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

Michele Nawata, Chris M. Wood, Michael J. O'Donnell

Research output: Contribution to journalArticle

58 Citations (Scopus)

Abstract

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, Rh30like) expressed in Xenopus oocytes, using the radiolabeled ammonia analogue [14C] methylamine, and the scanning ion electrode technique (SIET). All of the trout Rh proteins, except Rh30-like, facilitated methylamine uptake. Uptake was saturable, with Km values ranging from 4.6 to 8.9 mmoll-1. Raising external pH from 7.5 to 8.5 resulted in 3- to 4-fold elevations in Jmax values for methylamine; Km 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. NH4CI inhibited methylamine uptake in oocytes expressing Rhbg1 or Rhcg2. When external pH was elevated from 7.5 to 8.5, the K1 for ammonia against methylamine transport was 35-40% lower when expressed as total ammonia or NH4+, but 5- to 6-fold higher when expressed as NH3. 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 Jmax 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 Km for total ammonia (560μmoll-1) lies within the physiological range for trout. The results are consistent with a model whereby NH4+ initially binds, but NH3 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.

Original languageEnglish
Pages (from-to)1049-1059
Number of pages11
JournalJournal of Experimental Biology
Volume213
Issue number7
DOIs
StatePublished - Apr 1 2010

Fingerprint

Oncorhynchus mykiss
teleost
Ammonia
electrodes
methylamine
rainbow
Oocytes
glycoproteins
Glycoproteins
Electrodes
electrode
oocytes
ammonia
Ions
ions
ion
Trout
uptake mechanisms
trout
methodology

Keywords

  • Ammonia transport
  • Gill
  • Rh glycoproteins
  • Trout
  • Xenopus oocytes

ASJC Scopus subject areas

  • Animal Science and Zoology
  • Ecology, Evolution, Behavior and Systematics
  • Molecular Biology
  • Physiology
  • Insect Science
  • Aquatic Science

Cite this

Functional characterization of Rhesus glycoproteins from an ammoniotelic teleost, the rainbow trout, using oocyte expression and SIET analysis. / Nawata, Michele; Wood, Chris M.; O'Donnell, Michael J.

In: Journal of Experimental Biology, Vol. 213, No. 7, 01.04.2010, p. 1049-1059.

Research output: Contribution to journalArticle

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abstract = "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, Rh30like) expressed in Xenopus oocytes, using the radiolabeled ammonia analogue [14C] methylamine, and the scanning ion electrode technique (SIET). All of the trout Rh proteins, except Rh30-like, facilitated methylamine uptake. Uptake was saturable, with Km values ranging from 4.6 to 8.9 mmoll-1. Raising external pH from 7.5 to 8.5 resulted in 3- to 4-fold elevations in Jmax values for methylamine; Km 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. NH4CI inhibited methylamine uptake in oocytes expressing Rhbg1 or Rhcg2. When external pH was elevated from 7.5 to 8.5, the K1 for ammonia against methylamine transport was 35-40{\%} lower when expressed as total ammonia or NH4+, but 5- to 6-fold higher when expressed as NH3. 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 Jmax 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 Km for total ammonia (560μmoll-1) lies within the physiological range for trout. The results are consistent with a model whereby NH4+ initially binds, but NH3 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.",
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N2 - 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, Rh30like) expressed in Xenopus oocytes, using the radiolabeled ammonia analogue [14C] methylamine, and the scanning ion electrode technique (SIET). All of the trout Rh proteins, except Rh30-like, facilitated methylamine uptake. Uptake was saturable, with Km values ranging from 4.6 to 8.9 mmoll-1. Raising external pH from 7.5 to 8.5 resulted in 3- to 4-fold elevations in Jmax values for methylamine; Km 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. NH4CI inhibited methylamine uptake in oocytes expressing Rhbg1 or Rhcg2. When external pH was elevated from 7.5 to 8.5, the K1 for ammonia against methylamine transport was 35-40% lower when expressed as total ammonia or NH4+, but 5- to 6-fold higher when expressed as NH3. 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 Jmax 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 Km for total ammonia (560μmoll-1) lies within the physiological range for trout. The results are consistent with a model whereby NH4+ initially binds, but NH3 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.

AB - 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, Rh30like) expressed in Xenopus oocytes, using the radiolabeled ammonia analogue [14C] methylamine, and the scanning ion electrode technique (SIET). All of the trout Rh proteins, except Rh30-like, facilitated methylamine uptake. Uptake was saturable, with Km values ranging from 4.6 to 8.9 mmoll-1. Raising external pH from 7.5 to 8.5 resulted in 3- to 4-fold elevations in Jmax values for methylamine; Km 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. NH4CI inhibited methylamine uptake in oocytes expressing Rhbg1 or Rhcg2. When external pH was elevated from 7.5 to 8.5, the K1 for ammonia against methylamine transport was 35-40% lower when expressed as total ammonia or NH4+, but 5- to 6-fold higher when expressed as NH3. 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 Jmax 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 Km for total ammonia (560μmoll-1) lies within the physiological range for trout. The results are consistent with a model whereby NH4+ initially binds, but NH3 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.

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