Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M403801200 on April 20, 2004

J. Biol. Chem., Vol. 279, Issue 25, 26351-26357, June 18, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/25/26351    most recent
M403801200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gkika, D.
Right arrow Articles by Bindels, R. J. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gkika, D.
Right arrow Articles by Bindels, R. J. M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

80K-H as a New Ca2+ Sensor Regulating the Activity of the Epithelial Ca2+ Channel Transient Receptor Potential Cation Channel V5 (TRPV5)*

Dimitra Gkika{ddagger}, Frank Mahieu§, Bernd Nilius§, Joost G. J. Hoenderop{ddagger}, and René J. M. Bindels{ddagger}

From the {ddagger}Department of Physiology, Nijmegen Centre for Molecular Life Sciences, University Medical Centre Nijmegen, NL-6500 HB Nijmegen, The Netherlands and §Department of Physiology, Campus Gasthuisberg, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium

Received for publication, April 6, 2004 , and in revised form, April 19, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The epithelial Ca2+ channel transient receptor potential cation channel V5 (TRPV5) constitutes the apical Ca2+ entry pathway in the process of active Ca2+ reabsorption. Ca2+ influx through TRPV5 is tightly controlled by modulators of Ca2+ homeostasis, including 1,25-dihydroxyvitamin D3 and dietary Ca2+. However, little is known about intracellular proteins that interact with TRPV5 and directly regulate the activation of this channel. By the use of cDNA microarrays, the present study identified 80K-H as the first protein involved in the Ca2+-dependent control of the epithelial Ca2+ channel TRPV5. 80K-H was initially identified as a protein kinase C substrate, but its biological function remains to be established. We demonstrated a specific interaction between 80K-H and TRPV5, co-localization of both proteins in the kidney, and similar transcriptional regulation by 1,25-dihydroxyvitamin D3 and dietary Ca2+. Furthermore, 80K-H directly bound Ca2+, and inactivation of its two EF-hand structures totally abolished Ca2+ binding. Electrophysiological studies using 80K-H mutants showed that three domains of 80K-H (the two EF-hand structures, the highly acidic glutamic stretch, and the His-Asp-Glu-Leu sequence) are critical determinants for TRPV5 activity. Importantly, inactivation of the EF-hand pair reduced the TRPV5-mediated Ca2+ current and increased the TRPV5 sensitivity to intracellular Ca2+, accelerating the feedback inhibition of the channel. None of the 80K-H mutants altered the TRPV5 plasma membrane localization nor the association of 80K-H with TRPV5, suggesting that 80K-H has a direct effect on TRPV5 activity. In conclusion, we report a novel function for 80K-H as a Ca2+ sensor controlling TRPV5 channel activity.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The maintenance of the body Ca2+ balance is of crucial importance for many vital physiological functions, including neuronal excitability, muscle contraction, and bone formation. The recent identification of two novel members of the transient receptor potential (TRP)1 superfamily, TRPV5 and TRPV6, has provided new insight into the molecular mechanisms controlling the extracellular Ca2+ balance (1–3). TRPV5 and TRPV6 are Ca2+-selective channels involved in transcellular Ca2+ transport across vitamin D3-sensitive epithelia, such as kidney, intestine, and placenta (4, 5). Furthermore, these two channels possess unique functional characteristics among the highly heterogeneous group of TRP channels (6). They exhibit a constitutively activated Ca2+ permeability, a high Ca2+ selectivity, and a Ca2+-dependent feedback mechanism regulating channel activity (7, 8).

TRPV5 activity is controlled by various regulatory mechanisms ranging from long-term transcriptional modulations to short-term direct physical interactions with intracellular factors. TRPV5 is regulated at the transcriptional level by 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), dietary Ca2+, and 17{beta}-estradiol (5, 9, 10). Previous studies have shown that these hormones and dietary Ca2+ normalize the hypocalcemia in 25-hydroxyvitamin D3-1{alpha}-hydroxylase knock-out mice, which is accompanied by an up-regulation of TRPV5 mRNA expression levels. Furthermore, TRPV5 modulation occurs at the post-translational level by controlling translocation of the channel from intracellular pools to the plasma membrane. Recently, the S100A10-annexin 2 complex was demonstrated to modulate the functional plasma membrane distribution of TRPV5 by direct association to the carboxyl (C)-terminal tail of the channel. Down-regulation of annexin 2 inhibited TRPV5-mediated currents in TRPV5-transfected cells (11). Finally, the terminal tails of the channel contain potential regulatory motifs, such as protein kinase C (PKC) phosphorylation sites, PDZ motifs, and ankyrin repeats (4). This suggests a regulatory role for the intracellular tails of TRPV5 in the (in)activation or the trafficking of the channel. In line with this observation, Niemeyer et al. (13) demonstrated that TRPV6 is competitively regulated by PKC and calmodulin.

The aim of the present study was to identify novel regulators of Ca2+ reabsorption via TRPV5. To this end, cDNA microarray experiments were performed to identify candidates that exhibit a similar transcriptional regulation to well known proteins involved in Ca2+ transport. Analysis of the data pointed to a PKC substrate, 80K-H, of which the biological function remains unclear. The putative role of this protein in active Ca2+ reabsorption was investigated by immunohistochemical, biochemical, and electrophysiological analysis of 80K-H interacting with TRPV5.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
DNA Constructs and cRNA Synthesis—TRPV5 constructs were generated as described previously (11). The coding sequence of 80K-H wild-type protein was amplified from total mouse kidney cDNA material, cloned into the pCINeo/IRES-GFP vector (14) as an EcoRI-NheI fragment, and subsequently subcloned into the pGEX6p-2 vector (Amersham Pharmacia Biotech AB, Uppsala, Sweden) and the pT7Ts vector (15). Using in vitro mutagenesis (QuickChange site-directed mutagenesis kit, Stratagene, La Jolla, CA), three mutants of 80K-H were generated in the pCINeo/IRES GFP vector: one mutant was generated by alanine substitution of the first two aspartates of each EF-hand structure (80K-HEF), the second mutant was generated by deletion of a stretch containing 21 glutamates (80K-H{Delta}Glu) and the third mutant was generated by deletion of the C-terminal His-Asp-Glu-Leu (HDEL) sequence (80K-H{Delta}HDEL). The obtained mutants were subsequently subcloned into the pT7Ts and pGEX6p-2 vectors. All constructs were verified by sequence analysis. pTLN and pT7Ts constructs were linearized, and cRNA was synthesized in vitro using SP6 and T7 RNA polymerase, respectively (16).

Real-time Quantitative PCR—80K-H mRNA expression level was quantified by real-time quantitative PCR in kidney of 8-week-old mice (strain C57BL/6) that had received a dietary supplementation with 100 pg/g body weight of 1,25-(OH)2D3 or 2% (w/v) Ca2+ (instead of 1.1% w/v for normal diet) (9). The primers used were 5'-GTC CCA ATG GCA GCT TTC A-3' and 5'-CCC GGC TGG AGA GGA TGT A-3' and the probe 5'-TGC ACC AAC ACT GGG TAC AAG CCC T-3'. Probes were labeled with the quencher dye 6-carboxytetramethylrhodamine (3'-ends) and the reporter dye 6-carboxyfluorescein (5'-ends) (Biolegio, Malden, The Netherlands). The expression level of hypoxanthine-guanine phosphoribosyl transferase was used as an internal control (10).

RT-PCR Analysis—Total RNA from mouse brain, eye, salivary tissue, thymus, heart, lung, liver, spleen, pancreas, stomach, duodenum, colon, kidney, prostate, testis, uterus, skin, skeletal muscle, and bone was isolated using TRIzol (GIBCO/BRL, Life Technologies, Breda, The Netherlands). Total RNA (2 µg) was subjected to reverse transcription using Moloney murine leukemia virus reverse transcriptase, and a PCR for 80K-H was performed in a species-conserved region using the primers 5'-TAC GTC TAC CGG CTT TGC C-3' and 5'-AGG TAC TCA CAG CGA CTG GG-3'.

Immunohistochemistry—Immunohistochemistry was performed as described (17). Briefly, mouse kidney sections were incubated for 16 h at 4 °C with affinity-purified guinea pig antiserum against TRPV5 (1:100) and rabbit antiserum against 80K-H (1:500) (18). To visualize TRPV5 and 80K-H, goat anti-guinea pig Alexa Fluor 488-conjugated antibody (1:300), a goat anti-rabbit Alexa Fluor 488- and 594-conjugated antibodies (1:300) (Molecular Probes, Eugene, OR) were used. All negative controls, including sections incubated with pre-immune serum or conjugated antibodies solely, were devoid of any staining.

Glutathione S-transferase (GST)-TRPV5 Fusion Protein and Interaction Assays—pGEX6p-2 constructs were transformed in Escherichia coli BL21 and GST-fusion proteins were expressed and purified according to the manufacturer's protocol (Amersham Pharmacia Biotech AB). [35S]Methionine-labeled 80K-H, 80K-HEF, and 80K-H{Delta}Glu were prepared using a reticulocyte lysate and added to GST or GST-TRPV5 fusion proteins immobilized on glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech AB) in PBS containing 1% (v/v) Triton X-100. After 2-h incubation at room temperature, the beads were washed extensively, and bound proteins were eluted with SDS-PAGE loading buffer, separated on 10% (w/v) SDS-PAGE gels, and visualized by autoradiography.

Injection of Xenopus Oocytes and Co-immunoprecipitation—Xenopus laevis oocytes were co-injected with 5 ng of hemagglutinin (HA)-tagged TRPV5 and 20 ng of 80K-H cRNA; after 48 h, injected and non-injected oocytes were subjected to total membrane isolation by centrifugation (19). The membrane fraction was incubated on ice for 60 min in lysis buffer (10 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.5% (v/v) Nonidet P-40, 1 mM CaCl2, 10% (w/v) sucrose). After centrifugation of the lysates, supernatants were incubated with anti-HA antibody (Sigma) or rabbit anti-80K-H antibody immobilized on protein A-agarose beads (Kem-En-Tec A/S, Copenhagen, Denmark) in the presence of 1% (w/v) bovine serum albumin for 16 h at 4 °C. Samples were washed three times with washing buffer (10 mM Tris-HCl, pH 7.6, 500 mM NaCl, 0.5% (v/v) Nonidet P-40, and 1 mM CaCl2). Immunoprecipitated proteins were eluted with SDS-PAGE loading buffer, separated on 10% (w/v) SDS-PAGE gel, and analyzed by immunoblotting using rabbit anti-80K-H (1:10,000) and anti-HA (1:4000) antibodies, respectively.

45Ca2+-binding Assay—To study the direct Ca2+-binding properties of 80K-H, GST-fused 80K-H proteins migrated on SDS-12% PAGE gel and were blotted to polyvinylidene difluoride membrane. Blotted proteins were visualized with Ponceau S staining (Sigma). 45Ca2+ binding was performed as described by Maruyama et al. (20). Briefly, polyvinylidene difluoride membranes were thoroughly washed and incubated for 10 min with 1 µCi/ml 45CaCl2 (PerkinElmer Life Sciences). After washing, the membrane was dried and 45Ca2+ binding was visualized by autoradiography.

Biotinylation Assay—TRPV5, 80K-H, 80K-HEF, 80K-H{Delta}Glu, and 80K-H{Delta}HDEL pCINeo/IRES-GFP constructs were transfected transiently in human embryonic kidney 293 (HEK293) cells. Cells were washed twice with ice-cold phosphate-buffered saline (PBS) containing 0.5 mM CaCl2 and 1 mM MgCl2 (PBS-CM) 48 h after transfection; surface biotinylation was performed by incubating the cells twice for 20 min at 4 °C with 1 mg/ml Sulfo-NHS-SS-Biotin (Pierce). Subsequently, cells were incubated for 5 min with quenching solution (50 mM NH4Cl in PBS-CM) at 4 °C and rinsed twice with cold PBS-CM. Cells were lysed with 1 ml of lysis buffer (150 mM NaCl, 20 mM Tris-HCl, pH 7.6, 5 mM EDTA, 0.5% (v/v) Triton X-100, 0.2% (w/v) bovine serum albumin) for 1 h on ice. Lysates were added to 30 µl of Immunopure Streptavidin beads (Pierce). After incubation for 16 h at 4 °C, the beads were washed twice with PBS-CM, twice with lysis buffer, and once with 10 mM Tris-HCl, pH 7.5. Finally, biotinylated proteins were eluted with SDS-PAGE loading buffer, separated on 10% (w/v) SDS-PAGE gel, and analyzed by immunoblotting with guinea pig antiserum against TRPV5 (1:1500) and rabbit antiserum against 80K-H (1:10000).

Electrophysiology—The full-length cDNAs encoding TRPV5 and 80K-H mutants were transiently transfected in HEK293, and patch-clamp experiments were performed in the whole-cell configuration, as described previously (11). Monovalent cation currents were measured in nominal divalent-free (DVF) solutions, in which Ca2+ and Mg2+ were omitted from Krebs solution. For measuring the Ca2+ currents, Krebs was used with 10 mM CaCl2 instead of 1 mM CaCl2. The internal (pipette) solution contained 10 mM BAPTA, 20 mM CsCl, 100 mM Csaspartate, 1 mM MgCl2, 4 mM Na2ATP, 10 mM HEPES-CsOH, pH 7.2. The Ca2+ concentration in the pipette was adjusted to 10 nM, 20 nM, 50 nM, 100 nM, 250 nM, or 1 µM by adding 0.747, 1.391, 2.880, 4.457, 6.682, or 8.916 mM CaCl2, respectively, in the presence of 10 mM BAPTA, as calculated by the CaBuf program.2 All experiments were performed at room temperature (20–22 °C). Reported current densities were calculated from the current at -80 mV during the ramp protocol for the Na+ currents or at the peak amplitude in the step protocol, respectively. The software package ASCD3 was used for analysis of the whole-cell data. All current amplitudes in the dose-response curves were normalized to -80 mV with 10 mM BAPTA in the pipette solution and fitted with a modified Hill function (using Origin version 7.0 software, OriginLab Corporation, Northampton, MA).

Statistical Analysis—In all experiments, the data are expressed as mean ± S.E. Overall statistical significance was determined by analysis of variance. In case of significance (p < 0.05), individual groups were compared by Student's t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of 80K-H as a Gene Regulated by 1,25-(OH)2D3 and Dietary Ca2+—Mice, in which the renal D3-1{alpha}-hydroxylase enzyme catalyzing the 1,25-(OH)2D3 synthesis was inactivated, exhibited undetectable levels of 1,25-(OH)2D3, resulting in severe hypocalcemia (21). We performed a microarray analysis on kidney cDNA of these 1,25-(OH)2D3-deficient mice, which were rescued by 1,25-(OH)2D3 supplementation or by dietary Ca2+ (9, 22). Analysis of the gene expression data provided information about RNA transcripts important for the maintenance of the Ca2+ balance and pointed to the 80K-H protein, which is a PKC substrate containing two putative Ca2+-binding sites. 80K-H was significantly down-regulated at the mRNA level in the absence of 1,25-(OH)2D3 (79% compared with control values in the presence of 1,25-(OH)2D3) and up-regulated by dietary Ca2+ supplementation (189% compared with the 1,25-(OH)2D3-deficient mice). The regulation of 80K-H at the transcription level was confirmed by quantitative real-time PCR analysis (from 100% to 157 ± 12% and 155 ± 7%, respectively; p < 0.05).

Localization of 80K-H in Ca2+-transporting Epithelia—Tissue distribution of 80K-H was first investigated by reverse transcription PCR on mouse tissues using primers located in a species-conserved region within the 80K-H C-terminal tail. The expected 80K-H amplicon of 242 bp was detected in all tissues, including Ca2+-transporting epithelia such as kidney and duodenum (Fig. 1A). Localization of 80K-H was further studied by immunohistochemistry of mouse kidney, where immunopositive staining for 80K-H and TRPV5 was present along the apical membrane of distal convoluted and connecting tubules (Fig. 1B).



View larger version (71K):
[in this window]
[in a new window]
  		FIG. 1.
Localization and tissue distribution of 80K-H. A, RNA was extracted from several mouse tissues and 80K-H expression was determined by RT-PCR. The 80K-H specific band was amplified in all analyzed tissues at the expected height of 242 bp. B, immunohistochemical analysis of 80K-H and TRPV5 in mouse kidney sections. Kidney sections were co-stained with antibodies against 80K-H and TRPV5.

 
Interaction of 80K-H with TRPV5—To examine the association of 80K-H with TRPV5, immunoprecipitation experiments were performed using the X. laevis oocyte expression system. Membrane extracts of X. laevis heterologously expressing TRPV5 and 80K-H were first immunoprecipitated with anti-80K-H antibody, and the precipitated proteins were analyzed by immunoblotting. The two specific bands of HA-tagged TRPV5, corresponding to the core and the non-glycosylated proteins (23), were detected in the precipitated complex (Fig. 2A). Likewise, this interaction was confirmed in the reverse manner by immunoprecipitation of TRPV5 from membrane extracts using the anti-HA antibody and subsequent immunoblotting with the anti-80K-H antibody (Fig. 2B). Ca2+-dependence of 80K-H association with TRPV5 was investigated by GST pull-down assays in the presence (1 mM CaCl2) and absence (5 mM EDTA) of Ca2+. 80K-H protein bound specifically to TRPV5 in both conditions because no interaction was observed with GST alone (Fig. 2C). Immunoblot analysis of mouse kidney lysates did not result in a specific immunopositive signal (data not shown), which is probably due to the relatively low abundance of these channels (24). Endogenous immunoprecipitation of the two proteins was, therefore, not possible.



View larger version (32K):
[in this window]
[in a new window]
  		FIG. 2.
Interaction of 80K-H with TRPV5. cRNA of 80K-H and HA-tagged TRPV5 was co-injected in X. laevis oocytes, and total membrane extracts were used for immunoprecipitation and subsequent immunoblot analysis. Immunoprecipitation was performed using beads to which the anti-80K-H (A) or the anti-HA (B) antibody was bound (IP), and co-immunoprecipitation of the 80K-H and TRPV5 proteins was detected by immunoblot analysis (Blot) using the respective antibody for each protein. The specific bands of each protein are depicted with black arrows on the side of the blots. C, the Ca2+ dependence of the 80K-H binding to TRPV5 was investigated by a GST pull-down assay. GST-fused TRPV5 C-terminal proteins were immobilized on glutathione-Sepharose 4B beads and subsequently incubated with in vitro translated [35S]methionine-80K-H in the presence and the absence of Ca2+. GST alone was used as a negative control. D, mapping of 80K-H interaction domain in TRPV5. GST-fusion proteins corresponding to various truncations of the TRPV5 C-terminal tail were assayed for 80K-H binding in a GST pull-down experiment. The 80K-H binding site was localized between amino acids 598 and 608 of TRPV5.

 
Mapping of the 80K-H-binding Site in TRPV5—To determine the 80K-H binding site in TRPV5, a series of deletion mutants between positions 598 and 680 of the TRPV5 C-terminal tail was constructed. Truncated forms of TRPV5 were expressed as GST-fusion proteins and tested for their interaction with in vitro translated 80K-H protein using pull-down experiments (Fig. 2D). The interaction between the two proteins was abolished when TRPV5 was truncated at position 603, whereas truncations at positions 680 up to 608 did not disturb the interaction with 80K-H. The sequence of TRPV5 protein corresponding to the binding site of 80K-H was restricted to the short region between the positions 598 and 608.

Ca2+-binding Capacity of 80K-H—80K-H contains two putative EF-hand structures and a highly negatively charged glutamate stretch that might be a Ca2+-interacting region (25). Ca2+-binding properties of 80K-H were investigated by performing a 45Ca2+ overlay assay, using 80K-H wild type and mutants of the two putative Ca2+-related regions (Fig. 3A). The first mutant was generated by substitution of the critical aspartates for alanine residues that impair Ca2+ coordination in the two EF-hands (80K-HEF mutant). The second mutant was generated by excision of the glutamate stretch (80K-H{Delta}Glu mutant). Pull-down experiments demonstrated that mutation of 80K-H, in the regions described above, did not disturb binding to the TRPV5 C-terminal tail (Fig. 3B). Subsequently, GST-fused proteins of 80K-H were separated by SDS-PAGE followed by blotting. An equal expression level of 80K-H proteins was verified by Ponceau S staining. After incubation with 45CaCl2, the capacity of the blotted proteins to bind 45Ca2+ was determined (Fig. 4). The amino acid substitutions in the two EF-hands of 80K-H abolished the Ca2+ binding, whereas deletion of the glutamate stretch did not interfere with Ca2+ binding. GST protein alone was used as a negative control.



View larger version (42K):
[in this window]
[in a new window]
  		FIG. 3.
TRPV5 binding analysis of the 80K-H mutants. A, schematic representation of the 80K-H structure containing the two EF-hand structures (white boxes), the glutamate stretch (diagonal dashed box), the HDEL sequence (black box), and the putative PKC phosphorylation sites (vertical lines), as well as the truncated regions of the protein by point mutation (stars) or deletion (scissors). B, the binding of 80K-H mutants to TRPV5 was analyzed by GST pull-down experiments. The immobilized C-terminal tail of TRPV5 GST-fusion protein was incubated with in vitro translated [35S]methionine-80K-H proteins (80K-H, 80K-HEF, 80K-H{Delta}Glu). GST alone was used as a negative control.

 



View larger version (117K):
[in this window]
[in a new window]
  		FIG. 4.
Ca2+ binding analysis of 80K-H mutants. Purified GST-fused 80K-H, 80K-HEF, and 80K-H{Delta}Glu proteins were separated by SDS-PAGE and blotted. Equal levels in protein expression were visualized with Ponceau S staining (left panel), and Ca2+-binding capacity was demonstrated by incubation of the blot with 45CaCl2 as described under "Experimental Procedures" and followed by autoradiography (right panel). GST protein was used as a negative control.

 
Cell-surface-associated Expression of 80K-H and TRPV5—TRPV5 plasma membrane expression was examined in HEK293 cells transfected with TRPV5 and 80K-H with a biotinylation method using Sulfo-NHS-SS-Biotin (Pierce). TRPV5 was expressed at the plasma membrane of HEK293 cells when transfected alone and together with 80K-H. Co-expression of TRPV5 and 80K-H mutants did not significantly affect the amount of TRPV5 expression on the plasma membrane fraction. Non-transfected cells were used as a negative control (Fig. 5A). In the same experiment, the association of 80K-H with TRPV5 channels located in the plasma membrane was studied. Interestingly, 80K-H and mutants were detected in the biotinylated membrane fraction when co-expressed with TRPV5 (Fig. 5B). The endogenously and heterologously expressed 80K-H was not detected at the plasma membrane fraction in the absence of TRPV5, despite their abundant expression in the cell lysates (Fig. 5B, right panel). Thus, it could be assessed that the expression of 80K-H at the plasma membrane depends on the presence of TRPV5.



View larger version (53K):
[in this window]
[in a new window]
  		FIG. 5.
Cell-surface expression of TRPV5 and 80K-H. HEK293 cells were transfected with TRPV5 alone or in combination (+) with 80K-H, 80K-HEF, 80K-H{Delta}Glu, or 80K-H{Delta}HDEL. The extracellular parts of the proteins were biotinylated, precipitated with streptavidin-agarose beads, and immunoblotted for TRPV5 (A) and 80K-H (B). A sample of the lysed cells (Lysates) was immunoblotted in parallel to visualize the amount of expressed proteins. Both non-transfected cells (NT) and cells transfected with 80K-H only (B, right panel) did not show cell-surface expression.

 
Effect of Mutations in 80K-H on TRPV5 Channel Activity—The effect of endogenous 80K-HWT and mutants on TRPV5 activity was determined by whole-cell patch-clamp analysis in transiently transfected HEK293 cells (Fig. 6). In line with previous results, TRPV5 conducted Na+ and Ca2+ currents, including large monovalent currents, strong inward rectification, and high Ca2+ selectivity over Na+ (7). Cells expressing either TRPV5 alone or in combination with 80K-HWT displayed similar current densities and electrophysiological properties: Na+ current at -80 mV 441 ± 149 pA/pF (n = 4) versus 562 ± 169 pA/pF (n = 6), and Ca2+ peak current 337 ± 109 pA/pF (n = 4), compared with 299 ± 162 pA/pF (n = 4) for cells expressing TRPV5 alone or TRPV5 together with 80K-HWT, respectively. Thus, expression of 80K-HWT did not significantly alter TRPV5 channel activity (p > 0.1). Co-expression of TRPV5 with the 80K-HEF, 80K-H{Delta}Glu, and 80K-H{Delta}HDEL mutants had no significant effect upon the shape or amplitude of the current in a nominally DVF solution (p > 0.8, p > 0.8, and p > 0.1, respectively, compared with 80K-HWT) (Fig. 6, A and C). In the presence of extracellular Ca2+, TRPV5 became Ca2+-selective, and the inward Ca2+ currents exhibited pronounced inactivation during hyperpolarizing steps (Fig. 6B). In contrast to endogenous 80K-HWT, the 80K-H mutants had profound effects upon the TRPV5 currents measured in the presence of 10 mM extracellular Ca2+ (Fig. 6B). Both 80K-HEF and 80K-H{Delta}Glu reduced the peak inward current by ~60% (p < 0.05 for both) (Fig. 6, B and D). These mutants had no effect on the extent of Ca2+ current inactivation, which was quantified as the ratio between the peak current and the current at the end of the 3-s voltage step (Fig. 6E). 80K-H{Delta}HDEL did not affect the peak inward current (p > 0.9), but significantly delayed the Ca2+ current inactivation (p < 0.5) (Fig. 6, B, D, and E).



View larger version (41K):
[in this window]
[in a new window]
  		FIG. 6.
Effect of 80K-H mutants on TRPV5 activity. A, whole-cell current-voltage relations measured from 400-ms voltage ramps (interval = 5 s) in nominally DVF solution in HEK293 cells overexpressing either TRPV5 alone (80K-HWT) or TRPV5 in combination with the indicated 80K-H mutants. B, inward Ca2+ currents measured with 10 mM extracellular Ca2+ during a 3-s step to -100 mV from a holding potential of +70 mV. C, average current densities at -80 mV in nominally DVF solution for cells expressing either TRPV5 alone (80K-HWT) or TRPV5 in combination with the indicated 80K-H mutants. D, average peak current density during the voltage step in 10 mM Ca2+. E, comparison of the averaged ratio of the Ca2+ current at the beginning and the end of the 3-s voltage step in 10 mM extracellular Ca2+ for TRPV5 alone or in combination with the different 80K-H mutants. D and E: *, p < 0.05, significant from 80K-HWT control currents.

 
Modulation of TRPV5 Sensitivity for Intracellular Ca2+ by 80K-HEF—Currents through TRPV5 are inhibited by increasing the intracellular Ca2+ concentration ([Ca2+]i) (8). A 40% reduction was observed in TRPV5 expressing cells dialyzed with a solution containing 50 nM free Ca2+ when compared with an intracellular solution containing 10 mM BAPTA and no added Ca2+ (Fig. 7A). In contrast, the currents of cells co-expressing TRPV5 and 80K-HEF in nominally DVF solution were nearly blocked with 50 nM Ca2+ in the pipette, indicative of a higher sensitivity to intracellular Ca2+ than cells expressing TRPV5 solely (Fig. 7B). Fig. 7C displays normalized inward Na+ currents as a function of [Ca2+]i for cells expressing TRPV5 in the absence and presence of 80K-HEF. Ca2+ concentrations for half-maximal inhibition (IC50) of inward current were 72 ± 12 nM and 12 ± 2 nM for TRPV5 alone and in combination with 80K-HEF, respectively. All other 80K-H mutants had no significant effect on the Ca2+ sensitivity of TRPV5 (data not shown).



View larger version (16K):
[in this window]
[in a new window]
  		FIG. 7.
Mutation of the EF-hand structure in 80KH increases the Ca2+ sensitivity of TRPV5. Current-voltage relations in nominally DVF solution measured in cells expressing TRPV5 alone (A) or co-expressed with 80K-HEF (B) and dialysed with a pipette solution containing either <1 nM or 50 nM free Ca2+. Dose-response curves showing the effect of intracellular Ca2+ on the inward current density in nominally DVF solution (C). Values were normalized to the average current density measured with a pipette solution containing 10 mM BAPTA and no added Ca2+ (1279 ± 208 pA/pF, n = 27 for TRPV5 and 1502 ± 243 pA/pF, n = 10 for 80K-HEF).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The present study demonstrates a new function for the 80K-H protein as a Ca2+-dependent regulator of TRPV5 channel activity. This conclusion is based upon the following observations: (i) 80K-H and TRPV5 genes are similarly controlled by common regulators of Ca2+ homeostasis; (ii) 80K-H and TRPV5 co-localize in the distal part of the nephron; (iii) 80K-H forms a heteromeric complex with TRPV5 along the plasma membrane and a distinct region of the TRPV5 C-terminal tail is important for this binding; and (iv) the two EF-hand structures of 80K-H control TRPV5 activity in a Ca2+-dependent way.

Function of 80K-H—To date, the biological function of 80K-H is poorly understood, and there are no indications for a role in Ca2+ homeostasis or ion channel regulation. However, several studies propose a role in cell activation pathways through receptors located at the plasma membrane. 80K-H was initially identified as a PKC substrate and was subsequently associated with intracellular signaling (26). Tyrosine residues of 80K-H are phosphorylated upon ligand binding to the fibroblast growth factor receptor 3 and activate the mitogen-activated protein kinase pathway (27, 28). 80K-H is also implicated in signaling and auto-regulation of the advanced glycosylation end products receptor (29), which participates in endocytic and cell-activation pathways and is linked to nephropathy in type 1 diabetic patients (30). In addition, 80K-H homologous proteins have been identified, such as the vacuolar system-associated protein 60 which plays a role in membrane-trafficking events (31) and the non-catalytic {beta} subunit of the endoplasmic reticulum (ER) enzyme glucosidase-II GII{beta} involved in the protein folding via deglycosylation (18, 32).

80K-H Gene Expression Is Regulated by 1,25-(OH)2D3 and Dietary Ca2+—80K-H was identified using cDNA microarray analysis as a transcript down-regulated in 1,25-(OH)2D3-deficient mice (22). Normalization of the severe hypocalcemia in these mice by 1,25-(OH)2D3 or dietary Ca2+ supplementation was accompanied by an increase of the 80K-H mRNA expression. These two key regulators of Ca2+ (re)absorption are important for the transcriptional modulation of renal proteins participating in the mechanism of transcellular Ca2+ transport, including the epithelial Ca2+ channel TRPV5, calbindin-D28K, Na+/Ca2+ exchanger NCX1, and the Ca2+-ATPase PMCA1b (9).

80K-H Interacts with TRPV5—80K-H was ubiquitously expressed in mouse tissues and displayed consistent co-localization with TRPV5 along the apical membrane of distal convoluted and connecting tubules (17, 33). Co-localization of 80K-H and TRPV5 in the same cellular compartments substantiates the physiological relevance of the demonstrated interaction between these two proteins. Indeed, a short peptide sequence between amino acids 598 and 608 of the TRPV5 C-terminal tail is necessary for 80K-H binding. This particular region of TRPV5 is highly conserved among all identified species (4). This domain could play an important role in the regulation of the channel via protein-protein interaction, because the S100A10-annexin 2 complex binds here (11). Biotinylation assays on cells co-expressing 80K-H and TRPV5 confirmed cell-surface expression of both proteins. This observation, together with the binding data, demonstrates the participation of 80K-H in a heteromeric complex with TRPV5 that is located at the plasma membrane. Importantly, 80K-H was not expressed at the plasma membrane in the absence of TRPV5, indicating a unique role of this protein in TRPV5-expressing tissues. Likewise, a tissue-dependent activity could explain the implication of 80K-H in diverse biological processes, ranging from vesicular transport and ER quality control to receptor signaling and channel regulation (18, 29, 32).

Critical Regions in 80K-H for TRPV5 Activity—80K-H sequence analysis predicts several potential threonine and tyrosine phosphorylation sites, two EF-hand domains, a highly acidic domain, and a potential ER-targeting sequence (18). The overall structure of 80K-H (Fig. 3A) suggests two potential Ca2+-interacting motifs, including a pair of EF-hand motifs and the acidic stretch of glutamates. In this study, we demonstrated that 80K-H coordinates Ca2+ with the EF-hand pair, whereas the highly negatively charged poly-glutamate stretch does not account for Ca2+ binding. Thus, 80K-H can be distinguished from ER proteins that contain similar acidic stretches with low Ca2+ affinity (34). In this respect, deletion of the 80K-H putative ER-targeting signal (HDEL) did not disturb routing of the 80K-H-TRPV5 complex to plasma membrane. 80K-H{Delta}HDEL had no effect upon TRPV5-mediated Ca2+ currents; surprisingly, it increased the fraction of open channels. At this point, it is difficult to explain the action of the HDEL motif as a delay factor in the decay of TRPV5 Ca2+ currents. These data can not exclude the possibility that the HDEL sequence constitutes an ER retention or retrieval signal (35), but they clearly support the hypothesis that motifs like the yeast HDEL or the respective mammalian KDEL are not sufficient for ER localization (36). Mutation of the EF-hand pair and the acidic glutamate region of 80K-H resulted in a dramatic decrease of TRPV5-mediated Ca2+ influx without disturbing the association of 80K-H to TRPV5. Additionally, the plasma membrane localization of 80K-H and TRPV5, together with the fact that the Na+ current density was not altered, excludes the possibility of a routing defect. Previous findings have also indicated other EF-hand proteins, such as calmodulin, to be involved in the activation and inactivation process of ion channels and most interestingly, TRP channels (13, 37). Moreover, the enriched glutamate stretch could provide an acidic milieu essential for phosphorylation of the 80K-H PKC substrate. Evolutionary and functional studies showed that variation in length and acidity of similar domains are related to the degree of phosphorylation needed for receptor desensitization (38, 39).

80K-H as Ca2+ Sensor—A major question that emerges from this study is how 80K-H controls the TRPV5-mediated Ca2+ influx. Electrophysiological studies demonstrated that Ca2+ influx through TRPV5 induces a feedback inhibition controlled by the ambient Ca2+ concentration in a micro domain near the inner mouth of the channel (8). 80K-HEF significantly reduced the amount of intracellular Ca2+ needed for the induction of TRPV5 feedback inhibition, but did not delay the recovery of the channel. Therefore, 80K-H could act as a Ca2+-sensing subunit of TRPV5 and facilitate Ca2+ influx through TRPV5 after binding Ca2+, situated in the proximity of the inner mouth of the channel. In addition, our findings indicate roles for the glutamate stretch and the HDEL sequence of 80K-H in the inactivation and restoration processes of TRPV5 Ca2+ currents, but these need further investigation. 80K-H could have a multifaceted mechanism regulating TRPV5 Ca2+-mediated influx. A dual function has also been proposed for the Ca2+ sensor calmodulin, according to which calmodulin mediates both Ca2+-dependent facilitation and inactivation of L-type Ca2+ channels (40). Finally, a Ca2+-sensing role of this PKC substrate can be postulated if we take into account the involvement of 80K-H in signal transduction cascades. In contrast to the Ca2+ buffers parvalbumin, calretinin, and calbindin-D28K, the Ca2+ sensors calmodulin and troponin C do not only bind Ca2+ next to the inner mouth of channels, but also activate various downstream effectors (12).

In conclusion, our findings describe 80K-H as a new Ca2+ sensor and regulator of the epithelial Ca2+ channel TRPV5. Given the wide tissue distribution and the functional diversity of 80K-H, this novel functional feature of 80K-H could also apply to other Ca2+-related physiological processes.


   FOOTNOTES
 
* This work was supported by Grants Zon-Mw 016.006.001, Zon-Mw 902.18.298, NWO-ALW 810.38.004, and NWO-ALW 805.09.042 from the Dutch Organization of Scientific Research, by the Belgian Federal Government, the Flemish Government, and the Onderzoeksraad KU Leuven (GOA 99/07, F.W.O. G.0136.00; F.W.O. G.0172.03, Interuniversity Poles of Attraction Program, IUAP, GOA 2004/07). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: 160 Cell Physiology, University Medical Centre Nijmegen, P.O. Box 9101, NL-6500 HB Nijmegen, The Netherlands. Tel: 31-24-361-4211; Fax: 31-24-361-6413; E-mail: r.bindels{at}ncmls.kun.nl.

1 The abbreviations used are: TRP, transient receptor potential; TRPV5, transient receptor potential cation channel, subfamily vanilloid, member 5; HDEL, His-Asp-Glu-Leu; C, carboxyl; PKC, protein kinase C; PBS, phosphate-buffered saline; HA, hemagglutinin; HEK293, human embryonic kidney 293; PBS, phosphate-buffered saline; DVF, divalent-free; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; GST, glutathione S-transferase; ER, endoplasmic reticulum. Back

2 By G. Droogmans, KU Leuven; available on the World Wide Web at ftp://ftp.cc.kuleuven.ac.be/pub/droogmans/CaBuf.zip. Back

3 By G. Droogmans, KU Leuven; available on the World Wide Web at ftp://ftp.cc.kuleuven.ac.be/pub/droogmans/winascd.zip. Back


   ACKNOWLEDGMENTS
 
We thank Drs. C. W. Arendt and H. L. Ostergaard (Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Canada) for kindly providing the 80K-H antibody (anti-80.2), S. Hoefs for her assistance in the X. laevis oocytes injections, and M. Goossens and D. Lamers for their help with cloning of the constructs.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hoenderop, J. G., van Leeuwen, J. P., van der Eerden, B. C., Kersten, F. F., van der Kemp, A. W., Merillat, A. M., Waarsing, J. H., Rossier, B. C., Vallon, V., Hummler, E., and Bindels, R. J. (2003) J. Clin. Invest. 112, 1906-1914[CrossRef][Medline] [Order article via Infotrieve]
  2. Hoenderop, J. G., van der Kemp, A. W., Hartog, A., van de Graaf, S. F., van Os, C. H., Willems, P. H., and Bindels, R. J. (1999) J. Biol. Chem. 274, 8375-8378[Abstract/Free Full Text]
  3. Peng, J. B., Chen, X. Z., Berger, U. V., Vassilev, P. M., Tsukaguchi, H., Brown, E. M., and Hediger, M. A. (1999) J. Biol. Chem. 274, 22739-22746[Abstract/Free Full Text]
  4. Hoenderop, J. G., Vennekens, R., Muller, D., Prenen, J., Droogmans, G., Bindels, R. J., and Nilius, B. (2001) J. Physiol. 537, 747-761[Abstract/Free Full Text]
  5. van Cromphaut, S., Dewerchin, M., Hoenderop, J. G., Stockmans, I., van Herck, E., Kato, S., Bindels, R. J., Collen, D., Carmeliet, P., Bouillon, R., and Carmeliet, G. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 13324-13329[Abstract/Free Full Text]
  6. Clapham, D. E., Runnels, L. W., and Strubing, C. (2001) Nat. Rev. Neurosci. 2, 387-396[Medline] [Order article via Infotrieve]
  7. Vennekens, R., Hoenderop, J. G., Prenen, J., Stuiver, M., Willems, P. H., Droogmans, G., Nilius, B., and Bindels, R. J. (2000) J. Biol. Chem. 275, 3963-3969[Abstract/Free Full Text]
  8. Nilius, B., Prenen, J., Vennekens, R., Hoenderop, J. G., Bindels, R. J., and Droogmans, G. (2001) Cell Calcium 29, 417-428[CrossRef][Medline] [Order article via Infotrieve]
  9. Hoenderop, J. G., Dardenne, O., van Abel, M., van der Kemp, A. W., Van Os, C. H., St-Arnaud, R., and Bindels, R. J. (2002) FASEB J. 16, 1398-1406[Abstract/Free Full Text]
  10. van Abel, M., Hoenderop, J. G., Dardenne, O., St-Arnaud, R., Van Os, C., van Leeuwen, J. P., and Bindels, R. J. (2002) J. Am. Soc. Nephrol. 13, 2102-2109[Abstract/Free Full Text]
  11. van de Graaf, S. F., Hoenderop, J. G., Gkika, D., Lamers, D., Prenen, J., Rescher, U., Gerke, V., Staub, O., Nilius, B., and Bindels, R. J. (2003) EMBO J. 22, 1478-1487[CrossRef][Medline] [Order article via Infotrieve]
  12. Budde, T., Meuth, S., and Pape, H. C. (2002) Nat. Rev. Neurosci. 3, 873-883[CrossRef][Medline] [Order article via Infotrieve]
  13. Niemeyer, B. A., Bergs, C., Wissenbach, U., Flockerzi, V., and Trost, C. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 3600-3605[Abstract/Free Full Text]
  14. Trouet, D., Nilius, B., Voets, T., Droogmans, G., and Eggermont, J. (1997) Pflügers Arch. 434, 632-638[CrossRef][Medline] [Order article via Infotrieve]
  15. Krieg, P. A., and Melton, D. A. (1984) Nucleic Acids Res. 12, 7057-7070[Abstract/Free Full Text]
  16. Hoenderop, J. G., van der Kemp, A. W., Hartog, A., van Os, C. H., Willems, P. H., and Bindels, R. J. (1999) Biochem. Biophys. Res. Commun. 261, 488-492[CrossRef][Medline] [Order article via Infotrieve]
  17. Hoenderop, J. G., Hartog, A., Stuiver, M., Doucet, A., Willems, P. H., and Bindels, R. J. (2000) J. Am. Soc. Nephrol. 11, 1171-1178[Abstract/Free Full Text]
  18. Arendt, C. W., and Ostergaard, H. L. (1997) J. Biol. Chem. 272, 13117-13125[Abstract/Free Full Text]
  19. de Jong, J. C., Willems, P. H., Mooren, F. J., van Den Heuvel, L. P., Knoers, N. V., and Bindels, R. J. (2003) J. Biol. Chem. 278, 24302-24307[Abstract/Free Full Text]
  20. Maruyama, K., Mikawa, T., and Ebashi, S. (1984) J. Biochem. (Tokyo) 95, 511-519[Abstract/Free Full Text]
  21. Dardenne, O., Prud'homme, J., Arabian, A., Glorieux, F. H., and St-Arnaud, R. (2001) Endocrinology 142, 3135-3141[Abstract/Free Full Text]
  22. Hoenderop, J. G., Chon, H., Gkika, D., Bluyssen, H. A., Holstege, F. C., St-Arnaud, R., Braam, B., and Bindels, R. J. (2004) Kidney Int. 65, 531-539[CrossRef][Medline] [Order article via Infotrieve]
  23. Hoenderop, J. G., Voets, T., Hoefs, S., Weidema, F., Prenen, J., Nilius, B., and Bindels, R. J. (2003) EMBO J. 22, 776-785[CrossRef][Medline] [Order article via Infotrieve]
  24. Nijenhuis, T., Hoenderop, J. G., van der Kemp, A. W., and Bindels, R. J. (2003) J. Am. Soc. Nephrol. 14, 2731-2740[Abstract/Free Full Text]
  25. Baksh, S., and Michalak, M. (1991) J. Biol. Chem. 266, 21458-21465[Abstract/Free Full Text]
  26. Hirai, M., and Shimizu, N. (1990) Biochem. J. 270, 583-589[Medline] [Order article via Infotrieve]
  27. Goh, K. C., Lim, Y. P., Ong, S. H., Siak, C. B., Cao, X., Tan, Y. H., and Guy, G. R. (1996) J. Biol. Chem. 271, 5832-5838[Abstract/Free Full Text]
  28. Kanai, M., Goke, M., Tsunekawa, S., and Podolsky, D. K. (1997) J. Biol. Chem. 272, 6621-6628[Abstract/Free Full Text]
  29. Li, Y. M., Mitsuhashi, T., Wojciechowicz, D., Shimizu, N., Li, J., Stitt, A., He, C., Banerjee, D., and Vlassara, H. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 11047-11052[Abstract/Free Full Text]
  30. Poirier, O., Nicaud, V., Vionnet, N., Raoux, S., Tarnow, L., Vlassara, H., Parving, H. H., and Cambien, F. (2001) Diabetes 50, 1214-1218[Abstract/Free Full Text]
  31. Brule, S., Rabahi, F., Faure, R., Beckers, J. F., Silversides, D. W., and Lussier, J. G. (2000) Biol. Reprod. 62, 642-654[Abstract/Free Full Text]
  32. Trombetta, E. S., Simons, J. F., and Helenius, A. (1996) J. Biol. Chem. 271, 27509-27516[Abstract/Free Full Text]
  33. Loffing, J., Loffing-Cueni, D., Valderrabano, V., Klausli, L., Hebert, S. C., Rossier, B. C., Hoenderop, J. G., Bindels, R. J., and Kaissling, B. (2001) Am. J. Physiol. 281, F1021-F1027
  34. Michalak, M., Corbett, E. F., Mesaeli, N., Nakamura, K., and Opas, M. (1999) Biochem. J. 344, Pt. 2, 281-292[CrossRef][Medline] [Order article via Infotrieve]
  35. Jackson, M. R., Nilsson, T., and Peterson, P. A. (1990) EMBO J. 9, 3153-3162[Medline] [Order article via Infotrieve]
  36. Pagny, S., Cabanes-Macheteau, M., Gillikin, J. W., Leborgne-Castel, N., Lerouge, P., Boston, R. S., Faye, L., and Gomord, V. (2000) Plant Cell 12, 739-756[Abstract/Free Full Text]
  37. Scott, K., Sun, Y., Beckingham, K., and Zuker, C. S. (1997) Cell 91, 375-383[CrossRef][Medline] [Order article via Infotrieve]
  38. Small, K. M., Brown, K. M., Forbes, S. L., and Liggett, S. B. (2001) J. Biol. Chem. 276, 4917-4922[Abstract/Free Full Text]
  39. Madsen, O., Willemsen, D., Ursing, B. M., Arnason, U., and de Jong, W. W. (2002) Mol. Biol. Evol. 19, 2150-2160[Abstract/Free Full Text]
  40. Lee, A., Scheuer, T., and Catterall, W. A. (2000) J. Neurosci. 20, 6830-6838[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
K. Kawaai, C. Hisatsune, Y. Kuroda, A. Mizutani, T. Tashiro, and K. Mikoshiba
80K-H Interacts with Inositol 1,4,5-Trisphosphate (IP3) Receptors and Regulates IP3-induced Calcium Release Activity
J. Biol. Chem., January 2, 2009; 284(1): 372 - 380.
[Abstract] [Full Text] [PDF]

Home page
 PhysiologyHome page
J. G. J. Hoenderop and R. J. M. Bindels
Calciotropic and Magnesiotropic TRP Channels
Physiology, February 1, 2008; 23(1): 32 - 40.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
S. F. J. van de Graaf, J. G. J. Hoenderop, and R. J. M. Bindels
Regulation of TRPV5 and TRPV6 by associated proteins
Am J Physiol Renal Physiol, June 1, 2006; 290(6): F1295 - F1302.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
S. A. Oak, C. Tran, G. Pan, M. Thamotharan, and S. U. Devaskar
Perturbed skeletal muscle insulin signaling in the adult female intrauterine growth-restricted rat
Am J Physiol Endocrinol Metab, June 1, 2006; 290(6): E1321 - E1330.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
D. Gkika, C. N. Topala, J. G. J. Hoenderop, and R. J. M. Bindels
The immunophilin FKBP52 inhibits the activity of the epithelial Ca2+ channel TRPV5
Am J Physiol Renal Physiol, May 1, 2006; 290(5): F1253 - F1259.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
S. F. J. van de Graaf, Q. Chang, A. R. Mensenkamp, J. G. J. Hoenderop, and R. J. M. Bindels
Direct Interaction with Rab11a Targets the Epithelial Ca2+ Channels TRPV5 and TRPV6 to the Plasma Membrane
Mol. Cell. Biol., January 1, 2006; 26(1): 303 - 312.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
J. G. J. Hoenderop, B. Nilius, and R. J. M. Bindels
Calcium Absorption Across Epithelia
Physiol Rev, January 1, 2005; 85(1): 373 - 422.
[Abstract] [Full Text] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
J. G.J. Hoenderop and R. J.M. Bindels
Epithelial Ca2+ and Mg2+ Channels in Health and Disease
J. Am. Soc. Nephrol., January 1, 2005; 16(1): 15 - 26.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
C. G. Levine, D. Mitra, A. Sharma, C. L. Smith, and R. S. Hegde
The Efficiency of Protein Compartmentalization into the Secretory Pathway
Mol. Biol. Cell, January 1, 2005; 16(1): 279 - 291.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/25/26351    most recent
M403801200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gkika, D.
Right arrow Articles by Bindels, R. J. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gkika, D.
Right arrow Articles by Bindels, R. J. M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement