Lessons in waste management: Deciphering the dual role of Crustacean Rh1 protein in ammonia and CO2 excretion

In this issue of Acta Physioloigica, Quijada-Rodriguez and colleuges1 artfully combine empirical evidence with a suite of investigative tools to explore the dual capacity of Crustacean Rh1 (CmRh1) protein as a NH3 and CO2 channel in the European green crab (Carcinus maenas). Life is a complex balance of biochemical processes, and the regulation of ammonia and CO2 is critical for homeostasis in aquatic organisms. Both are natural waste products of cellular metabolism, yet their accumulation within tissues can be detrimental to cellular function and overall physiology. Due to its small molecular size, traditional views for transmembrane NH3/NH4+ transport propose (i) passive, gradient-driven diffusion of gaseous NH3 across lipid membranes and (ii) mediated movement of ionic NH4+ through promiscuous transport via K+ channel/transporters, although most of the transport occurs as NH3 at physiological pH. However, this paradigm radically shifted with the discovery of bacterial Amt/Met ammonium transporters and the closely related family of Rh glycoproteins. First identified in 1939 in connection with a severe case of post-stillborn spousal blood transfusion rejection2; it was only recently that Rh glycoproteins (a.k.a. SLC42 family of solute-linked transport carriers) were characterized as ammonia transport facilitators.3 Since then, they have been proven to be a critical player in transmembrane ammonia transport and are implicated in a wide array of physiological roles and indications, such as chronic kidney disease and renal tubular acidosis. The interplay between Rh glycoproteins and other putative acid/base/ion balance transporters is exemplified by the Rh “metabolon”—a multi-protein complex that includes Rh proteins and Na+/H+ exchangers (NHEs) that is proposed to help energize Na+ uptake for freshwater teleosts,4 with NHEs themselves also being recently posited as potential facilitators of ammonium transport,5 further upending previously held views on transport of NH4+ beyond K+ pathways. Like ammonia, CO2 transport has long been considered a passive process across lipid membranes and the identity of specific transmembrane facilitators of CO2 transport remains elusive and even somewhat contentious. Previous studies have hinted at the involvement of Rh proteins in CO2 transport in mammals,6 fishes,7 and even corals and algae.8, 9 Despite Crustacean Rhp1 being among the first Rh homologs cloned in aquatic organisms,10 it is only now that Quijada-Rodriguez et al.1 with their recent contribution bring to light the multiple potential roles of the Crustacean specific isoform Rh1 protein, in not only gaseous ammonia excretion, but now perhaps CO2 excretion as well. Quijada-Rodriguez and colleagues do not claim to be the first to propose a role for Rh glycoproteins in CO2 transport.6 However, their contribution stands out for its comprehensive use of both classical and modern comparative physiological approaches. To address their hypothesis of a dual-function Rh protein, they have employed a suite of techniques including cloning and site-directed mutagenesis, complementation, and radiolabeled flux experiments using heterologous expression in yeast and Xenopus oocytes. Additionally, they have utilized qPCR, Western blotting, and immunostaining to thoroughly investigate expressional patterns. The authors focus on a Rh-like homolog from the green crab and their utilization of a cross-species phylogenetic analysis of the Rh superfamily serves a dual purpose in their study. Initially, they categorize their cloned target, CmRh1, within the Rhp1 subfamily—a group previously linked to ammonia excretion in the swimming crab (Portunus trituberculatus) by Si and colleagues in 2018.11 More critically, they delineate the various animal isoforms within the Rh superfamily, identifying those known to be able to transport NH3 and/or CO2. This phylogenetic tree not only emphasizes the specific functions of these isoforms but also underscores the nascent state of research into dual roles in transport in the Rh family. Through functional assays using heterologous expression of wild-type CmRh1 in Xenopus oocytes, the authors demonstrate both ammonia and CO2 transport capacity. Most importantly, the authors show that transport of both CO2 and NH3 can be convincingly abolished when oocytes are instead injected with a mutant form of CmRh1 (D180N) , a site known to be important in substrate binding. Through qPCR analysis, the authors demonstrate that CmRh1 is predominantly expressed in the gills of C. maenas, with notable differences between the anterior and posterior gills. Specifically, Western blotting reveals an enriched anti-CmRh1 signal in the anterior gills. They also observe that exposure to high PCO2 levels (3 mmHg) leads to a reduction in CmRh1 mRNA expression in the anterior gills, while no significant changes are noted in the posterior gills. A similar, albeit less pronounced, attenuation in gill mRNA expression occurs in response to high environmental ammonia (HEA). Furthermore, immunostaining patterns reveal apical localization of Rh, corresponding with the expression of membrane-bound carbonic anhydrase (CA) within cells that are also abundant in Na+/K+-ATPase (NKA). Given the noted dynamics of CmRh1 at the transcriptional and protein expression levels in response to HEA, a plausible hypothesis is that these dynamic shifts are a result of a specific subcellular re-organizations of Rh expression, with Rh being removed from the apical membrane to regulate inward diffusion of ammonia. Such subcellular reorganization has been demonstrated in the gills of the highly ammonia-tolerant hagfish (Eptatretus stoutii) with stark reductions in apical expression of Rh in response to HEA.5 Taken together, the results from these two studies indicate that exposure to external HEA leads to dynamic subcellular regulation on the cell surface, and further hint at the existence of some form of an NH3 and/or NH4+ sensing mechanism, which has yet to be elucidated. Earlier studies on algal (Chlamydomonas reinhardtii) Rh variants successfully demonstrated changes in protein expression in response to elevated CO2.9 While Quijada-Rodriguez and colleagues1 were unable to observe any expressional responses to high PCO2 in their immunoblotting, a crucial caveat is that their whole-animal experiment was limited to a single exposure time-point for both HEA and high CO2 exposures. This singular temporal focus may not fully capture the dynamic nature of the Rh response, and thus, the possibility exists that Rh proteins respond differently over time to elevated CO2 compared with their response to HEA. Clearly, a detailed temporal analysis in future studies would offer valuable insights into the adaptive mechanisms and recovery processes of Crustaceans (or hagfish) in response to high CO2. Moving forward, the path forward is clear: A deeper dive into the mechanisms of Rh protein-mediated CO2 transport is essential. This exploration must extend across a diverse range of species, using the current study's phylogenetic framework as a roadmap to confirm or disconfirm the universality of dual-function Rh across the tree of life. In conclusion, Quijada-Rodriguez et al.'s study is an important step toward unraveling the capacities of Rhp1 in Crustacean physiology, and their results should encourage further examination for this capacity in other taxa. Overall, their contribution sets the stage for subsequent research that will undoubtedly refine our understanding of Rh glycoproteins and their intricate role in waste management. Alexander M. Clifford: Conceptualization; writing – original draft; writing – review and editing; investigation. There are no relevant acknowledgements for this article. The author declares no conflict of interest. Data sharing not applicable to this article as no datasets were generated or analysed during the current study.