Ammonia excretion in Caenorhabditis elegans: Physiological and molecular characterization of the rhr-2 knock-out mutant

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Abstract

Previous studies have shown the free living soil nematode Caenorhabditis elegans (N2 strain) to be ammonotelic. Ammonia excretion was suggested to take place partially via the hypodermis, involving the Na+/K+-ATPase (NKA), V-ATPase (VAT), carbonic anhydrase, NHX-3 and a functional microtubule network and at least one Rh-like ammonia transporter RHR-1. In the current study, we show that a second Rh-protein, RHR-2, is highly expressed in the hypodermis, here also in the apical membrane of that tissue. To further characterize the role of RHR-2 in ammonia excretion, a knock-out mutant rhr-2 (ok403), further referred to as ∆ rhr-2, was employed. Compared to wild-type worms (N2), this mutant showed a lower rate of ammonia excretion and a lower hypodermal H+ excretion rate. At the same time rhr-1, nka, vat, and nhx-3 showed higher mRNA expression levels when compared to N2. Also, in contrast to N2 worms, ∆ rhr-2 did not show enhanced ammonia excretion rates when exposed to a low pH environment, suggesting that RHR-2 represents the apical NH3 pathway that allows ammonia trapping via the hypodermis in N2 worms. A hypothetical model for the mechanism of hypodermal ammonia excretion is proposed on the basis of data in this and previous investigations.

Introduction

Transmembrane transport of ammonia,1 the primary end product of protein catabolism (Campbell, 1991), serves essential physiological functions that include the uptake of nitrogen in animals living on a nitrogen poor diet (Weihrauch, 2006), the adjustment of acid–base homeostasis (Fehsenfeld and Weihrauch, 2012, Weiner and Verlander, 2013) and the excretion of nitrogenous waste in ammonotelic animals, such as fish and aquatic invertebrates (Larsen et al., 2014, Weihrauch et al., 2009, Wright and Wood, 2009). Ammonia exists in a pH-dependent equilibrium of NH3 and NH4+ and occurs predominantly in its ionic form at physiological pH (7.2–8), given its pKa of approximately 9.3 (Cameron and Heisler, 1983). Due to its similar size to K+ ions when hydrated (Weiner and Hamm, 2007), NH4+ can serve as a substrate for numerous K+ transporting proteins and enzymes that includes the Na+/K+-ATPase (Cruz et al., 2013, Skou, 1960, Wood et al., 2013), H+/K+-ATPase (Swarts et al., 2005), K+ channels (Choe et al., 2000), and Na+/K+/2Cl cotransporters (Good et al., 1984). In addition, NH4+ may also substitute for alkali metal ions in cation/H+ exchangers (Blaesse et al., 2010). The gaseous form of ammonia, NH3, on the other hand, cannot be actively transported across biomembranes, but requires a partial pressure gradient (∆ PNH3) and ideally a NH3-permeable channel for transmembrane passage. In animals, such NH3-channels have been identified to be glycosylated proteins belonging to the Rhesus-protein family. These Rh-proteins are well conserved within the animal kingdom (Huang and Peng, 2005) and are predicted to form trimeric complexes in vivo, wherein each monomer allows the passage of NH3 (Gruswitz et al., 2010, Marini et al., 2000). Since their discovery by Marini and coworkers (Marini et al., 2000), numerous studies have shown the importance of Rh-proteins in acid–base regulation and epithelial ammonia transport processes, which have been extensively reviewed (Huang and Ye, 2010, Nakhoul and Lee Hamm, 2013, Weihrauch et al., 2009, Weiner and Verlander, 2013, Wright and Wood, 2009, Wright et al., 2014). In many ammonia transporting epithelia, including the mammalian distal nephron, fish gills, and anal papillae of dipteran insect larvae, two Rh-proteins are found, one localized on the apical and the other on the basolateral cell membrane, respectively. In a recent study, the ammonia excretion mechanism across the hypodermis of the soil dwelling nematode Caenorhabditis elegans was investigated (Adlimoghaddam et al., 2015). As in insects (Weihrauch et al., 2011), C. elegans also expresses two Rh-proteins, named RHR-1 and RHR-2. RHR-1 is expressed in many tissues of C. elegans but predominantly in the hypodermis (Ji et al., 2006). Moreover, RHR-1 shares high sequence similarity to other vertebrate and invertebrate Rh-proteins and has been shown to function as an ammonia transporter when expressed in yeast (Adlimoghaddam et al., 2015). The latter study also suggested that some ammonia is actively transported from the body fluids via the Na+/K+-ATPase into the cytoplasm of the hypodermal syncytium. A portion of the cytoplasmic ammonia is then believed to be trapped into acidified vesicles, which are then transported to the apical membrane for exocytotic release (Adlimoghaddam et al., 2015). The lack of transcriptional alterations in response to internal ammonia loading due to feeding suggested that RHR-1 serves as a housekeeping gene and is likely localized to the basal membrane of the hypodermis. In contrast, feeding induced an up-regulation of rhr-2 transcripts (see also Fig. 7) (Adlimoghaddam et al., 2015). However, due to its unknown tissue localization and function, the role of the second Rh-protein, RHR-2, in the ammonia transport processes of the worm remains unresolved.

Our earlier study suggested an ammonia trapping mechanism across the apical membrane of the hypodermis of the worm (Adlimoghaddam et al., 2015). NH3 channels in the apical membrane of the hypodermis are thus required for this mechanism to operate.

In the current investigation, tissue localization and potential functions of RHR-2 was investigated through the use of transgenic GFP (green fluorescence protein)-constructs as well as transport studies and gene-expression analysis in rhr-2 knock-out mutant, ∆ rhr-2.

Section snippets

Nematode cultures

The hermaphrodite wild-type C. elegans strain (N2) and RB651 rhr-2 (ok403) knock out worm (Δrhr-2) strain were obtained from the Caenorhabditis Genetics Center (CGC, University of Minnesota, Minneapolis) and maintained as described earlier (Adlimoghaddam et al., 2015). Strains were maintained in the laboratory on Nematode Growth Medium (NGM) seeded with Escherichia coli OP50 at 16 °C according to Brenner (Brenner, 1974). After revitalization, worms were washed from the plates with M9 buffer (in

Results

GFP expression activated by the rhr-2 promoter indicated that RHR-2 is expressed predominantly in the hypodermis, with additional abundance observed in the ventral nerve cord and body wall muscles (Fig. 1A). Wild-type transgenic animal expressing the RHR-2::GFP fusion protein expressed under its own promoter showed strong GFP expression in the hypodermis of adult C. elegans (Fig. 1B) as well as mid-staged larvae (Fig. 1C–F) (Altun and Hall, 2009a). In these transgenic animals the body wall

Discussion

This paper examined the role of RHR-2 in hypodermal ammonia excretion in C. elegans. As shown in Fig. S1, RHR-2 shares a high level of sequence homology (44% identity in amino acid sequence) to the functionally characterized RHR-1 ammonia transporter in C. elegans and contains, in common with RHR-1 and other ammonia transporting Rh-proteins, the conserved amino acid residues crucial for ammonia conductance (Adlimoghaddam et al., 2015, Zidi-Yahiaoui et al., 2009). Accordingly, an NH3 transport

Conclusion

Our data indicate a central role for RHR-2 in hypodermal ammonia excretion and suggest strongly that RHR-2 resides in the apical membrane of this tissue and possibly also in the basolateral membrane. Moreover, experiments employing the ∆ rhr-2 mutant verified the importance of other transporters, such as RHR-1, Na+/K+-ATPase, V-ATPase and NHX-3 in ammonia excretion, as suggested also in earlier studies. A working model for the hypodermal ammonia excretion mechanisms incorporates findings from

Acknowledgments

This work was supported by NSERC Canada Discovery Grants to D.W. (355891), M.J.O. (05359), J.K. (435974), D.M. (386532), and J.R.T. (418503). D.W. is also supported by Canada Foundation for Innovation (CFI). J.R.T. is also supported by the Canada Research Chairs program and CFI.

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