Functional characterization of Rhesus glycoproteins from an ammoniotelic teleost, the rainbow trout, using oocyte expression and SIET analysis

Abstract

SUMMARYRecent experimental evidence from rainbow trout suggests that gill ammonia transport may be mediated in part via Rhesus (Rh) glycoproteins. In this study we analyzed the transport properties of trout Rh proteins (Rhag, Rhbg1, Rhbg2, Rhcg1, Rhcg2, Rh30-like) expressed in Xenopus oocytes, using the radiolabeled ammonia analogue [ . Raising external pH from 7.5 to 8.5 resulted in 3-to 4-fold elevations in J max values for methylamine; K m values were unchanged when expressed as total or protonated methylamine. Efflux of methylamine was also facilitated in Rh-expressing oocytes. Efflux and influx rates were stimulated by a pH gradient, with higher rates observed with steeper H + gradients. NH 4 Cl inhibited methylamine uptake in oocytes expressing Rhbg1 or Rhcg2. When external pH was elevated from 7.5 to 8.5, the K i for ammonia against methylamine transport was 35-40% lower when expressed as total ammonia or NH 4 + , but 5-to 6-fold higher when expressed as NH 3 . With SIET we confirmed that ammonia uptake was facilitated by Rhag and Rhcg2, but not Rh30-like proteins. Ammonia uptake was saturable, with a comparable J max but lower K m value than for total or protonated methylamine. At low substrate concentrations, the ammonia uptake rate was greater than that of methylamine. The K m for total ammonia (560moll -1 ) lies within the physiological range for trout. The results are consistent with a model whereby NH 4 + initially binds, but NH 3 passes through the Rh channels. We propose that Rh glycoproteins in the trout gill are low affinity, high capacity ammonia transporters that exploit the favorable pH gradient formed by the acidified gill boundary layer in order to facilitate rapid ammonia efflux when plasma ammonia concentrations are elevated.

Aedes aegypti Rhesus glycoproteins contribute to ammonia excretion by larval anal papillae

“…Recently, studies in mammals and fish have implicated Rh proteins in the recruitment of NH 4 + as a substrate for net NH 3 transport (Nawata et al, 2010;Baday et al, 2015 Fig. 7.…”

Revisiting the effects of crowding and feeding in the gulf toadfish, Opsanus beta: the role of Rhesus glycoproteins in nitrogen metabolism and excretion

“…Acidification of the gastric lumen to a pH of 3 (Taylor and Grosell, 2006) and elevation of plasma ammonia tõ 350moll -1 create an even stronger gradient for ammonia secretion into the stomach. Given that piscine Rh proteins function as bidirectional transporters (Nawata et al, 2010a) and that Rhcg1 mRNA expression in the stomach was significantly higher in fed fish, toadfish may manage the post-prandial nitrogen load by increasing ammonia secretion into the stomach. This trend carries over into the intestine, where mild post-prandial acidification [pH ~7.5; (Taylor and Grosell, 2006)] coupled with the elevated circulating ammonia concentration creates an NH 3 partial pressure gradient for the movement of ammonia into the intestine.…”

“…As NH 3 diffuses across the gill and boundary layer, it is trapped as NH 4 + (Wright et al, 1986;Wright et al, 1989). The identification and characterization of Rhag, Rhbg, and splice variants Rhcg1 and Rhcg2 in the gill of rainbow trout has established a role for Rh glycoproteins as NH 3 conduits in this freshwater model Nawata and Wood, 2009;Nawata et al, 2010a) (reviewed by Wright and Wood, 2009). Less is known about mechanisms of branchial ammonia transport in marine teleosts.…”

Rh glycoprotein expression is modulated in pufferfish (Takifugu rubripes) during high environmental ammonia exposure

“…Indeed, Marini et al were able to show that RhAG and RhCG expressed in Mepdeficient yeast mediated the uptake of ammonia as well as the efflux of the ammonium analogue, methylamine (Marini et al, 2000). This was followed by numerous other functional studies that provided evidence for the transport of ammonia or methylamine through mammalian (reviewed by Javelle et al, 2007) and fish (Nakada et al, 2007;Nawata et al, 2010) Rh proteins expressed in heterologous systems. Very recently, it was demonstrated that purified RhCG reconstituted into liposomes functions as an NH 3 channel (MouroChanteloup et al, 2010).…”