13.1. Introduction

The epithelial calcium (Ca²⁺) channels TRPV5 and TRPV6 are members of the transient receptor potential (TRP) channel family TRPV (“V” for vanilloid) subgroup. TRPV5 and TRPV6 play major roles in the maintenance of blood Ca²⁺ levels in higher organisms. Both channels exhibit similarities in many ways, as they share a high level (75%) of amino acid identity, comparable functional properties, and similar mechanisms of regulation. Also, they were discovered using similar cloning strategies [1,2]. Yet, their physiological contributions toward maintaining a systemic calcium balance are distinct. In addition, the following three key features distinguish TRPV5 and TRPV6 from other members of the TRP superfamily of cation channels: (1) high selectivity for Ca²⁺ over other cations, (2) apical membrane localization in Ca²⁺-transporting epithelial tissues, and (3) responsiveness to 1,25-dihydroxyvitamin D₃ (1,25[OH]₂D₃) [3,4]. These features make TRPV5 and TRPV6 ideally suited to facilitate intestinal absorption and renal reabsorption of Ca²⁺, serving as apical Ca²⁺ entry channels in transepithelial Ca²⁺ transport [5,6]. A major difference between the properties of TRPV5 and TRPV6 lies in their tissue distribution: TRPV5 is predominantly expressed in the distal convoluted tubules (DCT) and connecting tubules (CNT) of the kidney, with limited expression in extrarenal tissues [1,7]. In contrast, TRPV6 exhibits a broader expression pattern, showing prominent expression in the intestine with additional expression in the kidney [8–10], placenta, epididymis, exocrine tissues (i.e., pancreas, prostate, salivary gland, sweat gland), and a few other tissues [11–13]. Thus, while TRPV5 plays a key role in determining the level of urinary Ca²⁺ excretion, the physiological roles of TRPV6 are not limited to intestinal Ca²⁺ absorption. Much progress has recently been made in understanding the roles of TRPV5 and TRPV6 channels in the kidney [14], intestine [15], placenta [16], and epididymis [17]. However, their roles in other organs have as yet not been fully investigated.

In this chapter, we review the current status of our knowledge of the physiological and pathological roles of TRPV5 and TRPV6 and discuss a variety of techniques that have led to a deeper understanding of these channels. We review the identification strategies of TRPV5 and TRPV6 in searches for Ca²⁺ absorption channels, as well as specific techniques used to reveal their key features. These include radiotracer Ca²⁺ uptake and electrophysiology procedures, structure-function studies, methods to identify regulatory interacting partners, genetically engineered animals, strategies to study the role of TRPV6 in cancers, procedures for the development of small-molecule modulators of TRPV6 and TRPV5, the evaluation of variations/mutations in humans, and 3D structural determination. For additional information about TRPV5 and TRPV6, we would like to refer the interested reader to other comprehensive review articles [3–6,18].

13.2. Ca²⁺ Transport across Epithelia

Epithelia form barriers to separate cells from the internal and external environment, allowing specific exchange of nutrients and other substances across epithelial cells. Among the many electrolytes, Ca²⁺ is an important intracellular messenger and a major component of the mineral phase of bones and teeth. Thus, at the cellular level, the cytosolic Ca²⁺ concentration is kept low (around 100 nM at rest), whereas its concentration in the extracellular fluids (ECF), such as blood, is maintained at a much higher level and within a relatively narrow limit (around 1 mM) [19]. The bone, which contains roughly 99% of the Ca²⁺ in the human body, is subject to constant Ca²⁺ exchange with the ECF. Any reduction in the extracellular Ca²⁺ concentration ([Ca²⁺]_o) in the ECF is being monitored by the G-protein-coupled Ca²⁺-sensing receptor (CaSR) in the parathyroid gland, triggering secretion of the parathyroid hormone (PTH) [19]. The PTH increases bone resorption and renal reabsorption of Ca²⁺ and stimulates the hydroxylation of 25-hydroxyvitamin D₃ [25(OH)₂D₃] in the proximal tubule of the kidney to form 1,25(OH)₂D₃ [20]. An important role of this activated form of vitamin D is the stimulation of intestinal Ca²⁺ absorption via the nuclear vitamin D receptor (VDR) [21]. Several organs and hormones work in concert to maintain stable levels of Ca²⁺ in the ECF. The intestinal and renal handling of Ca²⁺ is important as the intestine determines how much Ca²⁺ enters the body and the kidney determines how much Ca²⁺ is removed from the body.

Intestinal absorption and renal reabsorption of Ca²⁺ are similar processes, involving Ca²⁺ transport across the epithelia from the luminal side to the blood side [22,23]. In general, Ca²⁺ ions can cross the epithelia through the paracellular route, as well as the transcellular route (Figure 13.1). When [Ca²⁺]_o is higher than that of the plasma, luminal Ca²⁺ predominantly enters the intestine via the paracellular route through tight junctions between the epithelial cells (Figure 13.1a). However, this route does not operate in the absence of favorable transepithelial [Ca²⁺] gradients. Thus, when luminal [Ca²⁺]_o is lower than that in the plasma, Ca²⁺ will need to be actively absorbed across the epithelia via the transcellular route. For this to occur, Ca²⁺ first enters the cells across the apical membranes passively through TRPV6. This is followed by binding of Ca²⁺ to calbindin-D_9K and transfer to the basolateral membrane. Basolateral exit is then mediated by the plasma membrane Ca²⁺ ATPase (PMCA), that is, by primary active transport, at the expense of stored ATP [22].

Figure 13.1

Mechanisms of Ca²⁺ transport in the intestine and kidney.

Similar transport mechanisms also take place in the kidney during Ca²⁺ reabsorption (Figure 13.1b). Based on our current knowledge from animal studies, up to 95% of filtered Ca²⁺ is being reabsorbed mainly via the paracellular route in renal proximal tubules and loops of Henle, while about 5%–10% of Ca²⁺ reabsorption is delayed till the distal tubules, where reabsorption occurs via the transcellular route and in a regulated manner in order to fine-tune the whole-body calcium homeostasis. At this point, it is worthy to note that TRPV6 in the human kidney likely plays an additional role in transcellular Ca²⁺ transport in specific parts of the tubular system (see Section 13.8 for further details). Similar to the intestine, the transcellular route in the kidney involves passive Ca²⁺ entry across the apical membranes through TRPV5, transfer to the basolateral membrane after binding to calbindin-D_28K, and basolateral exit through the PMCA (primary active transport) or the Na⁺/Ca²⁺ exchanger NCX1 (at the expense of the inwardly directed Na⁺ gradient) [22,23].

Passive Ca²⁺ transport across the apical membrane via TRPV5 or TRPV6 is favored by the inwardly directed electrochemical gradient for Ca²⁺, which is warranted by the low cytosolic Ca²⁺ levels of approximately 100 nM. As already noted, once Ca²⁺ ions have entered the epithelial cell, they bind to calbindins during Ca²⁺ uptake (Figure 13.1). Two types of calbindins with different molecular weight and number of Ca²⁺-binding EF hands, calbindin-D_9K and calbindin-D_28K, are expressed in the Ca²⁺-transporting epithelial cells in the mammalian intestine and kidney, respectively [24]. They belong to a large family of high-affinity Ca²⁺-binding proteins (K_d = 10^-8 - 10^-6 M) featuring EF-hand structural motifs [25]. Calbindins and likely other Ca²⁺-binding proteins maintain the free cytosolic Ca²⁺ concentrations below toxic levels and help regulate TRPV5 and TRPV6 through their feedback mechanisms [26]. However, if apical Ca²⁺ entry exceeds the capacity of calbindins, Ca²⁺ will bind to calmodulin (CaM), resulting in a Ca²⁺ feedback inhibition of TRPV5 or TRPV6 channels (see Figure 13.1 on the right; also see Section 13.4.4).

In addition to the intestine and kidney, maternal-fetal transport of Ca²⁺ involves an active mechanism, as the serum [Ca²⁺] is higher in fetuses than in mothers [27]. In humans, Ca²⁺ is needed for the rapid growth of fetal skeleton, especially in the third trimester. The syncytiotrophoblast layer is a specialized epithelium responsible for the exchange of Ca²⁺ along with other nutrients and gases between mother and fetus [28]. Ca²⁺ transport in human syncytiotrophoblasts has been the subject of many studies [29]. These Ca²⁺ channels are insensitive to L-type voltage-gated Ca²⁺ channel modulators but sensitive to Mg²⁺ and ruthenium red [30], resembling the properties of TRPV6.

Active Ca²⁺ transport also takes place in specialized organs to create local environments where [Ca²⁺] needs to be controlled. For example, the luminal [Ca²⁺]_o is lower in the distal portion of the epididymis [31]. Recent studies revealed that this lowered [Ca²⁺] is maintained by TRPV6-mediated Ca²⁺ transport into the epithelial cells of the epididymis and that this is important for preserving optimal motility of sperm and optimal male fertility [17]. Therefore, TRPV6 of the epididymis and the progesterone-regulated CatSper channel of sperm [32] both cooperate to ensure maximal motility of the sperm and fertility (see Section 13.5.4 for further details).

Another example is the observation that [Ca²⁺] in the vestibular endolymph is only one-fourth of that of the perilymph (∼1 mM). Indeed, [Ca²⁺]_o is important in regulating the hair-bundle stiffness and gating-spring integrity in hair cells of the inner ear [33]. Active transcellular Ca²⁺ transport by TRPV5 and TRPV6 is present in the epithelial cells of the inner ear and maintains the low [Ca²⁺] of the vestibular endolymph [34,35].

The glandular tissue is also formed by epithelial cells. The release of content from secretory granules, which are loaded with Ca²⁺, is accompanied by the loss of Ca²⁺ [36]. Given that exocrine glands secrete large amounts of fluid, a Ca²⁺ reuptake mechanism may be useful to supply Ca²⁺ released in the apical pole of exocrine cells back into new secretory vesicles.

Lastly, carcinomas are cancer cells that originate from epithelial cells, and altered Ca²⁺ homeostasis is a characteristic feature of carcinomas. This may be related to cancer cell proliferation, survival, and metastasis formation [37–39]. The development of specific small-molecule inhibitors targeting Ca²⁺ channels overexpressed in carcinomas may offer novel strategies for cancer treatment [40,41] (see Section 13.7).

13.3. Identification of TRPV5 and TRPV6 by Expression Cloning

TRPV5 and TRPV6 were identified in 1999 by Bindels and colleagues and by our group from rabbit kidney cells and rat duodenum, respectively [1,2]. Both groups used expression cloning with radiotracer ⁴⁵Ca²⁺ uptake assays, screening intestinal or renal cDNA libraries for the ability of clones to induce Ca²⁺ uptake in complementary RNA (cRNA)-injected Xenopus laevis oocytes. This approach has proven very useful in the identification of novel transporters, channels, and receptors [42]. Before the epithelial Ca²⁺ channels were identified, calbindins were known to facilitate the diffusion of Ca²⁺ from the apical to basolateral membranes, followed by cellular exit via Na⁺/Ca²⁺ exchangers and/or PMCAs. However, what mediated Ca²⁺ entry across the plasma membrane remained elusive for a long time [22,24]. To increase our chances for successfully cloning TRPV6, we fed rats a Ca²⁺-deficient diet for 2 weeks to boost the expression of Ca²⁺-reabsorptive gene products. Also, we isolated mRNA from the duodenum and cecum of the rat intestine, the sites where Ca²⁺ absorption primarily occurs. A twofold increase in radiotracer uptake was observed in oocytes injected with mRNA samples from the duodenum and cecum compared to control uninjected oocytes. We then fractionated the duodenal mRNA by preparative gel electrophoresis and tested the ability of the mRNA samples of different size fractions to stimulate Ca²⁺ uptake. An increase in Ca²⁺ uptake was observed in a 3 kb fraction. This fraction was used to make a cDNA library, and the library was divided into smaller pools of about 300 clones. Plasmid DNA was isolated from each pool and reverse-transcribed into capped cRNA. These cRNAs were then tested for their ability to stimulate Ca²⁺ uptake in injected oocytes. Once a positive pool was identified, it was divided into even smaller pools, and the earlier mentioned process was repeated in an iterative way, until a single positive clone was identified [2]. A similar approach was used to identify TRPV5 from rabbit kidney [1]. Because the distal tubule represents a very short portion of the nephron, cells from CT and cortical collecting ducts were isolated from rabbit kidney using a monoclonal antibody against these tubule segments to obtain enough mRNA for expression cloning of TRPV5 [1].

The identified rabbit TRPV5 and rat TRPV6 cDNAs encode proteins of 730 and 727 amino acid residues, respectively. In vivo evidence exists that the translation of TRPV6 protein may start from an upstream non-AUG codon. This does not change the TRPV6 function but may provide an additional scaffold for channel assembly [43]. TRPV5 was originally named epithelial Ca²⁺ channel (ECaC) [1] and TRPV6 Ca²⁺ transport protein subtype 1 (CaT1) [2]. The two proteins share 74% amino acid sequence identity. At the time of cloning, only two cloned transport proteins shared amino acid sequence similarity to TRPV5 and TRPV6: the capsaicin receptor TRPV1, a heat-activated cation channel that contributes to the sensations of pain and temperature [44], and OSM-9, a Caenorhabditis elegans protein involved in olfaction, mechanosensation, and olfactory adaptation [45]. The mammalian TRPV family now includes six members, whereby TRPV5 and TRPV6 share approximately 40%–45% amino acid sequence identity with the other four members [8]. While TRPV5 and TRPV6 represent specialized epithelial Ca²⁺ transport proteins, the other four members are nonselective cation channels that serve as sensors and can be activated by physical cues such as heat (TRPV1-TRPV3) or tonicity (TRPV4) [46].

13.4. Ca²⁺ Transport Properties Uncovered by Various Approaches

13.4.3. Characterization of TRPV5 and TRPV6 by Patch Clamping

The properties of TRPV5 and TRPV6 at a microscopic level were investigated in great detail using the patch-clamp technique, which is discussed in more detail elsewhere in this book (see Chapter 1). Briefly, Na⁺ currents of TRPV5 were detected when divalent cations (e.g., Ca²⁺ and Mg²⁺) were removed from the extracellular solution [49]. Unitary Na⁺ conductance of TRPV5 and TRPV6 was subsequently determined [50,51]. Both TRPV5 and TRPV6 turned out to be highly selective for Ca²⁺, with Ca²⁺ to Na⁺ permeability ratios (P_Ca:P_Na) of over 100 for both channels [50,51]. Together with site-directed mutagenesis studies, the key residue for Ca²⁺ permeation and Mg²⁺ blockade was identified as aspartate 542 in TRPV5 [52,53] (shown in Figure 13.2 as the site for Ca²⁺ permeation).

Figure 13.2

Model of the human TRPV5 channel based on the rat TRPV6 structure (PDB ID: 5IWK). (a) Tilted sideways view with one monomer of the tetrameric unit shaded in blue. Approximate position of the membrane is indicated. (b) Cutaway view of two oppositely positioned (more...)

13.4.6. Additional Experimental Considerations

As shown in Figure 13.3, all of the aforementioned approaches have been utilized in evaluating channel function and regulation of TRPV5 and TRPV6. While radiotracer ⁴⁵Ca²⁺ uptake is a sensitive approach that directly measures Ca²⁺ influx, it cannot reveal detailed kinetics of channel activity. Two-electrode voltage-clamp studies reveal charge movement and voltage-dependence of membrane transport processes. However, its application is mostly limited to Xenopus oocytes. The patch-clamp approach offers detailed channel activity measurements, and it is also well suited to uncover the mechanisms of channel regulation. However, most patch-clamp experiments of TRPV5 and TRPV6 were done in divalent cation-free bathing solutions. Such experimental conditions might miss channel regulatory mechanisms that are Ca²⁺ dependent, for example. Furthermore, single-channel activities using Ca²⁺ as charge carrier have not been demonstrated for TRPV5 or TRPV6 so far. Nevertheless, data from this array of techniques are complementary and provide a well-documented understanding of the biophysical properties and regulatory aspects of TRPV5 and TRPV6.

Figure 13.3

Major approaches to assess TRPV5 and TRPV6 function.

13.5. Physiological Roles Revealed Using Genetically Engineered Animal Models

Genetically engineered mouse models were instrumental for exploring the physiological roles of TRPV5 and TRPV6. These include gene knockout (KO) models for TRPV5 [14] and TRPV6 [15], chemically induced gene mutations in TRPV5 [74], transgenic overexpression of TRPV6 in the small intestine [75], and a knock-in mouse model of TRPV6 [17]. These animal models and the findings from these models are summarized in the following sections.

13.5.2. TRPV6 as a Central Component in Vitamin D-Regulated Active Ca²⁺ Absorption

While TRPV6 is considered the key apical channel for active Ca²⁺ absorption, a certain level of active Ca²⁺ absorption also occurs in the absence of TRPV6 [77]. Furthermore, the stimulating effect of 1,25(OH)₂D₃ on intestinal Ca²⁺ absorption was still partially present in TRPV6 KO mice [78]. This raised the question as to what extent vitamin D-regulated transcellular Ca²⁺ absorption depends on TRPV6 expression. To address this question, Fleet and colleagues developed a transgenic mouse model in which intestinal epithelium-specific expression of human TRPV6 is driven by the villin gene promoter [75]. This approach directly demonstrated that TRPV6 functions as an apical Ca²⁺ transporter and intestinal absorption was elevated 300% in mice with intestine-specific transgenic expression of TRPV6 (TRPV6TG). In this study, TRPV6TG and VDRKO mice were also crossed to generate TRPV6TG-VDRKO mice.

The TRPV6TG mice on chow diet (0.72% calcium) exhibited elevated serum and urinary Ca²⁺ levels, poor growth, and soft tissue calcification. Lowering the calcium level in the diet to 0.25% normalized the serum Ca²⁺ levels. Femur bone mineral density of TRPV6TG mice was increased by 28% compared to normal mice. The 1,25(OH)₂D₃ level was reduced by 56% likely due to the 98% lower CYP27B1 mRNA in the kidney of TRPV6TG mice. The reduced CYP27B1 mRNA level is likely a result of reduced PTH levels (46% lower). The phenotype of TRPV6TG mice is largely opposite to that of the TRPV6 KO mice, confirming the importance of TRPV6 in the intestine facilitating vitamin D-regulated transcellular Ca²⁺ absorption.

The key regulator of intestinal Ca²⁺ absorption is 1,25(OH)₂D₃. TRPV6 expression is strongly regulated by vitamin D as the TRPV6 gene promoter has multiple vitamin-responsive elements [79]. In VDRKO mice, duodenal TRPV6 mRNA is reduced down to 5%–10% compared to wild-type mice [21,80]. VDRKO mice fed a diet with slightly reduced calcium content (0.5% calcium, standard commercial chow diet contained 0.72% calcium) for 10 weeks were hypocalcemic, with drastically elevated serum 1,25(OH)₂D₃ and PTH levels, reduced bone mass, and decreased intestinal Ca²⁺ absorption [75].

In TRPV6TG-VDRKO mice, the serum Ca²⁺ remained elevated when kept on 0.5% calcium diet, serum 1,25(OH)₂D₃ and PTH levels were very close to that of the wild type, bone mass was close to that in the TRPV6TG mice, and intestinal Ca²⁺ reabsorption remained elevated [75]. Thus, overexpression of TRPV6 in the intestine is sufficient to overcome the deficiency caused by the reduction of intestinal Ca²⁺ absorption in the absence of VDR. The results of this study also show that, once TRPV6 is restored, other components relevant to transcellular Ca²⁺ transport such as levels of calbindin-D_9K, 1,25(OH)₂D₃, and PTH are normalized in the absence of VDR [75]. Taken together, given the results from studies of TRPV6 KO, VDRKO, and TRPV6TG mice (Figure 13.4), TRPV6 is likely a key element in vitamin D-regulated Ca²⁺ transport. Given the importance of Ca²⁺ absorption, it is also unlikely that TRPV6 is the only Ca²⁺ channel for intestinal Ca²⁺ absorption. The presence of a vitamin D-regulated active Ca²⁺ transport mechanism in the intestine in mice lacking TRPV6 might be due to physiological adaptation, that is, upregulation of alternative, compensatory Ca²⁺ transport pathways. Also epigenetic and genetic alterations in the TRPV6 KO mice cannot be ruled out. The abovementioned studies suggest that an alternative transcellular Ca²⁺ entry pathway might exist in the intestine that is regulated by 1,25(OH)₂D₃. Further studies will be required to identify this putative additional transport mechanism in the intestinal brush border membrane.

Figure 13.4

Ca²⁺ homeostasis in mice with genetically engineered TRPV6 and VDR. Shown are changes in Ca²⁺ homeostasis-related phenotypes including serum levels of 1,25(OH)₂D₃ and PTH, intestinal Ca²⁺ absorption, renal Ca²⁺ reabsorption, and bone mineral density in (more...)

13.6. Proteins that Regulate TRPV5 and TRPV6

A number of proteins have been identified that interact and/or regulate TRPV5 and TRPV6 (see examples in Figure 13.2b). These proteins were identified because (1) they interact with TRPV5 or TRPV6 as detected by yeast two-hybrid screening, (2) they are expressed in the same tissues or cells, or (3) they are relevant to Ca²⁺ absorption or reabsorption in a physiological or pathological context [3]. Most of these proteins were identified for TRPV5. However, since TRPV6 and TRPV5 are structurally and functionally similar, they generally have analogous roles in regulating TRPV6, provided that they are adequately coexpressed.

13.7. Potential Use of TRPV6 in Therapy and Development of Chemical Modulators

13.7.2. Development of Chemical Modulators of TRPV6

13.7.2.1. Development of Antiproliferative Compound TH-1177

Strategies to inhibit Ca²⁺ entry into electrically non-excitable cells by small-molecule compounds were addressed by Gray and colleagues in the early 2000s [40]. These investigators synthesized compounds such as TH-1177 and its stereoisomer TH-1211 that share similarities with known Ca²⁺ channel blockers derived from the molecules dihydropyridine, benzothiazepine, and phenylalkylamine (Figure 13.5a; compounds 1 and 2). In vitro testing revealed that TH-1177 was effective in blocking Ca²⁺ influx in prostate cancer cells. Efforts toward the identification of the molecular target of TH-1177 revealed that the 25B splice variant of the Ca_v3.2 T-type Ca²⁺ channel (also known as CACNA1H) is inhibited by TH-1177, whereas its stereoisomer TH-1211 had no effect. Moreover, these investigators postulated that increased expression levels and activity of T-type Ca²⁺ channels are likely responsible for Ca²⁺ influx and stimulation of proliferation in cancer cells. Indeed, TH-1177 reduced the proliferative rate of human prostate cancer cells LNCaP and PC-3 in vitro, with a potency that correlates with its inhibition of Ca²⁺ influx. Furthermore, in xenograft studies, administration of TH-1177 to nude mice inoculated with PC-3 human prostate cancer cells led to a significant dose-dependent increase in longevity. Paradoxically, T-type Ca²⁺ channels are actually known to open during membrane depolarization and thus are expected to mediate Ca²⁺ influx into cells after an action potential or in response to depolarizing signals, whereas the epithelial Ca²⁺ channel TRPV6 channels would seem more appropriate to facilitate Ca²⁺ entry into cancer cells, as they are open at the negative resting potentials of these cells. Thus, while TH-1177 is well established as an inhibitor of Ca²⁺ entry in prostate cancer cells, it is reasonable to speculate that it might inhibit TRPV6 in these cells as well.

Figure 13.5

Chemical structures of known TRPV6 inhibitors: (a) heterocyclic compounds and (b) 2-APB analogs.

13.7.2.2. Development of TRPV6 Inhibitor “Compound #03”

To address the earlier question and to develop additional inhibitors of TRPV6 and also TRPV5, Landowski and colleagues synthesized compounds similar to TH-1177, (i.e., compounds #02, #03, #05, #06, and #09) [41]. Together with the antifungal agents econazole and miconazole that weakly inhibit TRPV6 [16,141,142] and TH-1177, these newly synthesized compounds were tested for their inhibitory effect on TRPV6 [41] in Xenopus oocytes expressing TRPV6 or TRPV5 and measuring ⁴⁵Ca²⁺ influx. Figure 13.5a shows the chemical structure of “compound #03” (compound 3), as well as those of econazole and miconazole (compounds 4 and 5). The compounds were also tested using LNCaP prostate cancer and T47D breast cancer cells, studying their effects on proliferation. In LNCaP cells, TRPV6 is believed to be the most abundantly expressed Ca²⁺ entry channel, and it was shown that there is a negligible expression of TRPV5 in these cells [126]. Indeed, TH-1177 inhibited TRPV6 (and also TRPV5)-mediated Ca²⁺ uptake in oocytes, suggesting that the molecular target of TH-1177 in prostate cancer cells is TRPV6. However, since it is well established that TH-1177 mediates inhibition of Ca²⁺ entry via T-type Ca²⁺ channels and given that this compound also inhibits Ca²⁺ entry into cells that do not express TRPV6 [143], it is possible that, in prostate cancer cells, TH-1177 inhibits both T-type and TRPV6 Ca²⁺ channels.

Compound #03 of the study by Landowski and colleagues was found to be the most potent and selective inhibitor of TRPV6, significantly inhibiting prostate cancer cell proliferation in vitro, with an IC₅₀ value of 0.44 μM [41]. Likewise, TRPV6-specific siRNAs were able to reduce proliferation. Indeed, it has been demonstrated that TRPV6 controls Ca²⁺ entry and proliferation in LNCaP cells (see Section 13.7.2.5 for further details).

13.7.2.3. Effects of Estrogen-Receptor Blocker Tamoxifen on TRPV6 Function

Looking for additional inhibitors, Bolanz et al. [69] demonstrated that the estrogen-receptor prodrug tamoxifen (Figure 13.5a, compound 6) that is currently used for treatment of breast cancer also inhibits TRPV6 in Xenopus oocytes, with an IC₅₀ of 7.5 μM based on Ca²⁺ uptake studies. This finding is interesting as it may help explain why certain ER-negative breast tumors favorably respond toward tamoxifen treatment.

13.7.2.4. Ligand-Based Virtual Screening (LBVS) to Develop Improved TRPV6 Inhibitors

All of the abovementioned agents are still relatively weak TRPV6 inhibitors, and several of them, including econazole and miconazole, are rather unspecific. Thus, to push forward the development of more effective TRPV6 inhibitors, Reymond and colleagues employed the LBVS approach [68]. For this, the publicly available ZINC database was exploited (Department of Pharmaceutical Chemistry at the University of California, San Francisco), a curated collection of commercially available chemical compounds prepared especially for virtual screening, allowing commercially available compounds to be tested without having to synthesize them first. Specific LBVS tools that have been developed by Reymond and colleagues were used to enable efficient exploitation of the ZINC database [68]. The strategy was to initially search for analogs of the abovementioned TRPV6 inhibitors, that is, TH-1177, compound #03, econazole, miconazole, and tamoxifen. Starting with these inhibitors, a pharmacophore shape similarity search for analogs in the ZINC database was performed, followed by purchase of selected virtual hits, to form small, focused libraries of a few hundred compounds. These were then experimentally tested using HEK293 cells stably transfected with TRPV6, measuring Ca²⁺ or Cd²⁺ uptake using the fluorophore calcium-5 and the FLIPR Tetra microplate fluorescence reader, in order to identify positive hits. Hits were used for a second round of LBVS, and the resulting improved compounds hits were further optimized by chemical synthesis and structure-activity relationship studies.

The most effective inhibitor cis-22a (IC₅₀ value of 0.12 μM) was further investigated (Figure 13.5a, compound cis-7). It showed high selectivity against other Ca²⁺ channels and related TRP targets. The antiproliferative activity of cis-22a on TRPV6 expressing T47D human breast cancer cells was determined and compared to that of SKOV3 ovarian carcinoma cells that do not express TRPV6. Treatment of T47D cells with cis-22a decreased cellular proliferation by 20% at 5 μM (IC₅₀ = 25 μM), a concentration sufficient to block TRPV6-mediated Ca²⁺ influx, while SKOV3 cells were unaffected. By contrast, the less potent diastereomer, trans-22a (Figure 13.5a, compound trans-7), had no significant effect on proliferation in these cells lines. The selective but significantly smaller effect of cis-22a compared to siRNA knockdown (resulting in 50% reduction in cell growth) may suggest that TRPV6 expression affects cell growth by mechanisms other than reduced Ca²⁺ uptake. In addition, it is possible that the TRPV6 inhibitor only works if its expression in the plasma membrane is activated as part of SOCE, as recently described by Prevarskaya and colleagues [127]. Therefore, further studies are needed to evaluate the beneficial effects of this compound on tumor growth and metastasis formation in prostate and breast cancer.

13.7.2.5. Analogs of 2-Aminoethyl Diphenylborinate (2-APB) as Potential Modulators of TRPV6 Function

2-APB is known to influence SOCE via Orai Ca²⁺ channels with different effects in different cell lines. Recent studies revealed that 2-APB also affects TRP channels, including TRPV6.

As already alluded, Prevarskaya’s group demonstrated that TRPV6 translocates to the plasma membrane via an Orai1-mediated mechanism, controlling cancer cell survival [127]. According to their proposal, TRPV6 switches in prostate cancer cells from its constitutive activity to a store-operated mode. This switch is proposed to occur through STIM1, Orai1, and TRPC1-evoked translocation of TRPV6 to the plasma membrane, involving the Ca²⁺/Annexin I/S100A11 pathway. As TRPV6 is virtually absent in healthy prostate, it is upregulated de novo in prostate cancer cells, where it changes its constitutively active role, supplying intracellular Ca²⁺ in a store-operated mode that is used in cancer cells to increase cell survival.

To investigate whether 2-APB analogs modulate TRPV6 function, a SAR study was conducted by Lochner and colleagues [144] (see Figure 13.5b for the chemical structures of relevant analogs) in an attempt to develop synthetic analogs that are more potent and more specific for TRPV6, compared to SOCE. The synthesis of a small library of 2-APB analogs was made, and the effects of the compounds were tested in HEK293 cells. To test the effect on human TRPV6, transiently expressing HEK293 cells were used. To test the effect on SOCE, measurements were performed in HEK293 cells in the absence of TRPV6 and the compounds were administered together with 1 μM thapsigargin, in order to activate SOCE. Several 2-APB analogs were discovered that were more potent inhibitors of TRPV6 than 2-APB. However, in general, they inhibited SOCE as well. Analog 19b (Figure 13.5b; compound 13) was an exception as it exhibits an approximately 2.5-fold higher selectivity for TRPV6 compared to SOCE. It appears that if steric bulk around the central boron atom or rigidity is increased, for example, as in compounds 2j, 6a, 6b, and 11 (compounds 9–12 in Figure 13.5b), TRPV6 inhibitory activity is lost, while SOCE inhibitory activity is retained. The most beneficial effects were achieved when only one of the phenyl rings of 2-APB was replaced by a 2-methylthiophene (compound 19b).

Interestingly, 2-APB and its amino alcohol variants turned out to be rather unstable compounds since they rapidly hydrolyze and transesterify in solution, which complicates the analysis of their pharmacological effects and their use as therapeutic compounds.

Taken together, further studies are needed to evaluate the therapeutic potential of 2-APB analogs and to determine whether such compounds are suitable for developing therapeutic applications, targeting specifically the TRPV6 Ca²⁺ channel. An additional issue to be addressed may be cellular acidification caused by 2-APB at high concentrations [145].

13.8. TRPV5 and TRPV6 Mutations and Kidney Stone Diseases

Kidney stone disease is a common condition with an incidence of approximately 5%–10% in each population. Although it is known that the environmental factors such as Ca²⁺ intake are important for the stone formation, 40% of stone-forming patients have family history, suggesting that there should be genetic factors as well. Several lines of evidence including ones from a stone-forming rat (GHS rat) suggest that kidney stone disease is a polygenic disease. Hypercalciuria (higher urine Ca²⁺ level) is a common risk factor for stone formation [6].

There are three types of hypercalciuria: (1) absorptive hypercalciuria caused by excess amount of intestinal Ca²⁺ absorption, (2) resorptive hypercalciuria that is derived from abnormal bone metabolism, and (3) renal leak hypercalciuria caused by a decreased Ca²⁺ reabsorption in the kidney. However, a hypercalciuria patient may have abnormal Ca²⁺ absorption and reabsorption and bone resorption at the same time. Since TRPV5 is suggested to be the molecular identity of the apical Ca²⁺ entry pathway for final renal Ca²⁺ reabsorption, and TRPV5 KO mice exhibited renal leak hypercalciuria-like phenotypes [14], it was reasonable to predict that TRPV5 mutations cause kidney stone disease with a renal Ca²⁺ leak. However, Muller et al. reported nine families with no association between TRPV5 gene and autosomal dominant hypercalciuria [146]. This is probably because the renal leak mutations are normally recessive mutations. However, TRPV5 cannot be excluded as a candidate gene for hypercalciuria. The same group also investigated 20 renal leak hypercalciuric patients, and 4 nonsynonymous polymorphisms were found. However, no association study was done in this report [147]. Later on, other groups investigated 365 kidney stone patients in Taiwan and found that the frequency of R154H (rs4236480) polymorphism in the TRPV5 gene was higher in the patients with multiple stones [148], indicating that the TRPV5 gene is somehow linked to renal stone formation.

Recently, the association between kidney stones and a novel TRPV5 gene mutation (L530R) was identified [149]. A genome-wide association study (GWAS) of 2636 Icelanders with kidney stones led to the identification of defects of the following genes that are responsible for kidney stone formation: (1) ALPL (tissue nonspecific alkali phosphatase for maintaining urine pyrophosphate that inhibits stone formation), (2) SLC34A1 (Na/Pi cotransporter for renal phosphate reabsorption), (3) claudin-14 (CLDN14), (4) CaSR, and (5) TRPV5. Claudin-14 and CaSR are thought to be important for renal Ca²⁺ handling to excrete excess Ca²⁺ via the paracellular pathway. With respect to the L530R mutation in the TRPV5 gene, it might represent a loss-of-function mutation, because it is localized in the pore loop of the TRPV5 channel. Thus, so far only one mutation of the TRPV5 gene has been identified that is associated with kidney stone disease, most likely resulting in renal leak hypercalciuria.

There are no further reports thus far showing an association of TRPV5 gene mutations with other human diseases. However, with respect to single nucleotide polymorphisms, 154H and 563T were more frequently distributed among African population compared to that of 154R or 563A. Renkema and colleagues showed that there were no functional differences in these polymorphisms with respect to Na⁺ currents, Ca²⁺ currents, and [Ca²⁺]_i-dependent inactivation in patch-clamp recordings [147]. On the contrary, when using the in Xenopus oocyte expression system, an increased ⁴⁵Ca²⁺ uptake in the 563T variant was reported [150]. Computational modeling based on the structure of TRPV1 revealed that the A563T variation induces structural, dynamical, and electrostatic changes in the TRPV5 pore, providing structural insights into the altered function of this variant [151]. The 154H variant exhibited the lowest Ca²⁺ uptake activity among five TRPV5 variants; however, the data did not reach statistical significance compared to the reference 154R variant [150]. In preliminary experiments, we found decreased ⁴⁵Ca uptake in the 154H variant but not in A563T, without changing membrane surface expression of TRPV5 proteins (Y. Suzuki et al., unpublished data). Interestingly, there appears to be an association between R154H and bone density of the femoral neck and the tibial epiphysis in adults, most likely due to impaired osteoclastic bone resorption (A. Pasch, Y. Suzuki, and M.A. Hediger, unpublished data). These results and also data from TRPV5 KO mice [152] suggest that variants of the TRPV5 gene are risk factors for impaired bone quality. Genetic variants of TRPV5 might contribute to differences in bone quality and strength among different populations.

Given the numerous evidence, including the study of TRPV6 KO mice that lack TRPV6 function results in impaired intestinal Ca²⁺ absorption [15], TRPV6 is believed to be a key component of apical Ca²⁺ entry during intestinal absorption of Ca²⁺ from food and drinking water. Therefore, it is reasonable to speculate that genetic variations of the TRPV6 gene are associated with absorptive hypercalciuria, one of the major risk factors of kidney stone formation. Indeed, this is the case: a gain-of-function haplotype of TRPV6 gene has been identified from sequencing of 170 Ca²⁺-stone formers in Switzerland [153]. The frequency of this haplotype was statistically higher in the Ca²⁺-stone formers compared to normal individuals, suggesting that the haplotype might be a risk factor for kidney stone patients with absorptive hypercalciuria. Another line of evidence that stems from molecular evolution highlights the functional significance of this haplotype: it has been reported that this very same haplotype reflects a positive selection during human evolution [154,155] and that this “derived TRPV6 form” provided an evolutionary advantage for the survival of humans in response to certain past environmental changes, when compared to the ancestral form. An improvement of [Ca²⁺]_i-dependent inactivation has been suggested in the derived TRPV6 form, compared to the ancestral form [154], which is consistent with the observed increase in ⁴⁵Ca uptake in heterologous expression systems [155].

It is interesting to note that TRPV6 KO mice exhibit hypercalciuria [15]. This suggests that TRPV6 might also be involved in renal Ca²⁺ reabsorption. In fact, TRPV6 mRNA level appears to be higher than that of TRPV5 in the human kidney [8]. Also, prominent TRPV6 protein expression has been detected in both the proximal tubule and distal tubule in human kidney [10]. African-Americans exhibit lower urinary Ca²⁺ excretion and lower incidence of kidney stone disease [156]. The high prevalence of the ancestral haplotype of TRPV6 gene in African descendants suggest that TRPV6 may play a role in increased Ca²⁺ reabsorption in African populations.

13.9. Concluding Remarks

A variety of approaches have been utilized to achieve a better understanding of Ca²⁺ transport in epithelial cells, starting with expression cloning and moving all the way to in-depth cellular and biophysical analysis, generation of genetically engineered mouse models of TRPV5 and TRPV6, 3D structural studies, and chemical approaches. The remarkable Ca²⁺-selectivity and distinct Ca²⁺-dependent inactivation mechanisms involving PI(4,5)P₂ are examples of what patch-clamp techniques could achieve in understanding these channels. Genetically engineered animal models not only provided the expected roles of TRPV5 and TRPV6 in intestinal absorption and renal reabsorption of Ca²⁺ but also revealed unique roles of the TRPV6 channel in maternal-fetal Ca²⁺ transport, male fertility, and auditory system. The growing evidence for the involvement TRPV6 in cancer proliferation and metastasis formation generates pharmaceutical interest. New approaches, such as ligand-based virtual screening, have been utilized to design inhibitors as a first step toward therapeutic developments. The novel crystal structure of TRPV6 will be instrumental to our understanding of the Ca²⁺ transport mechanisms and regulation and help refine the chemical design of TRPV6 modulators. As the involvement of genetic mutations/variations of TRPV5 and TRPV6 in human health is emerging, the integration of multidisciplinary approaches will further advance our knowledge and help develop new strategies for the treatment of kidney stone disease and cancer.

Acknowledgments

We thank our colleagues for their contributions to the research projects related to this book chapter. Our research was supported by the National Institute of Diabetes and Digestive and Kidney Diseases (R01DK072154), the American Heart Association National Center (0430125N), the Greater Southeast Affiliate (09GRNT2160024), and the Swiss National Center for Competence in Research, NCCR TransCure (www.transcure.org). Gergely Gyimesi acknowledges the COFUND Marie Curie international fellowship program, IFP TransCure.

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Ji-Bin Peng

Division of Nephrology

Nephrology Research and Training Center

and

Department of Urology

University of Alabama at Birmingham

Birmingham, Alabama

Yoshiro Suzuki

Division of Cell Signaling

National Institutes of Natural Sciences

and

Department of Physiological Sciences

SOKENDAI (The Graduate University for Advanced Studies)

Okazaki, Japan

Gergely Gyimesi

Institute of Biochemistry and Molecular Medicine

and

Swiss National Center of Competence in Research

TransCure

University of Bern

Bern, Switzerland

Matthias A. Hediger

Institute of Biochemistry and Molecular Medicine

and

Swiss National Center of Competence in Research

TransCure

University of Bern

Bern, Switzerland