc could beexplained by the observation that equimolar calcium significantlydiminished zinc influx via TRPV6 and that distilled water containscalcium in the micromolar range. Our data suggest that hTRPV6 canconduct zinc and cadmium in mammalian expression systems.

Our finding contradicts previously published data in Xenopusoocytes [1,10]. However, there are several important differencesbetween the two model systems. Firstly, TRPV6 can form hetero-oligomers with other TRP channels. Because different TRP channelsare expressed and available for this interaction in amphibian andmammalian cells, permeation properties of TRPV6 can vary con-siderably. Secondly, there could be different regulatory proteins ofTRPV6 or the expression pattern of the same proteins may be dis-tinct in frog oocytes. Thirdly, the different electrochemical gradientfor zinc and cadmium could also explain this inconsistency betweenthe previous and current observations.

Ragozzino et al. reported that zinc permeated nicotinic acetyl-choline receptors while blocking sodium influx through thechannels [27]. Indeed, our data show that zinc evoked significantlysmaller currents and fluorescence signals compared to calcium orcadmium, suggesting that, while zinc permeates, it can interactwith TRPV6 and inhibit its permeability. The transient nature ofthe inward Zn2+ current further supports the idea that zinc ionsinteract with TRPV6 channels, blocking the permeation pathway.Previous studies indicate that zinc ions might also regulate Ca2+

uptake into cells through other entry mechanisms, such as nico-tinic acetylcholine receptor channels, voltage-gated channels, or

ATP-gated P2X purinergic receptor channels [27–29]. In our study,both inward Ca2+ current and 45Ca uptake were inhibited by zincat high micromolar or millimolar concentration. Interestingly, zincat low micromolar concentrations increased TRPV6-mediated 45Cauptake and inward Ca2+ currents, indicating that there are two zinc

52 G. Kovacs et al. / Cell Calcium 49 (2011) 43–55

Fig. 10. Inward currents obtained from EYFP-hTRPV6 expressing HEK-293 cells in the presence of different test cations. Currents were measured every 5 s at −80 mV duringvoltage ramps ranging from −110 to 90 mV in 200 ms. Cells were initially bathed in nominally divalent free solution. Test cations were administered at the time point indicated(arrows). The dashed lines represent zero current. (A) Time course of 2 mM Ca2+ evoked current that reached a peak value within 70 s and subsequently decreased until asteady-state level was reached. Only hTRPV6 expressing cells exhibited the inward current. Representative traces are shown. (B) Original traces demonstrating current–voltagerelationship in the absence (control) and presence of 2 mM Ca2+, measured at time point indicated in A (empty triangles). The Ca2+ current is inwardly rectifying and reversesat more positive potentials compared to control current. (C) Time course of 2 mM Zn2+ evoked inward current that peaked within 30 s then decayed rapidly below controlcurrent values. Note that Zn2+ reduced basal inward currents in non-transfected cells suggesting that this inhibitory effect was independent of hTRPV6. Representative tracesare shown. (D) Time course of 2 mM Cd2+ evoked inward current that peaked within 50 s and subsequently decreased to a steady-state level. Representative traces are shown.(E) Summarized data showing the maximal change in current amplitude detected at −80 mV during voltage ramps in the presence of different divalent cations. Positivev s comn sentet sing cr e abse

bsoft

alues represent an increase, while negative values represent a decrease in current= 6 (Co2+) and for non-transfected cells n = 4 for each test cation. Results are pre

he presence of extracellular Zn2+. In these experiments, Zn2+ was applied in increaepresent mean ± SEM of 4–7 cells and are plotted relative to currents elicited in th

inding sites in the TRPV6 structure: an activatory, high affinityite and an inhibitory, low affinity site. This phenomenon was alsobserved in the case of TRPA1, another member of the TRP super-amily [30]. Thus, we suggest that zinc might play a pivotal role inhe regulation and fine tuning of TRPV6 channel-mediated transep-

pared to control values. For transfected cells n = 10 (Ca2+), n = 8 (Zn2+), n = 6 (Cd2+),d as mean ± SEM. (F) Inward Ca2+ current amplitude in TRPV6 expressing cells inoncentrations (from 2 �M to 2000 �M), prior to the addition of 2 mM Ca2+. Pointsnce of Zn2+ (Icalcium, dashed line) in the same cells.

ithelial Ca2+ transport in various organs, such as the intestine andplacenta.

Cadmium currents and fluorescence signals were similar tothose evoked by calcium. A technical advantage of cadmium is that,since the KD value of Fura-2 and Newport Green DCF for cadmium

G. Kovacs et al. / Cell Calcium 49 (2011) 43–55 53

Fig. 11. Zinc and cadmium influx through TRPV6 overexpressed in MCF-7 human breast cancer cells.R y elevat Cd2+ wc n zincs . *P < 0

imeai

mmuihmfccut

Ft4

es

epresentative traces show that overexpression of TRPV6 in MCF-7 cells significantlhe increase of Fura-2 ratio in response to administration of 1 mM Zn2+ or 250 �Mells. Summarized data show that TRPV6 induced a ∼6 and ∼12 times increase iummarized data of at least 60 individual cells of 6 separate experiments per group

s much lower (these dyes are much more sensitive to cadmium),easuring cadmium influx provides a very sensitive method of

xamining TRPV6 activity. Additionally, because cadmium does notctivate calmodulin at higher micromolar concentrations, it furthermproves the usefulness of the assay to study TRPV6 [31].

Our 45Ca uptake experiments showed that both zinc and cad-ium are effective blockers of calcium uptake via TRPV6 atillimolar concentrations. In contrast, mercury had no effect on

ptake. Note that we used a 10 �M concentration of mercuryn the Fura-2 studies because at higher concentrations mercuryas been shown to induce calcium release from the endoplas-ic reticulum (ER) and mitochondria [32,33]. Calcium release

rom the ER by 100 �M mercury could lead to lower ER cal-ium levels, which would in turn activate store-operated calciumhannels at the plasma membrane. This could explain why 45Captake via TRPV6 is increased by mercury at higher concentra-ion. The regulation of TRPV6 activity by ER calcium levels, at least

ig. 12. Time course for 45Ca uptake in EYFP-hTRPV6-transfected and non-ransfected HEK293 cells.5Ca uptake experiments demonstrated increased calcium uptake in EYFP-hTRPV6-xpressing cells. Calcium uptake was saturated at about 10 min. For further inhibitortudies, a 2 min incubation time was chosen (n = 3).

tes basal intracellular calcium (Panels A and B). Furthermore, they also present thatas strikingly higher in hTRPV6-overexpressing cells compared to non-transfectedand cadmium permeability, respectively (Panels C and D). Bar graphs represent.05

under our experimental conditions, is also implicated by theseresults.

We also tested how the extracellular calcium concentrationaffects zinc and cadmium influx through TRPV6. We observedthat zinc but not cadmium influx can be significantly diminishedby equimolar extracellular calcium concentration. Further exper-iments are required to elucidate the interaction between zinc,cadmium, and calcium influxes via TRPV6.

Lanthanides have been reported to be potent inhibitors ofmany calcium channels and pumps by binding to the calcium-binding sites of these proteins. As expected, lanthanides alsoblocked TRPV6 activity efficiently above a low micromolar range.Interestingly, the Fura-2 ratio increased much faster in TRPV6-expressing cells when we introduced lanthanum or gadolinium.Because there was extracellular calcium outside only in smallamounts, and 45Ca uptake via TRPV6 and endogenous calciumchannels was inhibited by lanthanides, this increase cannot rep-resent a calcium influx from the extracellular space. Although,gadolinium and lanthanum efficiently block plasma membranecalcium ATPase (PMCA), the activity of PMCA should be lowbecause experiments were performed in nominally calcium-free buffer. Since we only analyzed cells that were able topump out calcium and reduce intracellular calcium when extra-cellular calcium was reduced from millimolar to micromolarlevels, these results could not be derived from apoptotic cellsin which cation influx would increase non-specifically. Keep-ing these arguments in mind, our results provide evidence thatTRPV6 is also permeable to lanthanides but to a much smallerextent.

We examined the influx of three other essential trace metals viaTRPV6, namely manganese, cobalt, and nickel, as well as by mea-suring the difference of the rate of quenching of Fura-2 intensity atits isobestic wavelength in transfected and non-transfected cells.We found that TRPV6 is permeable to manganese but not to cobalt

54 G. Kovacs et al. / Cell Calcium 49 (2011) 43–55

Fig. 13. Effect of several di- and trivalent cations on calcium uptake via EYFP-hTRPV6 expressed in HEK293 cells.S (Panee iated

oi

ibci

ibdoosaichb

acpd

ttmiap

ummarized data showing that lanthanides (Panel D) and cadmium, as well as zincarth (Panel A) and transitional (Panel C) metal elements did not block TRPV6-med

r nickel. All three divalent cations resulted in slight or mediumnhibition of TRPV6-mediated 45Ca uptake.

These findings could be especially important for placental phys-ology. Since TRPV6 is highly expressed in the placenta, it mighte the main route for the nourishment of the fetus not only withalcium but also with trace elements. Yhus TRPV6, could greatlynfluence the transplacental transport of trace elements.

Furthermore, under calcium-restricted dietary conditions, thencreased expression of duodenal TRPV6 could enhance suscepti-ility to heavy metal poisoning. In mice kept on a calcium-deficientiet, cadmium accumulation in the liver and intestinal expressionf TRPV6 were both significantly increased compared to animalsn a normal diet [23,24]. Moreover, cadmium inhibited vitamin-D-ensitive calcium uptake in rat intestine when animals were kept onlow but not on a normal calcium diet [34]. As mentioned above

n mice, TRPV6 was found to play a major role in intestinal cal-ium absorption under low dietary calcium conditions [8,9] and inuman intestinal expression of TRPV6 was shown to be regulatedy 1,25-dihydroxy-vitamin-D [35].

Dietary calcium also affects intestinal absorption of heavy met-ls in humans. An example of chronic cadmium poisoning was thease in the Itai-itai disease that affected a particularly sensitiveopulation in the Toyama district in Japan that lived on calcium-eficient diet and consumed poisoned rice [36].

In summary, we provide evidence that expression of TRPV6 athe plasma membrane increases the transport of other essential

race elements such as zinc and manganese, as well as toxic heavy

etals such as cadmium, lanthanum, and gadolinium. Further stud-es are required to investigate the significance and toxicologicalspects of TRPV6 as a multimetal ion channel in the gut and thelacenta.

l B) are potent inhibitors of TRPV6. Except for manganese, the investigated alkalinecalcium uptake effectively.

Acknowledgements

The authors would like to thank Maria Feher for her excellentassistance in cell culture, and for Leah Witton for her helpful com-ments on the manuscript.

This study was funded by the Swiss National Science Foundation(Hediger), Novartis Foundation (Hediger), Marie Curie Reintegra-tion Grant (Hediger), and Hungarian Scientific Research FundK79189 (Zsembery).

References

[1] J.B. Peng, X.Z. Chen, U.V. Berger, et al., Molecular cloning and characterizationof a channel-like transporter mediating intestinal calcium absorption, J. Biol.Chem. 274 (1999) 22739–22746.

[2] M.J. Gunthorpe, C.D. Benham, A. Randall, J.B. Davis, The diversity in the vanil-loid (TRPV) receptor family of ion channels, Trends Pharmacol. Sci. 23 (2002)183–191.

[3] P. Lu, S. Boros, Q. Chang, R.J. Bindels, J.G. Hoenderop, The {beta}-glucuronidaseklotho exclusively activates the epithelial Ca2+ channels TRPV5 and TRPV6,Nephrol. Dial. Transplant. 23 (2008) 3397–3402.

[4] R. Moreau, G. Daoud, R. Bernatchez, L. Simoneau, A. Masse, J. Lafond, Calciumuptake and calcium transporter expression by trophoblast cells from humanterm placenta, Biochim. Biophys. Acta (BBA) - Biomembr. 1564 (2002) 325–332.

[5] M. Irnaten, N. Blanchard-Gutton, B.J. Harvey, Rapid effects of 17[beta]-estradiolon epithelial TRPV6 Ca2+ channel in human T84 colonic cells, Cell Calcium 44(2008) 441–452.

[6] Y. Suzuki, C.S. Kovacs, T. Hitomi, J.B. Peng, C.P. Landowski, M.A. Hediger, Calciumchannel TRPV6 is involved in murine maternal–fetal calcium transport, J. BoneMiner. Res. 23 (2008) 1249–1256.

[7] S. Heche, L. Takser, F. Lebellego, et al., Alteration of calcium homeostasis inprimary preeclamptic syncytiotrophoblasts: effect on calcium exchange in pla-centa, J. Cell Mod. Med. (2010).

[8] L. Lieben, B.S. Benn, D. Ajibade, et al., Trpv6 mediates intestinal calcium absorp-tion during calcium restriction and contributes to bone homeostasis, Bone,Corrected Proof 47 (2010) 301–308.

Calciu

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

G. Kovacs et al. / Cell

[9] G.D. Kutuzova, F. Sundersingh, J. Vaughan, et al., TRPV6 is not required for1+¦,25-dihydroxyvitamin D3-induced intestinal calcium absorption in vivo,Proc. Natl. Acad. Sci. 105 (2008) 19655–19659.

10] J.B. Peng, X.Z. Chen, U.V. Berger, et al., Human calcium transport protein CaT1,Biochem. Biophys. Res. Commun. 278 (2000) 326–332.

11] B. Thyagarajan, V. Lukacs, T. Rohacs, Hydrolysis of phosphatidylinositol 4,5-bisphosphate mediates calcium-induced inactivation of TRPV6 channels, J. Biol.Chem. 283 (2008) 14980–14987.

12] T. Voets, A. Janssens, J. Prenen, G. Droogmans, B. Nilius, Mg2+-dependent gatingand strong inward rectification of the cation channel TRPV6, J. Gen. Physiol. 121(2003) 245–260.

13] A. Qiu, C. Hogstrand, Functional characterisation and genomic analysis of anepithelial calcium channel (ECaC) from pufferfish, Fugu rubripes, Gene 342(2004) 113–123.

14] C. Hogstrand, P.M. Verbost, S.E. Bonga, C.M. Wood, Mechanisms of zinc uptakein gills of freshwater rainbow trout: interplay with calcium transport, Am. J.Physiol. Regul. Integr. Comp. Physiol. 270 (1996) R1141–R1147.

15] A. Qiu, C.N. Glover, C. Hogstrand, Regulation of branchial zinc uptake by1[alpha],25-(OH)2D3 in rainbow trout and associated changes in expressionof ZIP1 and ECaC, Aquat. Toxicol. 84 (2007) 142–152.

16] H. Gunshin, T. Noguchi, H. Naito, Effect of calcium on the zinc uptake by brushborder membrane vesicles isolated form the rat small intestine, Agric. Biol.Chem. 55 (1991) 2813–2816.

17] R.F.P. Bertolo, W.J. Bettger, S.A. Atkinson, Calcium competes with zinc for achannel mechanism on the brush border membrane of piglet intestine, J. Nutr.Biochem. 12 (2001) 66–72.

18] V. Lehen’kyi, B. Beck, R. Polakowska, et al., TRPV6 is a Ca2+ entry channel essen-tial for Ca2+-induced differentiation of human keratinocytes, J. Biol. Chem. 282(2007) 22582–22591.

19] O.P. Hamill, A. Marty, E. Neher, B. Sakmann, F.J. Sigworth, Improved patch-clamp techniques for high-resolution current recording from cells and cell-freemembrane patches, Pflugers Arch. Eur. J. Physiol. 391 (1981) 85–100.

20] M. Bodding, Voltage-dependent changes of TRPV6-mediated Ca2+ currents, J.Biol. Chem. 280 (2005) 7022–7029.

21] G. Zhu, Y. Zhang, H. Xu, C. Jiang, Identification of endogenous outward currentsin the human embryonic kidney (HEK 293) cell line, J. Neurosci. Methods 81(1998) 73–83.

22] K.A. Bolanz, G.G. Kovacs, C.P. Landowski, M.A. Hediger, Tamoxifen inhibitsTRPV6 activity via estrogen receptor independent pathways in TRPV6-expressing MCF-7 breast cancer cells, Mol. Cancer Res. 7 (2009) 2000–2010.

[

[

m 49 (2011) 43–55 55

23] K.S. Min, H. Ueda, T. Kihara, K. Tanaka, Increased hepatic accumulation ofingested Cd is associated with upregulation of several intestinal transportersin mice fed diets deficient in essential metals, Toxicol. Sci. 106 (2008) 284–289.

24] K.S. Min, H. Ueda, K. Tanaka, Involvement of intestinal calcium transporter 1and metallothionein in cadmium accumulation in the liver and kidney of micefed a low-calcium diet, Toxicol. Lett. 176 (2008) 85–92.

25] M. Bodding, V. Flockerzi, Ca2+ dependence of the Ca2+-selective TRPV6 chan-nel, J. Biol. Chem. 279 (2004) 36546–36552.

26] L. Yue, J.B. Peng, M.A. Hediger, D.E. Clapham, CaT1 manifests the pore proper-ties of the calcium-release-activated calcium channel, Nature 410 (2001) 705–709.

27] D. Ragozzino, A. Giovannelli, V. Degasperi, F. Eusebi, F. Grassi, Zinc permeatesmouse muscle ACh receptor channels expressed in BOSC 23 cells and affectschannel function, J. Physiol. 529 (2000) 83–91.

28] J. Magistretti, L. Castelli, V. Taglietti, F. Tanzi, Dual effect of Zn2+ on multi-ple types of voltage-dependent Ca2+ currents in rat palaeocortical neurons,Neuroscience 117 (2003) 249–264.

29] E.M. Schwiebert, L. Liang, N.-L. Cheng, et al., Extracellular zinc and ATP-gatedP2X receptor calcium entry channels: new zinc receptors as physiological sen-sors and therapeutic targets, Purinergic Signal. 1 (2005) 299–310.

30] H. Hu, M. Bandell, M.J. Petrus, M.X. Zhu, A. Patapoutian, Zinc activates damage-sensing TRPA1 ion channels, Nat. Chem. Biol. 5 (2009) 183–190.

31] S.H. Chao, Y. Suzuki, J.R. Zysk, W.Y. Cheung, Activation of calmodulin by vari-ous metal cations as a function of ionic radius, Mol. Pharmacol. 26 (1984) 75–82.

32] B. Burlando, M. Bonomo, E. Fabbri, F. Dondero, A. Viarengo, Hg2+ signaling introut hepatoma (RTH-149) cells: involvement of Ca2+-induced Ca2+ release,Cell Calcium 34 (2003) 285–293.

33] E. Chavez, J.A. Holguin, Mitochondrial calcium release as induced by Hg2+, J.Biol. Chem. 263 (1988) 3582–3587.

34] D.L. Hamilton, M.W. Smith, Inhibition of intestinal calcium uptake by cadmiumand the effect of a low calcium diet on cadmium retention, Environ. Res. 15(1978) 175–184.

35] S. Balesaria, S. Sangha, J.R.F. Walters, Human duodenum responses to vitaminD metabolites of TRPV6 and other genes involved in calcium absorption, Am. J.Physiol. Gastrointest. Liver Physiol. 297 (2009) G1193–G1197.

36] Aoshima Keiko, Epidemiology of renal tubular dysfunction in the inhabitants ofa cadmium-polluted area in the Jinzu River basin in Toyama Prefecture, TohokuJ. Exp. Med. 152 (1987) 151–172.