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Molecular And Genomic |
1 Department of Molecular and Cellular Physiology
2 Departments of Genetics, Medicine, and Molecular Biophysics & Biochemistry, and Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06510, USA
3 Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, PA 15261, USA
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Abstract |
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(Received 22 November 2005;
accepted after revision 14 December 2005;
first published online 15 December 2005)
Corresponding author S. Hebert: Department of Molecular and Cellular Physiology, Yale University School of Medicine, New Haven, CT 06510, USA. Email: steven.hebert{at}yale.edu
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Introduction |
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Insights into the molecular mechanisms underlying the kidney's ability to maintain NaCl and K+ homeostasis have been obtained in part by studying rare Mendelian diseases that show derangements in renal NaCl and K+ handling as their primary phenotypes (Lifton et al. 2001). Recent genetic analysis identified WNK1 and WNK4 (encoded by PRKWNK1 and PRKWNK4, respectively) as kinases with the properties of integrative regulators of several renal ion transport pathways (Wilson et al. 2001). A unique substitution of cysteine for lysine at a highly conserved residue in the catalytic domain characterizes the WNK kinase family (hence WNK, with no lysine (=K)) (Xu et al. 2000; Min et al. 2004). Only four WNK kinases exist in the human genome (Wilson et al. 2001; Verissimo & Jordan, 2001); each shares significant homology in the serine–threonine kinase domain, an autoinhibitory domain, two putative coiled-coil domains, and a short acidic domain. Mutations in PRKWNK4, encoding WNK4, cause pseudohypoaldosteronism type II (PHAII), an autosomal dominant disease featuring hypertension and hyperkalaemia due to a coupled increase in renal NaCl reabsorption and deficiency in renal K+ secretion (Wilson et al. 2001). Physiological analysis has shown that wild-type WNK4, via catalytic and non-catalytic mechanisms, inhibits NCC and ROMK1, and activates paracellular Cl– flux (Wilson et al. 2003; Yang et al. 2003; Kahle et al. 2003, 2004a,b; Yamauchi et al. 2004; Golbang et al. 2005). PHAII-causing missense WNK4 mutations cluster within a 10 aa acidic domain conserved among all WNKs; these mutations have sharply divergent effects on these downstream targets. PHAII-causing mutations eliminate inhibition of NCC and augment paracellular Cl– conductance, while simultaneously enhancing inhibition of ROMK1. Together, these effects help to explain the increase in NaCl reabsorption and the decrease in K+ secretion seen in affected PHAII patients (Wilson et al. 2003; Yang et al. 2003; Kahle et al. 2003, 2004a,b; Yamauchi et al. 2004; Golbang et al. 2005). Intronic deletions that increase expression of WNK1 also cause PHAII (Wilson et al. 2001). Recent evidence has shown that WNK1 can reverse WNK4's inhibition of NCC (Yang et al. 2003) and increase the activity of ENaC (Naray-Fejes-Toth et al. 2004; Xu et al. 2005) in oocytes and mammalian cells.
We recently demonstrated that WNK3, a novel WNK kinase family member, localizes to ion-transporting epithelia and GABAergic neurones (Rinehart et al. 2005; Kahle et al. 2005). WNK3 stimulates SLC12A cotransporters that drive Cl– influx (NCC and the Na+–K+–2Cl– cotransporters), but inhibits SLC12A cotransporters that drive Cl– efflux (K+–Cl– cotransporters KCC1 and KCC2) (Rinehart et al. 2005; Kahle et al. 2005). Silencing WNK3's kinase activity reverses its effect at each of these targets. WNK3's ability to regulate multiple ion transporters that are coexpressed and coordinately function in the same cells (e.g. NKCC1 and KCC1 in epithelial and red blood cells, NKCC1 and KCC2 in GABAergic neurones) suggests it is a key integrator of these transport systems (Rinehart et al. 2005; Kahle et al. 2005). In this report, we show that WNK3 is expressed in CD principal cells and specifically regulates the kidney-specific K+ channel ROMK1 in Xenopus laevis oocytes. These data, along with the known action of WNK3 on the renal-specific NCC, suggest WNK3 is a component of a regulatory signalling pathway that determines the balance between renal NaCl reabsorption and K+ secretion.
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Methods |
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Antibodies used were: anti-WNK3 (Rinehart et al. 2005; Kahle et al. 2005), anti-ZO-1 (Choate et al. 2003), anti-aquaporin-2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), and affinity-purified secondary antibodies conjugated to the CY2 (green) and CY3 (red) fluors (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA).
Immunolocalization
Animal studies are in accordance with the humane practices of animal care established by the Yale Animal Care and Use Committee. Mice were killed by cervical dislocation and excised kidneys were prepared and sectioned as described (Choate et al. 2003). Slides were stained with primary and secondary antibodies, and visualized by immunofluorescence microscopy as described (Choate et al. 2003). Results were consistent in three different mice.
cDNA constructs pSPORT–ENaC-
, pSPORT–ENaC-ß, and pSPORT–ENaC-
have been previously described (Shimkets et al. 1997). WNK3 (pGH19–WNK3), kinase-inactive WNK3 (pGH19–WNK3 kin–), and a PHAII-like WNK3 mutant (pGH19–WNK3 PHAII) have been previously described (Rinehart et al. 2005; Kahle et al. 2005). pSPORT–eGFP–ROMK1 was previously published (Kahle et al. 2003). WNK3 was subdivided into the kinase domain (KD, bases 1–1251) and the regulatory domain (RD, bases 1252–5229). Each piece was subcloned into pGH19 with 5'-EcoRI and 3'-Xho1 restriction sites.
Paracellular studies
A cDNA encoding full-length human WNK3 (Rinehart et al. 2005; Kahle et al. 2005), tagged at the carboxyl terminus with haemagglutinin (HA), was subcloned into the pTRE2hyg expression vector (BD Biosciences Clontech, Palo Alto, CA, USA) and transfected into MDCK II tet-off cells (Clontech). Clones showing absence of WNK3 expression in the presence of doxycycline and robust expression following the elimination of doxycycline were chosen for further study. Current–voltage (I–V) relationships were acquired by voltage-clamping monolayers between –50 and +50 mV, and transepithelial resistance (TER) was calculated from Ohm's law. Dilution potential measurements were obtained as described (Kahle et al. 2004b). The ion permeability ratio (PCl/PNa) for the monolayer was calculated from the dilution potential by using the Goldman-Hodgkin-Katz equation. The absolute permeabilities of Na+ (PNa) and Cl– (PCl) were calculated by using a simplified Kimizuka-Koketsu equation. Transepithelial resistance (TER) and dilution potential measurements were performed in triplicate on each of three different wild-type WNK3 clonal cell lines in at least three independent experiments; results from independent clones showed no significant differences and were treated as independent measures. TER and dilution potentials are shown as raw data from a representative experiment. All data are expressed as means ±S.E.M., and statistical comparisons between groups are made by paired Student's t test (induced versus uninduced).
Functional assays with ENaC
Oocytes were injected with the cRNA of ENaC, ß, and subunits alone or together with wild-type WNK3 cRNA, incubated for 2–3 days, and amiloride-sensitive whole-cell Na+ currents were measured by two-electrode voltage clamp as previously described (Shimkets et al. 1997). To prevent Na+ loading and therefore prolong oocyte viability, oocytes were kept in incubation solution containing 1 µM amiloride prior to experiments. ENaC currents were calculated as the difference in whole-cell current before and after the addition of 10 µM amiloride to the bathing solution at a holding membrane potential of 100 mV. Results are expressed as the mean ±S.E.M. and statistical comparisons between groups were made by a paired t test. Data of a representative experiment are shown. Two separate experiments using oocytes from different frogs yielded similar results.
Functional assays with ROMK1
Oocytes were injected with ROMK1 cRNA alone or together with wild-type or mutant WNK3 cRNAs, incubated for 2–3 days, and Ba2+-sensitive whole-cell K+ currents were measured by two-electrode voltage clamp as previously described (Kahle et al. 2003). All reported K+ currents refer to Ba2+-sensitive currents. For I–V plots, K+ currents at each voltage represent the mean ±S.E.M. of at least seven oocytes for each group from a representative experiment. For statistical comparisons of K+ currents between groups of oocytes, we analysed K+ currents at +40 mV using two-tailed Student's t tests; these comparisons included data from three independent experiments from different frogs. In each experiment, > seven oocytes were analysed for each group.
Functional assays with KCNQ1/KCNE1
Oocytes were injected with KCNQ1 cRNA alone or together with KCNE1 and/or WNK3 cRNAs, incubated for 2 days, and whole-cell K+ currents were measured by two-electrode voltage clamp as previously described (Kahle et al. 2003). For I–V plots, K+ currents at each voltage represent the mean ±S.E.M. of at least seven oocytes for each group from a representative experiment. For statistical comparisons of K+ currents between groups of oocytes, we analysed K+ currents at +40 mV using two-tailed Student's t tests; these comparisons included data from three independent experiments from different frogs. In each independent experiment, > seven oocytes were analysed for each injected group.
Membrane surface expression studies
Oocytes were injected with enhanced green fluorescent protein (eGFP)-tagged ROMK1 cRNA alone or together with wild-type or mutant WNK3 cRNAs, incubated for 2 days, and membrane surface expression of eGFP–ROMK1 was assayed by laser-scanning confocal microscopy as previously described and validated (O'Connell et al. 2005). Total membrane fluorescence intensity was calculated for each imaged oocyte using SigmaScan Pro software (Systat Software Inc., Point Richmond, CA, USA) as previously described (Wilson et al. 2003). Four total experiments were performed; > 15 oocytes were injected per experimental group, and each experiment used oocytes from a different frog. A representative experiment is shown. The significance of differences between groups was assessed by two-tailed Student's t test.
Cell-attached patch-clamp analysis
After injection, oocytes were immersed in a hyperosmotic solution for 1–2 min (mM: 200 N-methyl-D-glucamine, 2 KCl, 1 MgCl2, 10 EGTA, 10 Hepes, adjusted to pH 7.4 with HCl) and vitelline membranes were removed using forceps. Electrodes were pulled from borosilicate glass capillaries (Sutter Instrument Co., Novato, CA, USA) on a Narishige PP-83 (Japan) and polished (Narishige microforge, MF-83, Japan). Electrodes had a tip resistance of 8–9 M
when filled with pipette solution (mM: 150 KCl, 1.0 MgCl2, 1.0 CaCl2, 5 Mes/Tris, adjusted to pH 7.4 with either Mes or Tris buffer). Mg2+-free bath solutions were used in all experiments and perfused by a multibarrel quick-exchange solution system (Model SF-77B, Warner, Hamden, CT, USA). Bath solutions contained (mM) 150 KCl, 2 EDTA, 10 Mes/Tris, and were adjusted to pH 7.4 with Mes or Tris. Single channel currents were recorded in the cell-attached configuration by an EPC-7 amplifier (Heka Elektronik, Lambrecht/Pfalz, Germany) and passed through an 8-pole Bessel filter at 1000 Hz (Warner Instruments, Hamden, CT, USA). Data were acquired via a DigiData 1200 Series digital-to-analog converter (Axon Instruments, Union City, CA, USA), driven by commercial software (pCLAMP Clampex 8.20, Axon Instruments). Currents were analysed off-line with Clampfit 8.20 (Axon Instruments), Fetchan 6.04 (Axon Instruments), and GraphPad Prism 4 (GraphPad Software Inc., San Diego, CA, USA).
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Results |
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Previous work reported that WNK3 is expressed along the length of the nephron (Rinehart et al. 2005). To explore the localization of WNK3 in the CD in more detail, we co-stained mouse kidney sections with a specific anti-WNK3 antibody together with an antibody that serves as a marker of the cortical (CCD) and medullary (OMCD) CD. WNK3 is expressed in both of these tubule segments (Fig. 1). In the CCD and OMCD, WNK3 expression is greater in principal cells than in intercalated cells, as demonstrated by more intense WNK3 immunostaining in cells that express AQP2 (a principal cell marker) versus non-AQP2-expressing cells (i.e. intercalated cells; Fig. 1A–D). In both CCD principal cells and OMCD cells, WNK3 predominantly localizes to intercellular junctions, demonstrated by co-staining experiments with an antibody against zona-occludens-1 (ZO-1), a tight-junction marker (Fig. 1E–G). Because WNK3 is expressed in CD principal cells, and other WNK kinases regulate distal nephron NaCl and K+ transport pathways, we investigated whether WNK3 regulates ENaC, the paracellular pathway, and/or ROMK1, mediators of NaCl and K+ handling in CD principal cells.
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Type II Madin-Darby canine kidney cells (MDCK II) are a useful model to study the intrinsic properties and regulation of paracellular ion flux because of their low transcellular ionic conductance and relatively ‘leaky’ (high conductance) tight junctions (Van Itallie & Anderson, 2004). WNK4 stimulates paracellular Cl– conductance in tetracycline-responsive MDCK II cells, where it physically associates with, and phosphorylates, claudins – pore-forming tight junction proteins (Yamauchi et al. 2004; Kahle et al. 2004b). Since WNK3, like WNK4, is expressed at tight junctions in the CD, we generated MDCK II cells with tetracycline-responsive WNK3 expression and tested the effects of WNK3 on cell monolayer transepithelial resistance and dilution potential, indices of paracellular ion conductance and selectivity, respectively (Fig. 2A–D). Similar to WNK4 (Yamauchi et al. 2004; Kahle et al. 2004b), WNK3 localizes to cell–cell borders in MDCK II cells (Fig. 2B and C). However, in contrast to WNK4 (Kahle et al. 2004b), induction of WNK3 expression does not alter the paracellular properties of MDCK II monolayers (Fig. 2D). Thus, WNK3 is not a modulator of paracellular ion flux in MDCK II cells.
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WNK1 increases the activity of ENaC in Xenopus laevis oocytes and mammalian cells (Xu et al. 2005; Naray-Fejes-Toth et al. 2004); there are no reports of ENaC regulation by WNK4. We directly tested whether WNK3 regulates ENaC activity in a coexpression assay in oocytes, a widely used system for characterizing the function and regulation of this Na+ channel (Shimkets et al. 1997; Valentijn et al. 1998; Friedrich et al. 2003). In the absence of coexpressed , ß, and ENaC subunits, amiloride-inhibitable Na+ current in oocytes was very low (< 0.1 µA; Fig. 3A). Co-expression of the three ENaC subunits without WNK3 gave a significant amiloride-sensitive Na+ current (Fig. 3A); however, this Na+ current was unaffected by coexpression of WNK3 (Fig. 3A). Western blots of homogenates from oocytes co-injected with ENaC and WNK3 revealed robust WNK3 expression (data not shown). We conclude that WNK3 does not affect ENaC activity in oocytes.
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We tested the effect of WNK3 on the activity of ROMK1 by performing co-expression studies in oocytes. Injection of cRNA encoding ROMK1 tagged with enhanced green fluorescent protein (eGFP–ROMK1) produced a large Ba2+-sensitive K+ whole-cell current that was markedly inhibited by addition of WNK3 (Fig. 4A–D; 
5.5-fold reduction at +40 mV, P < 0.0001). We observed comparable inhibition with injection of untagged ROMK1 (data not shown), as eGFP–ROMK1 and untagged ROMK1 have identical functional properties (O'Connell et al. 2005).
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2-fold greater inhibition relative to wild-type WNK3 at +40 mV, P < 0.0001). These findings indicate that inhibition of ROMK1 by WNK3 does not require WNK3 kinase activity, and that this Asp mutation enhances WNK3's effect on ROMK1. Kinases alter ROMK1 activity by altering its single channel properties and/or by reducing the number of channels at the cell surface (Hebert et al. 2005). To investigate the mechanism for WNK3's action on ROMK1, we first quantified membrane surface expression of eGFP–ROMK1 in oocytes in the presence or absence of WNK3. Wild-type WNK3 caused a marked reduction in expression of eGFP–ROMK1 at the plasma membrane (Fig. 4E–F; P < 0.0001). This reduction was not due to diminished total cellular expression of ROMK1, because Western blotting of whole-oocyte homogenates showed no difference in levels of eGFP–ROMK1 in the presence of WNK3 (data not shown). In concordance with the current data, kinase-inactive WNK3 reduced eGFP–ROMK1 surface expression more than wild-type kinase (Fig. 4E–F; 2-fold greater inhibition than wild-type WNK3, P < 0.0001). Consistent with WNK3 inhibiting ROMK1 primarily through protein trafficking, WNK3 did not alter ROMK1 single channel properties (conductance or open/closed probability), as determined by single-channel patch clamping experiments in oocytes (Fig. 5). These data show that the effect of wild-type and mutant WNK3 on ROMK1 activity can be fully accounted for by alteration of the expression of this channel at the plasma membrane.
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2-fold greater inhibition of K+ current (P < 0.0001) and 2-fold greater reduction in surface expression (P < 0.0001) compared with wild-type WNK3). This corresponds to an overall 85% reduction in K+ current and 90% reduction in surface expression compared with oocytes expressing eGFP–ROMK1 alone (Fig. 4C). These data demonstrate for the first time the importance of the conserved acidic domain in members of the WNK family other than WNK4. Recently, Yang et al. (2005) demonstrated that WNK4's inhibitory effect on NCC is mediated by the WNK4 carboxyl terminus (Yang et al. 2005). We explored whether WNK3's effect on ROMK1 may similarly depend on its C-terminal domain. To investigate this possibility, we constructed a WNK3 mutant that contains the WNK3 N-terminal kinase domain only (aa 1–417; WNK3-KD) or the WNK3 C-terminal regulatory domain only (aa 418–1742; WNK3-RD). WNK3-RD inhibited ROMK1 K+ currents similar to wild-type WNK3 (Figs 6, P < 0.0001). In contrast, WNK3-KD lost its ability to inhibit ROMK1 activity (Fig. 6). These data demonstrate that the WNK3 C-terminal regulatory domain is sufficient to confer WNK3's inhibitory effect on ROMK1.
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Discussion |
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Although WNK3 regulates both ROMK1 and NCC, there are considerable differences in the mechanisms of inhibition. WNK3's activation of NCC is dependent on its catalytic activity (Rinehart et al. 2005). Conversely, WNK3's inhibition of ROMK1 does not require WNK3's kinase activity, as a full-length kinase-dead WNK3 mutant and a WNK3 mutant containing only the C-terminal regulatory domain can still inhibit channel activity. These actions are reminiscent of WNK4 on these same targets: WNK4 inhibition of NCC requires its catalytic activity, while WNK4's inhibition of ROMK1 is independent of its kinase activity. Catalytic and non-catalytic functions in a single WNK kinase are not uncommon; WNK1 (Xu et al. 2005) and WNK4 (Wilson et al. 2003; Kahle et al. 2003) both have effects mediated by protein–protein interactions independent of their catalytic activities at downstream targets. WNK3's ability to regulate an electroneutral cotransporter (NCC) and a K+ channel (ROMK1) with different effects (stimulation/inhibition) and mechanisms of regulation (catalytic and non-catalytic) suggest a means to achieve independent modulation of ROMK1 and NCC activities.
Since WNK3 alters the expression of ROMK1 at the plasma membrane, but not total ROMK1 protein expression, we hypothesize WNK3 is either enhancing retrieval of channels from the cell surface or inhibiting the forward trafficking of newly synthesized channels to the membrane. WNK4 inhibits ROMK1 activity by increasing the clathrin-coated pit-mediated endocytosis of channels from the plasma membrane (Kahle et al. 2003). WNK3 could be functioning in parallel to or in series with WNK4, facilitating the internalization of K+ channels in a clathrin-dependent manner. Conversely, WNK3 could affect forward-trafficking of ROMK1, regulating the insertion of channels into the membrane from the endoplasmic reticulum (ER) or subapical vesicle pools that contain ‘dormant’ channels. In this light, it is interesting that phosphorylation of serine 44 in the cytoplasmic N-terminus of ROMK1 modulates channel surface expression by increasing channel delivery to the plasma membrane consequent to the suppression of a C-terminal ER retention signal (O'Connell et al. 2005). This phosphorylation switch of the ROMK1 ER retention signal could provide a pool of mature and properly folded channels for rapid delivery to the plasma membrane (O'Connell et al. 2005). WNK3, in a manner independent of its catalytic activity, might facilitate the dephosphorylation of serine 44 or another critical serine/threonine residue to retain ROMK1 in the ER, perhaps via association with phosphatases. Indeed, catalytically inactive WNK3 decreases the phosphorylation of NKCC1 on threonines that are necessary for the plasmalemmal translocation of this cotransporter (Kahle et al. 2005). These issues will require future investigation.
The effects of the WNK3 kinase-inactivating point mutation on ROMK1 and NCC are intriguing, and may mimic a normal in vivo phenomenon, perhaps achieved by a kinase regulator binding to or dissociating from WNK3 (Fig. 8). In its active state, WNK3 could stimulate NCC-mediated NaCl reabsorption and inhibit ROMK1-mediated K+ secretion; inactive WNK3 might simultaneously inhibit NCC and increase ROMK1 inhibition. When activated, WNK3 may be important for restoring intravascular volume while simultaneously defending serum K+ in the presence of high circulating aldosterone – thus controlling two effects of aldosterone (NCC NaCl reabsorption and ROMK1 K+ secretion) separately. In its inactive state, WNK3 may mediate basal repression of these NaCl reabsorption and K+ secretion pathways. Identifying the proximate factors that alter WNK3 activity in vivo, and thereby modulate these different functional states, will be important in future work. The similar effects of the PHAII-causing mutation in WNK4 and the homologous ‘PHAII-like’ mutation in WNK3 compared to kinase-dead WNK3 on ROMK1 (all mutants show enhanced channel inhibition relative to wild-type kinase) suggest that mutations in the conserved acidic domain in WNKs might alter catalytic activity. Moreover, the ‘availability’ of the acidic domain may also be regulated by interactions with other proteins, and this interaction could alter the ability of the WNK3 kinase to regulate ROMK1.
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It is likely that WNK3 is a component of a WNK1/WNK4 signalling network necessary for renal NaCl and K+ homeostasis. Different members of the WNK family can interact, both physically and catalytically (Lenertz et al. 2005). For example, WNK1 has been shown to phosphorylate WNK4 and impair WNK4's regulation of NCC (Yang et al. 2003; Yang et al. 2005) and WNK1 and WNK3 have been found in the same physical complex in human cells (Vitari et al. 2005). The fact that WNK3 affects some downstream targets differently from other WNKs (e.g. WNK3 stimulates NCC, WNK4 inhibits NCC; WNK3 has no effect on ENaC, WNK1 stimulates ENaC), but has similar effects at other targets (both WNK3 and WNK4 inhibit ROMK1), suggests that different WNKs may be recruited individually or in combination to execute a very specific response to a discrete physiological stimulus. Moreover, the multitiered nature of the WNK signalling pathway, which includes the related STE20-family kinases OSR1 and PASK (Vitari et al. 2005; Gagnon et al. 2006), could provide a means for amplification and transduction of upstream signals into rapid and robust downstream effects.
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Footnotes |
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 A. Zagorska, E. Pozo-Guisado, J. Boudeau, A. C. Vitari, F. H. Rafiqi, J. Thastrup, M. Deak, D. G. Campbell, N. A. Morrice, A. R. Prescott, et al. Regulation of activity and localization of the WNK1 protein kinase by hyperosmotic stress J. Cell Biol., January 1, 2007; 176(1): 89 - 100. [Abstract] [Full Text] [PDF]
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J. L. Pluznick and S. C. Sansom BK channels in the kidney: role in K+ secretion and localization of molecular components Am J Physiol Renal Physiol, September 1, 2006; 291(3): F517 - F529. [Abstract] [Full Text] [PDF]
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closed) was 1.52 ± 0.05 ms (n= 3) and the mean open time (
), and 34.4 ± 1.2 pS (n= 5) for eGFP–ROMK1 plus WNK3 (•) when measured between –100 and –40 mV. These data show that WNK3 does not alter the conductance or open/closed probability of ROMK1, and suggest that ROMK1 regulates ROMK1 primarily through protein trafficking, because WNK3 does not affect the total protein expression of ROMK1 in oocyte homogenates as detected by Western blotting (data not shown).
