Publications

• Purification and functional characterization of aquaporin-8.

  Liu K., Nagase H., Huang C.G., Calamita G., Agre P.

  <h4>Background information</h4>Aquaporins (AQPs) are a family of channels permeable to water and some small solutes. In mammals, 13 members (AQP0-AQP12) have been found. AQP8 is widely distributed in many tissues and organs. Previous studies in frog oocytes suggested that AQP8 was permeable to wat ... >> More

  <h4>Background information</h4>Aquaporins (AQPs) are a family of channels permeable to water and some small solutes. In mammals, 13 members (AQP0-AQP12) have been found. AQP8 is widely distributed in many tissues and organs. Previous studies in frog oocytes suggested that AQP8 was permeable to water, urea and ammonium, but no direct characterization had yet been reported.<h4>Results</h4>We expressed recombinant rAQP8, hAQP8 and mAQP8 (rat, human and mouse AQP8 respectively) in yeast, purified the proteins to homogeneity and reconstituted them into proteoliposomes. Although showing high sequence similarity, AQP8 proteins from the three species had to be purified with different detergents prior to reconstitution. In stopped-flow studies, all three AQP8 proteoliposomes showed water permeability, which was inhibited by mercuric chloride and rescued by 2-mercaptoethanol. rAQP8 and hAQP8 proteoliposomes did not transport glycerol or urea but were permeable to formamide, which was also inhibited by mercuric chloride. In the oocyte transport assay, hAQP8-injected oocytes showed significantly higher [14C]methylammonium uptake than water-injected oocytes.<h4>Conclusions</h4>In the present study, we successfully purified rAQP8, hAQP8 and mAQP8 proteins and characterized their biochemical and biophysical properties. All three AQP8 proteins transport water. rAQP8 and hAQP8 are not permeable to urea or glycerol. Moreover, hAQP8 is permeable to ammonium analogues (formamide and methylammonium). Our results suggest that AQP8 may transport ammonium in vivo and physiologically contribute to the acid-base equilibrium. << Less

  Biol. Cell 98:153-161(2006) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Structural determinants of NH3 and NH4+ transport by mouse Rhbg, a renal Rh glycoprotein.

  Abdulnour-Nakhoul S., Le T., Rabon E., Hamm L.L., Nakhoul N.L.

  Renal Rhbg is localized to the basolateral membrane of intercalated cells and is involved in NH₃/NH₄⁺ transport. The structure of Rhbg is not yet resolved; however, a high-resolution crystal structure of AmtB, a bacterial homolog of Rh, has been determined. We alig ... >> More

  Renal Rhbg is localized to the basolateral membrane of intercalated cells and is involved in NH₃/NH₄⁺ transport. The structure of Rhbg is not yet resolved; however, a high-resolution crystal structure of AmtB, a bacterial homolog of Rh, has been determined. We aligned the sequence of Rhbg to that of AmtB and identified important sites of Rhbg that may affect transport. Our analysis positioned three conserved amino acids, histidine 183 (H183), histidine 342 (H342), and tryptophan 230 (W230), within the hydrophobic pore where they presumably serve to control NH₃ transport. A fourth residue, phenylalanine 128 (F128) was positioned at the upper vestibule, presumably contributing to recruitment of NH₄⁺ We generated three mutations each of H183, H342, W230, and F128 and expressed them in frog oocytes. Immunolabeling showed that W230 and F128 mutants were localized to the cell membrane, whereas H183 and H342 staining was diffuse and mostly intracellular. To determine function, we compared measurements of NH₃/NH₄⁺ and methyl amine (MA)/methyl ammonium (MA⁺)-induced currents, intracellular pH, and surface pH (pHs) among oocytes expressing the mutants, Rhbg, or injected with H₂O. In H183 and W230 mutants, NH₄⁺-induced current and intracellular acidification were inhibited compared with that of Rhbg, and MA-induced intracellular alkalinization was completely absent. Expression of H183A or W230A mutants inhibited NH₃/NH₄⁺- and MA/MA⁺-induced decrease in pHs to the level observed in H₂O-injected oocytes. Mutations of F128 did not significantly affect transport of NH₃ or NH₄⁺ These data demonstrated that mutating H183 or W230 caused loss of function but not F128. H183 and H342 may affect membrane expression of the transporter. << Less

  Am. J. Physiol. 311:F1280-F1293(2016) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• NH3 and NH4+ permeability in aquaporin-expressing Xenopus oocytes.

  Holm L.M., Jahn T.P., Moeller A.L., Schjoerring J.K., Ferri D., Klaerke D.A., Zeuthen T.

  We have shown recently, in a yeast expression system, that some aquaporins are permeable to ammonia. In the present study, we expressed the mammalian aquaporins AQP8, AQP9, AQP3, AQP1 and a plant aquaporin TIP2;1 in Xenopus oocytes to study the transport of ammonia (NH3) and ammonium (NH4+) under ... >> More

  We have shown recently, in a yeast expression system, that some aquaporins are permeable to ammonia. In the present study, we expressed the mammalian aquaporins AQP8, AQP9, AQP3, AQP1 and a plant aquaporin TIP2;1 in Xenopus oocytes to study the transport of ammonia (NH3) and ammonium (NH4+) under open-circuit and voltage-clamped conditions. TIP2;1 was tested as the wild-type and in a mutated version (tip2;1) in which the water permeability is intact. When AQP8-, AQP9-, AQP3- and TIP2;1-expressing oocytes were placed in a well-stirred bathing medium of low buffer capacity, NH3 permeability was evident from the acidification of the bathing medium; the effects observed with AQP1 and tip2;1 did not exceed that of native oocytes. AQP8, AQP9, AQP3, and TIP2;1 were permeable to larger amides, while AQP1 was not. Under voltage-clamp conditions, given sufficient NH3, AQP8, AQP9, AQP3, and TIP2;1 supported inwards currents carried by NH4+. This conductivity increased as a sigmoid function of external [NH3]: for AQP8 at a bath pH (pH(e)) of 6.5, the conductance was abolished, at pH(e) 7.4 it was half maximal and at pH(e) 7.8 it saturated. NH4+ influx was associated with oocyte swelling. In comparison, native oocytes as well as AQP1 and tip2;1-expressing oocytes showed small currents that were associated with small and even negative volume changes. We conclude that AQP8, AQP9, AQP3, and TIP2;1, apart from being water channels, also support significant fluxes of NH3. These aquaporins could support NH4+ transport and have physiological implications for liver and kidney function. << Less

  Pflugers Arch. 450:415-428(2005) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Substrate specificity of Rhbg: ammonium and methyl ammonium transport.

  Nakhoul N.L., Abdulnour-Nakhoul S.M., Boulpaep E.L., Rabon E., Schmidt E., Hamm L.L.

  Rhbg is a nonerythroid membrane glycoprotein belonging to the Rh antigen family. In the kidney, Rhbg is expressed at the basolateral membrane of intercalated cells of the distal nephron and is involved in NH4+ transport. We investigated the substrate specificity of Rhbg by comparing transport of N ... >> More

  Rhbg is a nonerythroid membrane glycoprotein belonging to the Rh antigen family. In the kidney, Rhbg is expressed at the basolateral membrane of intercalated cells of the distal nephron and is involved in NH4+ transport. We investigated the substrate specificity of Rhbg by comparing transport of NH3/NH4+ with that of methyl amine (hydrochloride) (MA/MA+), often used to replace NH3/NH4+, in oocytes expressing Rhbg. Methyl amine (HCl) in solution exists as neutral methyl amine (MA) in equilibrium with the protonated methyl ammonium (MA+). To assess transport, we used ion-selective microelectrodes and voltage-clamp experiments to measure NH3/NH4+- and MA/MA+-induced intracellular pH (pH(i)) changes and whole cell currents. Our data showed that in Rhbg oocytes, NH3/NH4+ caused an inward current and decrease in pH(i) consistent with electrogenic NH4+ transport. These changes were significantly larger than in H2O-injected oocytes. The NH3/NH4+-induced current was not inhibited in the presence of barium or in the absence of Na+. In Rhbg oocytes, MA/MA+ caused an inward current but an increase (rather than a decrease) in pH(i). MA/MA+ did not cause any changes in H2O-injected oocytes. The MA/MA+-induced current and pH(i) increase were saturated at higher concentrations of MA/MA+. Amiloride inhibited MA/MA+-induced current and the increase in pH(i) in oocytes expressing Rhbg but had no effect on control oocytes. These results indicate that MA/MA+ is transported by Rhbg but differently than NH3/NH4+. The protonated MA+ is likely a direct substrate whose transport resembles that of NH4+. Transport of electroneutral MA is also enhanced by expression of Rhbg. << Less

  Am. J. Physiol. 299:C695-C705(2010) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.