Relative CO2/NH3 selectivities of AQP1, AQP4, AQP5, AmtB, and RhAG

Abstract

The water channel aquaporin 1 (AQP1) and certain Rh-family members are permeable to CO(2) and NH(3). Here, we use changes in surface pH (pH(S)) to assess relative CO(2) vs. NH(3) permeability of Xenopus oocytes expressing members of the AQP or Rh family. Exposed to CO(2) or NH(3), AQP1 oocytes exhibit a greater maximal magnitude of pH(S) change (DeltapH(S)) compared with day-matched controls injected with H(2)O or with RNA encoding SGLT1, NKCC2, or PepT1. With CO(2), AQP1 oocytes also have faster time constants for pH(S) relaxation (tau(pHs)). Thus, AQP1, but not the other proteins, conduct CO(2) and NH(3). Oocytes expressing rat AQP4, rat AQP5, human RhAG, or the bacterial Rh homolog AmtB also exhibit greater DeltapH(S)(CO(2)) and faster tau(pHs) compared with controls. Oocytes expressing AmtB and RhAG, but not AQP4 or AQP5, exhibit greater DeltapH(S)(NH(3)) values. Only AQPs exhibited significant osmotic water permeability (P(f)). We computed channel-dependent (*) DeltapH(S) or P(f) by subtracting values for H(2)O oocytes from those of channel-expressing oocytes. For the ratio DeltapH(S)(CO(2))*/P(f)*, the sequence was AQP5 > AQP1 congruent with AQP4. For DeltapH(S)(CO(2))*/DeltapH(S)(NH(3))*, the sequence was AQP4 congruent with AQP5 > AQP1 > AmtB > RhAG. Thus, each channel exhibits a characteristic ratio for indices of CO(2) vs. NH(3) permeability, demonstrating that, like ion channels, gas channels can exhibit selectivity.

Fig. 1.

Basis of surface pH changes. (A) CO₂ influx. At the outer surface of the membrane, the CO₂ influx creates a CO₂ deficit. The reaction HCO₃⁻ + H⁺ → CO₂ + H₂O, in part, replenishes the CO₂, raising pH_S. (B) NH₃ influx. The reaction NH₄⁺ → NH₃ + H⁺, in part, replenishes NH₃, lowering pH_S. (C–E) Representative pH_S transients from oocytes injected with H₂O or expressing AQP1 (record repeated in the 3 images), SGLT1, NKCC2, or PepT1. All data in C–E were obtained on the same day, from the same batch of oocytes, exposed first to CO₂/HCO₃⁻ and then (after CO₂ removal) to NH₃/NH₄⁺. Also shown in C are records with no oocyte present. Before and after solution changes, we retracted the pH electrode to the bulk extracellular solution (pH 7.50) for calibration. (F and G) Summary of extreme excursions of pH_S (ΔpH_S) for CO₂ and NH₃ data. (H and I) Representative pH_S transients from day-matched H₂O or AQP1 oocytes exposed to CO₂/HCO₃⁻ or 30 mM butyrate. (J and K) Summary of ΔpH_S for experiments like those in H or I. Values are means ± SE, with numbers of oocytes in parentheses. For F and G, statistical comparison between H₂O-injected controls and other oocytes (separately for CO₂ and NH₃ data) were made using a 1-way ANOVA for 5 groups, followed by Dunnett's multiple comparison. ΔpH_S values for H₂O, NKCC2, and PepT1 do not differ from one other. For J and K, statistical comparisons were made using unpaired 2-tailed t tests.

Fig. 2.

Carbonic anhydrase activities of Xenopus oocytes. CA activity was determined from membrane preparations of 100 oocytes injected with H₂O, 0.25 ng of cRNA encoding hCA IV, or 25 ng of cRNA encoding hAQP1. We divided each membrane preparation into aliquots containing 20 μg of total protein and performed a colorimetric CA assay on each aliquot (see SI Text). The sample mixtures containing CA-IV were run ± 100 μM methazolamide (MTZ), a CA inhibitor. Each N refers to a CA assay on 1 aliquot.

Fig. 3.

Surface pH changes caused by CO₂ and NH₃ influx in oocytes expressing gas-channel proteins. (A–E) Typical pH_S transients from oocytes injected with H₂O or expressing AQP1, AQP4, AQP5, WT AmtB or its inactive D160A mutant, or WT RhAG or its inactive D167A mutant. The protocol was the same as in Fig. 1. (F–J) Summary of extreme excursions of pH_S (ΔpH_S) caused by CO₂ influx. Each image represents mean values for day-matched oocytes. (K–O) Summary of ΔpH_S caused by NH₃ influx. Each image (F–O) represents mean values for day-matched oocytes. Some H₂O oocytes served as controls in more than 1 panel (total number of H₂O oocytes: 54 for CO₂, 61 for NH₃). Values are means ± SE, with numbers of oocytes in parentheses. For F–H and K–M, statistical comparisons were made using unpaired 2-tailed t tests. For I and J and N and O, statistical comparisons were made using 1-way ANOVAs for 3 groups, followed by Student–Newman–Keuls analyses.

Fig. 4.

Western blots testing cleavage of the AmtB signal peptide in Xenopus oocytes. (A) Wild-type AmtB vs. uncleavable A22K mutant. (B) Wild-type AmtB vs. AmtB with truncated signal peptide. Data are representative of 4 similar experiments. All constructs were His tagged at the C terminus and detected with an anti-His antibody.

Fig. 5.

Osmotic water permeabilities of Xenopus oocytes. P_f (cm/s) of oocytes injected with H₂O or cRNA encoding AQP1, AQP4, AQP5, AmtB, or RhAG. Values are means ± SE, with numbers of oocytes in parentheses. Statistical comparison between H₂O vs. AQP oocytes were made using unpaired 2-tailed t tests. Statistical comparisons among the 4 groups were made using a 1-way ANOVA, followed by Student–Newman–Keuls analyses.

Fig. 6.

Comparison of channel-dependent properties. (A) Indices of channel-dependent CO₂ permeability. For each ΔpH_S from a channel-expressing oocyte, we subtracted the mean, day-matched ΔpH_S for H₂O oocytes. Bars represent mean subtracted values, the channel-dependent ΔpH_S for CO₂, or (ΔpHS*)_CO2. Note: Oocytes in A are the same as those in B and C. (B) Indices of channel-dependent NH₃ permeability. We computed (ΔpH_S*)_NH3 using the same approach as in A. (C) Channel-dependent water permeabilities. For each P_f from a channel-expressing oocyte, we subtracted the mean, day-matched P_f for H₂O oocytes. Bars represent mean subtracted values, the channel-dependent P_f, or P_f*. (D) Indices of channel-dependent CO₂ and NH₃ permeability, normalized to P_f*. For each oocyte, we divided (ΔpH_S*)_CO2 and (ΔpH_S*)_NH3 by its P_f*. (E) Indices of gas selectivity. For each oocyte expressing AQP1, AmtB, or RhAG, we divided (ΔpH_S*)_CO2 by −(ΔpH_S*)_NH3. Because (ΔpH_S*)_NH3 was not significantly different from zero for oocytes expressing AQP4 or AQP5, we represent these ratios as “infinity.” Statistical comparisons were made using 1-way ANOVAs for 3 groups, followed by Student–Newman–Keuls analyses.