Regulation of TRPV5 and TRPV6 by associated proteins

【摘要】  The epithelial Ca 2+ channels TRPV5 and TRPV6 are the most Ca 2+ -selective members of the TRP channel superfamily. These channels are the prime target for hormonal control of the active Ca 2+ flux from the urine space or intestinal lumen to the blood compartment. Insight into their regulation is, therefore, pivotal in our understanding of the (patho)physiology of Ca 2+ homeostasis. The recent elucidation of TRPV5/6-associated proteins has provided new insight into the molecular mechanisms underlying the regulation of these channels. In this review, we describe the various means of TRPV5/6 regulation, the role of channel-associated proteins herein, and the relationship between both processes.

【关键词】  Rab calbindin KH BSPRY Klotho kidney

TRANSCELLULAR Ca 2+ (re)absorption is a pivotal process in the maintenance of extracellular Ca 2+ balance. It allows the organism to respond to fluctuations in dietary Ca 2+ and adapt to the body's demand during processes such as growth, pregnancy, lactation, and aging. The first step in transcellular transport is the influx of Ca 2+ across the apical membrane of the epithelial cell, which is a tightly controlled mechanism ( Fig. 1 ). Next, Ca 2+ enters the cell and is sequestered by specialized proteins called calbindins to maintain low cytosolic Ca 2+ concentrations. Subsequently, bound Ca 2+ diffuses to the basolateral side of the cell, where it is extruded into the bloodstream via the Na + /Ca 2+ exchanger (NCX1) and plasma membrane Ca 2+ -ATPase (PMCA1b). The molecular identification of two members of the transient receptor potential (TRP) superfamily, TRPV5 ( 28 ) and TRPV6 ( 48 ), boosted the research addressing the molecular mechanism of epithelial Ca 2+ transport. The physiological role of these channels has been substantiated in several mouse models of Ca 2+ -related disorders, including vitamin D deficiency rickets type I (VDDR-I) ( 13, 25 ) and vitamin D-resistant rickets type II ( 60 ) (VDDR-II). Furthermore, TRPV5 ( 29 ) and TRPV6 ( 3 ) knockout mice show significant disturbances in their Ca 2+ homeostasis. TRPV5 primarily fulfills the role as gatekeeper of epithelial Ca 2+ transport in the kidney ( 27 ), whereas TRPV6 forms the main Ca 2+ influx pathway in the small intestine ( 41 ). Therefore, detailed insight into the molecular regulation of TRPV5/6 is pivotal to our understanding of Ca 2+ homeostasis.

Fig. 1. Integrated model of active epithelial Ca 2+ transport. Ca 2+ enters the cell from the luminal side via TRPV5 and/or TRPV6, subsequently binds to calbindin-D 28K, and is extruded at the basolateral membrane by a Na + /Ca 2+ exchanger (NCX1) and/or a plasma membrane Ca 2+ -ATPase (PMCA1b). In this way, there is net Ca 2+ (re)absorption from the luminal space to the extracellular compartment. Ca 2+ (re)absorption is fine-tuned by calciotropic hormones. The active form of vitamin D [1,25(OH) 2 D 3 ] stimulates the individual steps of transcellular Ca 2+ transport by increasing the expression level of TRPV5/6, calbindins, and the extrusion systems.

STRUCTURAL FEATURES OF TRPV5 AND TRPV6

TRPV5 and TRPV6 display the highest sequence homology with the vanilloid receptor subfamily (TRPV) of TRP channels. These channels share a predicated topology consisting of large intracellular NH 2 - and COOH-terminal tails flanking six transmembrane segments (TM) and an additional hydrophobic stretch between TM5 and TM6, forming the pore-forming region ( Fig. 2 ). Several TRP channels contain ankyrin repeats in their NH 2 terminus. The number of ankyrin repeats range from 0 to 14, and the members of the TRPV subfamily have 3-4 repeats ( 12 ), varying slightly depending on the sequence-similarity algorithm used. TRPV5 and TRPV6 display several properties that distinguish these Ca 2+ channels from other TRP members. First, TRPV5 and TRPV6 are constitutively active at low intracellular Ca 2+ concentrations and physiological membrane potentials ( 65 ). Other members of the TRP family are activated by various stimuli, including ligand binding, heat, and cold ( 12 ). Second, the current-voltage relationships show strong inward rectification of TRPV5/6, at both the level of single channels and whole cells ( 26, 64, 65 ). Third, TRPV5 and TRPV6 are 100 times more selective for Ca 2+ than for Na +, making the epithelial Ca 2+ channels the most Ca 2+ -selective members of the TRP superfamily ( 65 ). In a physiological context, this property renders these channels particularly suited to transport Ca 2+ in the presence of Na +.

Fig. 2. Predicted topology of TRPV5/6 including the various binding sites identified for each associated protein. The extracellular loop between transmembrane domain (TM) 1 and TM2 is glycosylated. In the COOH terminus, Rab11, 80K-H, and S100A10 bind within a 30-amino acid-containing helical stretch juxtaposed to TM6. The S100A10-annexin 2 (Anx2) complex interacts with a conserved region within this stretch containing the VATTV amino acid sequence, whereas Rab11a and 80K-H share a binding site COOH terminally flanking this sequence. Na + /H + exchanger regulatory factor 2 (NHERF2) specifically interacts with TRPV5 at the extreme COOH terminus of this channel. Calmodulin (CaM) has multiple binding sites present in the NH 2 terminus, COOH terminus, and transmembrane region. The region between TM2 and TM3 of TRPV6 (indicated by vertical lines) is functionally linked to the fast Ca 2+ -dependent inactivation of this channel, suggesting the binding of an unidentified protein to this region.

Hoenderop and co-workers ( 30 ) recently demonstrated that the TRPV5/6 channel has a tetrameric architecture, where the four subunits surround a single pore. It was shown that TRPV5/6 can form homo- and heterotetramers. The functional properties of these tetramers, including Ca 2+ -dependent inactivation, cation selectivity, and pharmacological blockade, were dependent on the subunit ratio of TRPV5 and TRPV6 ( 30 ). Erler and co-workers ( 18 ) identified the ankyrin repeat at positions 116-191 of the NH 2 terminus as a stringent requirement for physical assembly of TRPV6 subunits. It was proposed that this repeat initiates a molecular zippering process that proceeds past the last ankyrin repeat and creates an intracellular anchor that is necessary for functional subunit assembly. Chang and co-workers ( 9 ) confirmed the important role of the NH 2 terminus in channel assembly in general, and in particular demonstrated that a region near the first ankyrin repeat (positions 64-77) is critical in the assembly process of TRPV5. However, Chang et al. observed slightly increased binding efficiencies using constructs including the first 162 amino acids, suggesting that the ankyrin repeat identified for TRPV6 might be also involved in TRPV5 multimerization. Interestingly, a structural model of the outer pore of TRPV5 and TRPV6 was recently provided by Dodier et al. ( 14 ) and Voets et al. ( 67 ), respectively. Mutation of a single aspartic residue at position number 542 (D542), a residue crucial for high-affinity Ca 2+ binding, altered the apparent pore diameter. This indicates that this residue lines the narrowest part of the pore ( 67 ). Furthermore, using cysteine-scanning mutagenesis these authors indicated that the main structural features of TRPV5 and TRPV6 are a pore helix followed by a nonhelical loop that forms the selectivity filter, similar to KcsA K + channels ( 16 ).

The biological activity of TRPV5/6 can be controlled by four categories of regulatory mechanisms. First, the expression level of TRPV5/6 is regulated by calciotropic hormones. Second, the activity of TRPV5/6 at the plasma membrane is subject to pH- and Ca 2+ -dependent regulatory mechanisms. Third, transport of TRPV5/6 to the luminal plasma membrane is essential to exert its biological activity. Fourth, the recent identification of diverse channel-associated proteins has provided important new insight into the regulation of TRPV5 and TRPV6. This review focuses on the role of channel-associated proteins in the regulation of TRPV5 and TRPV6 activity.

REGULATION OF CHANNEL ABUNDANCE

Various hormones are involved in the maintenance of extracellular Ca 2+ concentration, including vitamin D, parathyroid hormone (PTH), and estrogen. The physiological importance of vitamin D in Ca 2+ homeostasis is reflected by severe disorders like VDDR-I and VDDR-II, linked to alterations in vitamin D synthesis or the vitamin D receptor (VDR), respectively. Genetically modified mouse models of these diseases including VDR ( 31 ) and 25-hydroxyvitamin D 1 -hydroxylase (1 -OHase) knockout mice ( 55 ), in which the vitamin D system has been inactivated, have been created to systematically dissect the genetic regulation of Ca 2+ transport genes and their functional consequences with regard to transcellular Ca 2+ transport. Using these animal models, it was demonstrated that TRPV5 and TRPV6 are positively regulated by vitamin D in the kidney and intestine ( 5, 25, 57, 58, 60 ). These results provided novel insight into the molecular mechanisms of vitamin D efficacy and shed light on the hormonal regulation of TRPV5/6. In addition, it was shown that other hormones like estrogen ( 57, 58 ), PTH ( 59 ), and androgen ( 49 ) contribute significantly to the regulation of TRPV5 and TRPV6 expression levels. The regulation of channel abundance is elaborately discussed by Hoenderop et al. ( 26 ).

It has been described that the VDDR-I and VDDR-II phenotype can be rescued in animal models of these diseases that include a high-Ca 2+ diet ( 25, 37 ). Importantly, the reduced expression level of renal TRPV5, calbindin-D 28K, and NCX1 in the 1 -OHase knockout mice was restored by high dietary Ca 2+ intake ( 25 ), showing a vitamin D-independent effect of dietary Ca 2+ on the expression of renal Ca 2+ transport proteins. Similarly, the expression of intestinal Ca 2+ transport proteins, TRPV6, calbindin-D 9K, and PMCA1b, was normalized by this Ca 2+ diet ( 58 ). Importantly, the effects of this Ca 2+ diet on the expression of renal and intestinal Ca 2+ transport proteins were accompanied by normalization of plasma Ca 2+ concentration ( 25, 58 ). Comparable observations were made in VDR knockout mice where duodenal TRPV5 and TRPV6 mRNA levels were upregulated by dietary Ca 2+ ( 60 ). The molecular mechanism of this vitamin D-independent, Ca 2+ -sensitive pathway remains elusive.

CHANNEL ACTIVITY AT THE PLASMA MEMBRANE

Electrophysiological studies in human embryonic kidney (HEK293) cells heterologously expressing TRPV5 or TRPV6 showed that these channels are constitutively open at physiological membrane potentials ( 65 ). Unlike several other members of the TRP superfamily ( 12 ), there are no indications that TRPV5 and TRPV6 require either a stimulus or ligand to become active. However, the activity of these channels is regulated by various means. First, TRPV5 and TRPV6 are rapidly inactivated on an increase in intracellular Ca 2+ concentration, although the initial inactivation is faster in TRPV6 than in TRPV5 ( 42 ). The region between TM2 and TM3 was identified as a crucial domain in the fast inactivation of TRPV6 ( 42 ).

In addition, two domains in the COOH terminus of TRPV5 contribute to the Ca 2+ -dependent inactivation. Deletion of the last 30 amino acids of the COOH terminus of TRPV5 (G701X) significantly decreased Ca 2+ sensitivity ( 44 ). Detailed mutation analysis revealed that a region upstream in the COOH terminus (between E649 and C653) forms another critical stretch for Ca 2+ -dependent inactivation of TRPV5 ( 44 ).

A second factor involved in the regulation of TRPV5/6 is extracellular pH. Early studies in excitable tissues indicated that a pH decrease inhibits voltage-gated Na + channel activity ( 69 ). Similar findings were observed by Hess and collaborators ( 51 ) for L-type Ca 2+ channels. Changes in pH have been found to regulate a number of TRP channels. External acidic pH increases TRPV1 ( 7 ) currents and decreases the activity of TRPP2 ( 23 ), a distant TRP family member of TRPV5/6. Importantly, it was previously demonstrated that 45 Ca 2+ uptake in TRPV5-expressing Xenopus laevis oocytes is inhibited by acidification of the incubation medium ( 28, 47 ). Indeed, extracellular acidification reduced currents through TRPV5 carried by either monovalent or divalent cations ( 66 ).

Yeh et al. ( 71 ) demonstrated that mutation of the glutamate at position 522, preceding the pore region, to glutamine (E522Q) decreases the inhibition of the channel by extracellular protons. Therefore, this residue may act as the "pH sensor" of TRPV5 ( 71 ). Furthermore, they demonstrated that a rotation of the pore helix, mediated by intracellular protons, facilitates this inhibition of TRPV5 by extracellular acidification ( 70 ). It is well known that acidification of the apical medium inhibits transcellular Ca 2+ absorption across primary cultures of rabbit connecting tubule and cortical collecting duct cells ( 4 ). Extrapolating the pH dependence of TRPV5 to the in vivo situation suggests that inhibition of TRPV5 by extracellular pH may provide, at least in part, the molecular basis of acidosis-induced calciuresis.

A third level of control of channel activity is the recovery from Ca 2+ -dependent inactivation. This intriguing phenomenon seems to be mediated by a mechanism distinct from Ca 2+ -dependent inactivation ( 43 ). Nilius et al. ( 43 ) showed that recovery from inhibition occurred on washout of extracellular Ca 2+ (whole cell configuration) or by removal of Ca 2+ from the inner side of the channel (inside-out patches). However, this process does not simply correlate with the removal of intracellular Ca 2+, because full recovery occurs much later than restoration of the basal Ca 2+ level in non-Ca 2+ -buffered cells or after removal of Ca 2+ from the inner side of excised membrane patches ( 43 ). It is currently unknown whether the recovery reflects reopening of channels present in the plasma membrane or insertion of channels into the plasma membrane.

TRAFFICKING OF TRV5 AND TRPV6

TRPV5 channels are located in or near the apical plasma membrane in distal convoluted tubules (DCT) of the kidney. However, in connecting tubules (CNT) of the kidney, a significant subset of TRPV5 channels is located subapically, hinting at a shuttling mechanism of these channels to the plasma membrane ( 38 ). Because TRPV5 and TRPV6 are constitutively active at physiological membrane potentials, control of the number of channels at the cell surface is essential for efficient and balanced Ca 2+ transport. This is nicely illustrated by a recent elucidation of a novel mechanism of TRPV5 regulation by Chang and co-workers ( 10 ). Klotho, a novel hormone that has been linked to longevity (related to high levels of the protein) ( 34 ) and premature aging (associated with diminished Klotho levels) ( 33 ), was shown to hydrolyze extracellular sugar residues of TRPV5, resulting in cell surface-trapped TRPV5 ( 10 ). This ultimately results in increased Ca 2+ transport, which is in line with the observed effect of Klotho in Ca 2+ homeostasis ( 10 ).

Despite these recent developments, information about the mechanisms underlying the trafficking of TRPV5/6 to and from the plasma membrane remains scarce. In general, our understanding of regulated TRP trafficking is still in its infancy. To date, channel translocation to the plasma membrane has only been observed for TRPL ( 1 ), TRPV2 ( 32 ), TRPC5 ( 2 ), TRPC3 ( 53 ), and TRPC6 ( 8 ). However, these studies have initiated a discussion about the questions of whether TRP trafficking plays a general role in TRP regulation and about the relative contribution of changes in channel gating and channel trafficking ( 39 ).

REGULATION OF TRPV5 AND TRPV6 BY CHANNEL-ASSOCIATED PROTEINS

A number of regulatory proteins have recently been described that modify the biophysical, pharmacological, and expression properties of ion channels and transporters by direct interactions. These newly identified associated proteins have facilitated the elucidation of important molecular pathways modulating transport activity. Similarly, identification of the molecular players that associate with TRPV5 and TRPV6 could be pivotal in our understanding of the regulation of these channels. Until now, five regulatory proteins have been identified that associate with TRPV5 and/or TRPV6, i.e., S100A10-annexin 2, calmodulin (CaM), 80K-H, Na + /H + exchanger regulatory factor 2 (NHERF2), and Rab11a ( 17, 22, 35, 40, 46, 61, 62 ).

S100A10/Annexin 2

S100A10 (also known as p11 or annexin 2 light chain) was identified as an auxiliary protein for TRPV5 and TRPV6 using a yeast two-hybrid system ( 62 ). S100A10 is a 97-amino acid protein member of the S100 superfamily that is present in a large number of organisms, including vertebrates, insects, nematodes, and plants. S100A10 is a distinct member of this family, because its two EF hands carry deletions and substitutions that render it Ca 2+ insensitive. S100A10 is predominantly present as a heterotetrameric complex with annexin 2, which has been implicated in several biological processes, including endocytosis, exocytosis, and membrane-cytoskeleton interactions ( 19, 20 ). Annexin 2 interacts with actin and is postulated to bind the cytoplasmic face of membrane rafts to stabilize these domains, thereby providing a link to the actin cytoskeleton ( 20 ). Van de Graaf et al. ( 62 ) provided the first evidence of a regulatory role for the S100A10-annexin 2 heterotetramer in vitamin D-mediated Ca 2+ (re)absorption in general and in TRPV5 and TRPV6 functioning in particular. It was shown that S100A10, annexin 2, and TRPV5 or TRPV6 are coexpressed in Ca 2+ -transporting cells of the kidney and small intestine ( 62 ). The association of S100A10 with TRPV5 and TRPV6 was restricted to a short peptide sequence, VATTV, located in the COOH termini of these channels ( Fig. 2 ). This stretch is conserved among all identified species of TRPV5 and TRPV6. Interestingly, the TTV sequence in the S100A10-binding site resembles an internal type I PDZ consensus binding sequence, which is S/TXV ( 54 ). However, S100A10 does not contain PDZ domains, indicating that TRPV5-S100A10 interaction has a different nature. The first threonine of the S100A10 interaction motif is a crucial determinant for binding. Furthermore, the activity of TRPV5 and TRPV6 is abolished when this particular threonine is mutated, demonstrating that this motif is essential for channel function. Malfunctioning of these mutant channels is accompanied by a major disturbance in their subcellular localization, indicating that the S100A10-annexin 2 heterotetramer facilitates the translocation of TRPV5 and TRPV6 channels to the plasma membrane. The importance of annexin 2 in this process was demonstrated by a small-interference RNA-based approach. Downregulation of annexin 2 that significantly inhibits the currents through TRPV5 and TRPV6 indicates that annexin 2, in conjunction with S100A10, is crucial for TRPV5 activity. Interestingly, an association of annexin 2 with TRPV5 was only shown in the presence of S100A10, demonstrating that annexin 2 binds indirectly to the channel, with S100A10 most likely operating as a molecular bridge between TRPV5 and annexin 2. In line with the submembranous localization of annexin 2 and its postulated function in organizing membrane domains, these findings provide the first functional evidence for a regulatory role of annexin 2 controlling Ca 2+ channel trafficking and, therefore, Ca 2+ homeostasis. Interestingly, previous studies indicated that the background K + channel (TASK1) is associated with S100A10 via its COOH-terminal sequence SSV ( 21 ). The S100A10 interaction promotes the translocation of TASK1 to the plasma membrane, producing functional K + channels ( 21 ). This sequence resembles the binding motif in TRPV5 and TRPV6 identified in the present study, suggesting a shared structural S100A10-binding pocket. However, this motif is absent in the tetrodotoxin-insensitive, voltage-gated Na + channel (Nav1.8), which has been shown to bind S100A10 via its NH 2 terminus ( 50 ). The binding of S100A10 is essential for plasma membrane trafficking of this Na + channel ( 45 ). Finally, Donier et al. ( 15 ) have recently demonstrated the association of S100A10 with an acid-sensing ion channel (ASIC1) and confirmed this association in rat dorsal root ganglion neurons by coimmunoprecipitation. Taken together, these findings show that the S100A10-annexin 2 complex is a significant component for the trafficking of ion channels to the plasma membrane.

CaM is a ubiquitous protein, well known to be involved in Ca 2+ -dependent feedback regulation of several ion channels ( 36 ). Niemeyer and co-workers ( 40 ) initially demonstrated that TRPV6 interacts with CaM in a Ca 2+ -dependent manner. CaM was shown to bind the CaM-binding regions in the COOH and NH 2 of TRPV5 and TRPV6 as well as the transmembrane domain of TRPV6 in a Ca 2+ -dependent manner ( 35, 40 ). Removal of the CaM-binding site in the COOH terminus of TRPV6 resulted in a significant reduction of the slow component of channel inactivation, revealing a role of CaM in TRPV6 regulation ( 40 ). Furthermore, HEK293 cells heterologously coexpressing Ca 2+ -insensitive CaM mutants along with TRPV5 or TRPV6 showed a significantly reduced Ca 2+ current through TRPV6, whereas no effect was demonstrated on TRPV5 ( 35 ). This effect was localized to the high-Ca 2+ -affinity EF-hand structures of CaM. These data demonstrated a regulatory role of CaM in TRPV6-mediated Ca 2+ influx. It remains to be established whether CaM functions as a general Ca 2+ sensor in TRPV5/6 channels or, alternatively, might explain the differences in Ca 2+ -dependent inactivation between TRPV5 and TRPV6.

80K-H

In a microarray screen designed to identify proteins that respond similarly to vitamin D and/or altered dietary Ca 2+ intake as TRPV5, Gkika et al. ( 22 ) identified 80K-H. 80K-H was originally cloned as a PKC substrate of 80 kDa and was subsequently associated with intracellular signaling ( 52 ). However, these studies did not resolve the biological function of this protein. 80K-H contains two putative EF-hand structures, a highly negatively charged glutamate stretch, which might be a Ca 2+ -interacting region, and a putative ER-targeting signal (HDEL). Using glutathione S -transferase (GST) pull-down assays and coimmunoprecipitations, a physical interaction between 80K-H and TRPV5 was demonstrated. Furthermore, both proteins colocalized in a subset of tubular segments in the kidney and show similar transcriptional regulation by vitamin D and dietary Ca 2+ ( 22 ). Electrophysiological studies using 80K-H mutants showed that three domains of 80K-H (the 2 EF-hand structures, the glutamate stretch, and the HDEL sequence) are critical determinants of TRPV5 activity. 80K-H directly binds Ca 2+, which is abolished on inactivation of its two EF-hand structures. Importantly, inactivation of the EF-hand pair also reduces the TRPV5-mediated Ca 2+ current and increased the TRPV5 sensitivity to intracellular Ca 2+, accelerating the feedback inhibition of the channel. Therefore, it is hypothesized that 80K-H acts a Ca 2+ sensor to regulate TRPV5 activity at the plasma membrane.

NHERF2 and Serum- and Glucocorticoid-Indicible Kinase

Embark and co-workers ( 17 ) demonstrated that TRPV5 activity increases on coexpression with the PDZ domain containing protein NHERF2 and serum- and glucocorticoid-indicible kinase 1 (SGK1) or SGK3 in X. laevis oocytes. Coexpression of TRPV5 with NHERF2 or SGK only did not stimulate TRPV5-mediated currents, indicating that NHERF2 and SGK operate codependently ( 17 ). Deletion of the second but not the first PDZ domain in NHERF2 abrogates the stimulating effect of SGK/NHERF2 on TRPV5 activity ( 46 ). Furthermore, it was demonstrated that TRPV5 activity could not be stimulated using a kinase-dead point mutant of SGK1 (K127N), suggesting a phosphorylation-mediated effect ( 17 ). Using GST pull-down and overlay assays, the specific interaction of NHERF2 with the last three amino acids (YHF) of the COOH terminus of TRPV5 was demonstrated (Ref. 46 and van de Graaf SF, Hoenderop JG, van der Kemp AW, Gisler SM, and Bindels RJ, unpublished observations). Furthermore, it was shown that TRPV6 does not bind NHERF2 ( 46 ). These findings suggest that regulation of the epithelial Ca 2+ channels by NHERF2-SGK is limited to TRPV5 and operates via direct interaction with the channel. The coexpression of SGK1 with NHERF2 also stimulates the activity of ROMK1, a K + channel involved in renal K + handling. This results from a stabilization of ROMK1 in the plasma membrane ( 72 ). Therefore, it is postulated that NHERF2/SGK stimulates the activity of TRPV5 via a comparable mechanism.

Rab11a

Van de Graaf et al. ( 61 ) recently identified Rab11a as a novel TRPV5- and TRPV6-associated protein. Rab11a is a small GTPase involved in trafficking via recycling endosomes ( 6, 68 ). Although the role of Rab GTPases in protein trafficking has long been recognized, the underlying mechanism is far from understood. Rab11a colocalizes with TRPV5 and TRPV6 in Ca 2+ -transporting epithelial cells of the kidney. Here, both TRPV5 and Rab11a are present in vesicular structures underlying the apical plasma membrane ( 61 ). Using a combination of GST pull-down and coimmunoprecipitation assays, a direct interaction between Rab11a and the epithelial Ca 2+ channels was demonstrated, hinting at a novel mechanism of Rab11a-mediated trafficking ( 61 ). Other Rab GTPases, including Rab7 and Rab22b, did not bind TRPV5/6, indicating the specificity of the interaction. Association of cargo with Rab GTPases has recently received much attention with the identification of an association between Rab3 and the polymeric IgA receptor ( 63 ) and between Rab11a and the tromboxane A 2 receptor ( 24 ). The binding of TRPV5/6 to Rab11a would provide the first evidence for an ion channel directly associating with a Rab GTPase. Furthermore, it was demonstrated that TRPV5 and TRPV6 preferentially interact with Rab11a in its GDP-bound conformation. Expression of a mutant Rab11a protein, locked in the GDP-bound state, results in a marked decrease in channels at the cell surface, indicating a direct role of Rab11a in the trafficking of TRPV5/6 toward the plasma membrane. Therefore, it is likely that TRPV5/6 channels, present on the (apical) plasma membrane, are being exchanged with TRPV5/6 channels from the intracellular (recycling) endosomes in a Rab11-dependent manner.

COORDINATED REGULATION OF CHANNEL EXPRESSION, TRAFFICKING, AND ACTIVITY

As described above, TRPV5/6 channels are controlled by mechanisms that target their expression, trafficking, or activity. The identification and characterization of channel-associated proteins reveal two interesting relationships between TRPV5/6-regulatory mechanisms. First, various observations suggest that S100A10/annexin 2 and Rab11a employ identical pathways to regulate TRPV5/6. For instance, Zobiack and co-workers ( 73 ) have demonstrated that the annexin 2-S100A10 complex controls the distribution of Rab11-positive endosomes. These endosomes have previously been identified as recycling endosomes, involved in the transport of internalized membrane proteins back to the cell surface ( 6 ). Furthermore, annexin 2 was shown to be enriched in these Rab11-containing structures ( 56 ). Therefore, impaired transport of TRPV5 from recycling endosomes to the plasma membrane could explain the inhibitory effect of annexin 2 downregulation on TRPV5/6-mediated currents. This suggests that two crucial pathways controlling the cell surface expression of TRPV5 and TRPV6 intersect.

Second, it was recently demonstrated that S100A10 and annexin 2 are coregulated by the calciotropic hormone vitamin D in a similar fashion as TRPV5 ( 62 ). This indicates that vitamin D exerts its physiological function by concertedly regulating the expression of the apical Ca 2+ influx proteins and stimulation of the insertion of these proteins into the plasma membrane. Similarly, Chen and co-workers ( 11 ) recently demonstrated that Rab11 is regulated in an estrogen-dependent manner. Finally, 80K-H responds like TRPV5 to alterations in vitamin D levels and dietary Ca 2+ intake ( 22 ). Together, this suggests that changes in channel expression, trafficking to the plasma membrane, and channel activity are modulated in a well-orchestrated manner to ultimately control the activity of TRPV5/6.

In summary, the identification of TRPV5/6 binding partners and characterization of their functional importance is an emerging field that has significantly contributed to our understanding of the regulation of apical Ca 2+ influx in epithelial cells. Some of the newly identified proteins operate at the plasma membrane, whereas others are involved in intracellular traffic pathways leading to and from this membrane. These findings provide new information about the physiological function of the channel-interacting proteins and point to novel mechanisms regulating apical Ca 2+ influx in Ca 2+ -transporting epithelia. Identification of the proteins that interact with TRPV5/6 is only a first step in a long endeavor. One of the major challenges of the future is to establish a comprehensive view of TRPV5/6-interacting proteins and their relative contribution to channel regulation. Most importantly, to fully understand the regulation of TRPV5/6, the relationship between protein interactions and signaling processes involved in epithelial Ca 2+ transport has to be revealed.

GRANTS

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, NWO 812-08.002, NWO Talent stipendium S91-282), the Stomach, Liver, Intestine Foundation (MWO 03-19), the Human Frontiers Science Program (RGP32/2004), and the Dutch Kidney Foundation (C10.1881 and C03.6017).

【参考文献】
  Bahner M, Frechter S, Da Silva N, Minke B, Paulsen R, and Huber A. Light-regulated subcellular translocation of Drosophila TRPL channels induces long-term adaptation and modifies the light-induced current. Neuron 34: 83-93, 2002.

Bezzerides VJ, Ramsey IS, Kotecha S, Greka A, and Clapham DE. Rapid vesicular translocation and insertion of TRP channels. Nat Cell Biol 6: 709-720, 2004.

Bianco S, Peng JB, Takanaga H, Kos CH, Crescenzi A, Brown EM, and Hediger MA. Mice lacking the epithelial calcium channel CaT1 (TRPV6) show a deficiency in intestinal calcium absorption depite high plasma levels of 1, 25-dihydroxyvitamin D. FASEB J 18: A706, 2004.

Bindels RJ, Hartog A, Abrahamse SL, and Van Os CH. Effects of pH on apical calcium entry and active calcium transport in rabbit cortical collecting system. Am J Physiol Renal Fluid Electrolyte Physiol 266: F620-F627, 1994.

Bouillon R, Van Cromphaut S, and Carmeliet G. Intestinal calcium absorption: molecular vitamin D mediated mechanisms. J Cell Biochem 88: 332-339, 2003.

Casanova JE, Wang X, Kumar R, Bhartur SG, Navarre J, Woodrum JE, Altschuler Y, Ray GS, and Goldenring JR. Association of Rab25 and Rab11a with the apical recycling system of polarized Madin-Darby canine kidney cells. Mol Biol Cell 10: 47-61, 1999.

Caterina MJ and Julius D. The vanilloid receptor: a molecular gateway to the pain pathway. Annu Rev Neurosci 24: 487-517, 2001.

Cayouette S, Lussier MP, Mathieu EL, Bousquet SM, and Boulay G. Exocytotic insertion of TRPC6 channel into the plasma membrane upon G q protein-coupled receptor activation. J Biol Chem 279: 7241-7246, 2004.

Chang Q, Gyftogianni E, van de Graaf SF, Hoefs S, Weidema FA, Bindels RJ, and Hoenderop JG. Molecular determinants in TRPV5 channel assembly. J Biol Chem 279: 54304-54311, 2004.

Chang Q, Hoefs S, van der Kemp AW, Topala CN, Bindels RJ, and Hoenderop JG. The -glucuronidase klotho hydrolyzes and activates the TRPV5 channel. Science 310: 490-493, 2005.

Chen D, Ganapathy P, Zhu LJ, Xu X, Li Q, Bagchi IC, and Bagchi MK. Potential regulation of membrane trafficking by estrogen receptor via induction of rab11 in uterine glands during implantation. Mol Endocrinol 13: 993-1004, 1999.

Clapham DE. TRP channels as cellular sensors. Nature 426: 517-524, 2003.

Dardenne O, Prud'homme J, Arabian A, Glorieux FH, and St.-Arnaud R. Targeted inactivation of the 25-hydroxyvitamin D 3 -1 -hydroxylase gene (CYP27B1) creates an animal model of pseudovitamin D-deficiency rickets. Endocrinology 142: 3135-3141, 2001.

Dodier Y, Banderali U, Klein H, Topalak O, Dafi O, Simoes M, Bernatchez G, Sauve R, and Parent L. Outer pore topology of the ECaC-TRPV5 channel by cysteine scan mutagenesis. J Biol Chem 279: 6853-6862, 2004.

Donier E, Rugiero F, Okuse K, and Wood JN. Annexin II light chain p11 promotes functional expression of acid-sensing ion channel ASIC1a. J Biol Chem 280: 38666-38672, 2005.

Doyle DA, Morais Cabral J, Pfuetzner RA, Kuo A, Gulbis JM, Cohen SL, Chait BT, and MacKinnon R. The structure of the potassium channel: molecular basis of K + conduction and selectivity. Science 280: 69-77, 1998.

Embark HM, Setiawan I, Poppendieck S, Van de Graaf SFJ, Boehmer C, Palmada M, Wieder T, Gerstberger R, Cohen P, Yun CC, Bindels RJM, and Lang F. Regulation of the epithelial Ca 2+ channel TRPV5 by the NHE regulating factor NHERF2 and the serum and glucocorticoid inducible kinase isoforms SGK1 and SGK3 expressed in Xenopus oocytes. Cell Physiol Biochem 14: 203-212, 2004.

Erler I, Hirnet D, Wissenbach U, Flockerzi V, and Niemeyer BA. Ca 2+ -selective transient receptor potential V channel architecture and function require a specific ankyrin repeat. J Biol Chem 279: 34456-34463, 2004.

Gerke V, Creutz CE, and Moss SE. Annexins: linking Ca 2+ signalling to membrane dynamics. Nat Rev Mol Cell Biol 6: 449-461, 2005.

Gerke V and Moss SE. Annexins: from structure to function. Physiol Rev 82: 331-371, 2002.

Girard C, Tinel N, Terrenoire C, Romey G, Lazdunski M, and Borsotto M. p11, An annexin II subunit, an auxiliary protein associated with the background K + channel, TASK-1. EMBO J 21: 4439-4448, 2002.

Gkika D, Mahieu F, Nilius B, Hoenderop JG, and Bindels RJ. 80K-H as a new Ca 2+ sensor regulating the activity of the epithelial Ca 2+ channel transient receptor potential cation channel V5 (TRPV5). J Biol Chem 279: 26351-26357, 2004.

Gonzalez-Perrett S, Batelli M, Kim K, Essafi M, Timpanaro G, Moltabetti N, Reisin IL, Arnaout MA, and Cantiello HF. Voltage dependence and pH regulation of human polycystin-2-mediated cation channel activity. J Biol Chem 277: 24959-24966, 2002.

Hamelin E, Theriault C, Laroche G, and Parent JL. The intracellular trafficking of the G protein-coupled receptor TP depends on a direct interaction with Rab11. J Biol Chem 280: 36195-36205, 2005.

Hoenderop JG, Dardenne O, Van Abel M, Van Der Kemp AW, Van Os CH, St-Arnaud R, and Bindels RJ. Modulation of renal Ca 2+ transport protein genes by dietary Ca 2+ and 1,25-dihydroxyvitamin D 3 in 25-hydroxyvitamin D 3 -1 -hydroxylase knockout mice. FASEB J 16: 1398-1406, 2002.

Hoenderop JG, Nilius B, and Bindels RJ. Calcium absorption across epithelia. Physiol Rev 85: 373-422, 2005.

Hoenderop JG, Nilius B, and Bindels RJ. Molecular mechanism of active Ca 2+ reabsorption in the distal nephron. Annu Rev Physiol 64: 529-549, 2002.

Hoenderop JG, van der Kemp AW, Hartog A, van de Graaf SF, van Os CH, Willems PH, and Bindels RJ. Molecular identification of the apical Ca 2+ channel in 1, 25-dihydroxyvitamin D 3 -responsive epithelia. J Biol Chem 274: 8375-8378, 1999.

Hoenderop JG, van Leeuwen JP, van der Eerden BC, Kersten FF, van der Kemp AW, Merillat AM, Waarsing JH, Rossier BC, Vallon V, Hummler E, and Bindels RJ. Renal Ca 2+ wasting, hyperabsorption, and reduced bone thickness in mice lacking TRPV5. J Clin Invest 112: 1906-1914, 2003.

Hoenderop JG, Voets T, Hoefs S, Weidema F, Prenen J, Nilius B, and Bindels RJ. Homo- and heterotetrameric architecture of the epithelial Ca 2+ channels TRPV5 and TRPV6. EMBO J 22: 776-785, 2003.

Hughes MR, Malloy PJ, Kieback DG, Kesterson RA, Pike JW, Feldman D, and O'Malley BW. Point mutations in the human vitamin D receptor gene associated with hypocalcemic rickets. Science 242: 1702-1705, 1988.

Kanzaki M, Zhang YQ, Mashima H, Li L, Shibata H, and Kojima I. Translocation of a calcium-permeable cation channel induced by insulin-like growth factor-I. Nat Cell Biol 1: 165-170, 1999.

Kuro-o M, Matsumura Y, Aizawa H, Kawaguchi H, Suga T, Utsugi T, Ohyama Y, Kurabayashi M, Kaname T, Kume E, Iwasaki H, Iida A, Shiraki-Iida T, Nishikawa S, Nagai R, and Nabeshima YI. Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature 390: 45-51, 1997.

Kurosu H, Yamamoto M, Clark JD, Pastor JV, Nandi A, Gurnani P, McGuinness OP, Chikuda H, Yamaguchi M, Kawaguchi H, Shimomura I, Takayama Y, Herz J, Kahn CR, Rosenblatt KP, and Kuro-o M. Suppression of aging in mice by the hormone Klotho. Science 309: 1829-1833, 2005.

Lambers TT, Weidema AF, Nilius B, Hoenderop JG, and Bindels RJ. Regulation of the mouse epithelial Ca 2+ channel TRPV6, by the Ca 2+ -sensor calmodulin. J Biol Chem 279: 28855-28861, 2004.

Levitan IB. It is calmodulin after all! Mediator of the calcium modulation of multiple ion channels. Neuron 22: 645-648, 1999.

Li YC, Bolt MJ, Cao LP, and Sitrin MD. Effects of vitamin D receptor inactivation on the expression of calbindins and calcium metabolism. Am J Physiol Endocrinol Metab 281: E558-E564, 2001.

Loffing J, Loffing-Cueni D, Valderrabano V, Klausli L, Hebert SC, Rossier BC, Hoenderop JG, Bindels RJ, and Kaissling B. Distribution of transcellular calcium and sodium transport pathways along mouse distal nephron. Am J Physiol Renal Physiol 281: F1021-F1027, 2001.

Montell C. Exciting trips for TRPs. Nat Cell Biol 6: 690-692, 2004.

Niemeyer BA, Bergs C, Wissenbach U, Flockerzi V, and Trost C. Competitive regulation of CaT-like-mediated Ca 2+ entry by protein kinase C and calmodulin. Proc Natl Acad Sci USA 98: 3600-3605, 2001.

Nijenhuis T, Hoenderop JG, van der Kemp AW, and Bindels RJ. Localization and regulation of the epithelial Ca 2+ channel TRPV6 in the kidney. J Am Soc Nephrol 14: 2731-2740, 2003.

Nilius B, Prenen J, Hoenderop JG, Vennekens R, Hoefs S, Weidema AF, Droogmans G, and Bindels RJ. Fast and slow inactivation kinetics of the Ca 2+ channels ECaC1 and ECaC2 (TRPV5 and TRPV6). Role of the intracellular loop located between transmembrane segments 2 and 3. J Biol Chem 277: 30852-30858, 2002.

Nilius B, Prenen J, Vennekens R, Hoenderop JG, Bindels RJ, and Droogmans G. Modulation of the epithelial calcium channel, ECaC, by intracellular Ca 2+. Cell Calcium 29: 417-428, 2001.

Nilius B, Weidema F, Prenen J, Hoenderop JG, Vennekens R, Hoefs S, Droogmans G, and Bindels RJ. The carboxyl terminus of the epithelial Ca 2+ channel ECaC1 is involved in Ca 2+ -dependent inactivation. Pflügers Arch 445: 584-588, 2003.

Okuse K, Malik-Hall M, Baker MD, Poon WY, Kong H, Chao MV, and Wood JN. Annexin II light chain regulates sensory neuron-specific sodium channel expression. Nature 417: 653-656, 2002.

Palmada M, Poppendieck S, Embark HM, van de Graaf SF, Boehmer C, Bindels RJ, and Lang F. Requirement of PDZ domains for the stimulation of the epithelial Ca 2+ channel TRPV5 by the NHE regulating factor NHERF2 and the serum and glucocorticoid inducible kinase SGK1. Cell Physiol Biochem 15: 175-182, 2005.

Peng JB, Chen XZ, Berger UV, Vassilev PM, Brown EM, and Hediger MA. A rat kidney-specific calcium transporter in the distal nephron. J Biol Chem 275: 28186-28194, 2000.

Peng JB, Chen XZ, Berger UV, Vassilev PM, Tsukaguchi H, Brown EM, and Hediger MA. Molecular cloning and characterization of a channel-like transporter mediating intestinal calcium absorption. J Biol Chem 274: 22739-22746, 1999.

Peng JB, Zhuang L, Berger UV, Adam RM, Williams BJ, Brown EM, Hediger MA, and Freeman MR. CaT1 expression correlates with tumor grade in prostate cancer. Biochem Biophys Res Commun 282: 729-734, 2001.

Poon WY, Malik-Hall M, Wood JN, and Okuse K. Identification of binding domains in the sodium channel Na(V)1.8 intracellular N-terminal region and annexin II light chain p11. FEBS Lett 558: 114-118, 2004.

Prod'hom B, Pietrobon D, and Hess P. Interactions of protons with single open L-type calcium channels. Location of protonation site and dependence of proton-induced current fluctuations on concentration and species of permeant ion. J Gen Physiol 94: 23-42, 1989.

Sakai K, Hirai M, Minoshima S, Kudoh J, Fukuyama R, and Shimizu N. Isolation of cDNAs encoding a substrate for protein kinase C: nucleotide sequence and chromosomal mapping of the gene for a human 80K protein. Genomics 5: 309-315, 1989.

Singh BB, Lockwich TP, Bandyopadhyay BC, Liu X, Bollimuntha S, Brazer SC, Combs C, Das S, Leenders AG, Sheng ZH, Knepper MA, Ambudkar SV, and Ambudkar IS. VAMP2-dependent exocytosis regulates plasma membrane insertion of TRPC3 channels and contributes to agonist-stimulated Ca 2+ influx. Mol Cell 15: 635-646, 2004.

Songyang Z, Fanning AS, Fu C, Xu J, Marfatia SM, Chishti AH, Crompton A, Chan AC, Anderson JM, and Cantley LC. Recognition of unique carboxyl-terminal motifs by distinct PDZ domains. Science 275: 73-77, 1997.

St.-Arnaud R, Messerlian S, Moir JM, Omdahl JL, and Glorieux FH. The 25-hydroxyvitamin D 1- -hydroxylase gene maps to the pseudovitamin D-deficiency rickets (PDDR) disease locus. J Bone Miner Res 12: 1552-1559, 1997.

Trischler M, Stoorvogel W, and Ullrich O. Biochemical analysis of distinct Rab5- and Rab11-positive endosomes along the transferrin pathway. J Cell Sci 112: 4773-4783, 1999.

Van Abel M, Hoenderop JG, Dardenne O, St. Arnaud R, Van Os CH, Van Leeuwen HJ, and Bindels RJ. 1,25-dihydroxyvitamin D 3 -independent stimulatory effect of estrogen on the expression of ECaC1 in the kidney. J Am Soc Nephrol 13: 2102-2109, 2002.

Van Abel M, Hoenderop JG, van der Kemp AW, van Leeuwen JP, and Bindels RJ. Regulation of the epithelial Ca 2+ channels in small intestine as studied by quantitative mRNA detection. Am J Physiol Gastrointest Liver Physiol 285: G78-G85, 2003.

Van Abel M, Hoenderop JGJ, Van Leeuwen HJ, and Bindels RJM. Downregulation of calcium transporters in kidney and duodenum by the calcimimetic compound NPS R-467. J Am Soc Nephrol 14, 2003.

Van Cromphaut SJ, Dewerchin M, Hoenderop JG, Stockmans I, Van Herck E, Kato S, Bindels RJ, Collen D, Carmeliet P, Bouillon R, and Carmeliet G. Duodenal calcium absorption in vitamin D receptor-knockout mice: functional and molecular aspects. Proc Natl Acad Sci USA 98: 13324-13329, 2001.

Van de Graaf SF, Chang Q, Mensenkamp AR, Hoenderop JG, and Bindels RJ. Direct interaction with Rab11a targets the epithelial Ca 2+ channels TRPV5 and TRPV6 towards the plasma membrane. Mol Cell Biol 26: 303-312, 2006.

Van de Graaf SF, Hoenderop JG, Gkika D, Lamers D, Prenen J, Rescher U, Gerke V, Staub O, Nilius B, and Bindels RJ. Functional expression of the epithelial Ca 2+ channels (TRPV5 and TRPV6) requires association of the S100A10-annexin 2 complex. EMBO J 22: 1478-1487, 2003.

Van IJzendoorn SC, Tuvim MJ, Weimbs T, Dickey BF, and Mostov KE. Direct interaction between Rab3b and the polymeric immunoglobulin receptor controls ligand-stimulated transcytosis in epithelial cells. Dev Cell 2: 219-228, 2002.

Vassilev PM, Peng JB, Hediger MA, and Brown EM. Single-channel activities of the human epithelial Ca 2+ transport proteins CaT1 and CaT2. J Membr Biol 184: 113-120, 2001.

Vennekens R, Hoenderop JG, Prenen J, Stuiver M, Willems PH, Droogmans G, Nilius B, and Bindels RJ. Permeation and gating properties of the novel epithelial Ca 2+ channel. J Biol Chem 275: 3963-3969, 2000.

Vennekens R, Prenen J, Hoenderop JG, Bindels RJ, Droogmans G, and Nilius B. Modulation of the epithelial Ca 2+ channel ECaC by extracellular pH. Pflügers Arch 442: 237-242, 2001.

Voets T, Janssens A, Droogmans G, and Nilius B. Outer pore architecture of a Ca 2+ -selective TRP channel. J Biol Chem 279: 15223-15230, 2004.

Wang X, Kumar R, Navarre J, Casanova JE, and Goldenring JR. Regulation of vesicle trafficking in madin-darby canine kidney cells by Rab11a and Rab25. J Biol Chem 275: 29138-29146, 2000.

Woodhull AM. Ionic blockage of sodium channels in nerve. J Gen Physiol 61: 687-708, 1973.

Yeh BI, Kim YK, Jabbar W, and Huang CL. Conformational changes of pore helix coupled to gating of TRPV5 by protons. EMBO J 24: 3224-3234, 2005.

Yeh BI, Sun TJ, Lee JZ, Chen HH, and Huang CL. Mechanism and molecular determinant for regulation of rabbit transient receptor potential type 5 (TRPV5) channel by extracellular pH. J Biol Chem 278: 51044-51052, 2003.

Yun CC, Palmada M, Embark HM, Fedorenko O, Feng Y, Henke G, Setiawan I, Boehmer C, Weinman EJ, Sandrasagra S, Korbmacher C, Cohen P, Pearce D, and Lang F. The serum and glucocorticoid-inducible kinase SGK1 and the Na + /H + exchange regulating factor NHERF2 synergize to stimulate the renal outer medullary K + channel ROMK1. J Am Soc Nephrol 13: 2823-2830, 2002.

Zobiack N, Rescher U, Ludwig C, Zeuschner D, and Gerke V. The annexin 2/S100A10 complex controls the distribution of transferrin receptor-containing recycling endosomes. Mol Biol Cell 14: 4896-4908, 2003.

作者单位：Department of Physiology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands