标题: WNK4 enhances TRPV5-mediated calcium transport: potential role in hypercalciuria [打印本页] 作者: 轻羽 时间: 2009-4-22 09:46 标题: WNK4 enhances TRPV5-mediated calcium transport: potential role in hypercalciuria
作者：Yi Jiang, William B. Ferguson, and Ji-Bin Peng作者单位：Nephrology Research and Training Center, Division of Nephrology, Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama I9 C6 A- E; R3 Q: Z 6 n7 I3 p' ~2 Y , ^8 s1 h/ R' l: z( }: k$ h6 Z
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- W% _4 i6 [, ? 【摘要】! T5 Q: D" L! [, E
The epithelial Ca 2 channel TRPV5 serves as a gatekeeper for active Ca 2 reabsorption in the distal convoluted tubule and connecting tubule of the kidney. WNK4, a protein serine/threonine kinase with gene mutations that cause familial hyperkalemic hypertension (FHH), including a subtype with hypercalciuria, is also localized in the distal tubule of the nephron. To understand the role of WNK4 in modulation of Ca 2 reabsorption, we evaluated the effect of WNK4 on TRPV5-mediated Ca 2 transport in Xenopus laevis oocytes. Coexpression of TRPV5 with WNK4 resulted in a twofold increase in TRPV5-mediated Ca 2 uptake. The increase in Ca 2 uptake was due to the increase in surface expression of TRPV5. When the thiazide-sensitive Na -Cl - cotransporter NCC was coexpressed, the effect of WNK4 on TRPV5 was weakened by NCC in a dose-dependent manner. Although the WNK4 disease-causing mutants E562K, D564A, Q565E, and R1185C retained their ability to upregulate TRPV5, the blocking effect of NCC was further strengthened when wild-type WNK4 was replaced by the Q565E mutant, which causes FHH with hypercalciuria. We conclude that WNK4 positively regulates TRPV5-mediated Ca 2 transport and that the inhibitory effect of NCC on this process may be involved in the pathogenesis of hypercalciuria of FHH caused by gene mutation in WNK4. , |! e$ h) E" y' r { 【关键词】 epithelial calcium channel CaT calcium reabsorption WNK TRPV / q# w# G$ c: o5 O A NOVEL SERINE / THREONINE protein kinase family characterized by the absence of a conserved lysine residue in the kinase domain is formed by WNK [with no lysine (K)] kinases ( 34 ). Mutations in WNK1 and WNK4 genes, two members of the family of four WNK genes, which are linked to familial hyperkalemic hypertension (FHH, also known as pseudohypoaldosteronism type II or Gordon's syndrome) ( 32 ), highlight the physiological significance of these kinases. Mutations in WNK1 are deletions in the first intron, which result in overexpression of WNK1. Mutations in WNK4 are missense mutations, most of which are clustered in a small region rich in negatively charged amino acids after a coiled-coil stretch following its kinase domain, with the exception of R1185C, which is located close to the carboxy terminus. FHH patients carrying WNK1 or WNK4 mutations have common manifestations, including hyperkalemia, hypertension, mild metabolic acidosis, low renin, and normal glomerular filtration rate ( 1, 22 ). Despite the aforementioned similarities in the two forms of FHH, there is a significant difference in Ca 2 metabolism between FHH patients carrying WNK1 gene mutations and those carrying WNK4 gene mutations. Affected patients carrying the Q565E mutation in the WNK4 gene exhibit marked hypercalciuria compared with unaffected subjects in the same family ( 21, 22 ). In contrast, in FHH patients with a WNK1 intronic deletion mutation, urinary Ca 2 content is similar to that of the unaffected subjects ( 1 )." h' B& N8 Z, q+ P" s
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WNK4 has been shown to be a multifunctional protein regulating renal ion transport. WNK4 decreases activity of the thiazide-sensitive Na -Cl - cotransporter (NCC, also known as TSC and NCCT) ( 33, 36 ), the renal outer medullary K (ROMK) channel ( 16 ), and TRPV4, an osmolarity-sensitive Ca 2 -permeable channel ( 7 ). On the other hand, WNK4 increases paracellular Cl - permeability, likely through phosphorylation of claudins ( 15, 35 ). The disease-causing Q565E mutant exhibits impaired ability to suppress NCC ( 36 ) and enhanced ability to suppress the ROMK channel ( 16 ). These findings explain the hypertension and hyperkalemia characteristic of FHH; however, the mechanism underlying hypercalciuria in patients carrying the Q565E mutation of the WNK4 gene is unclear. It is possible that the enhanced NCC-mediated Na transport in patients with FHH might negatively affect Ca 2 transport in the distal tubule; alternatively, disease-causing mutants of WNK4 may affect a Ca 2 transporter or channel in the distal tubule, as suggested by Mayan et al. ( 21 ). & v* H; f; \# P1 r2 J! E/ g4 ~! | ; v* q7 N( R+ x6 SThe distal tubule of the kidney, where WNK4 is localized, is important in Ca 2 reabsorption. The vitamin D-responsive transcellular Ca 2 transport pathway in this segment makes the distal tubule a determinant of the final Ca 2 excretion into the urine. The recently identified Ca 2 -selective channel TRPV5 [transient receptor potential, vanilloid subfamily, member 5; also known as epithelial Ca 2 channel (ECaC)] is a key player in the distal tubule for Ca 2 transport ( 11, 12, 25 ). An understanding of the role of WNK4 in regulation of TRPV5 may shed light on the regulation of Ca 2 reabsorption in the distal tubule under pathophysiological conditions.0 ~9 G) C) g# ]: g- L I/ }: {
6 C. J, @ Z2 [% g0 hHere we report that WNK4 positively regulated TRPV5 by increasing the surface abundance of TRPV5. Moreover, the positive effect of WNK4 on TRPV5 was greatly reduced when NCC was coexpressed. These findings suggest that WNK4 may be an important modulator of the inverse relation between Na and Ca 2 reabsorption in the distal tubule and also help explain the hypercalciuria in FHH patients carrying the Q565E mutation in the WNK4 gene.; q" M/ q% T ^: ]5 P
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MATERIALS AND METHODS$ n% ]7 R% J* r
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cDNA constructs. Human TRPV5 and TRPV6 cDNAs were described previously ( 24 ). Human WNK4 cDNA was provided by Dr. Xavier Jeunemaitre, and rat WNK1 cDNA was provided by Dr. Melanie H. Cobb ( 34 ). Mouse NCC cDNA (IMAGE:4237274, GenBank accession no. BC038612 ) was purchased from Open Biosystems (Huntsville, AL). For experiments using Xenopus laevis oocytes, the cDNA was subcloned into the X. laevis oocyte expression vector pIN, which we constructed as an entry vector for Gateway cloning technology (Invitrogen, Carlsbad, CA). For detection of TRPV5 in X. laevis oocytes, a yellow fluorescent protein (YFP) was incorporated into the amino or carboxy terminus of TRPV5. WNK4 and NCC mutants were generated using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) following the manufacturer's instruction. All the mutants were confirmed by sequencing. . c$ W0 _$ k+ ~# ?5 c+ w " f2 S9 x) C% y! _; e+ NCa 2 uptake in X. laevis oocytes. In vitro transcription, injection of the resultant capped synthetic RNAs (cRNAs) into oocytes, and Ca 2 uptake assay in oocytes were conducted as described previously ( 26 ). The animal protocol was approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham. cRNA for TRPV5 was injected at 6.25 ng/oocyte, and TRPV6, WNK1, WNK4, and NCC were injected at 12.5 ng/oocyte. When a combination of two or three cRNAs was required, the cRNAs were mixed, such that the concentration of individual cRNA was maintained. Defolliculated X. laevis oocytes were kept at 18°C in Barth's solution supplemented with 2 mM pyruvate, penicillin (10,000 U/l), and streptomycin and gentamicin (each at 10 mg/l). In later experiments, 0.5 x L-15 medium (Invitrogen) supplemented with 5% heat-inactivated horse serum, penicillin (10,000 U/l), streptomycin (10 mg/l), and amphotericin B (25 µg/l) were also used. Uptake experiments were carried out at room temperature (24°C) for 30 min. Standard uptake solution contained (in mM) 100 NaCl, 2 KCl, 1 MgCl 2, 1 CaCl 2 (including 45 CaCl 2 at 10 µCi/ml), and 10 HEPES (pH 7.5). After uptake, oocytes were washed six times with ice-cold standard uptake solution without 45 CaCl 2 and then dissolved in 10% SDS. The incorporated 45 Ca was determined using a scintillation counter. Ca 2 uptake data are presented as mean values from at least three experiments with seven to nine oocytes per group, with the standard error of the mean used as the index of dispersion.! D* Q ~ e- F3 F% f0 `
* y W% H7 s# N. ?Two-microelectrode voltage-clamp technique. Two-microelectrode voltage-clamp experiments were performed as described previously ( 26 ). The resistance of microelectrodes filled with 3 M KCl was 0.5-5 M. In experiments involving voltage jumping or holding, currents and voltages were digitized at 0.3 and 200 ms/sample, respectively. After 3 min of stabilization of membrane potential following impalement with the microelectrodes, the oocyte was clamped at the holding potential of -50 mV. Then 100-ms voltage pulses between -160 and 60 mV, in 20-mV increments, were applied, and steady-state currents were obtained as the average values 80-95 ms after initiation of the voltage pulses. The standard perfusion solution contained (in mM) 100 choline chloride, 2 KCl, 1 MgCl 2, and 10 HEPES, with pH adjusted to 7.5 with Tris base and HCl. One-half of the choline chloride was substituted with NaCl (final concentration 50 mM) when Na -evoked current was tested.1 p) ?* A1 Q m9 a$ c
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Patch-clamp recording. All patch-clamp ( 9 ) recordings were in the cell-attached configuration. The pipette solution contained (in mM) 100 NaCl, 5 EGTA, and 10 HEPES, with pH adjusted to 7.5 with Tris base, as previously reported ( 30 2 G ) seals were obtained for all recordings, and all experiments were performed at room temperature (24°C). Pipettes were made from Kimax-51 capillary tubing that was pulled and polished using equipment from Narishige Scientific Instruments (Tokyo, Japan). Only patches with five or fewer channels were used, because single-channel amplitudes were difficult to separate when more than five channels were present. All data were recorded using an EPC-7 amplifier (List Electronics) with gain set at 100 mV/pA and a total filtering of 2.3 kHz. Data were digitized (Digidata 1322A, Axon Instruments, Sunnyvale, CA) and sampled at 50 µs using pClamp 9 software (Axon Instruments). Recordings were later refiltered at 1 kHz, and the baseline was manually adjusted to cancel any baseline drift. Single-channel amplitudes were measured using a histogram of the data and confirmed by manual verification of the single-channel amplitudes. A half-amplitude protocol was used to calculate probability of channel opening with pClamp 9 software. Data are means ± SD from at least three experiments.% g( w' Y) R& s. O; {) ^
8 N- d( i4 P6 ~Surface expression measurement. X. laevis oocytes were injected with cRNA of TRPV5 tagged with YFP in the carboxy terminus (TRPV5-YFP) alone or together with cRNA of wild-type WNK4 or the D321A mutant. Membrane surface levels of TRPV5-YFP were assayed by Leica DMIRBE laser-scanning confocal microscopy 2 days after injection. Excitation was performed at 488 nm with an argon ion laser, and emissions were captured using a x 5 objective lens. Identical brightness and contrast settings were applied to all measurements in an experiment, and images were captured at the equatorial plane of each oocyte. Three independent experiments were performed using oocytes from different frogs. Data were obtained with Leica Confocal Software version 2.0 and analyzed using MetaMorph 6.2r4 software (Universal Imaging). Values are means ± SE of 20-23 oocytes from 3 frogs. 1 s2 P; E* `& {: d3 W, W O3 }, H$ z9 l0 [: ?5 r- ]6 [/ t9 E, r
Cell surface biotinylation and Western blot analysis. At 2 days after injection with cRNA, oocytes were washed with PBS five times on ice and then incubated with Sulfo-NHS-SS-Biotin (1 mg/ml; Pierce Biotechnology, Rockford, IL) in PBS for 1 h at 4°C with end-to-end shaking. Oocytes were washed five times and then quenched with 100 mM glycine for 1 h at 4°C. Oocytes were lysed with lysis buffer [100 mM NaCl, 20 mM Tris·Cl, and 1% Triton X-100 (pH 7.6)], and the yolk was removed by centrifugation at 3,000 g for 10 min. The supernatant was incubated with immobilized NeutrAvidin beads (Pierce Biotechnology) for 2 h at 4°C. The beads were centrifuged, and the supernatants were removed. The beads were washed three times with Tris-buffered saline. Biotinylated proteins were eluted from the beads by 1 x SDS-PAGE loading buffer with 50 mM DDT at 65°C for 10 min. Input cell lysates and biotinylated sample were subjected to SDS-PAGE. Proteins were transferred from the gel onto nitrocellulose membranes and blocked with 5% nonfat dry milk in PBS containing 0.05% Tween 20 (PBS-T) for 1 h. TRPV5 protein was probed by anti-TRPV5 antibody (Alpha Diagnostics International, San Antonio, TX; 1:1,000 dilution) at 4°C overnight. After four 5-min washes in PBS-T, the membrane was probed with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Pierce Biotechnology; 1:1,000 dilution) at room temperature for 1 h and washed four times for 5 min each in PBS-T. The TRPV5 band signal was developed using SuperSignal West Pico chemiluminescent substrate (Pierce Biotechnology). Polyclonal antibodies against WNK1 and WNK4 raised in rabbits (Alpha Diagnostics International) were utilized to confirm the expression of WNK1 and WNK4 with a procedure similar to that described above. 3 y; ~; t# E& k l9 m V6 {- I- M( H9 @
Glycosylation analysis. Lysates of X. laevis oocytes expressing TRPV5 were treated with peptide: N -glycosidase F (PNGase F) or endoglycosidase H (Endo H) following the manufacturer's instructions (New England Biolabs, Beverly, MA). Equal amounts of lysates were denatured at 65°C for 10 min. After addition of 1:10 (vol/vol) of the appropriate 10 x reaction buffer (and 10% NP-40 for the PNGase F reaction) and 2 µl (1,000 U) of PNGase F, Endo H, or water (for control reactions), samples in 40 µl of total reaction volume were incubated at 37°C for 1-2 h. After the incubation, an equal volume of 2 x sample buffer was added to each reaction. The resultant samples were analyzed by SDS-PAGE and Western blot. j2 T& _+ f- m& L3 B0 O- S) W4 T; C
4 D) X5 C$ K0 [* o& ?RESULTS ! m0 e" ~% L# H, l' f % B& o) |: [% H. Q- c' ]5 \/ ] @) r* BWNK4 increased TRPV5-mediated Ca 2 transport. FHH patients carrying the WNK4 Q565E mutation exhibit hypercalciuria ( 22 ). Expression of WNK4 in the distal tubule ( 32 ) raises the possibility that WNK4 is a modulator of transcellular Ca 2 transport, and WNK4 mutation may impair the normal Ca 2 reabsorption in the distal tubule ( 21 ). Inasmuch as TRPV5 is a key player in the Ca 2 transport pathway in the distal tubule, we examined whether TRPV5 could be regulated by WNK4 in the X. laevis oocyte expression system. When human TRPV5 was expressed in X. laevis oocytes, it stimulated Ca 2 uptake over that in the control oocytes 2 days after injection ( Fig. 1 A ). Ca 2 uptake was increased by 97.6 ± 22.7% in oocytes expressing TRPV5 WNK4 over that in oocytes expressing TRPV5, whereas WNK4 did not stimulate a significant increase in Ca 2 uptake over that in the control oocytes ( Fig. 1 A ). The reason for the variation in the amplitude of increase in different batches of oocytes is unknown. Figure 1 A summarizes data from five experiments showing that the effect was statistically significant ( P . d! A1 a I- N: K4 Y& U ; z8 d g/ ~. D0 o, MFig. 1. Effects of WNK kinases on TRPV5- and TRPV6-mediated Ca 2 uptake in X. laevis oocytes. A : top, TRPV5-mediated Ca 2 uptake was increased by 97.6 ± 22.7% when WNK4 was coexpressed; WNK4 alone did not stimulate significant Ca 2 uptake over that in oocytes mock-injected with water. Bottom, expression of WNK4 detected by Western blot analysis 2 days after cRNA injection. Values are means ± SE of 5 independent experiments. B : top, effects of WNK1 or WNK4 on TRPV5 or TRPV6. Inset : TRPV6-mediated Ca 2 uptake in X. laevis oocytes. Bottom, expression of WNK4 and WNK1 detected by Western blot analysis 2 days after cRNA injection (12.5 ng each of WNK1, WNK4, and TRPV6 cRNA and 6.25 ng of TRPV5 cRNA). Amount of each cRNA remained unchanged in coinjection experiments. Ca 2 uptake experiments were performed 2 days after injection. Data from 3 independent experiments are presented as percentage of Ca 2 uptake of TRPV5 and TRPV6. * P - {! @2 T: r. I8 X V% T! L) |* m! ?$ W3 E
The effect of WNK4 on TRPV5 appeared to be specific. WNK4 had little effect on TRPV6-mediated Ca 2 uptake ( Fig. 1 B ), although TRPV6 [also known as CaT1 ( 26 )] shares 75% amino acid identity with TRPV5. On the other hand, WNK1, another member of the WNK kinase family, did not significantly increase TRPV5- and TRPV6-mediated Ca 2 uptake ( Fig. 1 B ). The expression of WNK1 in oocytes was also confirmed by Western blot analysis ( Fig. 1 B, bottom ). A major WNK1 band slightly higher than the 250-kDa marker was observed in groups of oocytes injected with WNK1 cRNA. Smaller bands of less intensity were also observed. These bands may result from alternative translation start sites and/or degradation of the WNK1 protein. This antibody detected no bands corresponding to the size of WNK1 in the control oocytes without injection of WNK1 cRNA. ' t. W# A: i* T# ~9 A: Q- l% J( |! a4 ~( D
Surface abundance of TRPV5 was increased by WNK4. For a constitutively active channel such as TRPV5, protein expression at the plasma membrane is an important determinant of the overall channel activity. To evaluate the extent to which the surface level of TRPV5 is altered by WNK4, we used confocal microscopy, as described by Hoover et al. ( 13 ), to test the surface level of TRPV5 in X. laevis oocytes. To detect TRPV5 protein, we made TRPV5 constructs tagged with YFP in amino and carboxy termini of TRPV5 (YFP-TRPV5 and TRPV5-YFP), respectively. We evaluated the effects of YFP tagging on the function of TRPV5 and the action of WNK4 on tagged TRPV5 ( Fig. 2, A-C ). Carboxy-terminal tagging of YFP appeared to have no major effect on TRPV5-mediated Ca 2 uptake and Na -evoked current, whereas amino-terminal tagging with YFP significantly decreased both ( Fig. 2, A and B ). The magnitude of the increase in Ca 2 uptake by WNK4 was significantly decreased by amino-terminal, but not carboxy-terminal, YFP tagging of TRPV5 ( Fig. 2 C ). The TRPV5-YFP exhibited a plasma membrane distribution in oocytes 2 days after injection ( Fig. 2 D ). When wild-type WNK4 was coexpressed with the TRPV5-YFP, the membrane fluorescence intensity significantly increased ( Fig. 2 D ). By quantification of the fluorescence on the surface of oocytes, a 75.4 ± 15.1% increase in the fluorescence level on the oocyte surface was detected in the oocytes expressing TRPV5-YFP WNK4 over the oocytes expressing TRPV5-YFP ( Fig. 2 E ). This magnitude of WNK4-induced increase in surface fluorescence was comparable to that in TRPV5-mediated (70.7 ± 15.1%) and TRPV5-YFP-mediated (63.5 ± 25.8%) Ca 2 uptake in the same batches of oocytes ( Fig. 2 C ). Water-injected control oocytes exhibited no detectable fluorescence on the oocyte surface (data not shown).* s5 m# f* `, H: g! d9 o* A
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Fig. 2. Surface TRPV5 was increased in the presence of WNK4. A : effect of yellow fluorescent protein (YFP) tagging in the amino or carboxy terminus of TRPV5 (YFP-TRPV5 and TRPV5-YFP, respectively) on TRPV5-mediated Ca 2 uptake. * P 4 j: o9 Q: p9 l- d8 h- p. o6 T; a' z) b/ _% G7 K" q
To further confirm the increase of TRPV5 abundance within the plasma membrane by WNK4, we utilized a surface biotinylation approach to evaluate the level of TRPV5 expressed on the plasma membrane in oocytes expressing TRPV5 and TRPV5 WNK4. TRPV5 antibody detects three major bands representing different stages of protein glycosylation ( Fig. 3 A ). Two dense bands ( bands A and B ) migrated below 75 and 100 kDa, respectively, and sometimes a much weaker band ( band C ) beneath band A was also visible. Bands A and B were sensitive to PNGase F, which removes oligosaccharide chains completely. After PNGase F digestion, all bands were reduced to the size of band C, suggesting that band C represents an unglycosylated form of TRPV5. In contrast to band A, band B was resistant to Endo H, which removes unprocessed core oligosaccharide chains without affecting fully glycosylated complex oligosaccharides ( Fig. 3 A ). Therefore, band A represents core-glycosylated TRPV5, and band B represents mature TRPV5 within the plasma membrane and could be labeled with membrane-impermeable Sulfo-NHS-SS-Biotin ( Fig. 3 B ). Interestingly, when WNK4 was coexpressed, the fully glycosylated form of TRPV5 ( band B ) was significantly increased, whereas the core-glycosylated form was not much changed ( Fig. 3 C ). This predicts that mature TRPV5 on the plasma membrane should be increased in the presence of WNK4. Indeed, this was the case ( Fig. 3 D ). e9 U0 \4 y* y3 D6 ]3 A0 J( t% a l3 e
Fig. 3. WNK4 increased fully glycosylated TRPV5 on the plasma membrane. A : left, 3 major forms of TRPV5 proteins were detected in X. laevis oocyte system [mature (fully glycosylated, band B ), immature core glycosylated ( band A ), and nonglycosylated ( band C )]. Right, bands A and B were sensitive to N -glycosidase F (PNGase F), and band B, but not band A, was resistant to endoglycosidase H (Endo H). B : mature form of TRPV5 expressed on the plasma membrane of oocytes detected by biotinylation appeared on band B. C : fully glycosylated form of TRPV5 ( band B ) was increased when WNK4 was coexpressed in X. laevis oocytes. A nonspecific band at 53 kDa serves as an indication of equal loading. Note increase in the mature form ( band B ) in the presence of WNK4 compared with the absence of WNK4. D : biotinylated TRPV5 at the oocyte surface was increased in the presence of WNK4. ' o8 L6 t7 A0 M% l) v0 V$ q1 m+ @! N1 ?* F5 E0 E
TRPV5 channel properties appeared to be unaltered by WNK4. The increase of TRPV5 abundance on the plasma membrane by WNK4 may account for the increase in TRPV5-mediated Ca 2 uptake in the oocyte; however, it is also possible that the channel properties could be affected by WNK4. Therefore, we used electrophysiology techniques to examine whether channel properties of TRPV5 were altered in the presence of WNK4.: J+ i8 F9 }/ S& j% ~
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Using the voltage-clamp technique, we examined the current-voltage ( I-V ) relation of the Na -evoked currents in oocytes expressing TRPV5 or TRPV5 WNK4 ( Fig. 4, A and B ). The Na -evoked currents in oocytes expressing WNK4 were indistinguishable from those in the control oocytes ( Fig. 4 A ). Na evoked inward rectifying currents in oocytes expressing TRPV5, as reported previously ( 24 ). A significant increase in current amplitude was evident at negative potentials when WNK4 was coexpressed with TRPV5 ( Fig. 4 A ). Nevertheless, the I-V relation of TRPV5-mediated Na current remained unchanged on coexpression of WNK4 ( Fig. 4 B ). This suggests that the TRPV5 channel property was not altered by WNK4.3 {1 k0 X e0 y+ e- h/ S7 D' g1 I
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Fig. 4. Channel properties of TRPV5 appear to be unaltered by WNK4. A : I-V plots of Na -evoked currents in water-injected control oocytes (Water) or oocytes expressing WNK4, TRPV5, or TRPV5 WNK4. Values are means ± SE from 3 experiments with a total of 11-18 oocytes in each group. B : normalized I-V plots of Na -evoked current in oocytes expressing TRPV5 with and without WNK4. Mean values of TRPV5 currents with and without WNK4 at different potentials from A were normalized as percentage of respective mean current value at -160 mV in each group. C : representative traces of activity for oocytes injected with TRPV5 or TRPV5 WNK4 at negative pipette potential (- V p ) of -50 and -100 mV. D : I-V plots of oocytes injected with TRPV5 alone or TRPV5 WNK4. Values are means ± SD from 3-5 recordings at each point. Single-channel conductance for TRPV5 (with Na as charge carrier) calculated from the slope is 85 ± 16 pS in the absence of WNK4 and 76 ± 15 pS in the presence of WNK4. 2 L% V, ~* b; ^7 ~' { 6 H1 Q: b- H, |We also examined channel properties of TRPV5 in the presence and absence of WNK4 using the patch-clamp technique under the cell-attached configuration with Na as charge carrier in the absence of extracellular divalent cations ( Fig. 4, C and D ). Although we observed no background channel activity in water-injected control oocytes under this condition, TRPV5 exhibited a rather high level of expression in X. laevis oocytes, resulting in too many channels in a single patch, as reported elsewhere ( 30 ). To reduce the number of channels in each patch, we lowered the amount of cRNA injected into each oocyte to 0.5-1 ng. The amount of WNK4 cRNA injected remained at 12.5 ng/oocyte. With recordings from at least five batches of oocytes, we observed an increase in apparent channel number in each patch from the group coexpressing TRPV5 and WNK4 over that in the group expressing TRPV5 alone: 2.5 ± 1.2 (13 recordings) vs. 1.5 ± 0.5 (8 recordings) channels/patch ( P % r( ]9 M: `# `8 x 3 O, f: a$ P: _, E) i$ sPositive effect of WNK4 on TRPV5 was abolished by the D321A mutation. The effect of WNK4 on ion channels and transporters may or may not be related to its intact kinase activity, as previously reported ( 16, 33, 37 ). To assess the extent to which the effect of WNK4 on TRPV5 requires the kinase activity of WNK4, a kinase-dead mutant (D321A) of WNK4 was employed. This aspartate residue is among the 12 residues that are invariant or nearly invariant in the kinase domain of the protein kinase superfamily ( 10 ). The murine version of this mutant (D318A) has been shown to abolish the inhibitory effect of WNK4 on NCC ( 33 ) but does not affect the action of WNK4 on the ROMK channel ( 16 ). Therefore, D321 may play a critical role in the kinase function of WNK4. Expression of the WNK4 D321A mutant in oocytes was not different from expression of wild-type WNK4 ( Fig. 5 B ). However, when the D321A mutant was coexpressed with TRPV5, it did not significantly increase TRPV5-mediated Ca 2 uptake 2 days after injection ( Fig. 5 A ). On the contrary, the WNK4 D321A mutant significantly inhibited TRPV5-mediated Ca 2 uptake 1 day after injection ( Fig. 5 A ). The mechanism underlying this effect was unclear. We sometimes observed a band identical in size to that of WNK4 in water-injected control oocytes ( Fig. 1 A, bottom ), especially when the film was overexposed ( Fig. 5 B ). In the absence of the sequence information of X. laevis WNK4, we could not determine whether the band reflects endogenously expressed WNK4. If it does, the expression of TRPV5 in oocytes might have been enhanced by the endogenous WNK4. In this case, the D321A mutant likely acted as a dominant-negative inhibitor of the endogenous WNK4. It is still unclear how this inhibitory effect diminished 2 days after injection. Consistent with Ca 2 uptake ( Fig. 5 A ), no effect of the D321A mutant on the surface expression of TRPV5-YFP was observed 2 days after injection ( Fig. 5 C ). We did not determine the surface expression of TRPV5-YFP at early time points, because the full maturation 1 day. Therefore, the WNK4-induced increase in TRPV5 plasma membrane expression is abolished by this kinase-dead mutation. _% k0 S @$ O' @ 2 u. j( R5 O5 s9 j" {Fig. 5. Kinase-dead D321A mutant of WNK4 failed to enhance TRPV5. A : D321A mutant significantly inhibited TRPV5-mediated Ca 2 uptake 1 day after cRNA injection (* P , j4 }- o4 W8 B. e9 i6 y
4 a" l8 C6 t2 nDisease-causing mutants of WNK4 retained their ability to regulate TRPV5. Hypercalciuria is a major manifestation in patients carrying the WNK4 Q565E mutation, and it has been suggested that WNK4 mutation might affect a Ca 2 transporter or channel in the distal tubule ( 21 ). We examined the extent to which the effects of WNK4 on TRPV5 are affected by disease-causing mutations in FHH patients in the X. laevis oocyte system. Using the QuikChange II Site-Directed Mutagenesis kit (Stratagene), we constructed WNK4 mutants ( 32 ), including E562K, D564A, Q565E, and R1185C. Given the possibility that WNK4 or its mutants may affect TRPV5 differently at different time points, we evaluated the effect of wild-type WNK4 and the disease-causing mutants 1, 2, and 3 days after injection of cRNA. In contrast to our expectation, all WNK4 mutants enhanced TRPV5-mediated Ca 2 uptake to an extent comparable to that of wild-type WNK4 ( Fig. 6 ). At 2 days after injection, the increases in Ca 2 uptake compared with the group treated with TRPV5 alone were 100.2 ± 23.3%, 106.4 ± 18.4%, 95.1 ± 17.9%, 94.7 ± 21.2%, and 74.7 ± 14.9% in groups coexpressed with wild-type WNK4, E562K D564A, Q565E, and R1185C, respectively. The differences between these disease-causing mutants and wild-type WNK4 are not statistically significant. At 3 days after injection, oocytes expressing TRPV5 together with wild-type WNK4 or its disease-causing mutants became much less healthy because of the overexpression of TRPV5 than those injected with TRPV5 alone. This resulted in scattered data distribution 3 days after injection. However, no statistical difference could be achieved between the group expressing TRPV5 with wild-type WNK4 and the groups expressing an individual mutant 1, 2, and 3 days after injection, whereas all these groups were statistically different from the group expressing TRPV5 alone 2 and 3 days after injection. d) z1 C: @" H. L. _2 C6 N" P+ ~& v2 x( Y* V1 x' f2 F/ Q
Fig. 6. Time-dependent effects of wild-type and disease-causing mutants of WNK4 on TRPV5. Each oocyte was injected with 6.25 ng of TRPV5 cRNA alone or 12.5 ng of WNK4 (wild-type or mutant) cRNA. Ca 2 uptake experiments were performed 1, 2, and 3 days after injection. Values are means ± SE from 5 experiments. . e) g9 x9 w3 W% _ 0 U2 ?! M# m& h n& g) T0 n) NEffect of WNK4 on TRPV5 was blocked by NCC. Ca 2 and Na reabsorptions are inversely related in the distal convoluted tubule (DCT) ( 4 ). NCC and TRPV5 are coexpressed in the late segment of the DCT ( 19 ). NCC is inhibited by WNK4 when expressed in X. laevis oocytes ( 33, 36 ) through a mechanism that involves direct binding of WNK4 to the carboxy-terminal domain of NCC ( 37 ). We therefore tested the extent to which the presence of NCC influences the regulatory effect of WNK4 on TRPV5. As shown in Fig. 7 A, the effect of WNK4 on TRPV5 was dose dependently blocked by NCC. The dose-dependent effect implied that the NCC protein level or the Na -Cl - transport activity was related to the inhibitory effect of NCC on WNK4.% I# m9 A5 O! d3 j
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Fig. 7. A : NCC blocked the effect of WNK4 on TRPV5 in a dose-dependent manner. B : disease-causing mutants of NCC (A586V, G739R, and R936Q) retained their ability to block the effect of WNK4 on TRPV5 as the wild-type NCC. Na -Cl - transport function was reportedly impaired in these mutants. C : blocking effect of NCC was more pronounced in the presence of Q565E mutant (Q565) than in the presence of wild-type WNK4 (WT WNK4). Q565E mutant and WT WNK4 increased TRPV5-mediated Ca 2 uptake to a similar extent in the absence of NCC. Enhancing effect on TRPV5 was blocked in the presence of WT WNK4 when NCC was coexpressed, and this blocking effect was more pronounced in the presence of the Q565E mutant of WNK4. Data are from 11 independent experiments. * P & [9 @2 v0 j x3 s
. \ t' x# |# L. v3 e: x) sBecause it has been shown that WNK4 activity is regulated by Na concentration ( 17 ), we tested whether the blocking effect is due to the Na -Cl - transport activity of NCC. We employed three disease-causing mutants of NCC (A586V, G739R, and R936Q) in Gitelman's syndrome. These mutants of NCC are associated with impaired Na -Cl - transport activity. G739R mutation in murine NCC corresponds to the G741R mutation in human NCC, which was shown to be a defect in plasma membrane targeting and exhibits no metolazone-sensitive Na uptake ( 5 ). A586V and R936Q mutants correspond to A585V and R935Q mutants in another murine NCC ( 28 ), which lacks the Q93 residue in the amino terminus of NCC compared with the NCC clone we used in this study. A585V and R936Q exhibits 6% and 36% of the Na transport activity of the wild-type NCC because of reduced abundance on the plasma membrane ( 28 ). As shown in Fig. 7 B, these mutants exhibited a blocking effect similar to that of wild-type NCC, suggesting that the blocking effect is not associated with the transport activity of NCC. Consistent with this result, the presence of the NCC blocker hydrochlorothiazide (100 µM) in the culture medium and in the uptake solution did not affect the blocking effect of NCC on the positive regulatory effect of WNK4 on TRPV5 (data not shown).) M( J% U4 J* d; X2 q+ ^2 @! M
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Patients carrying the Q565E mutation exhibit hypercalciuria ( 21 ); however, this mutant exhibited a similar ability to upregulate TRPV5 in X. laevis oocytes ( Fig. 6 ). In contrast, the Q565E mutant has been shown to be less effective in blocking NCC ( 33, 36 ). This was likely due to an increase in the protein level of NCC in the presence of the Q565E mutant compared with the wild-type WNK4. Because of the dose-dependent effect of NCC on blocking WNK4, an increase in NCC protein level will result in a stronger blocking effect on WNK4 and, in turn, a reduced Ca 2 transport mediated by TRPV5. Indeed, although wild-type WNK4 and the Q565E mutant increased TRPV5's activity to a similar extent in the absence of NCC, WNK4's regulatory effect on TRPV5 was significantly reduced by the Q565E mutation in the presence of NCC: 22.4 ± 4.3% vs. 4.9 ± 2.9% ( P 0 D3 x" n4 D+ k+ c) ~- \% F# y1 C, t+ E1 b+ p/ I
DISCUSSION/ q$ b% A0 T* g/ E7 V5 V1 Y
: t2 x% N; y) m! O% V3 G `4 xIn this study, we have demonstrated that WNK4 enhanced TRPV5-mediated Ca 2 uptake in X. laevis oocytes by increasing the surface level of TRPV5. This is in striking contrast to the inhibitory effects of WNK4 on NCC ( 33, 36 ), the ROMK channel ( 16 ), and TRPV4 ( 7 ). Furthermore, the positive effect of WNK4 on TRPV5 could be blocked by NCC dose dependently. Disease-causing mutants of WNK4 tested in this study remained capable of enhancing TRPV5 to an extent similar to wild-type TRPV5. However, in the presence of NCC, the WNK4 Q565E mutant exhibited an impaired ability to enhance TRPV5 compared with wild-type WNK4, suggesting reduced Ca 2 reabsorption under the disease condition.4 J# x, A2 k/ r E) q9 B$ \- t
. U$ ?# H6 V4 b0 a# z# LThe effect of WNK4 on TRPV5 exhibited a certain degree of specificity ( Fig. 1 B ). WNK4 did not significantly upregulate the closest TRPV5 homolog TRPV6, which is also expressed in the distal tubule. On the other hand, WNK1, a member of the WNK protein kinase family, had no significant effect on TRPV5, although it blocked the effect of WNK4 on TRPV5 to some extent. The specific effect of WNK4 on TRPV5 implies that this regulation is due to the intrinsic properties of TRPV5 and WNK4, rather than the exogenous expression in X. laevis oocytes. The opposite effects of WNK4 on TRPV5 and NCC observed in the oocyte system may therefore reflect the situation in the distal tubule.* n6 L2 r. u% W
6 `/ Q$ X# U# l- C0 J, W0 N3 v$ @' TIn addition to TRPV5 and NCC, the ROMK channel, the Na -K -Cl - cotransporter NKCC1 in the Cl - -secreting epithelia and the anion exchanger SLC26A6 (CFEX) in the intestine and pancreatic duct have been reported to be regulated by WNK4 ( 14, 16 ). TRPV4, a homolog of TRPV5, has also been recently shown to be inhibited by WNK4 ( 7 ). Among these transporters and ion channels, TRPV5 is the only ion channel that is upregulated by WNK4. The ability of WNK4 to regulate ion transporters/channels in different directions in the same experimental system also suggests that the direction of regulation resides in the transporter or the ion channel protein itself.8 A* n7 h' y' ^
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The mechanism by which WNK4 regulates the abundance of TRPV5 on the plasma membrane remains to be investigated. Regulation of surface abundance is a common feature of the action of WNK4 on ion channels and transporters, yet the mechanisms underlying effects of WNK on different ion transport proteins differ. The effect of WNK4 on NCC appears to be related to its kinase activity, because the "kinase-dead" WNK4 mutant abolished the regulation of WNK4 on NCC ( 33 ). Meanwhile, the regulation of the ROMK channel by WNK4 was not altered by the kinase-dead mutation; it was mediated by the clathrin-dependent endocytosis pathway ( 16 ). In addition, claudins appeared to be phosphorylated by WNK4 ( 35 ). The action of WNK4 on TRPV5 was abolished by a kinase-dead mutation ( Fig. 5 ), a feature shared with its action on NCC. However, a recent study indicates that the carboxy-terminal 222 amino acids of WNK4 outside the kinase domain are sufficient to inhibit NCC ( 37 ). Therefore, it is likely that the kinase-dead mutation renders the inhibitory domain of WNK4 inaccessible to NCC. Therefore, it is uncertain whether the regulation of TRPV5 by WNK4 is related to the kinase activity of WNK4. An alternative explanation could be that a domain required for the regulatory effect becomes inaccessible in the kinase-dead mutant. Preliminary results (unpublished observation) indicated that neither the kinase domain nor the carboxy-terminal region of WNK4 alone is capable of upregulating TRPV5, suggesting that the domain regulating TRPV5 may be different from that regulating NCC. In support of this idea, some disease-causing mutants of WNK4 exhibited impaired inhibitory effects on NCC ( 33, 36 ), while they retained their abilities to enhance TRPV5 ( Fig. 6 ). A recent study suggests that WNK4 enhances degradation of NCC through a lysosomal pathway in mammalian cells ( 2 ). This sheds new light on the cellular mechanisms of WNK4-mediated regulation of ion channels and transporters. The increase of a mature form of TRPV5 on the plasma membrane by WNK4 ( Fig. 3 C ) might be a result of increases in TRPV5 biosynthesis, delivery to the plasma membrane, or reduced degradation and retrieval of TRPV5 from the plasma membrane. WNK4 may play a key role in regulating one or more of these processes. Further studies are required to address how WNK4 affects TRPV5 differently from other ion channels and transporters.. Q- a" s+ a" Z+ h/ _2 |9 Z
4 `) B* g6 g2 s, E; g7 aMost recently, it has been shown that WNK1 and WNK4 phosphorylate the Ste20-related serine/threonine kinases SPAK and OSR1 ( 31 ) and, in turn, regulate the cation-Cl - cotransporters NKCC1, KCC2, and NCC ( 8 ). It is unknown whether TRPV5 could be regulated by these Ste20-related kinases and whether the effect of WNK4 on TRPV5 is mediated through these kinases, because the extent to which Ste20-related kinases are expressed in X. laevis oocytes endogenously is unclear. On the basis of the ability of the carboxy terminal without the kinase domain to regulate NCC ( 37 ), it is likely that WNK4 may regulate ion transport proteins at least in part independently of Ste20-related kinases. Nevertheless, it would be of interest to determine the roles of Ste20-related kinases in WNK4-mediated regulation of TRPV5 in future studies. : q0 N; I: b, B" I- P: N7 D% |; m7 O3 j; ]9 w* W# y
One of our motivations to study the effect of WNK4 on TRPV5 is that FHH patients carrying the WNK4 Q565E missense mutation exhibit hypercalciuria and low bone density ( 22 ), whereas patients carrying WNK1 intronic deletion mutations show normocalciuria ( 1 ). In the Israeli kindred with the Q565E WNK4 mutation, the urinary Ca 2 level of the 17 affected members was much higher than that of the 16 unaffected members (0.85 ± 0.27 vs. 0.28 ± 0.12 mmol Ca 2 /mmol creatinine) ( 21 ). In the French kindred with a WNK1 gene intronic deletion, however, there was no significant difference in urinary Ca 2 levels ( 1 ). The affected members of the Israeli family also exhibited lower serum Ca 2 levels than the unaffected members. Mayan and colleagues ( 21 ) suggested that the Ca 2 leak in the WNK4 Q565E patients could be a direct effect of the WNK4 Q565E mutation on a Ca 2 channel or transporter. Our result shows that disease-causing mutants E562K, D564A, Q565E, and R1185C retained their ability to regulate TRPV5 at levels comparable to wild-type WNK4 ( Fig. 6 ). On the basis of these results, it is unlikely that a direct action of the disease-causing WNK4 Q565E mutant on TRPV5 is responsible for the hypercalciuria observed in patients carrying this gene mutation (see below). However, this does not exclude the possibility that WNK4 mutations directly affect transcellular Ca 2 transport in the distal tubule. Other players in the transcellular Ca 2 transport pathway in the distal tubule, e.g., Na /Ca 2 exchanger 1 (NCX1) and calbindin D 28k, may be affected by disease-causing WNK4 gene mutations. The effects of WNK4 and its disease-causing mutants on these Ca 2 -transporting proteins remain to be determined. ' p& G5 B% C& b. g, A X' l5 X" x3 y$ q" e$ ]9 d7 ^; L
An interesting finding in this study is that NCC blocks the effect of WNK4 on TRPV5. The blocking effect is increased by an increase in the amount of NCC. This raises the possibility that an alteration of NCC level could affect TRPV5-mediated Ca 2 transport through the action of NCC on WNK4. The Q565E mutant of WNK4 is less capable of inhibiting NCC ( 33, 36 ): the protein level of NCC might be increased in the presence of the Q565E mutant compared with wild-type WNK4. This should lead to a stronger blocking effect on the action of WNK4 on TRPV5. Indeed, when NCC was coexpressed, TRPV5-mediated Ca 2 uptake was significantly lowered in the presence of the Q565E mutant compared with that in the presence of wild-type WNK4, despite their similar abilities to enhance the level of TRPV5 in the absence of NCC ( Fig. 7 C ). The effect in our oocyte experimental system was small. In the DCT, the amplitude of this effect should depend on relative abundance of the proteins involved. Because every 2 h all Ca 2 in the circulation is filtered and reabsorbed once, even a small effect could accumulate to have a greater effect. For instance, only 5-10% of Ca 2 load is reabsorbed in the DCT, yet mice lacking TRPV5 exhibited a sixfold increase in urinary Ca 2 excretion ( 12 ). Thus a small blockage of TRPV5 in the presence of the Q565E mutation could result in a more pronounced increase in urinary excretion. To some extent, this may contribute to the hypercalciuria observed in the FHH patients carrying the Q565E mutation.* a* F' S# n: Y+ x$ [& I9 ]! a- t. {
% s# U/ {, r, [. |" YIt is well known that Ca 2 and Na transport in the DCT are inversely related ( 6 ). TRPV5 is most abundantly expressed in the latter segment of DCT (DCT2) and is prominently localized on the apical membrane; the abundance decreases downward and the localization of TRPV5 progressively shifts to intracellular in the connecting tubule (CNT) ( 19 ). NCC is localized in both segments of the DCT and is gradually decreased toward the CNT ( 3, 20 ). The architecture of the DCT and the distribution of ion transporters in the DCT vary among species ( 18, 27 ). The human DCT resembles the mouse and rat DCT but differs from the rabbit DCT, which exhibits an abrupt transition between the DCT and the CNT in ion transporter expression ( 18 ). NCC and TRPV5 are coexpressed in DCT2 in mouse and rat, but not in rabbit ( 18 ). The distribution of TRPV5 in human kidney is not available; however, NCC appears to be expressed in DCT as well as in CNT ( 23 ). Therefore, overlapping distribution of NCC with TRPV5 is also expected in the human distal tubule. The ability of WNK4 to regulate TRPV5 and NCC in opposite directions suggests that WNK4 is an important modulator of Na and Ca 2 transport in DCT2, where NCC and TRPV5 are colocalized. In addition to a potential functional interaction between NCC and TRPV5 via alteration of intracellular Cl - by NCC and, in turn, electrical potential difference across the apical membrane, which affects TRPV5-mediated Ca 2 transport, protein abundance of NCC in DCT2 is likely an important factor in WNK4-mediated regulation of TRPV5. This is consistent with the observation that mice lacking NCC exhibit hypocalciuria ( 29 ). Thus an exquisite cross-talk mechanism between these transporters and their regulators likely exists in this important portion of the nephron. Our results provide evidence of interregulation between TRPV5 and NCC through their common regulator WNK4. Further studies are necessary to illustrate the detailed molecular mechanism of this cross talk, which should prove important in understanding the pathogenesis of relevant diseases such as FHH. 4 B2 l& `9 j. y* M: X- [: E4 x P5 j+ O; Q
GRANTS 5 T# [9 x- v* V* G$ `: |3 m, d: r; R/ U
This work was supported by American Heart Association Scientist Development Grant 0430125N. {5 I9 l ]5 N3 u& V5 s
% Q7 E1 F) F/ j4 _ACKNOWLEDGMENTS ! R4 J7 [1 q+ \8 U" @ G' C( J' l. F 6 u3 |" D6 W1 X0 NThe authors thank Drs. Anupam Agarwal and David G. Warnock for support, assistance, ongoing input, and critical reading of the manuscript, Shawn Williams for technical assistance with confocal microscopy, Dr. Xavier Jeunemaitre for the WNK4 cDNA, and Dr. Melanie Cobb for the WNK1 cDNA. : N [8 b% I$ A* K- r; g7 Q4 _$ G. O7 u 【参考文献】 8 b( C: u( @3 Q) T; \! i; | Achard JM, Warnock DG, Disse-Nicodeme S, Fiquet-Kempf BB, Corvol P, Fournier A, Jeunemaitre X. Familial hyperkalemic hypertension: phenotypic analysis in a large family with the WNK1 deletion mutation. Am J Med 114: 495-498, 2003. % i' E8 A' \* m5 N# _, Z, ?2 C/ g I% W8 @* l) r/ k
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