Functional characterization of Rhesus glycoproteins from an ammoniotelic teleost, the rainbow trout, using oocyte expression and SIET analysis.
Recent 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 [(14)C]methylamine, and the scanning ion electrode technique (SIET). All of the trout Rh proteins, except Rh30-like, facilitated methylamine uptake. Uptake was saturable, with K(m) values ranging from 4.6 to 8.9 mmol l(-1). 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 micromol 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.
Nawata
C Michele
CM
Department of Biology, McMaster University, 1280 Main St. West, Hamilton, Ontario, Canada L8S 4K1. nawatacm@mcmaster.ca
Wood
Chris M
CM
O'Donnell
Michael J
MJ
D000143:Acids; D000641:Ammonia / Q000378:metabolism*; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D001798:Blood Proteins / Q000378:metabolism*; D004566:Electrodes; D004594:Electrophysiology / Q000379:methods*; D029941:Fish Proteins / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D007700:Kinetics; D008744:Methylamines / Q000378:metabolism; D017686:Oncorhynchus mykiss / Q000378:metabolism*; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism*; D011522:Protons
D000143:Acids; D001798:Blood Proteins; D029941:Fish Proteins; D006023:Glycoproteins; D008744:Methylamines; D011522:Protons; D000643:Ammonium Chloride; D000641:Ammonia; C027451:methylamine