INVITED REVIEW

Tom Nijenhuis Æ Joost G. J. Hoenderop

Rene J. M. Bindels

TRPV5 and TRPV6 in Ca2+ (re)absorption: regulating Ca2+ entryat the gate

Received: 31 March 2005 / Accepted: 1 April 2005 / Published online: 26 July 2005� Springer-Verlag 2005

Abstract Many physiological functions rely on the exactmaintenance of body Ca2+ balance. Therefore, theextracellular Ca2+ concentration is tightly regulated bythe concerted actions of intestinal Ca2+ absorption, ex-change of Ca2+ to and from bone, and renal Ca2+

reabsorption. Renal distal convoluted and connectingtubular cells as well as duodenal epithelial cells areunique in their ability to mediate transcellular(re)absorption of Ca2+ at large and highly variable rates.Two members of the transient receptor potential (TRP)superfamily, TRP vanilloid (TRPV)5 and TRPV6, arespecialized epithelial Ca2+ channels responsible forthe critical Ca2+ entry step in transcellular Ca2+

(re)absorption in intestine and kidney, respectively. Be-cause transcellular Ca2+ transport is fine-tuned to thebody’s specific requirements, regulation of the trans-membrane Ca2+ flux through TRPV5/6 is of particularimportance and has, therefore, to be conspicuouslycontrolled. We present an overview of the currentknowledge and recent advances concerning the coordi-nated regulation of Ca2+ influx through the epithelialCa2+ channels TRPV5 and TRPV6 in transcellular Ca2+

(re)absorption.

Keywords Calcium transport Æ TRPV5 Æ TRPV6 ÆRegulation

Introduction

Maintenance of body Ca2+ homeostasis is of vitalimportance for many physiological functions includingintracellular signalling processes, neuronal excitability,

muscle contraction and bone formation. ExtracellularCa2+ concentration is regulated by a homeostaticmechanism tightly controlling the concerted actions ofintestinal Ca2+ absorption, exchange of Ca2+ to andfrom bone and renal Ca2+ reabsorption [1]. Renal distalconvoluted (DCT) and connecting tubular (CNT) cellsas well as intestinal epithelial cells are unique in theirability to mediate transcellular transport of Ca2+

between the luminal and basolateral compartments,whereas the free cytosolic Ca2+ concentration is gener-ally maintained at low resting values. The latter impli-cates that these cells are capable of sustaining large ratesof transcellular Ca2+ fluxes, dependent on the tightregulation of the plasma Ca2+ concentration, withoutinterfering with intracellular signalling. This transport ismediated by Ca2+ entry across the apical membranethrough the specialized epithelial Ca2+ channels, tran-sient receptor potential vanilloid 5 and 6 (TRPV5 andTRPV6), intracellular buffering and facilitated diffusionbound to Ca2+-binding proteins (calbindins) andextrusion across the basolateral membrane by a Na+/Ca2+ exchanger (NCX1) and/or plasma membraneCa2+-ATPase (Fig. 1) [1–3]. Regulation of Ca2+ influxacross the luminal membrane through TRPV5 andTRPV6 is particularly important, because it occursdown a steep concentration gradient and, therefore,these channels are generally considered as the gate-keepers of transcellular Ca2+ transport.

Two unique TRP channels facilitating epithelialCa2+ transport

The epithelial Ca2+ channel family is restricted to twodistinct members, TRPV5 and TRPV6, which are juxta-posed on the human chromosome 7q35 with a distance ofonly 22 kb, suggesting an evolutionary gene duplicationevent [4–6]. TRPV5 and TRPV6 were originally clonedfrom rabbit kidney and rat small intestine, respectively, andwere identified in many additional species including fish,rabbit, rat, mouse and human [4, 7]. TRPV5 and TRPV6

T. Nijenhuis Æ J. G. J. Hoenderop Æ R. J. M. Bindels (&)Department of Physiology, Nijmegen Centre for Molecular LifeSciences, Radboud University Nijmegen Medical Centre,P.O. Box 9101, 6500 HB Nijmegen, The NetherlandsE-mail: r.bindels@ncmls.ru.nlTel.: +31-24-3614211Fax: +31-24-3616413

Pflugers Arch – Eur J Physiol (2005) 451: 181–192DOI 10.1007/s00424-005-1430-6

are �730 amino acids, along with a predicted molecularmass around 83 kDa, containing six transmembrane (TM)segments and large cytosolic N- and C-tails, the formercontaining ankyrin repeats (Fig. 2a). A short hydrophobicstretch between TM5 and TM6 was predicted to bethe pore-forming region. Later studies showed that thesix-TM unit is one of four subunits presumed to surroundthe central pore in a tetrameric configuration (Fig. 2b) [8].Detailed expression and (co)localization studies suggestedthat TRPV5 comprises the epithelial Ca2+ channel pre-dominantly involved in renal transcellular Ca2+ transportin DCT/CNT, whereas TRPV6 was postulated to mediateintestinal Ca2+ absorption [2, 9–11]. TRPV5 and TRPV6are so far the only known highly Ca2+-selective channelsin the TRP superfamily. Even the closely related TRPVsubfamily members, which are all Ca2+ permeable,discriminate much less between divalent and monovalentcations [12–15]. Functional data on TRPV5 included45Ca2+ uptake measurements, electrophysiological datausing voltage-clamp and patch-clamp experiments andfluorimetric measurements in several mammalian cell lines.In short, the permeation sequence for the TRPV5/6 pore isCa2+> barium (Ba2+)> strontium (Sr2+)>Mn2+, andthe channels are inhibited in order of potency by lanthanum(La3+)> cadmium (Cd2+)>Mn2+. Permeability toNa+

was negligible in the situation where Ca2+ and Na+ wereboth present, whereas Ba2+ and Sr2+ did not affect theCa2+ influx. We have demonstrated that the molecular

determinants of the Ca2+ selectivity and permeation ofTRPV5/6 reside at a single aspartate residue (TRPV5-D542and TRPV6-D541) present in the pore-forming region [16,17]. A nearly complete inward rectification is anothercharacteristic hallmark of the TRPV5/6 channels in addi-tion to the high Ca2+ selectivity. This intrinsic rectificationof the channels is significantly reduced by neutralization ofD542/D541, indicating that this site is also involved inrectification [17]. Electrophysiological studies demonstratedthat the characteristics of TRPV6 are roughly comparableto those measured for TRPV5. However, differences existwith respect to Ca2+-dependent inactivation, Ba2+ selec-tivity and sensitivity for inhibition by the potent channelblocker ruthenium red. Importantly, the current throughTRPV5 and TRPV6 is carried exclusively by Ca2+ atphysiological extracellular Ca2+ concentrations. Thus,the functional properties of TRPV5 and TRPV6 and theirin vivo localization are in line with a role in epithelialCa2+ (re)absorption, providing the first line of evidencethat these channels are responsible for the Ca2+ influxstep in the process of transcellular Ca2+ transport[18, 19].

Subsequently, TRPV5 knockout (TRPV5�/�) micewere generated in our laboratory by ablation of theTRPV5 gene to substantiate the in vivo function of thischannel in Ca2+ (re)absorption [6]. TRPV5�/� miceshowed robust renal Ca2+ wasting, and micropunctureexperiments were performed to pinpoint the defective

Fig. 1 Transcellular Ca2+ transport in kidney and small intestine.Schematic representation of transcellular Ca2+ transport consistingof apical entry of Ca2+ through the epithelial Ca2+ channelstransient receptor potential vanilloid 5 and 6 (TRPV5 andTRPV6), cytosolic diffusion bound to Ca2+-binding proteins

(calbindins) and extrusion across the basolateral membrane by aNa+/Ca2+ exchanger (NCX1) and/or a plasma membrane Ca2+-ATPase (PMCA1b). Transcellular Ca2+ (re)absorption occurs inthe distal convoluted and connecting tubules in kidney involvingTRPV5 as well as in duodenum involving TRPV6

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site of Ca2+ reabsorption along the nephron (Fig. 3a).Quantitative free-flow collections of tubular fluid re-vealed unaffected Ca2+ absorption up to the last loop ofthe late proximal tubule, whereas Ca2+ delivery topuncturing sites within DCT/CNT was significantly en-hanced. Because K+ secretion occurs along the distalpart of the nephron, the luminal K+ concentrationserves as an indicator of the collection site in the distalconvolution. The inverse relationship between distalluminal K+ concentration and fractional Ca2+ deliveryindicated that active Ca2+ reabsorption in DCT/CNT iseffectively abolished in TRPV5�/� mice (Fig. 3b). Inaddition, the expression of proteins involved in trans-cellular Ca2+ transport, downstream from apical Ca2+

entry through TRPV5, was significantly reduced(Fig. 3c). In contrast, TRPV5 mRNA expression levelswere not affected in calbindin-D28K knockout mice [20,21]. Taken together, these findings confirm the crucialrole of TRPV5 as the gatekeeper of renal Ca2+ reab-sorption. Furthermore, TRPV5�/� mice displayed acompensatory intestinal Ca2+ hyperabsorption accom-panied by significantly enhanced duodenal TRPV6expression (Fig. 3d, e). These data suggest that, in con-trast to the kidney, TRPV6 is predominantly involved inCa2+ absorption in the small intestine. Recently, Hedi-ger and co-workers evaluated the functional role ofTRPV6 in Ca2+ absorption by inactivation of the mouseTRPV6 gene [22]. Initial data showed that TRPV6knockout (TRPV6�/�) mice placed on a Ca2+-deficientdiet display a consistent decrease in Ca2+ absorptionover time. These data indicate that TRPV6 indeedconstitutes the Ca2+-entry step in intestinal Ca2+

absorption. In addition, bone thickness was reduced inTRPV5�/� mice [6]. This bone phenotype could either

result from the renal Ca2+ wasting or indicate a directeffect of TRPV5 ablation on bone, because TRPV5 waspreviously shown to be expressed in this tissue [23].

Regulation of epithelial Ca2+ channels

Transcellular Ca2+ transport is a highly coordinated andregulated process, which can be fine-tuned to the body’sspecific requirements. In line with their central role in theaforementioned process, TRPV5/6 expression andchannel activity has to be conspicuously controlled. Thisregulation occurs at different levels, including (1) tran-scriptional and translational regulation, (2) the tetra-metric channel stoichiometry, (3) trafficking of channelsto and from the plasma membrane and (4) modulationof channel activity at the plasma membrane (Fig. 4).

Transcriptional and translational regulation

Parathyroid hormone

Parathyroid hormone (PTH) and the biologically activeform of vitamin D [1,25-(OH)2D3] are the main calcio-tropic hormones controlling Ca2+ balance [1, 24, 25].The parathyroid gland plays a key role through thecapacity to sense minute changes in the extracellularCa2+ concentration. Upon a decrease in the extracellu-lar Ca2+ level, PTH is secreted into the circulation, andthen acts primarily on the kidney and bone, where itactivates the PTH/PTHrP receptor [26]. Early studiesusing micropuncture and cell preparations demonstratedthat PTH directly stimulates active Ca2+ reabsorptionin the distal part of the nephron [27, 28]. In addition to

Fig. 2 Structural organization of TRPV5 and TRPV6. Theepithelial Ca2+ channels consist of six transmembrane (TM)domains, one putative pore-forming region between TM5 and

TM6 and large cytosolic C- and N-terminal tails containingankyrin repeats (a). The six-TM units are presumed to surrounda central pore in a (homo- or hetero-) tetrameric configuration (b)

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enhancing tubular Ca2+ reabsorption, PTH stimulatesthe activity of 25-hydroxyvitamin-D3-1a-hydroxylase(1a-OHase), a crucial enzyme in the biosynthesis of 1,25-(OH)2D3 and, thereby, increases the 1,25-(OH)2D3-dependent absorption of Ca2+ in the small intestine [29].Preliminary studies from our lab demonstrated thatparathyroidectomy in rats results in decreased serumPTH levels and hypocalcaemia, which was accompaniedby decreased TRPV5 mRNA levels and protein abun-dance [30]. Supplementation with PTH restored serumCa2+ concentrations and TRPV5 abundance in kidney,suggesting that PTH affects renal Ca2+ handlingthrough the regulation of TRPV5. Further studies areneeded to evaluate the effects of PTH on TRPV5/6expression in more detail.

Vitamin D

Various studies have provided evidence that the expres-sion of TRPV5 and TRPV6 is tightly controlled by 1,25-

(OH)2D3 [31–42]. Analysis of putative promoter regionsof human and murine TRPV5/6 genes revealed potentialvitamin D-response elements [32, 41]. Studies in severalcell models suggested that 1,25-(OH)2D3 enhances epi-thelial Ca2+ channel expression [38, 40]. Woods et al. [40]showed that upon addition of 1,25-(OH)2D3 to Caco-2cells, TRPV6 expression is enhanced within 24 h, pre-ceding an increase in calbindin expression levels. In vivo,the vitamin D sensitivity of epithelial Ca2+ channelexpression was first determined when repletion of vita-min D3-depleted rats normalized the plasma Ca2+ con-centration and increased the amount of TRPV5 mRNAand protein expression in the kidney [32]. In mice, asingle dose of 1,25-(OH)2D3 up-regulated renal TRPV5mRNA expression and TRPV6 mRNA levels in duode-num, illustrating the genomic vitamin D response [34,41]. Time-dependent studies following a single dose of1,25-(OH)2D3 showed that induction of duodenalTRPV6 mRNA occurs within 3–6 h and precedes thestimulation of intestinal Ca2+ absorption [35]. Several

Fig. 3 Characteristics ofTRPV5 knockout (TRPV5�/�)mice. Micropunctureexperiments measuringfractional Ca2+ delivery to thelast surface loop of proximaltubule (LPT), distalconvolution (DC) and urine (U)in TRPV5+/+ and TRPV5�/�

mice (a). Association betweenK+ concentration in distalconvoluted tubular fluid andfractional Ca2+ delivery to thispuncturing site (b). Renalcalbindin-D28K (CaBP28K) andNCX1 mRNA expression levelsas determined by real-timequantitative polymerase chainreaction (PCR) analysis(c). Intestinal Ca2+ absorptionmeasured by changes in serumCa2+ after administration of 45

Ca2+ by oral gavage inTRPV5+/+ and TRPV5�/�

mice (d). Duodenal TRPV6 andcalbindin-D9K (CaBP9K)mRNA expression levels asdetermined by real-timequantitative PCR analysis (e)

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genetically modified mouse models in which vitamin Dregulation was inactivated were used to further study theregulation of TRPV5/6 by 1,25-(OH)2D3. In vitamin Dreceptor (VDR) knockout mice, duodenal TRPV6mRNA levels were significantly down-regulated andassociated with decreased intestinal Ca2+ absorptionand hypocalcemia [34, 43]. Targeted ablation of 1a-OHase in mice, resulting in impairment of 1,25-(OH)2D3

biosynthesis, was shown to result in severe hypocalcemiaand down-regulation of both renal TRPV5 and intestinalTRPV6 expression. Repletion with 1,25-(OH)2D3 re-stored Ca2+ channel expression levels and normalizedserum Ca2+ levels. Furthermore, additional studiesdemonstrated differential up-regulation of TRPV5/6 byvitamin D analogues, which were developed in an effortto treat secondary hyperparathyroidism without thehypercalcemic side-effects of conventional vitamin Dtreatment [44]. In particular, the time course of up-reg-ulation of TRPV5/6 correlated with serum levels of activevitamin D metabolites. Thus, the ameliorated calcemiceffects appeared to result from differential stimulation ofCa2+ channel expression. Taken together, the tran-scriptional and translational regulation of TRPV5 andTRPV6 by 1,25-(OH)2D3 was consistently established.

Dietary Ca2+

In addition to the calciotropic hormones, there areindications that Ca2+ itself can affect TRPV5/6 expres-sion. It is, however, difficult to study the effects of, e.g.hypocalcemia, without affecting serum 1,25-(OH)2D3

levels. In an effort to surpass this interrelationship,studies were performed in VDR�/� and 1a-OHase�/�

mice fed normal and high-Ca2+-rescue diets [31, 34].Importantly, high dietary Ca2+ intake restored the re-duced expression levels of renal TRPV5 and intestinalTRPV6 in 1a-OHase�/� mice, which was accompaniedby normalization of the plasma Ca2+. Furthermore,Dardenne et al. showed that these effects cured ricketsand osteomalacia, a hallmark of 1a-OHase�/� mice,resulting in a partial rescue of the bone phenotype [45]. Incontrast, the Ca2+-enriched rescue diet reduced theexpression of renal TRPV5 and calbindin-D28K in wild-type (1a-OHase+/�) mice that exhibit normal serumvitamin D and Ca2+ levels [31, 33, 34]. The latter illus-trated the physiological mechanism whereby plasmaCa2+ acts via a negative feedback mechanism, eventuallyleading to the suppression of 1a-OHase activity, whichdecreases expression of Ca2+ transport proteins andCa2+ reabsorption. Similarly, dietary rescue of thehypocalcemic state in VDR�/� mice up-regulated thesignificantly decreased duodenal TRPV6 mRNAexpression, whereas in wild-type mice high dietary Ca2+

content decreased TRPV6 expression due to a decrease ofserum 1,25-(OH)2D3 [34]. Thus, these studies showedthat Ca2+ supplementation can up-regulate TRPV5/6gene transcription in the absence of circulating 1,25-(OH)2D3. Hypothetically, Ca2+-responsive elements arepresent in the TRPV5/6 promoter regions, but detailedpromoter analyses are needed to investigate this vitaminD-independent Ca2+-sensitive regulation of TRPV5 andTRPV6.

Fig. 4 Overview of levels atwhich TRPV5 and TRPV6 areregulated. Regulation ofepithelial Ca2+ channels can,hypothetically, occur at thelevel of (1) transcription andtranslation, (2) the tetramericchannel stoichiometry, (3)intracellular trafficking ofchannels to the plasmamembrane and (4) regulation ofchannel activity at the plasmamembrane

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Oestrogens and androgens

It is generally accepted that oestrogens affect Ca2+

homeostasis. Postmenopausal oestrogen deficiency isassociated with increased renal Ca2+ loss, which can becorrected by oestrogen replacement therapy [46, 47].Furthermore, oestrogen receptors were shown to residein DCT/CNT [48]. Van Abel et al. [48, 49] showed thatin ovariectomized rats and 1a-OH�/� mice, 17b-estra-diol replacement therapy results in up-regulation ofrenal TRPV5 mRNA and protein levels as well asduodenal TRPV6 mRNA expression, accompanied bynormalization of plasma Ca2+ levels. These data dem-onstrated that oestrogens regulate the expression ofTRPV5/6 in a 1,25-(OH)2D3-independent manner. VanCromphaut and co-workers [50] reported that renalTRPV5 and duodenal TRPV6 expression was reduced inoestrogen receptor-a knockout mice and could beup-regulated by oestrogen treatment. Interestingly,Weber et al. [51] described an oestrogen-responsive ele-ment in the promoter sequence of the mouse TRPV6gene, which was, however, absent or not conserved inthe mouse TRPV5 gene. Taken together, oestrogenseems to harbour calciotropic hormone characteristics,which physiological relevance is readily exemplified bythe increased risk of osteoporosis in postmenopausalwomen. In addition, TRPV6 was shown to be expressedin murine prostate tissue, being up-regulated in prostatecancer cell lines and in vivo in prostate carcinoma cor-relating with tumour grade [52–54]. Peng and co-work-ers [52] showed that TRPV6 expression is particularlyincreased in androgen-sensitive as compared to andro-gen-insensitive prostate adenocarcinoma cell lines. Di-hydrotestosteron and androgen-receptor antagonistswere shown to regulate TRPV6 in these cell lines. Thus,in addition to oestrogens, androgens also appear toregulate TRPV6 expression. However, it is unclearwhether androgens control duodenal TRPV6 expressionand intestinal Ca2+ absorption. Fixemer and co-workers[55] showed that TRPV6 expression is absent in normalhuman prostate tissue, benign prostate hyperplasia andpremalignant prostatic lesions. However, TRPV6expression correlated with clinical progression scores,pathological stage and extraprostatic extension ofprostate cancer, suggesting TRPV6 could be used as aprognostic marker in prostate cancer.

Immunosuppressive agents

Various drugs used in clinical practice are known toaffect Ca2+ homeostasis, among which immunosup-pressants like the calcineurin inhibitors tacrolimus(FK506) and cyclosporine A as well as glucocorticoidssuch as prednisone and dexamethasone. These drugs areassociated with an increased bone turnover, a negativeCa2+ balance and hypercalciuria [56–58]. Recently, weshowed that FK506 treatment in mice reduced renalTRPV5 mRNA and protein expression associated with

renal Ca2+ wasting, suggesting that down-regulation ofCa2+ channel abundance provides the molecularmechanism for FK506-induced hypercalciuria [59]. Theimmunosuppressive action of tacrolimus depends on theinhibition of the Ca2+ -dependent phosphatase calci-neurin in T lymphocytes [60–62]. Calcineurin is notknown to be involved in renal Ca2+ reabsorption, butanother calcineurin inhibitor, cyclosporine A, increasedurinary Ca2+ excretion and decreased calbindin-D28K

protein levels [63, 64]. This suggested that calcineurininhibition may play a role in the impairment of Ca2+

reabsorption by this class of drugs. In addition, tacrol-imus binds to intracellular immunophilins called FK506-binding proteins (FKBPs), which were implicated as ionchannel regulators, e.g. the regulation of the Ca2+-per-meable Drosophila TRPL channel by FKBP4 [65–68]. Ofnote, microarray analysis showed that FKBP4 was reg-ulated by 1a-OHase ablation and Ca2+ supplementationin 1a-OHase�/� mice [69]. In contrast to FK506, theglucocorticoid agent dexamethasone increased renalTRPV5 as well as duodenal TRPV6 expression, sug-gesting that stimulation of corticoid receptors positivelyaffects TRPV5/6 transcription, counteracting the nega-tive Ca2+ balance during treatment with these com-pounds.

Thiazide diuretics

Thiazide diuretics are among the most commonly pre-scribed drugs employed in the treatment of arterialhypertension. These diuretics enhance renal Na+

excretion through inhibition of the Na+/Cl� cotrans-porter (NCC) present in the apical membrane of DCTcells [70]. In addition, these drugs are known to inducehypocalciuria and mutations in the gene encoding NCCwere shown to cause Gitelman’s syndrome, a recessivedisorder with a phenotype resembling chronic thiazideadministration including hypocalciuria [1, 71–80]. Thehypocalciuric effect was suggested to result from directstimulation of transcellular Ca2+ transport and, inparticular, apical Ca2+ entry through TRPV5 [81–85].Alternatively, hypocalciuria was proposed to result fromenhancement of passive paracellular Ca2+ reabsorptionin proximal tubules secondary to extracellular volume(ECV) contraction, distinct from any effect on trans-cellular Ca2+ transport [78, 86, 87]. Previously, wereported that thiazide-induced hypocalciuria occurs inspite of reduced renal expression of Ca2+ transportproteins in rat [78]. In addition, we showed that ECVcontraction mimics the hypocalciuria, and volumerepletion completely reverses thiazide-induced hypocal-ciuria in these rats. We recently demonstrated inmicropuncture experiments that reabsorption of Na+

and, importantly, Ca2+ in the proximal tubule isincreased during chronic hydrochlorothiazide (HCTZ)treatment, whereas Ca2+ reabsorption in DCT/CNTappeared unaffected [88]. Furthermore, we showed thatchronic HCTZ administration still induces hypocalciuria

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in TRPV5�/� mice, in which active Ca2+ reabsorption isabolished. HCTZ did not affect renal expression of theproteins involved in active Ca2+ transport, includingTRPV5 mRNA and protein expression in wild-typemice. Lee et al. confirmed that thiazide treatment in micedoes not affect renal TRPV5 expression, except whenthiazide treatment is combined with salt repletion [89].However, salt repletion alone induced TRPV5 mRNAexpression to a similar extent, suggesting that this effectis not thiazide-specific. Loffing and co-workers [90, 91]recently demonstrated that renal TRPV5 expression isunaffected in NCC knockout mice, an animal model forGitelman’s syndrome. In accordance, micropunctureexperiments in these mice showed that active Ca2+

reabsorption is unaltered in DCT/CNT and indicatedincreased fractional absorption of both Na+ and Ca2+

upstream of DCT [91]. These studies demonstrated thatthiazides do not directly affect TRPV5 expression andtranscellular Ca2+ reabsorption, whereas increasedpassive Ca2+ reabsorption in the proximal tubuleexplains the Ca2+-sparing during thiazide treatment andGitelman’s syndrome.

Tetrameric channel stoichiometry

Assembly domains

Another level at which epithelial Ca2+ channels may beregulated is during the assembly into a functionalchannel complex. By analogy with other cation channelsubunits comprising six transmembrane domains, TRPmembers are hypothesized to assemble into homo- and/or heterotetrameric complexes. Niemeyer et al. [92]identified the third ankyrin repeat being a stringentrequirement for physical assembly of TRPV6 subunits.Subsequent studies by our group showed that at leasttwo regions in the cytosolic tails are involved in channelassembly [93]. Physical interactions between the N-tail/N-tail, N-tail/C-tail and C-tail/C-tail were demonstratedby pull-down assays and co-immunoprecipitations.TRPV5 truncants lacking either the N-tail or C-tailshowed dominant-negative effects on channel activity.Detailed mapping assays identified two critical regionsin the N-tail and C-tail involved in assembly which,when mutated, abolished the interactions between thetails. In addition, the identified N-tail assembly domainincluded an ankyrin repeat, substantiating the involve-ment of these protein–protein binding modules inTRPV5 assembly. The importance of both the N and Ctermini in TRPV channel oligomerization, includingTRPV5 and TRPV6, was recently confirmed by Hellwiget al. [94].

Heterotetrameric TRPV5/6 channels

Recently, we confirmed by co-immunoprecipitations andmolecular mass determination of TRPV5 and TRPV6complexes using sucrose gradient sedimentation that

these channels can indeed form homo- and heterotetra-meric channel complexes [8]. Because TRPV5 andTRPV6 exhibit different channel kinetics, the influenceof the heterotetramer composition on channel propertieswas investigated [8]. When concatemeric channels wereconstructed consisting of four TRPV5 and/or TRPV6subunits, differences in the ratio of TRPV5 and TRPV6subunits resulted in mixed properties of TRPV5 andTRPV6 channels [8]. Recently, Hellwig and co-workersshowed by fluorescence resonance energy transfer andco-immunoprecipitation experiments that, whereas theother TRPV channels preferentially form homotetra-mers, TRPV5 and TRPV6 seem to be unique in formingheterotetramers [94]. Thus, regulation of the relativeexpression levels of TRPV5 and TRPV6 may be amechanism to fine-tune the Ca2+ transport kinetics inTRPV5/6-co-expressing tissues. Indeed, several studiesindicated that certain tissues co-express TRPV5 andTRPV6, including kidney and small intestine, whichwould allow oligomerization of these channels in vivo [1,10, 41, 95]. Recently, Clapham and co-workers producedan extensive analysis of TRP channel tissue distribution,showing that TRPV5 and TRPV6 are co-expressed inbrain, kidney, pancreas, small intestine, colon, prostateand testis (http://www.clapham.tch.harvard.edu).

Trafficking to the plasma membrane

S100A10-annexin 2 complex

Accurate trafficking of channels and transporters to theplasma membrane is essential for transcellular iontransport. Van de graaf et al. [96] provided the firstevidence of a regulatory role for the S100A10-annexin 2heterotetramer in the trafficking of TRPV5 and TRPV6.S100A10, a 97-amino acid protein member of the S100superfamily, was identified as an associated protein ofTRPV5 and TRPV6 using the yeast two-hybrid tech-nique. S100A10 is found tightly associated with annexin2, a member of the Ca2+ and phospholipids-bindingproteins which is implicated in numerous biologicalprocesses including exocytosis, endocytosis and mem-brane-cytoskeleton interactions [97]. The association ofS100A10 with TRPV5 and TRPV6 was restricted to ashort conserved peptide sequence located in the C-tail ofthese channels. The first threonine of this sequence wasidentified as a crucial amino acid for binding andchannel function. When this particular threonine wasmutated, the activity of TRPV5 and TRPV6 was abol-ished accompanied by a major disturbance in theirsubcellular localization. This indicated that theS100A10-annexin 2 heterotetramer facilitates the trans-location of TRPV5 and TRPV6 channels towards theplasma membrane. The importance of annexin 2 in thisprocess was demonstrated by showing that small inter-fering RNA-based down-regulation of annexin 2 sig-nificantly inhibits TRPV5 and TRPV6 currents. Thelatter demonstrated that annexin 2 in conjunction with

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S100A10 is crucial for TRPV5 and TRPV6 activity.Interestingly, S100A10 as well as annexin 2 appear to beregulated by 1,25-(OH)2D3, further supporting theimportance of the S100A10-annexin-2 complex in theregulation of 1,25-(OH)2D3-dependent TRPV5/6-medi-ated Ca2+ influx [96, 98].

Na+/H+ exchanger regulating factor 2 and serumand glucocorticoid-inducible kinase

Embark and co-workers [99, 100] recently demonstratedthat TRPV5 activity is increased upon co-expressionwith the Na+/H+ exchanger regulating factor 2(NHERF2) and the serum and glucocorticoid-induciblekinase (SGK1) in Xenopus laevis oocytes. Using GSTpull-down and overlay assays, the specific interaction ofNHERF2 with the last three amino acids in the C-tail ofTRPV5 was demonstrated (S.F. van de Graaf, unpub-lished results). This suggested that the NHERF2/SGK1protein pair affects TRPV5 in a direct fashion via pro-tein–protein interaction. The last three amino acids ofTRPV5 resemble a PDZ motif, which allows binding toPDZ domain-harbouring proteins, including NHERF2.Of note, co-expression of SGK1 with NHERF2 stimu-lated the activity of the renal outer medullary K+

channel (ROMK1), a K+ channel involved in renal K+

handling, by stabilization of ROMK1 in the plasmamembrane [101, 102]. Currently, it is not known whetheran identical mechanism is applicable for TRPV5. It istempting to speculate that a stimulatory effect onTRPV5 trafficking towards the plasma membranereflects SGK1-mediated phosphorylation of TRPV5, butthis also remains to be demonstrated.

Modulation of TRPV5/6 channel activityat the plasma membrane

Ca2+-dependent feedback inhibition

In mammalian cells heterogeneously overexpressing theepithelial Ca2+ channels, TRPV5 and TRPV6 are con-stitutively open at a low-intracellular Ca2+ concentrationand a negative membrane potential [103]. Extrapolatingthese findings to the in vivo situation suggests thatadditional regulatory factors or at least an increasedCa2+ buffer capacity is a necessity for TRPV5/6-expressing cells, in particular because TRPV5 andTRPV6 are controlled by Ca2+ -dependent feedbackinhibition. Both Ca2+ channels rapidly inactivate duringhyperpolarizing voltage-steps and this inhibition isreduced when Ca2+ is substituted for Ba2+ or Sr2+ ascharge carriers. The inactivation is dependent on theextracellular Ca2+ concentration and also occurs incells buffered intracellularly with 1,2-bis(o-aminophen-oxy)ethane-N,N,N¢,N¢-tetraacetic acid [103]. Ca2+ influxis a prerequisite for this phenomenon because theCa2+-impermeable D542A TRPV5 mutant lacks amonovalent current decay in response to repetitive

stimulation [104]. These data suggested that TRPV5/6activity is inhibited by Ca2+ influx through the channel,most likely by increasing the Ca2+ concentration in amicrodomain near the pore region, thereby inducingfeedback inhibition of the channel. This could be a cru-cial mechanism for the regulation of TRPV5 andTRPV6 at the plasma membrane under physiologicalconditions. Considering the high affinity mechanism ofCa2+-dependent TRPV5/6 inhibition, the presence ofintracellular Ca2+ buffering proteins such as calbindins,is of pivotal importance to maintain channel activity [18,105, 106].

Calmodulin

Ca2+-dependent regulation is not restricted to TRPV5/6, but also occurs in L-type and P/Q-type voltage-gatedCa2+ channels, where facilitation and inactivation ofthe current is mediated by the ubiquitously expressedCa2+-sensor calmodulin (CaM) [107, 108]. Niemeyeret al. [107] showed that CaM binds to the C-tail of hu-man TRPV6 in a Ca2+-dependent manner. In addition,Nilius et al. [109] demonstrated that the rabbit TRPV5C-tail is important for the Ca2+-dependent inactivationprocess. Recently, CaM was shown to bind to the CaM-binding motifs in the C- and N-tails of TRPV5 andTRPV6 as well as the transmembrane domain of TRPV6in a Ca2+-dependent fashion [110, 111]. Electrophysio-logical measurements of HEK293 cells heterologouslyco-expressing Ca2+-insensitive CaM mutants along withTRPV5 or TRPV6 revealed a significantly reduced in-ward Ca2+ current through TRPV6, whereas no effectwas demonstrated on TRPV5. This effect was localizedto the high Ca2+-affinity EF-hand structures of CaM.Thus, these data demonstrated a regulatory role ofCaM in TRPV6-mediated Ca2+ influx. It remains to beestablished whether CaM functions as a general Ca2+

sensor in TRPV5/6 channels or, alternatively, mightexplain the differences in Ca2+ -dependent inactivationbetween TRPV5 and TRPV6.

80K-H

In an effort to identify novel regulators of Ca2+ reab-sorption by TRPV5, we used cDNA microarray analysiswhich identified the protein kinase C substrate 80K-H asa potential associated protein [110]. In a recent study, wedemonstrated a specific interaction between 80K-H andTRPV5 and showed that a highly conserved short pep-tide sequence in the TRPV5 C-tail was necessary for80K-H binding [110]. Furthermore, 80K-H was shownto bind Ca2+ directly and inactivation of its two EF-hand structures totally abolished Ca2+ binding. Elec-trophysiological studies using 80K-H mutants showedthat three domains of 80K-H (the two EF-hand struc-tures, a highly acidic glutamic stretch and a His–Asp–Glu–Leu sequence) are critical determinants for TRPV5activity. Importantly, inactivation of the EF-hand pair

188

reduced the TRPV5-mediated Ca2+ current and in-creased the TRPV5 sensitivity for intracellular Ca2+,accelerating the feedback inhibition of the channel.None of the 80K-H mutants altered the TRPV5 plasmamembrane localization or the association of 80K-H withTRPV5, suggesting that 80K-H has a direct effect onTRPV5 activity at the plasma membrane. It was shownthat both proteins co-localized in the distal part of thenephron, indicating that regulation of TRPV5 by 80K-Hcould occur in vivo. Furthermore, parallel transcrip-tional regulation of both proteins by 1,25-(OH)2D3 anddietary Ca2+ was demonstrated [112]. Taken together,80K-H might act as novel Ca2+ sensor controllingTRPV5 channel activity at the plasma membrane.

pH

It is well known that metabolic acidosis and alkalosisinfluence Ca2+ homeostasis and studies using primarycultures of rabbit CNT and cortical collecting duct cellsindicated that acidification of the apical medium inhibitstranscellular Ca2+ absorption [2, 113, 114]. Therefore,pH might directly influence TRPV5/6 channel activity atthe apical plasma membrane. Indeed, TRPV5 expressingX. laevis oocytes showed decreased 45Ca2+ uptake whenthe incubation medium was acidified and extracellularacidification significantly reduced currents throughTRPV5 [115]. In addition, extracellular pH also affectedcurrent kinetics including extracellular Mg2+ blockadeand Ca2+ affinity. The mean current density decreasedat acidic pH and increased at alkaline pH. Recently, Yehand co-workers [115] showed that mutation of the Gluresidue at position 522 to Gln near the pore helix de-creased the inhibition of TRPV5 by extracellular acidi-fication. This Glu-522 in the extracellular loop betweenTM5 and the pore region, appears to mediate pH sen-sitivity and, therefore, acts as the pH sensor in TRPV5.The exact mechanism explaining these effects, possiblyby a conformational change upon titration of this aminoacid or owing to the neutralization of its negativecharge, is unknown. Alternatively, pH could also affectchannel insertion and/or retrieval from the plasmamembrane. Taken together, these data suggest that theluminal pH directly regulates Ca2+ entry through theepithelial Ca2+ channels in vivo.

Conclusion and future directions

This review has focused on the unique characteristicsand regulation of the two most Ca2+-selective TRPmembers, TRPV5 and TRPV6. These channels facilitatethe Ca2+ entry step in transcellular Ca2+ (re)absorptionin kidney and intestine, and thereby determine the netCa2+ flux to the extracellular compartment. Regulationof the membrane permeability for Ca2+ through TRPV5and TRPV6 is of particular physiological importance,and we have presented an overview of the current

knowledge of these regulatory processes. The tran-scriptional and translational regulation by 1,25-(OH)2D3, PTH, dietary Ca2+, oestrogens and theinfluence of drugs were established over the last years.Data on the mechanisms and importance of channelassembly and trafficking to the plasma membrane,including the role of auxiliary proteins, were recentlyinvestigated. The complex process from gene transcrip-tion to activation of channels at the plasma membraneremains, however, incompletely understood. Because theregulation at several discrete levels has now been dem-onstrated, future studies should focus on establishing acomprehensive and integrated model of TRPV5/6 reg-ulation and transcellular Ca2+ transport. Consequently,processes including trafficking of channels towards andfrom the plasma membrane have to be studied in detail.The role of the epithelial Ca2+ channels in human dis-ease also remains to be defined. The phenotypes ofTRPV5�/� and TRPV6�/� mice, including a profoundrenal Ca2+ leak and disturbed intestinal Ca2+ absorp-tion, suggest that these channels are interesting candi-date genes for several disorders including genetichypercalciuria, nephrolithiasis and Ca2+ malabsorptionsyndromes. Finally, the significant bone abnormalities inTRPV5�/� mice, along with the demonstration ofTRPV5 and TRPV6 expression in bone, suggest thatthese epithelial Ca2+ channels have additional physio-logical functions in bone dynamics.

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