We report here the application of a previously described method to directly determine the CO2 permeability (P(CO2)) of the cell membranes of normal human red blood cells (RBCs) vs. those deficient in aquaporin 1 (AQP1), as well as AQP1-expressing Xenopus laevis oocytes. This method measures the exchange of (18)O between CO2, HCO3(-), and H2O in cell suspensions. In addition, we measure the alkaline surface pH (pH(S)) transients caused by the dominant effect of entry of CO2 vs. HCO3(-) into oocytes exposed to step increases in [CO2]. We report that 1) AQP1 constitutes the major pathway for molecular CO2 in human RBCs; lack of AQP1 reduces P(CO2) from the normal value of 0.15 +/- 0.08 (SD; n=85) cm/s by 60% to 0.06 cm/s. Expression of AQP1 in oocytes increases P(CO2) 2-fold and doubles the alkaline pH(S) gradient. 2) pCMBS, an inhibitor of the AQP1 water channel, reduces P(CO2) of RBCs solely by action on AQP1 as it has no effect in AQP1-deficient RBCs. 3) P(CO2) determinations of RBCs and pH(S) measurements of oocytes indicate that DIDS inhibits the CO2 pathway of AQP1 by half. 4) RBCs have at least one other DIDS-sensitive pathway for CO2. We conclude that AQP1 is responsible for 60% of the high P(CO2) of red cells and that another, so far unidentified, CO2 pathway is present in this membrane that may account for at least 30% of total P(CO2).

The water channel aquaporin 1 (AQP1) and certain Rh-family members are permeable to CO 2 and NH3. Here, we use changes in surface pH (pH S) to assess relative CO2 vs. NH3 permeability of Xenopus oocytes expressing members of the AQP or Rh family. Exposed to CO 2 or NH3, AQP1 oocytes exhibit a greater maximal magnitude of pH S change (⌬pHS) compared with day-matched controls injected with H 2O or with RNA encoding SGLT1, NKCC2, or PepT1. With CO 2, AQP1 oocytes also have faster time constants for pH S relaxation ( pHs). Thus, AQP1, but not the other proteins, conduct CO 2 and NH3. Oocytes expressing rat AQP4, rat AQP5, human RhAG, or the bacterial Rh homolog AmtB also exhibit greater ⌬pH S(CO2) and faster pHs compared with controls. Oocytes expressing AmtB and RhAG, but not AQP4 or AQP5, exhibit greater ⌬pH S(NH3) values. Only AQPs exhibited significant osmotic water permeability (P f). We computed channel-dependent (*) ⌬pHS or Pf by subtracting values for H2O oocytes from those of channelexpressing oocytes. For the ratio ⌬pH S(CO2)*/Pf ‫ء‬ , the sequence was AQP5 > AQP1 Х AQP4. For ⌬pH S(CO2)*/⌬pHS(NH3)*, the sequence was AQP4 Х AQP5 > AQP1 > AmtB > RhAG. Thus, each channel exhibits a characteristic ratio for indices of CO 2 vs. NH3 permeability, demonstrating that, like ion channels, gas channels can exhibit selectivity.gas channel ͉ oocyte ͉ permeability ͉ signal peptide ͉ surface pH measurement G as transport through membranes is of fundamental importance for nutritive transport, photosynthesis, oxidative metabolism, and signaling. For most of the past century, we assumed that gas molecules cross biological membranes merely by diffusing through the lipid phase. This dogma was challenged by 2 observations: (i) Apical membranes of gastric-gland cells have no demonstrable permeability to CO 2 or NH 3 (1). (ii) Heterologous expression of the water channel aquaporin 1 (AQP1) increases the CO 2 permeability of Xenopus oocytes (2). Cooper and Boron (3) and Prasad et al. (4) confirmed and extended this observation. Uehlein (5) showed that an AQP plays a physiological role by enhancing CO 2 uptake by plants. Endeward et al. (6) demonstrated that AQP1 accounts for Ϸ60% of the CO 2 permeability of human red blood cells (RBCs). Molecular dynamics simulations suggest that CO 2 can pass through the 4 aquapores of an AQP1 tetramer (7) and especially through the central pore between the 4 monomers (7). Additional data indicate that AQP1 is permeable to nitric oxide (8), and that-when expressed in Xenopus oocytes (9, 10) or when reconstituted into planar lipid bilayers (11)-AQP1, AQP3, AQP8, AQP9, and the plant aquaporin TIP2;1 are all permeable to NH 3 .The AmtB/MEP/Rh proteins represent a second family of gas channels (12-15). Early work showed that AmtB and MEP transport NH 3 or NH 4 ϩ , thereby playing a nutritive role in archaea, bacteria, and fungi (16,17). The crystal structures of the bacterial AmtB (18-20) and Rh50 (21) and the fungal Amt-1 (22) are consistent with the idea that NH 3 passes through a pore in each m...

Others report that carbonic anhydrase II (CA II) binds to the C termini of the anion exchanger AE1 and the electrogenic Na/HCO 3 cotransporter NBCe1-A, enhancing transport. After injecting oocytes with NBCe1-A cRNA (Day 0), we measured NBC current (I NBC ) by two-electrode voltage clamp (Day 3), injected CA II protein ؉ Tris or just Tris (Day 3), measured I NBC or the initial rate at which the intracellular pH fell (dpH i /dt) upon applying 5% CO 2 (Day 4), exposed oocytes to the permeant CA inhibitor ethoxzolamide (EZA), and measured I NBC or dpH i /dt (Day 4). Because dpH i /dt was greater in CA II than Tris oocytes, and EZA eliminated the difference, injected CA II was functional. I NBC slope conductance was unaffected by injecting CA II. Moreover, EZA had identical effects in CA II versus Tris oocytes. Thus, injected CA II does not enhance NBC activity. In a second protocol, we made a fusion protein with enhanced green fluorescent protein (EGFP) at the 5 end of NBCe1-A and CA II at the 3 end (EGFP-e1-CAII). We measured I NBC or dpH i /dt (days 3-4), exposed oocytes to EZA, and measured I NBC or dpH i /dt (Day 3-4). dpH i /dt was greater in oocytes expressing EGFP-e1-CA II versus EGFP-e1, and EZA eliminated the difference. Thus, fused CA II was functional. Slope conductances of EGFP-e1-CAII versus EGFP-e1 oocytes were indistinguishable, and EZA had no effect. Thus, even when fused to NBCe1-A, CA II does not enhance NBCe1-A activity.The electrogenic Na/bicarbonate cotransporter (NBCe1 or e1) 3 plays a central role in HCO 3 Ϫ reabsorption and regulation of intracellular pH (1). The kidney-specific splice variant NBCe1-A is localized at the basolateral membrane of renal proximal tubule cells (2), where it mediates efflux of HCO 3 Ϫ (and/or CO 3 ϭ ). Cytoplasmic HCO 3 Ϫ arises from the intracellular hydration of CO 2 , which is catalyzed by the cytoplasmic enzyme carbonic anhydrase II (CA II) (3).Because the reports of Vince and Reithmeier (4 -6) that cytosolic CA II binds to the LDADD motif on the cytoplasmic C terminus of the Cl-HCO 3 exchanger AE1, Sterling et al. (7,8) have measured rates of intracellular pH (pH i ) change in HEK293 cells transiently transfected with AE1 and concluded that CA II enhances AE1-mediated HCO 3 Ϫ transport. The C termini of all three NBCe1 splice variants, i.e. NBCe1-A as well as the more universally expressed variant NBCe1-B (9, 10) and the brain-specific NBCe1-C (11), have two motifs similar to LDADD in AE1. Moreover, isothermal titration calorimetry and pulldown assays suggest that, at least under non-reducing conditions, the common C terminus of NBCe1-A/B interacts with CA II in vitro (12). Similarly, Pushkin and co-workers (12-15) working with a mouse proximal convoluted tubule (mPCT) cell line stably transfected with NBCe1-A, concluded that CA II enhances the current carried by NBCe1-A.For three reasons, we set out to verify the hypothesis that CA II enhances the activity of NBCe1-A. First, a pH i measurement, as in the AE1 study, is an indirect index of the rate of HCO 3 Ϫ ...