Aim

To determine whether the crustacean Rh1 protein functions as a dual CO₂ /ammonia transporter and investigate its role in branchial ammonia excretion and acid-base regulation.

Methods

Sequence analysis of decapod Rh1 proteins was used to determine the conservation of amino acid residues putatively involved in ammonia transport and CO₂ binding in human and bacterial Rh proteins. Using the Carcinus maenas Rh1 protein (CmRh1) as a representative of decapod Rh1 proteins, we test the ammonia and CO₂ transport capabilities of CmRh1 through heterologous expression in yeast and Xenopus oocytes coupled with site-directed mutagenesis. Quantitative PCR was used to assess the distribution of CmRh1 mRNA in various tissues. Western blotting was used to assess CmRh1 protein expression changes in response to high environmental ammonia and CO₂ . Further, immunohistochemistry was used to assess sub-cellular localization of CmRh1 and a membrane-bound carbonic anhydrase (CmCAg).

Results

Sequence analysis of decapod Rh proteins revealed high conservation of several amino acid residues putatively involved in conducting ammonia transport and CO₂ binding. Expression of CmRh1 in Xenopus oocytes enhanced both ammonia and CO₂ transport which was nullified in CmRh1 D180N mutant oocytes. Transport of the ammonia analog methylamine by CmRh1 is dependent on both ionized and un-ionized ammonia/methylamine species. CmRh1 was co-localized with CmCAg to the apical membrane of the crustacean gill and only experienced decreased protein expression in the anterior gills when exposed to high environmental ammonia.

Conclusion

CmRh1 is the first identified apical transporter-mediated route for ammonia and CO₂ excretion in the crustacean gill. Our findings shed further light on the potential universality of dual ammonia and CO₂ transport capacity of Rhesus glycoproteins in both vertebrates and invertebrates.