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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
Departments of Anesthesiology, Pharmacology, and Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee
Submitted 29 May 2007 ; accepted in final form 13 June 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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cell volume regulation; osmotic stress; osmoregulation
With no lysine (K) (WNK) protein kinases are serine/threonine protein kinases that lack a conserved lysine residue in the catalytic domain (57, 62). Humans have four WNK kinases, and rare mutations in WNK1 and WNK4 are linked to pseudohypoaldosteronism type II, an autosomal dominant form of hypertension (61). Common polymorphisms of WNK1 contribute to blood pressure variation in the general population (54). WNK1 and WNK4 control blood pressure by regulating the activity of ion transport pathways that mediate salt transport in distal renal tubules (27, 33). Several recent studies have demonstrated that WNK1 and WNK4 bind to, phosphorylate, and activate PASK and OSR1 (2, 19, 40, 58, 59).
The nematode C. elegans provides many advantages for identifying and characterizing components of cell volume and epithelial transport signaling pathways (3, 51). C. elegans inhabits surface soil and decaying organic matter that undergo periodic desiccation. Under laboratory conditions, worms can readily acclimate to growth agar containing up to 500 mM NaCl (30, 34). Because nematodes lack a rigid skeleton and are highly permeable to water, they initially lose up to 50% of their total body volume when exposed to extreme hypertonic stress. If the stress is nonlethal, the worms recover their volume within a few hours by yet to be characterized mechanisms (34).
We demonstrate here that GCK-3 is required for acute volume recovery and survival after exposure of C. elegans to extreme hypertonic stress. We also demonstrate that GCK-3 physically interacts with the single worm WNK kinase, WNK-1, and that the two kinases function together in a common signaling cascade. Phylogenetic analysis of kinase functional domains suggests that the interaction between GCK-VI and WNK kinases first occurred in an early metazoan. The evolution of WNK/GCK-VI signaling cascades may therefore have coincided with the need of multicellular animals to tightly regulate transepithelial transport processes that mediate systemic osmotic homeostasis. Our studies are the first to show directly the importance of GCK-VI kinases in whole animal osmoregulation, and they provide novel insights into the evolution and function of a conserved osmoregulatory protein kinase cascade, establishing a foundation for its further characterization in a genetically tractable model organism.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Generation of transgenes and transgenic worms. Transcriptional gck-3 green fluorescent protein (GFP) reporters were generated by a PCR-based method (24). Y59b::GFP was produced by PCR amplification of a 9.5-kb fragment containing the first intron of gck-3 and 7.8 kb of upstream genomic DNA (see Fig. 1A). The reverse primer in this PCR contained a 24-bp 5'-extension that is complementary to a C. elegans GFP expression vector (pPD95.75, Fire Lab Vector Kit, Adgene, Cambridge, MA). This fragment was fused with GFP and the unc-54 3'-untranslated region by a second round of PCR with nested primers. Y59a was produced by PCR amplification of a 13.7-kb fragment containing the entire gene upstream from gck-3, Y59A8B.22, and its upstream intergenic region (see Fig. 1A). Y59a and Y59b::GFP overlapped by 1.5 kb and were coinjected into N2 worms, using rol-6 (pRF4) as a transformation marker (39). A transcriptional GFP fusion containing the first intron of wnk-1 and 8 kb of upstream genomic DNA including the entire 4-kb intergenic region was generated by PCR (24) and injected into worms together with rol-6. Transgenic worms were imaged with a LSM510-Meta confocal microscope and a Plan-Neofluar x40/1.3 numerical aperture oil-objective lens (Carl Zeiss MicroImaging, Thornwood, NY).
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RNA interference. RNAi was performed by feeding worms strains of E. coli that are engineered to transcribe double-stranded RNA (dsRNA) homologous to a target gene (28). pPD128.110 is a derivative of pPD129.36 that contains the cDNA for GFP and was used as a control for nonspecific RNAi effects in all feeding experiments. The GCK-3 RNAi feeding vector was generated from a cDNA library and contained a fragment that corresponds to nucleotides 1010–1810 of gck-3 (GenBank accession no. AY741200). The WNK-1 RNAi feeding vector was obtained from a commercially available RNAi feeding library (Geneservice) and contained a fragment that corresponds to wnk-1 (GenBank accession no. NM_069202.4) nucleotides 3986–4760. BLAST searches of C. elegans genomic and expressed sequence tag databases failed to identify coding regions with nucleotide sequence homologous to the GCK-3 or WNK-1 RNAi constructs, indicating that off-target effects are unlikely.
All RNAi feeding vectors were maintained and expressed in HT115(DE3) E. coli as described previously (28). dsRNA feeding was carried out by placing eggs onto agar plates with a lawn of HT115(DE3) bacteria. The adults that developed from these eggs were then transferred to a second plate of HT115(DE3) bacteria and allowed to lay eggs overnight. The second or later generations of dsRNA-fed worms were used for all functional assays. Dual RNAi feeding experiments were performed by combining equal amounts of bacteria expressing dsRNA as described previously (1).
Quantitative PCR. Quantitative real-time PCR was used to measure knockdown efficiency as described by Choe et al. (10) with minor modifications. Total RNA was isolated from RNAi-treated worms with an RNAqueous-Micro kit (Ambion, Austin, TX), and cDNA was synthesized with Superscript III (Invitrogen, Carlsbad, CA). Quantitative real-time PCR was conducted in a Bio-Rad IQ5 Real-Time PCR system with IQ SYBR Green Supermix (Bio-Rad, Hercules, CA). The housekeeping ribosomal protein gene rpl-2 was used as an internal control.
Hypertonic stress assays.
Hypertonic NGM agar plates for RNAi studies contained 25 µg/ml carbenicillin and 1 mM isopropyl 
-D-thiogalactopyranoside to induce dsRNA synthesis plus 349 mM NaCl (400 mM total) or an osmotically equivalent amount of sorbitol (611 mM). The osmolality of the high-NaCl and sorbitol media was measured with a vapor pressure osmometer (Wescor, Logan, UT) in representative solutions that lacked agar and peptone. To measure survival, six populations of 8–17 L4 worms were transferred to hypertonic agar plates and the number of worms surviving was determined after various times for 72 h. Worms were considered alive if they displayed any muscle contractions in the pharynx or body in response to repeated prodding with an eyelash. Total body volume was measured by imaging individual worms with a Zeiss M2BIO stereo dissecting microscope (Kramer Scientific, Valley Cottage, NY) fitted with a DAGE-MTI (Michigan City, IN) CCD-100 camera. Worm width, which we define as the average of the widest points anterior and posterior to the vulva, and length were quantified with ImageJ 1.32j software (National Institutes of Health). Volume was calculated by assuming that body shape approximates a cylinder.
Yeast two-hybrid screen and glutathione S-transferase affinity assay. The bait plasmid for yeast two-hybrid screening was generated by Gateway cloning the carboxy terminus (amino acids 496–596) of GCK-3 into pDEST 32 (Invitrogen). Interactions between GCK-3 and proteins generated from a C. elegans cDNA library (Invitrogen) were identified, and prey clones were sequenced and tested for self-activation as described previously (13).
A glutathione S-transferase (GST)-GCK-3 fusion construct was generated by Gateway cloning amino acids 496–596 of GCK-3 into pDEST 27 (Invitrogen). V5 epitope-tagged WNK-1 was generated by cloning the entire open reading frame of WNK-1 (C46C2.1a) into pcDNA3.1/V5-His-TOPO (Invitrogen). Cell culture, transfection, and lysis were carried out as described previously (13). Briefly, Chinese hamster ovary (CHO) cells were cotransfected with either WNK-1 and GST or WNK-1 and GST-GCK-3 cDNAs. After 24 h, the cells were lysed and lysates were centrifuged for 20 min at 10,000 g and 4°C to remove cellular debris. Supernatants were incubated with glutathione-Sepharose 4B for 30 min, washed five times with 1 ml of PBS, resuspended in Laemmli buffer, and heated to 60°C for 60 min. V5-tagged WNK-1 was identified by Western blotting using an anti-V5 antibody (Invitrogen).
Statistical analysis. Statistical significance was determined with a Student's t-test when two means were compared and a one-way analysis of variance with a Dunnett or Tukey-Kramer post hoc test when three or more means were compared. P values of <0.05 were taken to indicate statistical significance.
Phylogenetic analysis. Basic local alignment search tool (BLAST) on the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/) was used to find the closest homologs of GCK-3 and WNK-1 in the genomes of Homo sapiens (human), Drosophila melanogaster (fly), C. elegans (worm), Arabidopsis thaliana (plant), and Saccharomyces cerevisiae (yeast). These proteins were aligned by ClustalW with Vector NTI Advance 10 (Invitrogen), and MEGA 3.1 software (32) was used to make phylogenetic trees with the neighbor-joining method and Poisson-corrected evolutionary distances (44). Branches were then tested for statistical significance by bootstrapping with 1,000 replicates. The location and homology of previously described functional domains in GCK-VI kinases (8, 40, 48, 58) and WNK kinases (62) were assessed from the ClustalW alignment.
BLAST searches of nonmetazoan eukaryote sequences (e.g., protozoans, plants, and fungi) were performed using the region around S325 (amino acids 278–346) and the interaction domain (amino acids 446–527) of human OSR1. BLAST searches for nonmetazoan eukaryotic WNKs were performed using the kinase domain (amino acids 221–479) of human WNK1.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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3.5 kb of genomic sequence upstream from the gene's start codon. About 15% of the genes in the worm genome are predicted to be expressed as multigene transcriptional units or operons (5). Characteristics of worm operons include short intergenic regions and trans-splicing of downstream mRNAs with SL2, a small nuclear ribosomal particle. Reexamination of gck-3 gene structure suggested to us that it may be part of an operon together with the nearest upstream gene, Y59A8B.22. gck-3 and Y59A8B.22 are separated by <1.7 kb of intergenic DNA, and gck-3 cDNAs are enriched with SL2 trans-spliced leaders twofold relative to SL1 (5). The presence of gck-3 in a possible operon suggested that its expression could be driven by the Y59A8B.22 promoter.
gck-3 and Y59A8B.22 together with their intergenic regions are too large to amplify as a single clone. Therefore, to assess whether gck-3 is regulated by the Y59A8B.22 promoter, we took advantage of the finding that DNA microinjected into C. elegans undergoes homologous recombination with high frequency (39). Two PCR fragments that overlapped by 1.5 kb were generated. One fragment was a fusion of GFP to the 9.5-kb region upstream from the second exon of gck-3 and was termed Y59b::GFP. The other fragment, termed Y59a, included Y59A8B.22 and its entire upstream intergenic region (Fig. 1A). We coinjected Y59b::GFP and Y59a into worms and allowed them to fuse via homologous recombination (39). Three independent, stable worm strains were generated. In addition to the excretory cell, the recombined GFP reporter was expressed in the excretory duct, hypodermis, spermatheca, body wall muscle, and nerve cord (Fig. 1, B–D). Stable lines resulting from injection of Y59b::GFP by itself expressed GFP only in the excretory cell (data not shown).
C. elegans serial analysis of gene expression (SAGE) databases (http://elegans.bcgsc.ca/home/sage.html) have also detected gck-3 expression in dissected gonad and fluorescence-activated cell sorter (FACS)-enriched populations of hypodermal cells, neurons, and muscle cells. While these results support our GFP reporter studies, definitive cellular and subcellular localization of GCK-3 will require development of suitable antibodies.
GCK-3 is required for survival during hypertonic stress. C. elegans normally inhabits surface soil and decaying vegetable matter environments that experience periodic desiccation (31). Under laboratory conditions, C. elegans is able to acclimate to and survive extreme hypertonic stress (30, 34, 35, 50, 60). The hypodermis and excretory cell play important roles in nematode osmotic homeostasis (18, 25, 46). Expression of GCK-3 in these tissues (Fig. 1, B–D) as well as recent studies demonstrating that members of the GCK-VI subfamily regulate osmotically sensitive ion transport mechanisms (2, 13, 14, 19, 40, 58, 59) suggested that GCK-3 may play a role in systemic osmoregulation.
Unfortunately, the one available mutant allele of gck-3, tm1223, is not null, and therefore is not useful for determining GCK-3 function. Analysis of this allele revealed that it is a deletion contained completely within intron 6 that does not prevent gck-3 expression (K. Choe, X. Yin, and K. Strange, unpublished results). We also attempted to isolate our own deletion allele of gck-3, but failed on three attempts. Therefore, we used RNAi to induce gck-3 knockdown. To test whether GCK-3 function is required for systemic osmoregulation, we fed RNAi-hypersensitive eri-1 worms (29) GFP or GCK-3 dsRNA-producing bacteria and quantified survival after transfer from normal NGM agar to NGM agar supplemented with 349 mM NaCl. As shown in Fig. 2A, >90% of GFP dsRNA-fed worms survived on high-NaCl agar for at least 3 days. These worms had no obvious morphological or behavioral defects and produced brood sizes that were only modestly reduced (30% reduction; data not shown) compared with worms on normal NGM. In sharp contrast, virtually all of the gck-3(RNAi) worms died within 48 h of transfer to high-NaCl agar. Mean ± SE survival of gck-3(RNAi) worms on NGM agar without added NaCl was 84 ± 2% (n = 5 populations of 8–11 worms) after 3 days. Therefore, GCK-3 is specifically required for survival of worms on hypertonic NaCl agar.
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GCK-3 regulates acute volume recovery.
When worms are exposed to hypertonic conditions, they initially shrink because of osmotic water loss (34). If shrinkage is excessive, the animals become paralyzed because of loss of turgor pressure. During a nonlethal hypertonic stress, the worms slowly recover their initial volume and regain motility (34) (Fig. 2B, top). To determine whether GCK-3 is required for acute volume recovery, we fed eri-1 worms GFP or GCK-3 dsRNA and quantified total body volume for 5 h after transfer to high-NaCl or sorbitol agar. As shown in Fig. 2, B and C, control GFP dsRNA-fed worms initially lost 30% of their total body volume in response to NaCl- or sorbitol-induced hypertonic stress. However, within 5 h, the animals had nearly fully recovered to their initial volume. In contrast, gck-3(RNAi) worms exposed to high-NaCl or sorbitol agar underwent a significantly (P < 0.02) greater initial shrinkage and showed <10% volume recovery after 5 h. The degree of volume recovery was not significantly different (P > 0.2) in response to NaCl- or sorbitol-induced hypertonic stress. These results demonstrate that GCK-3 plays an essential role in acute volume recovery after osmotically induced water loss. After 24 h, most gck-3(RNAi) worms had regained mobility on the high-sorbitol plates, indicating that volume recovery does eventually occur if the initial hypertonic stress is not lethal.
GCK-3 functions in the intestine and hypodermis to mediate acute volume recovery. Acute systemic volume recovery in C. elegans after hypertonic stress likely requires absorption of solutes and water from the external environment as well as inhibition of fluid excretion. To assess the role of the hypodermis and excretory cell, which are sites of GCK-3 reporter expression (Fig. 1) and are known to play roles in C. elegans osmoregulation (18, 25, 46), we carried out tissue-specific RNAi experiments. rde-1 (RNAi defective) encodes a protein involved in translation initiation (17, 53), and rde-1 loss-of-function mutants are strongly resistant to RNAi induced by dsRNA injection, feeding, or expression (17, 53). Tissue-specific rescue of the rde-1 loss-of-function allele ne219 allows RNAi to be targeted to a specific cell type (16). We selectively rescued rde-1(ne219) in the hypodermis and excretory cell with the promoters for dpy-7 and clh-4, respectively. Promoters for these genes have been used previously to drive transgene expression selectively in these tissues/cell types (see, e.g., Refs. 23, 42, 49, 50).
As shown in Fig. 3, rde-1(ne219) mutants fed gck-3 dsRNA-producing bacteria recover their volume and survive normally on high NaCl, indicating that the animals are resistant to RNAi. rde-1(ne219) worms in which the gene is rescued in the excretory cell also show normal volume recovery and survival. To verify that rde-1 had been rescued in this cell type, we fed Pclh-4::RDE-1 worms, which express GFP in the excretory cell, GFP dsRNA-producing bacteria. Excretory cell GFP expression was reduced in these animals (data not shown), demonstrating that the gene had been successfully rescued.
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The intestine of C. elegans (35) and other nematodes (18) is known to play a role in osmoregulation. While we did not see expression of the GCK-3 GFP reporter in the intestinal epithelium, this cannot be taken as definitive evidence that the kinase is not expressed in this tissue. GFP reporters may not be detected for a variety of reasons, including low expression levels, interference from autofluorescence, and improper protein processing (6). Given this, we examined the C. elegans SAGE databases (http://elegans.bcgsc.ca/home/sage.html). SAGE analysis of FACS-sorted intestinal cells indicates that gck-3 is expressed in the intestine. We therefore rescued rde-1(ne219) in intestinal cells with the intestine-specific promoter for nhx-2 (16, 41, 43). As shown in Fig. 3, GCK-3 RNAi in hypertonically stressed rde-1 intestinal rescue worms also reduced survival and volume recovery by about half that observed in control animals. Taken together, these results indicate that GCK-3-regulated excretory cell processes do not play a predominant role in acclimation to acute hypertonic stress. Instead, GCK-3-regulated solute and water transport mechanisms in the intestine and hypodermis mediate systemic volume recovery and survival after hypertonicity-induced water loss and shrinkage.
GCK-3 interacts with WNK-1. To identify proteins that may function in GCK-3 signaling cascades, we performed a yeast two-hybrid screen of 7 x 105 cDNA clones, using the carboxy terminus of the kinase (amino acids 496–596). Of 15 positive clones that we identified, 2 contained the carboxy terminus (amino acids 1011–1838) of a protein encoded by C46C2.1a (Fig. 4A), one of two potential splice variants of wnk-1. WNK-1 is a member of the with no lysine (K) family of serine/threonine kinases (37), which play important roles in regulating salt and water transport in the mammalian kidney (21, 22, 27).
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We confirmed the interaction between GCK-3 and WNK-1a by GST affinity assay. Total proteins from CHO cells coexpressing V5 epitope-tagged full-length WNK-1a and either GST alone or a GST-GCK-3 (amino acids 496–596) fusion protein were incubated with glutathione-Sepharose beads, washed, and Western blotted with an anti-V5 antibody. As shown in Fig. 4B, WNK-1a interacts with GST-GCK-3, but not with GST alone.
We cloned a full-length WNK-1 coding region from a cDNA library that matched WNK-1a. Alignments of C. elegans WNK-1a with the four human WNKs indicated that it is most similar to WNK1. Figure 5 shows an alignment of C. elegans WNK-1 with human WNK1. The most significantly conserved regions are the kinase and autoinhibitory domains.
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To determine the sites of WNK-1 expression, we generated a transcriptional reporter that included the entire intergenic region upstream from wnk-1 and its first intron. As shown in Fig. 6, Pwnk-1::GFP is expressed in the excretory cell, hypodermis, spermatheca, and body wall muscle, which are also sites of GCK-3 GFP reporter expression (Fig. 1, B–D). C. elegans SAGE databases (http://elegans.bcgsc.ca/home/sage.html) corroborated our finding of wnk-1 expression in hypodermal cells, neurons, and gonad and also indicated that the kinase is expressed in the intestine. As with GCK-3, definitive localization of WNK-1 expression will require generation of suitable antibodies.
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The effect of wnk-1 RNAi was qualitatively similar to that of gck-3 RNAi. WNK-1 knockdown significantly decreased survival and acute volume recovery in worms exposed to either high NaCl- or sorbitol-induced hypertonic stress (Fig. 7). As with GCK-3 RNAi (Fig. 2A), the effect of WNK-1 RNAi on survival was much more pronounced with high NaCl than with high sorbitol. Mean ± SE survival of wnk-1(RNAi) worms on control agar without added NaCl was 98 ± 2% (n = 6 populations of 8–11 worms) after 3 days. Therefore, WNK-1 is required for survival of worms under hypertonic conditions.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Several studies have demonstrated that PASK and OSR1 regulate SLC12 cotransporters (2, 14, 19, 40, 48, 58, 59), suggesting that these kinases may play a role in cell and systemic osmoregulation. In Drosophila, Fray is essential for neuronal development (36). However, little else is known about the in vivo physiological functions of GCK-VI kinases. Our studies are the first to demonstrate that this kinase family functions in whole animal osmotic homeostasis.
Our studies also provide the first detailed description of WNK-1 function in C. elegans. Like their mammalian homologs, WNK-1 and GCK-3 physically interact (Fig. 4), and silencing of wnk-1 or gck-3 gives rise to qualitatively similar phenotypes (Fig. 7). Knocking down expression of the kinases together has no additive effect on survival and acute volume recovery (Fig. 8), suggesting that they function in a common pathway. We postulate that WNK-1 functions upstream of GCK-3 in a manner similar to that proposed for its mammalian homologs (2, 19, 40, 58, 59).
Figure 9 shows a working model based on the results of this study. GCK-3 function in the hypodermis and intestine is required for acute volume recovery and survival under high-NaCl conditions (Fig. 3). These tissues are in direct contact with the external environment and likely mediate osmoregulatory exchanges of ions and water. Unfortunately, direct measurement of transepithelial transport in C. elegans has not been possible because of the animal's small size. However, isolated body wall sacs from the parasitic nematode Pseudoterranova decipiens, which are comprised primarily of muscle and hypodermis, maintain their volume when exposed to hypotonic media. Only the hypodermis is in direct contact with the environment, suggesting that this tissue mediates osmoregulatory ion and water transport (18). Blockage of the anus of P. decipiens disrupts whole animal osmotic balance, indicating that the intestine also functions as an osmoregulatory organ (18).
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Interestingly, the mortality of gck-3(RNAi) and wnk-1(RNAi) worms was much greater in animals exposed to high NaCl versus high sorbitol (Figs. 2A and 7A). This difference appears to be independent of volume recovery, since acute volume regulation is similarly impaired by high NaCl and sorbitol (Figs. 2B and 7B). This suggests that in addition to regulating solute and water uptake mechanisms required for acute volume recovery, GCK-3 and WNK-1 may also regulate transport processes responsible for excretion of a chronic NaCl load. Additionally, the kinases may regulate mechanisms that protect cells from the damaging effects of elevated salt levels.
The GCK-VI-WNK interaction evolved in early metazoans.
GCK-3 and WNK-1 are the only C. elegans GCK-VI and WNK kinases (Fig. 10, A and B). The catalytic domains of GCK-VI and WNK kinases show strong homology across all species (Figs. 10, C and D). Similarly, a threonine residue (T185), whose phosphorylation activates mammalian OSR1 (58), is also highly conserved (Fig. 10C), suggesting that activation of GCK-VI kinases may occur by a common mechanism.
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C. elegans and mammals are separated by hundreds of millions of years of evolution (4, 56). Our results thus demonstrate that the interaction between GCK-VI and WNK kinases is evolutionarily very ancient. Phylogenetic analysis (Fig. 10) suggests that the interaction between these kinases first evolved early in the metazoan lineage. Interestingly, this coincides with the evolution of multicellularity, polarized epithelia, and the need for complex coordination of epithelial ion transporters and channels to regulate the osmotic and ionic composition of extracellular fluids. GCK-VI and WNK kinases are expressed in many transporting epithelia (9, 26, 47, 48, 55) (Figs. 1 and 6). Because GCK-VI and WNK kinases regulate Cl– channels and cation-Cl– cotransporters (2, 13, 14, 19, 40, 58, 59), it has been suggested that they may coordinate epithelial apical and basolateral ion transport (13, 27, 52). We propose that the physical and functional interaction of WNK and GCK-VI kinases arose early and specifically in animal evolution as a mechanism to regulate epithelial transport processes and systemic salt and water balance, and that this interaction was conserved as metazoans radiated.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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