Geyer RR, Musa-Aziz R, Qin X, Boron WF. Relative CO2/NH3 selectivities of mammalian aquaporins 0 -9. Am J Physiol Cell Physiol 304: C985-C994, 2013. First published March 13, 2013; doi:10.1152/ajpcell.00033.2013.-Previous work showed that aquaporin 1 (AQP1), AQP4-M23, and AQP5 each has a characteristic CO2/NH3 and CO2/H2O permeability ratio. The goal of the present study is to characterize AQPs 0 -9, which traffic to the plasma membrane when heterologously expressed in Xenopus oocytes. We use video microscopy to compute osmotic water permeability (Pf) and microelectrodes to record transient changes in surface pH (⌬pHS) caused by CO2 or NH3 influx. Subtracting respective values for day-matched, H2O-injected control oocytes yields the channel-specific values Pf* and ⌬pHS*. We find that Pf* is significantly Ͼ0 for all AQPs tested except AQP6. (⌬pHS*)CO 2 is significantly Ͼ0 for AQP0, AQP1, AQP4-M23, AQP5, AQP6, and AQP9. (⌬pHS*)NH 3 is Ͼ0 for AQP1, AQP3, AQP6, AQP7, AQP8, and AQP9. The ratio (⌬pHS*)CO 2 /Pf* falls in the sequence AQP6 (ϱ)The ratio (⌬pHS*)CO 2 /(Ϫ⌬pHS*)NH 3 is indeterminate for both AQP2 and AQP4-M1. In summary, we find that mammalian AQPs exhibit a diverse range of selectivities for CO2 vs. NH3 vs. H2O. As a consequence, by expressing specific combinations of AQPs, cells could exert considerable control over the movements of each of these three substances.water transport; gas transport; gas channels; membrane protein; Xenopus oocytes THE FIRST GAS CHANNEL IDENTIFIED was the water channel aquaporin 1 (AQP1). The pioneering work of Agre and colleagues (51,52,74) showed that heterologously expressing AQP1 in Xenopus oocytes markedly increases osmotic water permeability (P f ). In later experiments in which they exposed oocytes to CO 2 , Nakhoul et al. (45) and Cooper and Boron (9) found that the expression of AQP1 also increases the rate at which intracellular pH (pH i ) declines due to the intracellular reactions:One stoppedflow study of AQP1 reconstituted into artificial lipid vesicles showed that AQP1 enhances CO 2 permeability (50), whereas another reached the opposite conclusion (71). However, Itel et al. (29) using an 18 O-exchange technique, recently showed that reconstituted AQP1 markedly increases CO 2 permeability. Moreover, several studies have shown that plant AQPs also function as CO 2 channels (31,32,66,67). Additional work has shown that AQP1 also conducts NH 3 (24,42,46) and nitric oxide (21).As an extension to the aforementioned pH i methodology for assessing the relative permeabilities of a cell membrane to dissolved gases, our laboratory exploited a principle revealed by earlier work in which investigators had monitored surface pH (pH S ) or tissue extracellular pH (pH o ) while exposing cells to CO 2 /HCO 3 Ϫ (11, 57) or NH 3 /NH 4 ϩ (8). The novelty of our approach was to push a relatively large pH-sensitive microelectrode up against the surface of an oocyte and to use transient changes in pH S to assess the membrane permeability of gases that alter pH (i.e., CO 2 and NH 3 ). Fo...

Typical 2-Cys Peroxiredoxins (2-Cys Prxs) reduce hydroperoxides with extraordinary rates due to an active site composed of a catalytic triad, containing a peroxidatic cysteine (CP), an Arg, and a Thr (or Ser). 2-Cys Prx are involved in processes such as cancer; neurodegeneration and host-pathogen interactions. During catalysis, 2-Cys Prxs switch between decamers and dimers. Analysis of 2-Cys Prx structures in the fully folded (but not locally unfolded) form revealed a highly conserved, non-conventional hydrogen bond (CH-π) between the catalytic triad Thr of a dimer with an aromatic residue of an adjacent dimer. In contrast, structures of 2-Cys Prxs with a Ser in place of the Thr do not display this CH-π bond. Chromatographic and structural data indicate that the Thr (but not Ser) destabilizes the decamer structure in the oxidized state probably through steric hindrance. As a general trend, mutations in a yeast 2-Cys Prx (Tsa1) favoring the dimeric state also displayed a decreased catalytic activity. Remarkably, yeast naturally contains Thr-Ser variants (Tsa1 and Tsa2, respectively) with distinct oligomeric stabilities in their disulfide states.

Mammalian glycosylated rhesus (Rh) proteins include the erythroid RhAG and the nonerythroid RhBG and RhCG. RhBG and RhCG are expressed in multiple tissues, including hepatocytes and the collecting duct (CD) of the kidney. Here, we expressed human RhAG, RhBG and RhCG in Xenopus oocytes (vs. H2O-injected control oocytes) and used microelectrodes to monitor the maximum transient change in surface pH (DpHS) caused by exposing the same oocyte to 5 % CO₂/33 mM HCO₃⁻ (an increase) or 0.5 mM NH₃/NH₄⁺ (a decrease). Subtracting the respective values for day-matched, H₂O-injected control oocytes yielded channel-specific values (*). (ΔpH*(S))(CO₂) and (-ΔpH*(S))(NH₃) were each significantly >0 for all channels, indicating that RhBG and RhCG--like RhAG--can carry CO₂ and NH₃. We also investigated the role of a conserved aspartate residue, which was reported to inhibit NH₃ transport. However, surface biotinylation experiments indicate the mutants RhBG(D178N) and RhCG(D177N) have at most a very low abundance in the oocyte plasma membrane. We demonstrate for the first time that RhBG and RhCG--like RhAG--have significant CO₂ permeability, and we confirm that RhAG, RhBG and RhCG all have significant NH₃ permeability. However, as evidenced by (ΔpH*(S))(CO₂)/ (-ΔpH*(S))(NH₃) values, we could not distinguish among the CO₂/ NH₃ permeability ratios for RhAG, RhBG and RhCG. Finally, we propose a mechanism whereby RhBG and RhCG contribute to acid secretion in the CD by enhancing the transport of not only NH₃ but also CO₂ across the membranes of CD cells.

Aquaporins and Rh proteins can function as gas (CO₂ and NH₃) channels. The present study explores the urea, H₂O, CO₂, and NH₃ permeability of the human urea transporter B (UT-B) (SLC14A1), expressed in Xenopus oocytes. We monitored urea uptake using [¹⁴C]urea and measured osmotic water permeability (Pf) using video microscopy. To obtain a semiquantitative measure of gas permeability, we used microelectrodes to record the maximum transient change in surface pH (ΔpHS) caused by exposing oocytes to 5% CO₂/33 mM HCO₃⁻ (pHS increase) or 0.5 mM NH₃/NH₄⁺ (pHS decrease). UT-B expression increased oocyte permeability to urea by >20-fold, and Pf by 8-fold vs. H₂O-injected control oocytes. UT-B expression had no effect on the CO₂-induced ΔpHS but doubled the NH₃-induced ΔpHS. Phloretin reduced UT-B-dependent urea uptake (Jurea*) by 45%, Pf* by 50%, and (- ΔpHS*)NH₃ by 70%. p-Chloromercuribenzene sulfonate reduced Jurea* by 25%, Pf* by 30%, and (ΔpHS*)NH₃ by 100%. Molecular dynamics (MD) simulations of membrane-embedded models of UT-B identified the monomeric UT-B pores as the main conduction pathway for both H₂O and NH₃ and characterized the energetics associated with permeation of these species through the channel. Mutating each of two conserved threonines lining the monomeric urea pores reduced H₂O and NH₃ permeability. Our data confirm that UT-B has significant H₂O permeability and for the first time demonstrate significant NH₃ permeability. Thus the UTs become the third family of gas channels. Inhibitor and mutagenesis studies and results of MD simulations suggest that NH₃ and H₂O pass through the three monomeric urea channels in UT-B.