TRPV5: A Ca²⁺ Channel for the Fine-Tuning of Ca²⁺ Reabsorption

Abstract

TRPV5 is one of the two channels in the TRPV family that exhibit high selectivity to Ca²⁺ ions. TRPV5 mediates Ca²⁺ influx into cells as the first step to transport Ca²⁺ across epithelia. The specialized distribution in the distal tubule of the kidney positions TRPV5 as a key player in Ca²⁺ reabsorption. The responsiveness in expression and/or activity of TRPV5 to hormones such as 1,25-dihydroxyvitamin D₃, parathyroid hormone, estrogen, and testosterone makes TRPV5 suitable for its role in the fine-tuning of Ca²⁺ reabsorption. This role is further optimized by the modulation of TRPV5 trafficking and activity via its binding partners; co-expressed proteins; tubular factors such as calbindin-D_28k, calmodulin, klotho, uromodulin, and plasmin; extracellular and intracellular factors such as proton, Mg²⁺, Ca²⁺, and phosphatidylinositol-4,5-bisphosphate; and fluid flow. These regulations allow TRPV5 to adjust its overall activity in response to the body’s demand for Ca²⁺ and to prevent kidney stone formation. A point mutation in mouse Trpv5 gene leads to hypercalciuria similar to Trpv5 knockout mice, suggesting a possible role of TRPV5 in hypercalciuric disorders in humans. In addition, the single nucleotide polymorphisms in Trpv5 gene prevalently present in African descents may contribute to the efficient renal Ca²⁺ reabsorption among African descendants. TRPV5 represents a potential therapeutic target for disorders with altered Ca²⁺ homeostasis.

1 Introduction

Ca²⁺ serves as an important intracellular and extracellular messenger, and it is indispensable for many physiological activities, such as muscle contraction, neuron excitability, and blood coagulation (Brown et al. 1995; Clapham 1995). For this reason, the plasma Ca²⁺ concentration is monitored by a Ca²⁺-sensing receptor, which controls the secretion of parathyroid hormone (PTH) (Brown 1991). PTH regulates the synthesis of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] (Henry 1985; Yang et al. 1999). PTH and 1,25(OH)₂D₃ regulate Ca²⁺ homeostasis via intestinal absorption and renal reabsorption, as well as deposition into and mobilization from the bone. In the intestine and kidney, Ca²⁺ is transported across epithelia through the tight junction between cells (paracellular pathway), or across the apical and basolateral membranes of epithelial cells (transcellular pathway) (Bronner 1998; Wasserman and Fullmer 1995). Only the transcellular pathway allows Ca²⁺ to be transported against an electrical and concentration gradient. The Ca²⁺-transporting epithelial cells in the intestinal and the kidney express proteins critical in the transcellular Ca²⁺ transport pathway. Ca²⁺ channels TRPV5 and TRPV6 in the apical membrane mediate Ca²⁺ entering into the cell (Hoenderop et al. 1999; Peng et al. 1999; Zhuang et al. 2002). The steep Ca²⁺ concentration gradient (approximately 1 mM extracellular vs. 0.1 μM intracellular) and the transmembrane potential (typically −60 mV inside the cell) allow Ca²⁺ to enter the cells via channels. Ca²⁺ exits the cell via high-affinity Ca²⁺–ATPase (PMCA) and/or Na⁺–Ca²⁺ exchanger 1 (NCX1) at the expense of energy stored in ATP or in Na⁺ concentration gradient (Wasserman et al. 1992). Ca²⁺-binding proteins (e.g., calbindins) are also expressed to keep the intracellular Ca²⁺ concentration ([Ca²⁺] _i ) low so that the driving force for Ca²⁺ entry is maintained and [Ca²⁺] _i will not rise to a level which is toxic to cells (Christakos et al. 1992; Lambers et al. 2006; Zheng et al. 2004). Although TRPV5 and TRPV6 were identified at a much later date compared to calbindins, PMCA, and NCX, their specific roles in the transcellular Ca²⁺ transport pathway energized the studies of Ca²⁺ absorption and reabsorption at molecular level since the cloning of TRPV5 and TRPV6. Compared to the broader distribution of TRPV6, TRPV5 is relatively confined to the kidney, where it is responsive to physiological signals to regulate Ca²⁺excretion into the urine.

2 Gene

TRPV5 was cloned as a result of searching for genes involved in Ca²⁺ reabsorption in kidney cells (Hoenderop et al. 1999). Using an expression cloning approach, Hoenderop and Bindels identified a cDNA from primary cells of rabbit connecting tubule (CNT) and cortical collecting duct (CCD) that stimulates ⁴⁵Ca²⁺ uptake in Xenopus laevis oocytes. The cDNA encodes a protein of 730 amino acid residues and was initially named as epithelial Ca²⁺ channel (ECaC) (Hoenderop et al. 1999). At the time of cloning, the only mammalian protein similar to TRPV5 was rat vanilloid receptor VR1 (TRPV1). Human, rat, and mouse TRPV5 share high levels of amino acid identity to rabbit TRPV5 (Peng et al. 2000a, 2001). The protein most similar to TRPV5 is TRPV6 (75 % amino acid identity), which was independently identified from rat duodenum using similar approach (Peng et al. 1999). TRPV5 shares approximately 40–45 % amino acid identity with other members of the TRPV family, including TRPV1 to TRPV4.

Trpv5 gene is located in human chromosome 7q35 side by side with TRPV6 gene in 7q33–34 (Muller et al. 2000; Peng et al. 2001). The human Trpv5 gene comprises 15 exons. Except for the first and last exons, exons 2–14 of Trpv5 and Trpv6 genes are identical in size (Peng et al. 2001). In fact, all genes of the TRPV family share a conserved gene structure (Peng et al. 2001). In addition to Trpv5 and Trpv6 in 7q34–35, genes for Trpv1, Trpv2, and Trpv3 are located in 17q11.2 to 17q13.2, suggesting recent gene duplication events in this subfamily. Fish and birds have only one Trpv6-like gene (Qiu and Hogstrand 2004; Yang et al. 2011), whereas mammalians have both Trpv5 and Trpv6 genes. Based on the Joint Genome Institute (JGI; http://genome.jgi-psf.org/) genome database for Xenopus tropicalis (Western clawed frog), five Trpv6-like genes are present. This is not restricted to Trpv6, as 6 Trpv4-like genes are present in X. tropicalis (Saito and Shingai 2006). Thus, it is likely that duplication events occurred in Trpv6 gene when amphibians and mammals were evolved, respectively (Fig. 1). The physiological roles of the Trpv6-like genes in X. tropicalis are unclear. In mammals, the duplicated gene became Trpv5 as it gained the ability for specialized expression in the distal tubule of kidney and properties for a finer regulation of channel function and trafficking.

Fig. 1

Trpv5 gene was likely produced by the duplication of Trpv6 gene during the process of evolution. Gene duplication events are indicated on the respective branches. WC frog, Western clawed frog (Xenopus tropicalis). Plus symbol indicates present, minus symbol indicates absent, and asterisk indicates that there are five genes similar to TRPV6 in Western clawed frogs

3 Tissue Distribution

In contrast to the broader tissue distribution of TRPV6, TRPV5 is rather specifically distributed to the kidney. Due to the high sequence similarity between TRPV5 and TRPV6, initial studies showed strong signals in Northern blot analyses with TRPV5 cDNA probe in rabbit and rat duodenum (Peng et al. 2000a; Hoenderop et al. 1999), but this is likely due to cross hybridization with TRPV6 mRNA, which is highly expressed in the duodenum (Peng et al. 2000a). Similarly, although TRPV5 is also expressed in the placenta, the mRNA abundance of TRPV5 is much lower than that of TRPV6 in human placenta (Peng et al. 2001).

Based on Expressed Sequence Tag (EST) database from the National Center for Biotechnology Information (NCBI), among 45 human organs listed, TRPV5 is detected in only two of them (numbers in brackets are tag/million): blood [8] and lung [2]. In contrast, TRPV6 is present in 13 organs/tissues: bladder [33], blood [16], brain [4], cervix [20], eye [9], intestine [8], lung [2], mammary gland [6], pancreas [4], placenta [215], prostate [47], testis [6], and trachea [19]. Both TRPV5 and TRPV6 are not detected in human kidney, indicating their number of EST is lower than 1/million. In mouse, among 47 organs/tissues listed, TRPV5 is present in 2, including embryonic tissue [1] and kidney [24], whereas TRPV6 is present in 9 of them including brain [10], embryonic tissue [2], extraembryonic tissue [120], lung [30], mammary gland [69], pancreas [37], salivary gland [103], skin [8], and thymus [8]. These EST profiles may not be accurate due to various reasons; however, they provide an unbiased overview of the gene expression. The expression profile of TRPV5 in mouse suggests a specific role of TRPV5 in the kidney.

In rabbit kidney, TRPV5 protein is expressed in the apical membrane of distal convoluted tubule (DCT), CNT, and CCD, where it co-localizes with calbindin-D_28k (Hoenderop et al. 1999). In rats, TRPV5 mRNA exhibits similar expression pattern as NCX1 and calbindin-D_28k (Peng et al. 2000a). In mouse kidney, TRPV5 is expressed in the late segment of DCT and CNT (Loffing et al. 2001).

In mice and rats, TRPV5 mRNA is much more abundant than that of TRPV6 in the kidney (Song et al. 2003; Van Cromphaut et al. 2007). TRPV5 mRNA level is approximately 10–20 times higher than that of TRPV6 in mouse kidney (Song et al. 2003). In contrast, we have observed much higher mRNA level of TRPV6 than TRPV5 in the human kidney (Peng et al. 2001). Our unpublished observations also indicated a higher mRNA level of TRPV6 in outer medulla than cortex of human kidney, opposite to that of TRPV5. Similarly, TRPV6 is also much abundant than TRPV5 in horse kidney (Rourke et al. 2010). Thus, TRPV6 may play more significant roles in some species such as humans and horses.

Even though TRPV5 is mainly expressed in the kidney, TRPV5 mRNA or protein has also been detected in other organs/tissues. TRPV5 is detected in mouse testis (Jang et al. 2012) and inner ear (Takumida et al. 2009) and rat testis, spermatogenic cells, sperm (Li et al. 2010), cochlea (Yamauchi et al. 2010), and osteoclast-like cells (Yan et al. 2011). In horse, TRPV5 mRNA is expressed in the duodenum and proximal jejunum (Rourke et al. 2010), and TRPV5 protein is detected in chondrocytes from the superficial zone of articular cartilage (Hdud et al. 2012). In human, TRPV5 protein and mRNA are present in placenta (Bernucci et al. 2006), osteoclasts (van der Eerden et al. 2005), retinal pigment epithelium (Kennedy et al. 2010), lymphocytes, Jurkat leukemia T cells (Vassilieva et al. 2013), and leukemia K562 cells (Semenova et al. 2009).

4 Structural Aspects

The mammalian TRPV5 proteins comprise around 730 amino acid residues (729, 731, 730, and 723 amino acids in human, horse, rabbit, rat, and mouse, respectively). Mammalian TRPV5 proteins consist of an intracellular N- and C-termini, six transmembrane domains, and a pore region between the last two transmembrane domains. In the intracellular portions of TRPV5, six ankyrin repeats are present in the N-terminal region, and a PDZ-binding motif is located in the C terminus (Phelps et al. 2008; de Groot et al. 2011b; Palmada et al. 2005; Jing et al. 2011). An N-linked glycosylation site is located in the first extracellular loop, and asparagine 358 (N358) is indispensable for the N-linked glycosylation of TRPV5 (Chang et al. 2005; Jiang et al. 2008). Putative protein kinase A (PKA) and protein kinase C (PKC) phosphorylation sites and calmodulin-binding sites are present within the N- and C-terminal regions (Hoenderop et al. 1999; Kovalevskaya et al. 2012). Some of these sites have already been shown to be important in regulating TRPV5.

Four TRPV5 molecules form a homotetramer with a single pore in the middle (Nilius et al. 2001b; Hoenderop et al. 2003b). Two critical motifs at positions 64–77 (part of the first ankyrin repeat) in the N terminus and at 596–601 in the C terminus of TRPV5 are needed for the formation of the functional channel complex (Chang et al. 2004). The N-terminal ankyrin repeats appear to be important for channel assembly. The first ankyrin repeat does not appear to be critical to the formation of TRPV5 tetramer; however, it is likely essential for correct folding of TRPV5 into a functional channel complex (de Groot et al. 2011b). In contrast, the third ankyrin repeat initiates a zippering process through the fifth ankyrin repeat, and they form an anchor for channel assembly (Erler et al. 2004). The tetramerization of TRPV5 allows the four aspartate 542 (D542) residues in pore-forming loop between transmembrane domain 5 and 6 to form a Ca²⁺-selective ring, which is also involved in Mg²⁺ blockade (Nilius et al. 2001b; Voets et al. 2004; Lee et al. 2005).

TRPV5 and TRPV6 appeared to form heterotetramers when four pieces of TRPV5 and TRPV6 were linked together and heterologously expressed in X. laevis oocytes (Hoenderop et al. 2003b). The more TRPV6 subunits presented in the heterotetramer, the more TRPV6 characteristics were exhibited (Hoenderop et al. 2003b). Other TRPV members preferentially form homomeric assembly; however, TRPV5 and TRPV6 can form heteromeric complex in HEK293 cells (Hellwig et al. 2005). This is likely due to the high degree of similarity between TRPV5 and TRPV6. Unlike in transfected cells, TRPV5 is likely outnumbered by TRPV6 in most cells. It is unclear whether heteromeric TRPV5 and TRPV6 complexes play a significant physiological role if they exist in vivo. In addition, TRPV5 and TRPV6 share certain structure–function similarities with TRPML3 (Grimm et al. 2007). They can form heteromers which display different features than the respective homomers (Guo et al. 2013). TRPML3 and TRPV5 are expressed in the kidney and inner ear (Takumida et al. 2009; Castiglioni et al. 2011); however, it is yet to be determined whether TRPML3 and TRPV5 form heteromeric complexes in native cells.

5 Biophysical Properties

5.1 Ca²⁺ Selectivity

TRPV5 is a Ca²⁺ permeant ion channel as it was identified as a result of increasing ⁴⁵Ca²⁺ uptake activity when expressed in Xenopus oocytes (Hoenderop et al. 1999). The most distinctive feature of TRPV5 is its Ca²⁺ selectivity. The permeability ratio between Ca²⁺ and Na⁺ (P _Ca:P _Na) is over 100 (Vennekens et al. 2000). The divalent cation selectivity profile of TRPV5 is Ca²⁺ > Mn²⁺ > Ba²⁺ ≈ Sr²⁺ (Vennekens et al. 2000). In the absence of divalent cation in the extracellular solution, TRPV5 allows monovalent cations to pass through. The permeation sequence for monovalent cations through TRPV5 is Na⁺ > Li⁺ > K⁺ > Cs⁺ > NMDG⁺ (Nilius et al. 2000). The Na⁺ current of TRPV5 correlates with the amplitude of Ca²⁺ uptake and is often used as a measure of TRPV5 channel activity. Some trivalent and divalent cations block current through TRPV5, and the sequence of block is Pb²⁺ = Cu²⁺ = Gd³⁺ > Cd²⁺ > Zn²⁺ > La³⁺ > Co²⁺ > Fe²⁺ > Fe³⁺ (Nilius et al. 2001a). TRPV5 is also sensitive to ruthenium red and econazole (Nilius et al. 2001a). TRPV5 and TRPV6 show difference in ruthenium red sensitivity: the IC₅₀ of ruthenium red for TRPV5 is around 121 nM, which is nearly100-fold lower than that for TRPV6 (Hoenderop et al. 2001b). Extracellular and intracellular Mg²⁺ blocks TRPV5 voltage dependently (Voets et al. 2001; Lee et al. 2005; Hoenderop et al. 2001b). D542 is the key residue to form the selectivity filter of TRPV5 and determines Ca²⁺ permeation and Mg²⁺ blockade (Nilius et al. 2001b; Lee et al. 2005; Dodier et al. 2007). The nonsynonymous single nucleotide polymorphism (SNP) A563T variation, close to D542, increases TRPV5 sensitivity to extracellular Mg²⁺, resulting in suppressed Na⁺ permeation through TRPV5 (Na et al. 2009). In clinical trials, urinary Ca²⁺ excretion is proportional to changes in magnesium excretion, and this is likely related to the blockade of TRPV5 by Mg²⁺ (Bonny et al. 2008).

TRPV5 currents exhibit strong inward rectification (Vennekens et al. 2000). Using TRPV6 as a model, Voets et al. demonstrated that intracellular Mg²⁺ acts as a permeant pore blocker and contributes to strong inward rectification of the channel (Voets et al. 2003). In addition, TRPV6 also exhibits intrinsic Mg²⁺-independent inward rectification for which the mechanism is not fully understood (Voets et al. 2003). Similar mechanisms are likely applicable to TRPV5 due to the high degree of similarity in structure and function between TRPV5 and TRPV6. Unitary channel activity of TRPV5 could be detected in the absence of extracellular divalent cations (Nilius et al. 2000). The single-channel conductance is 77.5 pS for rabbit TRPV5 using Na⁺ as a charge carrier (Nilius et al. 2000). Single-channel activity of TRPV5 was also detected using K⁺ as a charge carrier (Vassilev et al. 2001). In addition, subconductance state (partial channel opening) of TRPV5 was also observed (subconductance at 29 pS vs. full conductance at 59 pS at intracellular pH 7.4) (Cha et al. 2007).

5.2 pH Sensitivity

Another feature of TRPV5 is its pH sensitivity. In the initial characterization, TRPV5-mediated Ca²⁺ uptake was significantly inhibited when the pH value in the extracellular medium was lowered to 5.9 (Hoenderop et al. 1999). At single-channel level, both full and subconductance and their open probabilities were reduced by lower intracellular pH (Cha et al. 2007). On the other hand, high pH stimulates TRPV5-mediated Ca²⁺ uptake (Peng et al. 2000a). Low pH increases the blockage of monovalent cation current by extracellular Mg²⁺ (Vennekens et al. 2001). Glutamate 522 (E522) of rabbit TRPV5 acts as the extracellular pH sensor, and extracellular protons decrease the estimated diameter of channel pore to inhibit TRPV5 (Yeh et al. 2003, 2005). Glutamate 535 (E535) is also involved; however, its effect depends on E522 (Yeh et al. 2006). Meanwhile, intracellular protons also regulate TRPV5 (Yeh et al. 2005). Intracellular acidification promotes proton binding to lysine 607, intracellular pH sensor, and induces rotation of the pore helix, leading to decreases in the pore diameter, open probability, and single-channel conductance (Yeh et al. 2005; Cha et al. 2007).

In addition to channel property, pH may also affect the level of TRPV5 protein at the cell surface. Extracellular alkalization induces the recruitment of TRPV5 proteins to the cell surface from TRPV5-containing vesicles, and extracellular acidification leads to the retrieval of TRPV5 from plasma membrane (Lambers et al. 2007). Thus, TRPV5 could be regulated by pH in both channel activity and plasma membrane expression. Metabolic acidosis and alkalosis affect Ca²⁺ transport in the kidney (Sutton et al. 1979). Clinical trial data indicate that changes in urinary Ca²⁺ excretion and urine pH are inversely related (Bonny et al. 2008). The pH sensitivity of TRPV5 may contribute to the dysregulation of Ca²⁺ reabsorption under disturbance in acid–base balance.

5.3 Ca²⁺-Dependent Inactivation

High level of free Ca²⁺ is toxic to cells. The Ca²⁺-dependent inactivation limits the amount of Ca²⁺ ions that enter the cell through TRPV5 and prevents the toxic impact of Ca²⁺ overload in the cell. Calbindin-D_28k binds Ca²⁺ beneath the plasma membrane and therefore relieves the Ca²⁺-dependent inactivation of TRPV5 to some extent; it may also buffer free Ca²⁺ to limit the toxic effect (Lambers et al. 2006). TRPV5 exhibits Ca²⁺-dependent autoregulatory mechanisms, including fast inactivation and slow rundown (Vennekens et al. 2000). The C-terminal sequences in rabbit TRPV5 701–730 and 650–653 are critical determinants of Ca²⁺-dependent inactivation (Nilius et al. 2003). Compared to TRPV6, TRPV5 exhibit a much slower initial inactivation phase (Nilius et al. 2002). The first intracellular loop is critical to the kinetics of the initial phase in the inactivation process; three residues at positions 409, 411, and 412 in TRPV5 are critical for the delayed inactivation (Nilius et al. 2002). In addition, Q579 following the last transmembrane domain of TRPV5 is also important to the fast Ca²⁺-dependent inactivation kinetics of TRPV5 (Suzuki et al. 2002). The first intracellular loop of TRPV5 is capable of binding to CaM (Kovalevskaya et al. 2012), so is the C terminus of TRPV5 (de Groot et al. 2011a; Kovalevskaya et al. 2012). TRPV5 mutant deficient in interacting with CaM at C terminus (residues 696–729) exhibits diminished Ca²⁺-dependent inactivation (de Groot et al. 2011a). However, it is yet to be clarified what roles the other CaM-binding sites of TRPV5 play in the Ca²⁺-dependent inactivation mechanism. In addition, a helix-breaking mutation (M490P) in transmembrane domain 5 reduces Ca²⁺-dependent inactivation of TRPV5 and induces apoptosis due to Ca²⁺ overload in cells expressing TRPV5^M490P (Lee et al. 2010). Corresponding mutation in TRPML3 (A419P) leads to constitutive channel activity, which likely results in hair cell death in the inner ear and profound deafness and other abnormalities in mice homozygous in TRPML3^A419P (Grimm et al. 2007).

Although TRPV5 works constitutively in the kidney, its activity depends on the availability of phosphatidylinositol-4,5-bisphosphate (PIP₂). As the level of PIP₂ decreases, so does the activity of TRPV5 (Lee et al. 2005). PIP₂ activates TRPV5 in part by reducing conformational change-induced Mg²⁺ binding (Lee et al. 2005). Arginine 599 in mouse or rat TRPV5 (corresponding to R606 in rabbit and human TRPV5) in the “TRP domain” is likely the PIP₂-binding site (Rohacs et al. 2005). Elevation of [Ca²⁺] _i due to Ca²⁺ influx through TRPV5 may activate phospholipase C (PLC), which depletes PIP₂. Thus, removal of PIP₂ is an important Ca²⁺-dependent inactivation mechanism for TRPV5 as well as TRPV6 (Thyagarajan et al. 2008).

5.4 Responsiveness to Fluid Flow

Flow-stimulated K⁺ secretion in the distal nephron involves Ca²⁺-activated K⁺ channels. TRPV5 is expressed in the apical membrane in the distal nephron and therefore may provide a Ca²⁺ influx pathway to activate K⁺ channels. Recent study indicates that both TRPV5 and TRPV6 expressed in HEK cells are activated by shear force generated by fluid flow in physiological range (Cha et al. 2013). Flow-induced surge in TRPV5 or TRPV6 activity leads to the activation of co-expressed Slo1 maxi-K⁺ channel without affecting ROMK channel (Cha et al. 2013). Activation of maxi-K⁺ channel may counteract the depolarization due to Ca²⁺ influx and cause hyperpolarization which in turn would improve Ca²⁺ influx through TRPV5. N-linked glycosylation of the channels is involved in the responsiveness to fluid flow (Cha et al. 2013). The responsiveness of TRPV5 and TRPV6 to flow may have implications not only in flow-stimulated K⁺ secretion but also in thiazide-induced Ca²⁺ reabsorption.

6 Regulation by Interacting, Co-expressed, and Tubular Proteins

A number of proteins have been found to regulate TRPV5. These include TRPV5-interacting proteins, proteins co-expressed with TRPV5, and proteins in the tubular fluid (Table 1). Regulation of TRPV5 by some of the proteins is briefly summarized in the following.

Table 1 Proteins that regulate TRPV5

6.1 Regulation by Interacting Proteins

Some TRPV5-interacting proteins, such as S100A10, Rab11a, and NHERF2, regulate the trafficking and plasma membrane abundance of TRPV5. On the other hand, some TRPV5-interacting proteins, such as calbindin-D_28k, CaM, 80H-K, BSPRY, and FKBP52, regulate TRPV5 activity. It is worth noting that the C terminus of TRPV5 interacts with most of its binding partners, and the region from 598 to 608 in TRPV5 within the conserved “TRP domain” binds to S100A10, Rab11a, 80K-H, NHERF4, and CaM (Fig. 2). This region also contains the PIP₂-binding site (R606) and a pH sensor (K607). It is also not far away from histidine 711 (corresponding to H712 in rabbit TRPV5), a critical residue involved in the constitutive internalization of TRPV5 (de Groot et al. 2010). Thus, it is plausible that TRPV5-binding partners may affect TRPV5 function, membrane stability, and trafficking. However, it is not likely that these TRPV5-binding proteins regulate TRPV5 at the same time. It is possible that some of the proteins only regulate TRPV5 at a certain cell type, at a specific developmental stage, or at a state being stimulated by a hormone or other physiological cues. Most TRPV5-interacting proteins also interact with TRPV6 due to the conserved binding sites in the two channels. Thus, it is likely some of the TRPV5-interacting proteins may be more involved in regulating TRPV6 than TRPV5. Lastly, although overlapping expression with TRPV5 was found for most of the TRPV5-interacting proteins, they do express in cells that do not express TRPV5; they all have functions other than regulating TRPV5.

Fig. 2

Functional domains/sites in human TRPV5 (GenBank #: AF304464.1). Key residues, domains, and interacting motifs/sites for partners are indicated in the predicted structure of TRPV5. Factors with positive and negative effects on TRPV5 are labeled with green and red rectangles, respectively. See text for details

6.1.1 S100A10

S100A10 binds to a conserved motif of five residues (⁵⁹⁸VATTV⁶⁰²) in the C terminus of TRPV5 (van de Graaf et al. 2003). S100A10 is a member of the EF-hand containing S100 protein family, and it forms heterotetramer with annexin 2, a Ca²⁺, and phospholipid-binding protein in association with cytoskeleton underneath cell membrane (Gerke and Moss 2002). S100A10–annexin 2 complex is important for the plasma membrane localization of TRPV5 (van de Graaf et al. 2003). In addition, this complex may be involved in 1,25(OH)₂D₃-induced Ca²⁺ reabsorption as S100A10 is upregulated by 1,25(OH)₂D₃ (van de Graaf et al. 2003)_.

6.1.2 Rab11a

The small GTPase Rab11a is another TRPV5-interacting protein that regulates its trafficking to the plasma membrane (van de Graaf et al. 2006a). It binds to ⁵⁹⁶MLERK⁶⁰⁰ of mouse TRPV5 (603–607 in human TRPV5), following the S100A10-binding motif (van de Graaf et al. 2006a). Glycine substitution in the MLERK motif disrupts the binding of Rab11a to TRPV5 and the plasma membrane expression of TRPV5 (van de Graaf et al. 2006a). TRPV5 is one of a few cargo proteins that bind directly to the GDP-bound Rab proteins.

6.1.3 PDZ Proteins

The last four amino acids of TRPV5 and TRPV6 are PDZ (postsynaptic density-95, Drosophila discs-large protein, zonula occludens protein 1)-binding motifs. TRPV5 interacts with Na⁺/H⁺ exchanger regulating factors 2 (NHERF2) (Palmada et al. 2005). Removal of the last three amino acids of TRPV5 abolishes the interaction (van de Graaf et al. 2006b). TRPV5 also interacts with NHERF4; however, the interacting motif is located between residues 596 and 617 of mouse TRPV5 (603–624 of human TRPV5) (van de Graaf et al. 2006b). In contrast, NHERF4 interact with the last three amino acid residues of TRPV6 (Kim et al. 2007), suggesting it is likely TRPV6 is the real binding partner of NHERF4. The 4th PDZ domain of NHERF4 and the second PDZ domain of NHERF2 are essential for their interactions with TRPV5 (Kim et al. 2007; Palmada et al. 2005). NHERF2 is important to the effects of SGK1 and WNK4 on TRPV5 (Embark et al. 2004; Jing et al. 2011).

6.1.4 Calbindin-D_28k

Calbindins are Ca²⁺-binding proteins that are well known for their responsiveness to vitamin D and possible roles in Ca²⁺ absorption and reabsorption (Christakos et al. 1992). Calbindin-D_28k is co-expressed with TRPV5 in DCT and CNT (Loffing et al. 2001). Both N- and C-termini of TRPV5 interact with calbindin-D_28k in the absence but not in the presence of Ca²⁺ ions (Lambers et al. 2006). When [Ca²⁺] _i is low, calbindin-D_28k translocates toward the plasma membrane and associates with TRPV5. Calbindin-D_28k buffers Ca²⁺ close to the vicinity of the channel opening, thereby reducing local accumulation of free Ca²⁺ ions. After binding to Ca²⁺, calbindin-D_28k disassociates from TRPV5 and facilitates the diffusion of Ca²⁺ to the basolateral membrane (Lambers et al. 2006). Calbindin-D_28k expression in the kidney is greatly reduced in mice lacking TRPV5 (Hoenderop et al. 2003a). Mice lacking calbindin-D_28k do not show hypercalciuria, and mice lacking both TRPV5 and calbindin-D_28k do not exhibit more severe phenotype in Ca²⁺ homeostasis than mice lacking TRPV5 alone (Gkika et al. 2006a). These results indicate TRPV5 but not calbindin-D_28k is critical to Ca²⁺ reabsorption. Calbindin-D_28k acts as a dynamic Ca²⁺ buffer to avoid a sudden elevation of [Ca²⁺] _i ; however, it appears that its role could be compensated by other vitamin D-regulated Ca²⁺-binding proteins. The role of calbindin-D_28k in Ca²⁺ homeostasis could only be observed in the absence of the effects of vitamin D: mice lacking vitamin D receptor (VDR) and calbindin-D_28k display more severe hypercalciuria and secondary hyperparathyroidism than mice lacking VDR alone (Zheng et al. 2004).

6.1.5 Calmodulin

Being an intracellular Ca²⁺ sensor, calmodulin (CaM) is expected to play a role in modulating TRPV5 function. However, CaM antagonists calmidazolium R24571 and trifluoperazine only modestly inhibit TRPV5 at high concentrations (Nilius et al. 2001a). In addition, Ca²⁺-insensitive CaM mutants significantly reduced Na⁺ and Ca²⁺ currents of TRPV6 but not those of TRPV5 (Lambers et al. 2004). Yet, CaM is capable of binding to TRPV5. So far five CaM-binding sites in TRPV5 have been identified, including residues 696–712 (de Groot et al. 2011a; Kovalevskaya et al. 2012) and 591–612 (Kovalevskaya et al. 2012; Holakovska et al. 2011) in the C terminus, 401–428 in the first intracellular loop (Kovalevskaya et al. 2012), and 310–330 and 133–154 in the N-terminal region (Kovalevskaya et al. 2012). These CaM-binding sites display diversity in binding mode, stoichiometry, and affinity in interaction with CaM in vitro (Kovalevskaya et al. 2012). The first intracellular loop of TRPV5/6 plays a critical role in the fast and slow inactivation kinetics (Nilius et al. 2002), but it is unclear to what extent Ca²⁺/CaM is involved in this process. CaM negatively modulates TRPV5 activity by binding to the residues 696–729, and PTH-mediated PKA phosphorylation of the CaM-binding site reverses this action (de Groot et al. 2009, 2011a). In addition, phosphorylation of TRPV5 by PKC at serine 144 (S144) in the N-terminal CaM-binding site (133–154) results in decreased pore size and open probability in TRPV5 (Tudpor et al. 2012). This regulation is important in mediating the action of plasmin on TRPV5 (Tudpor et al. 2012).

6.1.6 80K-H

80K-H is a Ca²⁺-binding protein and a PKC substrate (Sakai et al. 1989). It was identified as a potential TRPV5-interacting protein because it was downregulated in 1,25(OH)₂D₃-deficient mice and was upregulated by dietary Ca²⁺ (Gkika et al. 2004). 80K-H-binding site in TRPV5 is located between residues 598 and 608 (Gkika et al. 2004). Co-expression of 80K-H did not alter the level of TRPV5 in the cell surface, but increased the sensitivity of TRPV5 to [Ca²⁺] _i (Gkika et al. 2004). Thus, 80K-H acts as a Ca²⁺ sensor to control the activity of TRPV5 in addition to CaM.

6.1.7 BSPRY

BSPRY (B-box and SPRY-domain-containing protein), a protein with unknown function, interacts with TRPV5 C terminus (van de Graaf et al. 2006c). It inhibits Ca²⁺ transport activity of TRPV5 without altering its surface level. BSPRY co-localized with TRPV5 in the DCT and CNT, and it is inversely regulated by 1,25(OH)₂D₃ (van de Graaf et al. 2006c). Thus, BSPRY adds a layer of regulation of TRPV5 by 1,25(OH)₂D₃.

6.1.8 FKBP52

Immunophilin FKBP52, one of the downstream targets of FK-506, co-localizes with TRPV5 in the DCT and CNT and inhibits TRPV5 activity in vitro (Gkika et al. 2006c). The peptidyl-propyl cis-trans isomerase activity of FKBP52 is essential to its inhibitory effect on TRPV5, and the inhibitory effect is reversed by the administration of FK-506. FKBP52 interacts with full-length TRPV5, but not its N- or C terminus (Gkika et al. 2006c). FKBP52 expression is decreased in Trpv5 KO mice, indicating a link between the two proteins (Gkika et al. 2006c). Since FK-506 induces hypercalciuria and causes a reduction in calbindin-D_28k and TRPV5 expression in the kidney (Nijenhuis et al. 2004; Lee et al. 2011; Aicher et al. 1997), further investigation is necessary to clarify the role of interaction between FKBP52 and TRPV5 in FK-506-induced hypercalciuria.

6.2 Regulation of TRPV5 by Co-expressed Proteins

In addition to the proteins that interact directly with TRPV5, proteins co-expressed with TRPV5 may also regulate TRPV5. These TRPV5-regulating proteins were studied because of physiological relevance, not as results of searching for TRPV5-binding partners. Transient protein interaction may or may not be involved in these regulations.

6.2.1 Klotho

Klotho functions to suppress aging process (Kuro-o et al. 1997). Klotho exists in membrane bound and circulating forms; it converts fibroblast growth factor (FGF) receptor FGFR1(IIIc) into a specific receptor for FGF 23 by binding with it (Urakawa et al. 2006). In the kidney, klotho is expressed in the DCT where it co-localizes with TRPV5 and increases TRPV5 activity in vitro (Chang et al. 2005; Lu et al. 2008; Cha et al. 2008a). The mechanism by which klotho regulates TRPV5 is not well understood. Because klotho exhibits β-glucuronidase activity (Tohyama et al. 2004), it was thought that klotho modifies TRPV5 glycan through this activity (Chang et al. 2005). Like klotho, β-glucuronidase also activates TRPV5 and TRPV6 but not related TRP channels TRPV4 and TRPM6 (Lu et al. 2008). Alternatively, klotho removes terminal sialic acids from their glycan chains of TRPV5 and exposes disaccharide galactose-N-acetylglucosamine, which binds to galactoside-binding lectin galectin-1 (Cha et al. 2008a). The galectin-1-linked TRPV5 proteins are likely resistant to endocytosis. However, complete removal of N-glycan by endoglycosidase-F also increases TRPV5 activity (Lu et al. 2008). However, galectin-3 but not galactin-1 co-localizes with TRPV5 in the DCT (Leunissen et al. 2013). Sialidase appears to increase TRPV5 activity by inhibiting lipid-raft-mediated endocytosis, and it does not discriminate N-glycan-deficient N358Q mutant and wild-type TRPV5 (Leunissen et al. 2013). Thus, klotho regulates TRPV5 in an N-glycan-dependent manner, whereas sialidase regulates TRPV5 in an N-glycan-independent and lipid-raft-mediated endocytosis-dependent manner (Leunissen et al. 2013). Although the mechanisms are still to be clarified, the regulation of TRPV5 by klotho represents a novel area of ion channel regulation. The dysregulation of TRPV5 may be responsible for the increased excretion of Ca²⁺ in Kl (klotho gene)-deficient mice, in which PTH-stimulated Ca²⁺ reabsorption in the CNT is impaired (Tsuruoka et al. 2006). Lower plasma klotho concentration is associated with older age and lower serum Ca²⁺ level and is an independent risk factor for mortality in community-dwelling adults of 65 years or older (Semba et al. 2011). Presumably reduced TRPV5 activity due to the lower klotho level likely contributes to the lower serum Ca²⁺ and may play a role in the aging process. Although klotho is considered a hormone, its action on TRPV5 from the luminal side is distinct from its hormonal action from interstitium. However, it could be considered as a factor in the tubular fluid similar to plasmin and uromodulin.

6.2.2 Tissue Transglutaminase

Tissue transglutaminase (tTG) catalyzes Ca²⁺-dependent covalent cross-linking of specific lysine and glutamine residues of substrate proteins (Lorand and Graham 2003). Calbindin-D_28K and S100A10, which regulate TRPV5, are substrates of tTG (Vig et al. 2007; van de Graaf et al. 2003; Ruse et al. 2001; Lambers et al. 2006). In addition, activity of TRPV5 is inhibited by extracellular tTG treatment in HEK-293 and in rabbit CNT/CCD cells (Boros et al. 2012). This is caused by the reduction in channel pore diameter in aggregated TRPV5 in the plasma membrane as a result of tTG activity. N-glycosylation-deficient TRPV5 mutant is insensitive to tTG (Boros et al. 2012). Klotho and tTG both require the presence of N-glycan of TRPV5 for their action, but they direct TRPV5 activity to opposite directions.

6.2.3 Tissue Kallikrein

Tissue kallikrein (TK) is a serine protease expressed in CNT (Figueroa et al. 1988). TK knockout (KO) mice exhibit hypercalciuria (Picard et al. 2005). Consistent with this, TRPV5 was shown to be regulated by TK via PLC/diacylglycerol/PKC pathway (Gkika et al. 2006b). TK enhances TRPV5-mediated Ca²⁺ influx by delaying its retrieval from the plasma membrane (Gkika et al. 2006b). This effect was abolished by S299A and S654A mutations, which may disrupt phosphorylation of TRPV5 by PKC (Gkika et al. 2006b), which increases TRPV5 activity by inhibiting its endocytosis from plasma membrane (Cha et al. 2008b).

6.2.4 SGK1/3

Co-expression of NHERF2 and serum/glucocorticoid regulated kinase 1 or 3 (SGK1/3) with TRPV5 enhances TRPV5 activity in X. laevis oocytes (Embark et al. 2004). However, NHERF2 or SGK1/3 alone does not alter TRPV5 activity (Embark et al. 2004). The second PDZ domain in NHERF2 is required for the stimulatory effect of SGK1/NHERF2 on TRPV5 (Palmada et al. 2005). In the SGK1 KO mice, TRPV5 protein abundance in the CNT was reduced; however, urinary Ca²⁺ excretion was also reduced (Sandulache et al. 2006). The increased Ca²⁺ reabsorption is likely due to a compensatory increase in the function of thick ascending limb in response to the salt loss in the aldosterone-sensitive distal nephron. The reduced expression of TRPV5 in the CNT of SGK1 KO mice is possibly a compensatory effect due to increased Ca²⁺ reabsorption in the thick ascending limb, not necessarily a result due to the lack of positive regulation of TRPV5 by SGK1 (Sandulache et al. 2006).

6.2.5 WNK Kinases

Point mutations in with-no-lysine (K) kinase 4 (WNK4) result in pseudohypoaldosteronism type II (PHAII, also known as familial hyperkalemia and hypertension or Gordon’s syndrome) (Wilson et al. 2001). Hypercalciuria was observed in PHAII patients with WNK4^Q565E mutation, leaving the possibility that WNK4 may regulate a Ca²⁺ channel or transporter in the kidney (Mayan et al. 2004). We found WNK4 increases the activity of TRPV5 channel by increasing the forward trafficking of the channel to the plasma membrane via the secretory pathway in X. laevis oocytes (Jiang et al. 2007, 2008). The positive effect of WNK4 on TRPV5 was greatly reduced when NCC was co-expressed (Jiang et al. 2007). Similar to WNK4, WNK3 also increases Ca²⁺ influx mediated by TRPV5 via a kinase-dependent pathway (Zhang et al. 2008). The effect of WNK4 on TRPV5 is enhanced and stabilized by NHERF2 (Jing et al. 2011). When the last two amino acid residues of TRPV5 were replaced by those of TRPV6, the effect of NHERF2 on TRPV5 was abolished (Jing et al. 2011). Thus, WNK4 and NHERF2 increase TRPV5 forward trafficking and membrane stability synergistically, leading to additive enhancement in TRPV5-mediated Ca²⁺ transport. On the other hand, Cha et al. found that WNK4 kinase enhances the endocytosis and decreases the plasma membrane abundance of TRPV5 in HEK293 cells (Cha and Huang 2010). The reason for the difference in WNK4-mediated regulation on TRPV5 is unclear.

6.2.6 Ubiquitin E3 Ligases

Nedd4-2 is an archetypal member of the ubiquitin E3 ligase family regulating cell surface stability of membrane proteins (Staub and Rotin 2006). Nedd4-2 is expressed in the DCT (Verrey et al. 2003) and CCD (Flores et al. 2005) in the kidney where TRPV5 is functionally expressed. When expressed in Xenopus oocytes, TRPV5/6-mediated Ca²⁺ uptake and Na⁺ current were decreased by Nedd4-2 and Nedd4 due to the reduction in TRPV5 protein level (Zhang et al. 2010). In all cases, Nedd4-2 exhibited stronger inhibitory effects than Nedd4 on both TRPV5 and TRPV6. WW1 and WW2 domains of Nedd4-2 may serve as a molecular switch to limit the ubiquitination of TRPV6/5 by the HECT domain (Zhang et al. 2010). Although Nedd4 and Nedd4-2 mediate TRPV5 degradation in vitro, it is unclear to what extent they do so in vivo. In addition, the degradation of TRPV5 is reduced by knocking down of Ubiquitin recoginition 4 (UBR4), another E3 ubiquitin ligase (Radhakrishnan et al. 2013). The physiological significance of these TRPV5 degradation pathways warrants further studies.

6.2.7 Ca²⁺-Sensing Receptor

The mRNA and protein of Ca²⁺-sensing receptor (CaR) are expressed in the DCT/CNT (Riccardi et al. 1996, 1998; Hoenderop et al. 1999). Activation of CaR increases TRPV5-mediated currents and elevates [Ca²⁺] _i in cells co-expressing TRPV5 and CaR (Topala et al. 2009). Phorbol-12-myristate-13-acetate (PMA)-insensitive PKC isoforms are likely involved in the signal pathway by which CaR stimulates TRPV5 (Topala et al. 2009). The stimulatory effect was abolished by mutation of two putative PKC phosphorylation sites, S299 and S654 in TRPV5, or by a dominant-negative CaR (R185Q) (Topala et al. 2009).

6.3 Regulation by Proteins in the Tubular Fluid

6.3.1 Plasmin

Plasminogen, which is elevated and can be filtered into the urine in nephrotic syndrome, is converted into active plasmin in the tubular fluid. When incubated with plasmin, either from commercial source or purified from nephrotic urine, TRPV5-mediated Ca²⁺ uptake in HEK-293 cells was inhibited (Tudpor et al. 2012). As a serine protease, plasmin does not cleave TRPV5 or alter TRPV5 surface abundance; instead, it affects TRPV5 by binding to protease-activated receptor-1 (PAR-1) (Tudpor et al. 2012). This activates the PAR-1/PLC/PKC pathway, which likely leads to phosphorylation of S144 within a CaM-binding site in the N terminus of TRPV5. The phosphorylation of S144 results in an alteration of CaM binding to TRPV5 and, in turn, a reduction in pore size and open probability (Tudpor et al. 2012). Suppression of TRPV5 by plasmin likely takes effect in nephrotic patients.

6.3.2 Uromodulin

Uromodulin (also known as Tamm–Horsfall glycoprotein/THP) is a urinary glycoprotein secreted by the thick ascending loop of Henle (Bachmann et al. 1990). Co-expression with uromodulin increases TRPV5 current density and surface abundance in HEK293 cells (Wolf et al. 2013). Acting from the tubular luminal side, uromodulin decreases caveolin-mediated endocytosis of TRPV5, and the level of TRPV5 is lower in uromodulin KO mice (Wolf et al. 2013). This result suggests that uromodulin may act as a physiological regulator of TRPV5 to prevent kidney stone formation.

7 Regulation by Hormones

Trpv5 expression is regulated under physiological conditions. Trpv5 expression in the kidney is upregulated under dietary Ca²⁺ restriction in mice (Song et al. 2003; Van Cromphaut et al. 2001); this regulation appears to be VDR dependent (Van Cromphaut et al. 2001). TRPV6 and TRPV5 in rat small intestine were decreased by immobilization and induced by endurance swimming, and a 1,25(OH)₂D₃-dependent pathway is likely involved in these changes (Sato et al. 2006; Teerapornpuntakit et al. 2009). TRPV5 was upregulated by high-salt intake and downregulated by low-salt intake or dehydration in rats; this was likely caused by the increase or decrease in the delivery of Ca²⁺ to the distal tubule in the kidney (Lee et al. 2012). Aging is associated with alterations in Ca²⁺ homeostasis, such as decreased Ca²⁺ absorption and increased urinary Ca²⁺ excretion. Duodenal TRPV5/6 mRNA level in adult (12-month old) rats was less than half of that in young (2-month old) rats (Brown et al. 2005). Trpv5 KO mice develop age-related hyperparathyroidism and osteoporotic characteristics earlier than wild-type mice, possibly due to the age-related vitamin D resistance and less robust compensatory expression of intestinal TRPV6 in older mice (van Abel et al. 2006). Thus, as Trpv5 KO mice age, vitamin D resistance prevents the animals to compensate the renal loss of Ca²⁺ through intestinal absorption, leading to a negative Ca²⁺ balance.

Hormonal regulation is at least in part behind the adaptation of TRPV5 expression in response to physiological conditions. Indeed, a number of hormones, including 1,25(OH)₂D₃, PTH, sex hormones, and vasopressin, have been found to regulate TRPV5 (Table 2). The effects of these hormones on TRPV5 are summarized briefly below.

Table 2 Hormones that regulate TRPV5

7.1 Vitamin D

In 1991, Bindels and colleagues reported that 1,25(OH)₂D₃ increases transcellular Ca²⁺ absorption in primary culture of rabbit kidney CCD (Bindels et al. 1991) where TRPV5 was later cloned (Hoenderop et al. 1999). After the identification, TRPV5 mRNA was shown to be upregulated by 1,25(OH)₂D₃ in the kidney of vitamin D-depleted rats (Hoenderop et al. 2001a). Moreover, a single injection of 1,25(OH)₂D₃ in vitamin D-deficient mice induced ~three- to fourfold increase in TRPV5 mRNA in the kidney that peaked at 12 h after injection following the peak of duodenal TRPV6 (6 h after injection) (Song et al. 2003). This suggests that the increase in TRPV5 in the kidney might be a secondary event due to the increased Ca²⁺ absorption. In mice deficient in 25-hydroxyvitamin D₃-1α-hydroxylase with undetectable level of 1,25(OH)₂D₃, the reduction of renal TRPV5 and hypocalcemia were normalized by high Ca²⁺ diet, suggesting that TRPV5 is upregulated by increased Ca²⁺ load to the distal tubule as a result of 1,25(OH)₂D₃-induced Ca²⁺ absorption (Hoenderop et al. 2002). Although potential vitamin D response elements (VDREs) have been identified in the promoter region of Trpv5 gene (Muller et al. 2000; Weber et al. 2001), their function has not been examined as those bona fide VDREs in Trpv6 gene promoter (Meyer et al. 2006). At least in mice duodenum, FGF-23 could reduce circulating 1,25(OH)₂D₃ and diminish 1,25(OH)₂D₃-induced surge of TRPV6 and TRPV5 (Khuituan et al. 2012).

7.2 Parathyroid Hormone

PTH regulates both the expression and activity of TRPV5. In parathyroidectomized rats and calcimimetic compound NPS R-467-infused mice that had a lower PTH level, renal TRPV5, calbindin-D_28k, and NCX1 levels were decreased; and they were restored by PTH supplementation (van Abel et al. 2005). The decrease in other Ca²⁺ transport proteins by PTH is likely secondary to the reduction of TRPV5-mediated Ca²⁺ influx (van Abel et al. 2005).

PTH is also capable of acutely increasing Ca²⁺ transport in the distal tubule (Bacskai and Friedman 1990). Both PKA and PKC are involved in this regulation (Friedman et al. 1996). Indeed, activation of TRPV5 by PTH via both PKA and PKC has been reported (de Groot et al. 2009; Cha et al. 2008b). PTH activates the cAMP–PKA signaling cascade and increases TRPV5 open probability via phosphorylation of threonine 709 (T709) in TRPV5 (de Groot et al. 2009). This regulation requires a strong buffering of intracellular Ca²⁺. However, the PKA phosphorylation site T709 is not conserved in human TRPV5. In addition to PKA, the increase of TRPV5 activity by heterogeneously expressed PTH receptor was prevented by PKC inhibitor (Cha et al. 2008b). Mutation of PKC phosphorylation sites S299/S654 in TRPV5 abolished the regulation. Caveolae-mediated endocytosis of TRPV5 appears to be inhibited by PTH via a PKC-dependent pathway (Cha et al. 2008b).

Besides vitamin D and PTH, calcitonin is a hormone that regulates Ca²⁺ homeostasis. However, calcitonin affects renal Ca²⁺ reabsorption mainly through the thick ascending limb (Elalouf et al. 1984; Di Stefano et al. 1990). TRPV5, which is mostly expressed in the DCT and CNT, is not involved in the effects of calcitonin on urinary excretion of Ca²⁺, Na⁺, and K⁺ as no difference was observed between wild-type and Trpv5 KO mice in this regard (Hsu et al. 2009).

7.3 Estrogen and Testosterone

As the most potent estrogen, 17β-estradiol increases TRPV5 mRNA and protein expression in the kidney (van Abel et al. 2002, 2003). Estrogen deficiency in aromatase-deficient mice results in decreased mRNA levels of renal TRPV5 and other Ca²⁺ transporters and Ca²⁺ wasting (Oz et al. 2007). In addition, 17β-estradiol (20–50 nM) rapidly increases TRPV5 current and [Ca²⁺] _i in rat CCD cells (Irnaten et al. 2009). Thus, estrogen acutely elevates TRPV5 activity and chronically increases its expression.

In contrast to the positive effects of estrogen, male hormone testosterone appears to have a negative effect on TRPV5. Male mice have higher urinary Ca²⁺ excretion than female mice (Hsu et al. 2010). Androgen deficiency increases renal TRPV5 mRNA and protein and decreases urinary Ca²⁺ excretion; these were normalized by testosterone treatment (Hsu et al. 2010). In addition, the negative effect of dihydrotestosterone on transcellular Ca²⁺ transport was demonstrated in primary rabbit CNT/CCD cells (Hsu et al. 2010).

7.4 Vasopressin

Arginine vasopressin stimulates transepithelial Ca²⁺ transport in primary cultures of rabbit CCD cells (van Baal et al. 1996). Vasopressin induces an increase in Ca²⁺ uptake and [Ca²⁺] _i in mpkDCT cells and freshly isolated late DCT and CNT cells, respectively, indicating that TRPV5 is a target of vasopressin (Diepens et al. 2004; Hofmeister et al. 2009).

8 Physiological Functions in Native Cells, Organs, Organ Systems

8.1 Ca²⁺ Reabsorption in the Kidney

TRPV5 is mainly distributed in the apical membrane of tubular cells in the DCT and CNT where it mediates the final reabsorption of Ca²⁺ via a transcellular pathway. TRPV5, calbindin-D_28k, NCX1, and PMCA are the major components of the pathway. Lines of evidence suggest that the expression of other Ca²⁺ transporters depends on TRPV5-mediated Ca²⁺ influx (van Abel et al. 2005; Hoenderop et al. 2003a). In this sense, TRPV5 is the rate-limiting component in the transcellular pathway of Ca²⁺ reabsorption.

TRPV5 is likely constitutively active in the apical membrane of tubular cells. At macroscopic level, TRPV5 acts as a facilitative transporter exhibiting saturable kinetics with apparent K _m values at sub-millimolar range, which are well suited for the luminal Ca²⁺ level in the distal tubule (Hoenderop et al. 1999; Peng et al. 1999, 2000a, b). The responsiveness of TRPV5 to vitamin D, PTH, Ca²⁺ load, pH, and tubular factors such as klotho, uromodulin, and plasmin makes TRPV5 well suited for the fine-tuning of Ca²⁺ reabsorption in the kidney.

8.2 Bone Resorption by Osteoclasts

TRPV5 also expresses in the ruffled border membrane of mouse osteoclasts (van der Eerden et al. 2005). Osteoclast numbers and area are increased, and paradoxically urinary bone resorption marker deoxypyridinoline was reduced in mice lacking TRPV5 (Hoenderop et al. 2003a). In vitro bone marrow culture system indicates that bone resorption by osteoclasts was impaired in Trpv5 KO mice (van der Eerden et al. 2005). These results suggest a malfunction of osteoclast to some degree in the absence of TRPV5. However, bone resorption inhibitor alendronate normalizes the reduced bone thickness in Trpv5 KO mice, even though it specifically increases bone TRPV5 expression (Nijenhuis et al. 2008). Furthermore, vitamin D analog ZK191784 partially restores the decreased bone matrix mineralization in Trpv5 KO mice, suggesting the bone phenotype of Trpv5 KO mice is secondary to the elevated 1,25(OH)₂D₃ (van der Eerden et al. 2013). Thus, TRPV5 plays a role in osteoclast function, but it is not absolutely required for bone reabsorption. The role of TRPV5 in osteoclast function is not well understood, but TRPV5 is involved in receptor activator of NF-κB ligand (RANKL)-induced rise in [Ca²⁺] _i in human osteoclasts (Chamoux et al. 2010). This process is likely a part of the negative feedback loop to terminate RANKL-induced bone resorption (Chamoux et al. 2010).

8.3 Function in the Inner Ear

The low Ca²⁺ concentration of mammalian endolymph in the inner ear is required for normal hearing and balance. Marcus and colleagues showed that TRPV5 and TRPV6 may play roles in the function of inner ear (Yamauchi et al. 2005, 2010; Wangemann et al. 2007; Nakaya et al. 2007). TRPV5 was detected in native semicircular canal duct (SCCD) epithelial cells, cochlear lateral wall, and stria vascularis of adult rats along with other Ca²⁺ transport proteins (Yamauchi et al. 2010). TRPV5 protein was localized close to the apical membrane of strial marginal cells and in outer and inner sulcus cells of the cochlea (Yamauchi et al. 2010). TRPV5 transcript was responsive to 1,25(OH)₂D₃ (Yamauchi et al. 2005); however, the protein level was not upregulated (Yamauchi et al. 2010). The levels of TRPV5 were decreased in the inner ear of older mice (Takumida et al. 2009). Mutations in pendrin (SLC26A4, an anion exchanger) cause the most common form of syndromic deafness. Reduced pH and utricular endolymphatic potential and increased Ca²⁺ concentration were found in pendrin KO mice (Wangemann et al. 2007; Nakaya et al. 2007). The reduced pH likely blocks the activity of TRPV5 and TRPV6, whose Ca²⁺ transport activity is reduced at low pH, similar to what was observed in primary SCCD cells (Nakaya et al. 2007). The elevation of endolymphatic Ca²⁺ level in pendrin KO mice may inhibit sensory transduction necessary for hearing and promote the degeneration of the sensory hair cells, which is necessary for the development of normal hearing (Wangemann et al. 2007).

8.4 Intestinal and Placental Ca²⁺ Transport

The levels of TRPV5 mRNA in intestine and placenta are much lower than TRPV6 (Peng et al. 2001). TRPV5 is regulated in the same direction with TRPV6 in the intestine in most cases, such as immobilization and exercise-induced alteration of gene expression (Teerapornpuntakit et al. 2009; Sato et al. 2006). In the placenta, Ca²⁺ transport in human syncytiotrophoblasts is insensitive to voltage and L-type Ca²⁺ channel modulators but is sensitive to TRPV5/6 blocker Mg²⁺ and ruthenium red (Moreau et al. 2002a). In cultured human trophoblasts isolated from term placenta, TRPV5 and TRPV6 expression correlated with the Ca²⁺ uptake potential along the differentiation of the trophoblasts (Moreau et al. 2002b). In contrast to the demonstrated role of TRPV6 in placental Ca²⁺ transport (Suzuki et al. 2008), the role of TRPV5 is unclear. TRPV5 likely plays a minor role in intestinal and placental Ca²⁺ transport due to its low levels of expression in these organs.

9 Lesson from TRPV5 Knockout Mice

The most distinctive features of Trpv5 KO mice include 2.9-fold elevation in serum 1,25(OH)₂D₃, sixfold increase in Ca²⁺ excretion, moderate reduced bone thickness in the femoral head, and normal level of plasma Ca²⁺ (Hoenderop et al. 2003a). In addition, polyuria and urine acidification were observed in Trpv5 KO mice (Hoenderop et al. 2003a). These alterations in renal function are caused by the activation of CaR by the increased tubular Ca²⁺ in the collecting duct and subsequently upregulated proton secretion by the H⁺–ATPase and reduced water reabsorption due to the downregulation of aquaporin 2 (Renkema et al. 2009b). These physiological adaptations reduce the risk of Ca²⁺ precipitations and stone formation. Intestinal Ca²⁺ absorption is increased in Trpv5 KO mice as duodenal TRPV6 and calbindin-D_9k mRNAs are increased due to the elevated 1,25(OH)₂D₃ level (Hoenderop et al. 2003a). However, the serum Ca²⁺ levels are largely normal, even though the PTH level is elevated in older Trpv5 KO mice (van Abel et al. 2006). The major phenotypes of Trpv5 KO mice are listed in Table 3.

Table 3 Phenotype of Trpv5 KO mice

Studies with Trpv5 KO mice reveal some important points in Ca²⁺ transport physiology: (1) the transcellular Ca²⁺ transport in the DCT and CNT, as represented by TRPV5, plays an important role in Ca²⁺ reabsorption. This is supported by the fact that removal of TRPV5 results in significant hypercalciuria. (2) TRPV5 is not indispensable to maintain a Ca²⁺ balance as long as vitamin D and related systems work normally. The body reset Ca²⁺ homeostasis by elevating the level of 1,25(OH)₂D₃, which increases intestinal Ca²⁺ absorption to compensate the renal loss of Ca²⁺ (Renkema et al. 2005). A new Ca²⁺ balance is achieved at higher intestinal absorption and higher urinary Ca²⁺ excretion in the absence of TRPV5. (3) The TRPV5 KO model shows an example that polyuria and reduced urinary pH are natural responses to increased Ca²⁺ excretion in order to prevent kidney stone formation. In addition, Trpv5 KO mouse is a useful model to clarify some basic physiological mechanisms. For example, with this model it has been shown that calcitonin or thiazide-induced hypocalciuria occurs independent of TRPV5; thus, the responsible mechanisms likely reside in more proximal segments of the nephron (Hsu et al. 2009; Nijenhuis et al. 2005). All the studies indicate that TRPV5 does not alter the bulk Ca²⁺ reabsorption; rather, it provides a key mechanism for the fine-tuning of Ca²⁺ reabsorption.

10 Role in Hereditary and Acquired Diseases

As we learned from Trpv5 KO mice, the lack of TRPV5 could be compensated through an elevation of 1,25(OH)₂D₃, which regulates other proteins (e.g., TRPV6) in maintaining Ca²⁺ homeostasis. Thus, humans with defect in TRPV5 may have altered Ca²⁺ homeostasis, but could be clinically asymptomatic except for hypercalciuria. However, no mutation in the exons of Trpv5 gene was found to be associated with autosomal dominant hypercalciuria (Muller et al. 2002). In another study involving 20 renal hypercalciuria patients, nonsynonymous variation of TRPV5 (A8V, R154H, and A561T) and synonymous variations were identified; however, these variants apparently do not alter the property of TRPV5 (Renkema et al. 2009a). Recently, S682P mutation of TRPV5 causes autosomal dominant hypercalciuria in mouse model (Loh et al. 2013). Interestingly, no significant functional differences were found between S682P and wild-type TRPV5 when expressed in Xenopus oocytes or HEK293 cells; the only difference was that S682P produced a lower baseline [Ca²⁺] _i than wild-type TRPV5 in HEK293 cells (Loh et al. 2013). However, mice with homozygous TRPV5^S682P did show reduced TRPV5 immunostaining in the kidney and phenotype resembling that of Trpv5 KO mice (Loh et al. 2013). This study suggests that mutations in TRPV5 could result in hypercalciuria; however, the functional changes of the TRPV5 mutations may not be detected in vitro. Thus, it remains to be further examined that mutations in Trpv5 gene cause hypercalciuric disorders in humans.

In addition to a possible role of TRPV5 in hypercalciuria, TRPV5 expression and potentially activity are altered in response to a number of diseases and therapeutic conditions (Table 4). Mechanisms for the change in TRPV5 expression are often not fully understood. TRPV5 expression could be regulated by hormones, chemicals, and likely intracellular or extracellular Ca²⁺. A change in Trpv5 expression likely leads to alteration in urinary Ca²⁺ excretion; on the other hand, the alteration of filtered Ca²⁺ load or reabsorption of Ca²⁺ elsewhere in the nephron leads to increased or decreased delivery of Ca²⁺ to the DCT and CNT and in turn causes an elevation or reduction in Trpv5 expression. If specific modulators for TRPV5 and TRPV6 could be developed, they could be useful to correct abnormality in Ca²⁺ homeostasis under disease or therapeutic conditions. For example, under disease conditions caused by vitamin D signaling defect, such as in vitamin D-dependent rickets, type 2a (Online Mendelian Inheritance in Man #277440), intestinal and renal TRPV5 and TRPV6 expressions are likely disrupted as demonstrated in the Vdr KO mice (Van Cromphaut et al. 2001). Enhancing TRPV5- and TRPV6-mediated Ca²⁺ absorption would be helpful to achieve a positive Ca²⁺ under this condition. Similarly, enhancers for TRPV5 and TRPV6 are desirable to prevent osteoporosis in women after the menopause due to reduced estrogen level (Oz et al. 2007; van Abel et al. 2002; Irnaten et al. 2009). On the other hand, TRPV6 inhibitors would be needed in preventing absorptive hypercalciuria and kidney stone disease, and TRPV5 stimulator would be useful in preventing kidney stone formation in patients with renal Ca²⁺ leak. Stimulators of TRPV5 would be also helpful in tacrolimus-induced hypercalciuria (Nijenhuis et al. 2004) or other conditions associated with reduced expression of TRPV5 in Table 4.

Table 4 Alteration of Trpv5 expression under disease and therapeutic conditions

11 Single Nucleotide Polymorphisms of TRPV5 in African Populations

TRPV5 and TRPV6 have been shown to have high frequency of SNPs in African populations. By analyzing SNP data from 24 African Americans and 23 European Americans in genes sequenced in SeattleSNPs, a 115-kb region in chromosome 7q34-35 of 4 contiguous genes, including EPHB6, TRPV6, TRPV5, and KEL4, was identified with features of a recent demographic selection (Stajich and Hahn 2005; Akey et al. 2004). TRPV6 SNPs defined by three nonsynonymous SNPs (C157R, M378V, and M681T) exhibit most striking footprint of positive selection (Akey et al. 2006; Hughes et al. 2008). In addition, four nonsynonymous SNPs in TRPV5 were identified by SeattleSNPs: three of them (A8V, A563T, and L712F) were only present in African Americans, not in European Americans; R154H is common in both populations. The nonsynonymous SNP variations in TRPV6 ancestral haplotype are conserved in other species; in contrast, the variations in TRPV5 are newly derived as they are not commonly present in other species surveyed including chimpanzee, dog, rat, and mouse, with the exceptions of 563T in dog and 8V in rat (Na et al. 2009). In addition, the nonsynonymous SNPs of TRPV5 are not associated with each other as are those in TRPV6. By expression in Xenopus oocytes, we found that two of the SNPs, A563T and L712F, significantly increased TRPV5-mediated Ca²⁺ uptake by approx. 50 % and 25 %, respectively (Na et al. 2009). For A563T variant, the increased Ca²⁺ uptake activity was not associated with increased protein abundance in the plasma membrane; rather it was associated with increased apparent K _m for Ca²⁺ and increased sensitivity to extracellular Mg²⁺, suggesting increased permeation of Ca²⁺ in the cation translocation pathway of the channel (Na et al. 2009). The A563 residue in the last transmembrane domain is 20 residues away from D542 residue in the Ca²⁺ filter in the pore. It is likely in the cation translocation path of the channel (Fig. 2).

African Americans exhibit lower urinary Ca²⁺ excretion than Caucasians (Braun et al. 2007; Pratt et al. 1996; Taylor and Curhan 2007), and the risk of kidney stone in African Americans is lower than that in Caucasians (Sarmina et al. 1987; Stamatelou et al. 2003). In addition, African Americans have higher bone mass (Bell et al. 1991) and lower incidence of osteoporosis-related fractures than whites (Bohannon 1999). Because of the high allele frequencies of TRPV5 and TRPV6 SNPs in African populations, these SNPs may contribute to the Ca²⁺ conservation mechanisms in African populations. Further population studies are necessary to clarify the relationship of SNPs in TRPV5 and TRPV6 and Ca²⁺ homeostasis in African populations.