Ca2+ is an essential ion in all organisms and many physiological functions in the body rely on the exact maintenance of the Ca2+ balance. The epithelial Ca2+ channels TRPV5 [TRP (transient receptor potential) vanilloid 5] and TRPV6 are the most Ca2+-selective members of the TRP superfamily and are generally considered as the gatekeepers of Ca2+ entry across epithelia. TRPV5 is involved in Ca2+ reabsorption from pro-urine, while TRPV6 has an essential role in intestinal Ca2+ uptake. These channels are the prime targets of calciotropic hormonal regulation, including vitamin D and parathyroid hormone. In addition, extra- and intra-cellular signalling by associated proteins and Ca2+ itself play key roles in TRPV5 and TRPV6 regulation. In this paper, we describe the present understanding of the concerted action of calbindin-D28k, klotho and BSPRY (B-box and SPRY-domain-containing protein) at different levels throughout the epithelial cell to control Ca2+ influx at the luminal entry gate.
Maintenance of body Ca2+ homoeostasis is of vital importance for many physiological functions including intracellular signalling processes, neuronal excitability, muscle contraction and bone formation. Extracellular Ca2+ concentration ([Ca2+]extracellular) is regulated by a homoeostatic mechanism tightly controlling the concerted actions of intestinal Ca2+ absorption, renal Ca2+ reabsorption and exchange of Ca2+ to and from bone . Transcellular Ca2+ (re)absorption is initially tightly regulated by the influx of Ca2+ across the luminal membrane of the epithelial cell. Next, Ca2+ enters the cell and is sequestered by calbindin-D28k or -D9k to maintain low cytosolic Ca2+ concentrations. Subsequently, calbindin-bound Ca2+ diffuses to the basolateral side of the cell, where it is extruded into the bloodstream via NCX1 (Na+/Ca2+ exchanger 1) and PMCA1b (plasma membrane Ca2+-ATPase; Figure 1). The influx of Ca2+ across the luminal membrane is mediated by specialized epithelial Ca2+ channels, i.e. TRPV5 [TRP (transient receptor potential) vanilloid 5] and TRPV6. The regulation of these channels is particularly important because it occurs down a steep concentration gradient of Ca2+ (see Figure 1). In addition, patch–clamp analysis demonstrated that the current–voltage relationship of TRPV5 and TRPV6 displays strong inward rectification, and the channels are characterized by a Ca2+ selectivity of more than 100 times over univalent cations. Therefore TRPV5 and TRPV6 are considered as the gatekeepers of transcellular Ca2+ transport [1–4].
Integrated model of active epithelial Ca2+ transport
TRPV5 and TRPV6 were originally cloned from rabbit kidney and rat intestine respectively and were subsequently identified in many additional species including fish, rabbit, mouse, rat and human [5,6]. TRPV5 and TRPV6 comprise approx. 730 amino acids, along with a molecular mass of approx. 83 kDa, containing six TM (transmembrane) segments and large cytosolic N- and C-terminal domains. A short hydrophobic stretch between TM5 and TM6 was predicted to be the pore-forming region. The six-TM unit is one of four subunits presumed to surround the central pore in a tetrameric configuration . The N-terminal domain contains ankyrin repeats that can mediate protein–protein interactions and it is suggested that the first ankyrin repeat is critical for functional channel multimerization . Detailed expression and (co)localization studies suggested that TRPV5 comprises the epithelial Ca2+ channel predominantly involved in renal transcellular Ca2+ transport in DCTs (distal convoluted tubules) and CNTs (connecting tubules), whereas TRPV6 was postulated to mediate duodenal Ca2+ absorption [2,9–11].
The electrophysiological properties of TRPV5 and TRPV6 have been studied extensively. Their characteristic inward rectifying currents can be blocked by Mg2+ in a voltage-dependent manner [12,13], which is removed upon neutralization of a single negatively charged amino acid residue crucial for high-affinity Ca2+ binding – Asp542 in TRPV5 and Asp541 in TRPV6 – within the pore region. This indicates that the aspartic residues Asp542 (TRPV5) and Asp541 (TRPV6) line the narrowest part of their pores. The pore diameter of TRPV6 was recently estimated at 5.4 Å (1 Å=0.1 nm) by cysteine scanning experiments .
The biological activity of TRPV5 and TRPV6 is a highly co-ordinated and regulated process that can be controlled by four categories of regulatory mechanisms: (i) the transcriptional and translational levels of TRPV5 and TRPV6 are regulated by calciotropic hormones including vitamin D, PTH (parathyroid hormone) and oestrogens; (ii) the activity of TRPV5 and TRPV6 at the plasma membrane is subject to pH- and Ca2+-dependent regulatory mechanisms; (iii) trafficking of TRPV5 and TRPV6 to the luminal membrane is essential to exert its biological activity; (iv) channel activity at the plasma membrane is modulated by different TRPV5- and TRPV6-associated proteins. The present review primarily focuses on the latest advances in channel-associated proteins that regulate TRPV5 and TRPV6 activity.
Dynamic control of channel activity by calbindin-D28K
Vitamin D-responsive epithelial cells are challenged by a high Ca2+ influx that is tightly regulated to maintain low non-toxic cytosolic Ca2+ concentrations. The apparent role of calbindin proteins has been shown by the involvement of these specialized Ca2+ buffering proteins to facilitate multiple steps in transcellular Ca2+ transport. First, by buffering intracellular Ca2+ in the immediate vicinity of the channel mouths of TRPV5 and TRPV6, they control a continuous flow of Ca2+ by preventing a negative feedback of free Ca2+ inhibiting channel activity [1,2]. Secondly, toxic levels of intracellular Ca2+ may arise during high rates of transcellular Ca2+ transport and therefore the cytosolic Ca2+ concentration has to be strictly regulated by specialized Ca2+-binding proteins [15–17]. Thirdly, Ca2+ that enters the cell at the luminal side will diffuse to the basolateral side without affecting other intracellular processes before it is extruded into the extracellular compartment via NCX1 and/or PMCA1b. Calbindins have been implicated to increase the diffusional range of Ca2+ by facilitating the transport of Ca2+ to the basolateral surface of the cell . In addition, an important role of calbindins in vitamin D-responsive transepithelial Ca2+ transport is supported by their consistent co-expression with the Ca2+-transport proteins TRPV5 and TRPV6, NCX1 and PMCA1b . Conversely, genetic ablation of TRPV5 in mice resulted in decreased expression of calbindin-D28K . As indicated, the intracellular Ca2+ concentration ([Ca2+]intracellular) in the close vicinity of the channel mouths of TRPV5 and TRPV6 tightly regulates their activities; however, how the Ca2+ concentration near the pore is regulated was until recently poorly understood. In addition to mathematical models  and co-ordinated regulation of renal Ca2+ transport proteins , Lambers et al.  provide the first experimental evidence that calbindin-D28K dynamically regulates the activity of the TRPV5 channel . In their study, Lambers et al.  demonstrate by evanescent-field life cell microscopy and protein-binding analysis that calbindin-D28K translocates to the plasma membrane and directly interacts with TRPV5 upon a decrease in the intracellular Ca2+ concentration. Furthermore, co-expression of TRPV5 and calbindin-D28K in HEK-293 cells (human embryonic kidney cells) significantly increased 45Ca2+ uptake in these cells. However, co-expression with the TRPV5-interacting Ca2+-insensitive mutant of calbindin-D28K, in which the Ca2+-binding EF-hand structures were inactivated (calbindin-D28KΔEF), abolishes this stimulatory effect. Moreover, calbindin-D28KΔEF competes with wild-type calbindin-D28K, resulting in a dominant-negative inhibition of transepithelial Ca2+ transport in primary rabbit CNTs and CCD (cortical collecting duct) cultures . Blockage of TRPV5 by Ruthenium Red eliminated PTH-stimulated Ca2+ transport in these primary cell cultures and simultaneously decreased the expression of calbindin-D28K. Likewise, van Abel et al.  showed that the magnitude of Ca2+ influx via TRPV5 predominantly controls the expression of calbindin-D28K. Together, this indicates that differential calbindin-D28K expression correlates with its capacity to dynamically buffer TRPV5-mediated Ca2+ influx. Lambers et al.  also show that the regulatory role of calbindin-D28K is a unique process in comparison with direct channel regulation via other Ca2+-binding proteins such as calmodulin [24,25] by measuring TRPV5 activity in whole cell patch–clamp configuration during different controlled intracellular Ca2+ concentrations, either in the presence of calbindin-D28K or calbindin-D28KΔEF. Ultraviolet light-induced uncaging of Ca2+ increased intracellular Ca2+ concentrations, revealing the negative feedback of Ca2+ on channel activity either in the presence of calbindin-D28K or calbindin-D28KΔEF, and indicated that calbindin-D28K does not directly affect channel inactivation characteristics. These data demonstrate that calbindin-D28K dynamically controls epithelial Ca2+ influx by tethering the TRPV5 channel to buffer free Ca2+ in the close vicinity of the pore.
Regulation of channel abundance at the cell surface by klotho
The body is enabled to fast and actively reabsorb Ca2+ when the extracellular Ca2+ concentration in the circulation decreases. As shown by calbindin-D28K functioning, the epithelial cell is well equipped to transport and sustain high rates of Ca2+ influx. A key component to create such elevated Ca2+ influx is to increase TRPV5 and TRPV6 channel abundance at the epithelial cell surface. TRPV5 is located in or near the apical membrane in DCTs of the kidney, whereas in CNTs a large subset of TRPV5 is located subapically . Therefore it is hypothesized that these channels are shuttled from intracellular vesicles into the plasma membrane. In addition, high Ca2+ influx may be obtained by prolonging the channel's durability at the cell surface before inactivation or internalization.
The recent elucidation of a novel mechanism of TRPV5 regulation by the anti-aging hormone klotho nicely illustrates the importance of controlling channel abundance at the cell surface. Chang et al.  showed that klotho, a vitamin D-controlled hormone discovered by Kuro-o [27,28], completely co-localized with TRPV5 at or near the apical membrane in DCTs and CNTs. In addition, it was found that klotho is present in urine, serum and cerebrospinal fluid , as well as in the supernatant obtained from transfected HEK-293 cells expressing klotho, suggesting that it may operate from the extracellular space to regulate transcellular Ca2+ transport. Expression of TRPV5 and klotho in HEK-293 cells, or direct application of klotho supernatant to TRPV5-expressing HEK-293 cells, stimulated TRPV5-mediated 45Ca2+ uptake , suggesting a physiological effect of klotho circulating in the bloodstream. Overexpression of klotho in mice significantly extended the life span and suppressed symptoms of aging , whereas klotho knockout mice displayed characteristics correlating with premature aging  and the pathophysiology of TRPV5 knockout mice (i.e. disturbed Ca2+ homoeostasis, vitamin D metabolism and bone abnormalities) . Further analysis of the molecular mechanism by which klotho stimulates Ca2+ influx included cell surface biotinylation experiments that revealed a significant increase in plasma membrane localization of TRPV5, while at the same time the total expression was not affected after klotho treatment. Interestingly, these effects could be mimicked by a purified β-glucuronidase, indicating that the enzymatic activity of klotho is responsible for the increased TRPV5 activity. Mutation of a predicted N-glycosylation site between TM regions 1 and 2 at the asparagine residue Asn358 in TRPV5 to a glutamine residue prevented TRPV5 glycosylation when expressed in HEK-293 cells. Cells expressing this mutant displayed normal 45Ca2+ uptake, which was unaffected by klotho treatment. Thus klotho may work by affecting the extracellular glycosylation status of TRPV5, entrapping the channels in the plasma membrane. Altogether, this results in increased Ca2+ influx from the lumen to preserve normal blood Ca2+ levels during periods of insufficient dietary Ca2+ by the reduction of Ca2+ loss via the urine. The enzymatic activity of klotho to regulate TRPV5 channel abundance at the cell surface from the extracellular site demonstrates a novel mechanism to control epithelial Ca2+ influx.
Modulation of TRPV5 channel activity at the plasma membrane by BSPRY (B-box and SPRY-domain-containing protein)
The contribution of Ca2+ uptake at the luminal membrane of the epithelial cell via TRPV5 is determined by the number of active TRPV5 channels at the cell surface. Whereas klotho entraps the channel at the cell surface and increases Ca2+ influx per cell, calbindin-D28K prevents accumulation of intracellular Ca2+ reaching toxic values by the efficient buffering and facilitated transport of Ca2+ to the basolateral side, where it is extruded into the bloodstream. In contrast, the molecular mechanisms underlying channel trafficking and regulation of its activity at the plasma membrane are poorly understood. Therefore the identification and functional characterization of TRPV5- and TRPV6-associated proteins with unknown or known functions in ion channel modulation or in vesicular trafficking might explain such a mechanism. TRPV5, like most of the TRP channels, mainly localizes to vesicular structures throughout the cytoplasm. The identifications of the S100A10–annexin 2 protein complex and Rab11a, which were proposed to be involved in vesicular trafficking, provided essential roles in TRPV5 and TRPV6 cell surface expression [32,33]. The S100A10–annexin 2 protein complex was shown to be involved in constitutive non-stimulated trafficking, whereas Rab11a was identified as a molecular switch involved in the recycling of TRPV5 channels from the plasma membrane.
With the recent identification of BSPRY  as a TRPV5- and TRPV6-interacting protein , further insight into the direct modulation of TRPV5 and TRPV6 activity at the plasma membrane was obtained. BSPRY showed complete co-localization with TRPV5 in Ca2+-transporting tubular segments of the kidney, and stable expression of BSPRY significantly inhibited Ca2+ influx in confluent monolayers of MDCK cells (Madin–Darby canine kidney cells) expressing TRPV5. In addition, via cell surface biotinylation experiments it was demonstrated that a significant fraction of BSPRY is present at the plasma membrane of HEK-293 cells co-expressing TRPV5 and BSPRY . These findings suggest that BSPRY is involved in inhibitory signalling cascades that modulate TRPV5 activity directly at the cell surface. Interestingly, BSPRY expression was inversely regulated by the calciotropic hormone vitamin D, as demonstrated by elevated mRNA expression of BSPRY in kidney samples of 25-hydroxyvitamin D3-1-α-hydroxylase knockout mice compared with wild-type mice . These knockout mice are unable to synthesize 1,25-dihydroxyvitamin D3 (1,25-dihydroxycholecalciferol), the biological form of vitamin D, and TRPV5 expression is significantly down-regulated in these mice [1,36]. Together, this suggests that vitamin D negatively regulates BSPRY expression while at the same time it stimulates Ca2+ influx via TRPV5.
As described above, the regulation of TRPV5 and TRPV6 is dependent on mechanisms that directly target channel expression or indirectly stimulate trafficking and activity of TRPV5 and TRPV6 at the luminal cell surface via associated proteins. In addition to the negative-feedback mechanism of Ca2+ itself, the efficient buffering and diffusion of free intracellular Ca2+ by calbindin-D28K demonstrates the ability of the epithelial cell to sustain high rates of transcellular Ca2+ transport. Furthermore, klotho entraps channels in the plasma membrane to increase durability and Ca2+ influx. Finally, it is suggested that BSPRY inversely regulates channel activity at the plasma membrane, depending on the serum level of vitamin D (Figure 1). These results explain that regulation of TRPV5 and TRPV6 is mediated at different sites throughout the epithelial cell, and that extracellular control makes a major contribution to the channels' activity. Previously identified interacting partners of TRPV5 and TRPV6 are also found to affect activity of the channels via distinct mechanisms. Functional expression of both TRPV5 and TRPV6 requires binding of the S100A10–annexin 2 protein complex. However, Ca2+ sensing to control the activity of the epithelial Ca2+ channels is differentially organized. The Ca2+ sensor 80K-H has been shown to affect TRPV5 activity [32,37], whereas regulation by the ubiquitous Ca2+ sensor calmodulin seems to be restricted to TRPV6 . Together, the concerted action of these associated proteins provides insight into the strict control of transcellular Ca2+ transport at the apical entry gate to determine the net active Ca2+ flux to the blood compartment. Unravelling the complete molecular mechanisms regulating TRPV5 and TRPV6 is challenging and may ultimately lead to a full understanding of the maintenance of body Ca2+ homoeostasis.
Cell and Molecular Biology of TRP Channels: Biochemical Society Focused Meeting held at University of Bath, U.K., 7–8 September 2006. Organized and Edited by D. Beech (Leeds, U.K.), B. Reaves (Bath, U.K.) and A. Wolstenholme (Bath, U.K.).
This work was supported by the Dutch Organization of Scientific Research (Zon-Mw 016.006.001, Zon-Mw 902.18.298, NWO-ALW 810.38.004, NWO-ALW 805-09.042, NWO-ALW 814-02.001 and NWO 812-08.002), the Stomach, Intestine, Liver Foundation (MWO 03-19), the Human Frontiers Science Program (RGP32/2004), and the Dutch Kidney Foundation (C10.1881 and C03.6017).