A novel method to determine the diffusional water permeability of oocyte plasma membranes

Measurements of the cell membrane diffusional water permeability (P_d) are important to characterize water passage across water channels and across the lipid bilayer component of the membrane. Existing methods for those measurements are involved; however, we report here a simple procedure to estimate P_d in Xenopus laevis oocytes and similar large cells. Due to the different densities of H₂O and D₂O (heavy water), an oocyte transferred from normal medium to a D₂O-based medium floats initially, but subsequently sinks when a certain amount of the water originally in them is replaced by the D₂O that diffuses in. We describe how the ‘flotation time’ (time that oocytes float in a heavy water solution before they start sinking) yields the P_d of the plasma membrane. Determination of P_d by this procedure and by the rate of tritiated water (T₂O) efflux give for P_d results which are very close: 2.2 ± 0.2 (n = 8) and 2.0 ± 0.1 (n = 6) μm/s, respectively (T = 10°C). Further-more, our method detects the increase in P_d elicited in oocytes by either expression of water channel proteins, or by treating them with the pore-forming antibiotic amphotericin B. This method appears useful to gauge the expression and function of pore-forming, water-permeable membrane proteins.     

Comparative Permeabilities of the Paracellular and Transcellular Pathways of Corneal Endothelial Layers

Layers of rabbit corneal endothelial cells were cultured on permeable inserts. We characterized the diffusional permeability of the cell layer to nonelectrolyte and charged molecules and compared the diffusional and filtration permeabilities of the paracellular and transcellular pathways. We determined the rates of diffusion of ³H- and ¹⁴C-labeled nonelectrolyte test molecules and estimated the equivalent pore radius of the tight junction. Negatively charged molecules permeate slower than neutral molecules, while positively charged molecules permeate faster. Palmitoyl-dl-carnitine, which opens tight junctions, caused an increase of permeability and equivalent pore radius. Diffusional water permeability was determined with ³H-labeled water; the permeabilities of the tight junction and lateral intercellular space were calculated using tissue geometry and the Renkin equation. The diffusional permeability (P _d ) of the paracellular pathway to water is 0.57 μm s⁻¹ and that of the transcellular path is 2.52 μm s⁻¹. From the P _d data we calculated the filtration permeabilities (P _f ) for the paracellular and transcellular pathways as 41.3 and 30.2 μm s⁻¹, respectively. In conclusion, the movement of hydrophilic molecules through tight junctions corresponds to diffusion through negatively charged pores (r = 2.1 ± 0.35 nm). The paracellular water permeability represents 58% of the filtration permeability of the layer, which points to that route as the site of sizable water transport. In addition, we calculated for NaCl a reflection coefficient of 0.16 ≤ σ_NaCl ≤ 0.33, which militates against osmosis through the junctions and, hence, indirectly supports the electro-osmosis hypothesis.     

Evidence for permanent water channels in the basolateral membrane of an ADH-sensitive epithelium

Summary The transepithelial water permeability in frog urinary bladder is believed to be essentially dependent on the ADH-regulated apical water permeability. To get a better understanding of the transmural water movement, the diffusional water permeability (P _d) of the basolateral membrane of urinary bladder was studied. Access to this post-luminal barrier was made possible by perforating the apical membrane with amphotericin B. The addition of this antibiotic increasedP _d from 1.12±0.10×10^–4 cm/sec (n=7) to 4.08±0.33×10^–4 cm/sec (n=7). The effect of mercuric sulfhydryl reagents, which are commonly used to characterize water channels, was tested on amphotericin B-treated bladders. HgCl₂ (10^–3 m) decreasedP _d by 52% andpara-chloromercuribenzoic acid (pCMB) (1.4×10^–4 m) by 34%. The activation energy for the diffusional water transport was found to increase from 4.52±0.23 kcal/mol (n=3), in the control situation, to 9.99±0.91 kcal/mol (n=4) in the presence of 1.4×10^–4 m pCMB. Our second approach was to measure the kinetics of water efflux, by stop-flow light scattering, on isolated epithelial cells from urinary bladders.pCMB (0.5 or 1.4×10^–4 m) was found to inhibit water exit by 91±2%. These data strongly support the existence of proteins responsible for water transport across the basolateral membrane, which are permanently present.     

Lack of Aquaporin 3 in bovine erythrocyte membranes correlates with low glycerol permeation

In general, erythrocytes are highly permeable to water, urea and glycerol. However, expression of aquaporin isoforms in erythrocytes appears to be species characteristic. In the present study, human (hRBC) and bovine (bRBC) erythrocytes were chosen for comparative studies due to their significant difference in membrane glycerol permeability.Osmotic water permeability (P_f) at 23 °C was (2.89 ± 0.37) × 10⁻² and (5.12 ± 0.61) × 10⁻² cm s⁻¹ for human and bovine cells, respectively, with similar activation energies for water transport. Glycerol permeability (P_gly) for human ((1.37 ± 0.26) × 10⁻⁵ cm s⁻¹) differed in three orders of magnitude from bovine erythrocytes ((5.82 ± 0.37) × 10⁻⁸ cm s⁻¹) that also showed higher activation energy for glycerol transport. When compared to human, bovine erythrocytes showed a similar expression pattern of AQP1 glycosylated forms on immunoblot analysis, though in slight higher levels, which could be correlated with the 1.5-fold larger P_f found. However, AQP3 expression was not detectable. Immunofluorescence analysis confirmed the absence of AQP3 expression in bovine erythrocyte membranes.In conclusion, lack of AQP3 in bovine erythrocytes points to the lipid pathway as responsible for glycerol permeation and explains the low glycerol permeability and high E_a for transport observed in ruminants.     

Aquaporin-11 containing a divergent NPA motif has normal water channel activity

Recently, two novel mammalian aquaporins (AQPs), AQPs 11 and 12, have been identified and classified as members of a new AQP subfamily, the “subcellular AQPs”. In members of this subfamily one of the two asparagine-proline-alanine (NPA) motifs, which play a crucial role in selective water conduction, are not completely conserved. Mouse AQP11 (mAQP11) was expressed in Sf9 cells and purified using the detergent Fos-choline 10. The protein was reconstituted into liposomes, which were used for water conduction studies with a stopped-flow device. Single water permeability (pf) of AQP11 was measured to be 1.72 ± 0.03 × 10^− 13 cm³/s, suggesting that other members of the subfamily with incompletely conserved NPA motifs may also function as water channels.     

Mercury chloride decreases the water permeability of aquaporin-4-reconstituted proteoliposomes

Background information. Mercurials inhibit AQPs (aquaporins), and site‐directed mutagenesis has identified Cys¹⁸⁹ as a site of the mercurial inhibition of AQP1. On the other hand, AQP4 has been considered to be a mercury‐insensitive water channel because it does not have the reactive cysteine residue corresponding to Cys¹⁸⁹ of AQP1. Indeed, the osmotic water permeability (P_f) of AQP4 expressed in various types of cells, including Xenopus oocytes, is not inhibited by HgCl₂. To examine the direct effects of mercurials on AQP4 in a proteoliposome reconstitution system, His‐tagged rAPR4 (rat AQP4) M23 was expressed in Saccharomyces cerevisiae, purified with an Ni²⁺‐nitrilotriacetate affinity column, and reconstituted into liposomes with the dilution method. Results. The water permeability of AQP4 proteoliposomes with or without HgCl₂ was measured with a stopped‐flow apparatus. Surprisingly, the P_f of AQP4 proteoliposomes was significantly decreased by 5 μM HgCl₂ within 30 s, and this effect was completely reversed by 2‐mercaptoethanol. The dose‐ and time‐dependent inhibitory effects of Hg²⁺ suggest that the sensitivity to mercury of AQP4 is different from that of AQP1. Site‐directed mutagenesis of six cysteine residues of AQP4 demonstrated that Cys¹⁷⁸, which is located at loop D facing the intracellular side, is a target responding to Hg²⁺. We confirmed that AQP4 is reconstituted into liposome in a bidirectional orientation. Conclusions. Our results suggest that mercury inhibits the P_f of AQP4 by mechanisms different from those for AQP1 and that AQP4 may be gated by modification of a cysteine residue in cytoplasmic loop D.     

Diffusive water permeability in isolated kidney proximal tubular cells: Nature of the cellular water pathways

Summary The diffusive water permeability (P _d ) of the plasma membrane of proximal kidney tubule cells was measured using a¹H-NMR technique. The values obtained for the exchange time (T _ex) across the membrane were independent of the cytocrit and of the Mn²⁺ concentration (in the range 2.5 to 5mm). At 25°C the calculatedP _d value was (per cm² of outer surface area without taking into account membrane invaginations) 197±17 m/sec. This value equals 22.3±1.9 m/sec when the invaginations are taken into account. Cell exposure to 2.5mm parachloromercuribenzenesulfonic acid,pCMBS, (for 20 to 35 min) reducedP _d to 45% of its control value. Fivemm dithiothreitol, DTT, reverted this effect. The activation energy for the diffusive water flux was 5.2±1.0 kcal/mol under control conditions. It increased to 9.1±2.2 kcal/mol in the presence of 2.5mm pCMBS. Using our previous values for the osmotic water permeability (P _os) in proximal straight tubular cells theP _os/P _d ratio equals 18±1, under control conditions, and 3.2±0.3 in the presence ofpCMBS. These experimental results indicate the presence of pathways for water, formed by proteins, crossing these membranes, which are closed bypCMBS. Assuming laminar flow (within the pore), fromP _os/P _d of 13 to 18 an unreasonably large pore radius of 12 to 15 Å is calculated which would not hinder cell entry of known extracellular markers. Alternatively, for a single-file pore, 11 to 20 would be the number of water molecules which would be in tandem inside the pore. The water permeability remaining in the presence ofpCMBS indicates water permeation through the lipid bilayer. There are similarities between these results and those obtained in human red blood cells and in the apical cell membrane of the toad urinary bladder.     

Investigation of γE-crystallin target protein binding to bovine lens alpha-crystallin by small-angle neutron scattering

α-Crystallin, one of the main constituent proteins in the crystalline lens, is an important molecular chaperone both within and outside the lens. Presently, the structural relationship between α-crystallin and its target proteins during chaperone action is poorly understood. It has been hypothesised that target proteins bind within a central cavity. Small-angle neutron-scattering (SANS) experiments in conjunction with isotopic substitution were undertaken to investigate the interaction of a target lens protein (γE-crystallin) with α-crystallin (α_H) and to measure the radius of gyration (Rg) of the proteins and their binary complexes in solution under thermal stress. The size of the α_H in D₂O incubated at 65 °C increased from 69 ± 3 to 81 ± 5 Å over 40 min, in good agreement with previously published small-angle X-ray scattering (SAXS) and SANS measurements. Deuterated γE-crystallin in H₂O buffer (γE_D/H₂O) and hydrogenous γE-crystallin in D₂O buffer (γE_H/D₂O) free in solution were of insufficient size and/or too dilute to provide any measurable scattering over the angular range used, which was selected primarily to investigate γE:α_H complexes. The evolution of the aggregation size/shape as an indicator of α_H chaperone action was monitored by recording the neutron scattering in different H:D solvent contrasts under thermally stressed conditions (65 °C) for binary mixtures of α_H, γE_H, and γE_D. It was found that Rg(α_H:γE_D/D₂O) > Rg(α_H:γE_H/D₂O) > Rg(α_H/D₂O) and that Rg(α_H:γE_H/D₂O) ≈ Rg(α_H/D₂O). The relative sizes observed for the complexes weighted by the respective scattering powers of the various components imply that γE-crystallin binds in a central cavity of the α-crystallin oligomer, during chaperone action.     

Water permeability properties of the ovarian oocytes from Bufo arenarum and Xenopus laevis: A comparative study

The water permeability properties of ovarian oocytes from Xenopus laevis and Bufo arenarum, a toad species found in the Buenos Aires region, were studied. We report that: (i) the water osmotic permeability (P _f, cm/sec × 10^–4) was significantly higher in Bufo (6°C=12.3±2.4; 18°C = 20.8±4.8) than in Xenopus oocytes (6°C=5.3±0.3; 18°C=6.2±1.6). The corresponding water diffusion permeability values (P _d, cm/sec × 10^–4) were: Xenopus = 2.3±0.3 (6°C) and 4.8±0.7 (18°C); Bufo=2.7±0.4 (6°C) and 6.0 ±0.5 (18°C). (ii) Amphotericin B increased the P _f and P _d values. The observed P _fP _d ratio was not significantly different from the expected results (n=3), after amphotericin B incorporation in both species. This means that the influence of unstirred layers and other potential artifactual compounds did not significantly affect our experimental results, (iii) Preincubation with gramicidin during 12 hr induced a clear increase in the oocyte volume. After that, a hypotonic shock only slightly increased the oocyte volume. Conversely, a hypertonic challenge induced a volume change significantly higher than the one observed in control conditions, (iv) Mercury ions did not affect the osmotic permeability in Xenopus oocytes but clearly inhibited, in a reversible way, the osmotic permeability in oocytes from B. arenarum. (v) Mercury ions did not reduce P _d values in either species, (vi) The P _fP _d values calculated from the differences observed in these parameters between both species were 11.9±5.1 at 18°C and 15.5±2.4 at 6°C. These numbers are similar to those previously reported in the case of membranes having water channels. From these results, we propose that water channels are present in the ovarian oocyte from B. arenarum but not in the ovarian oocyte from X. laevis.This work was supported by Fundación Antorchas, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina) and Universidad de Buenos Aires (UBA). It was developed in the frame of an INSERM (France)-CONICET cooperative program.     

Oxidative gating of water channels (aquaporins) in Chara by hydroxyl radicals

Hydroxyl radicals (*OH) as produced in the Fenton reaction (Fe²⁺ + H₂O₂ = Fe³⁺ + OH^– + *OH) have been used to reversibly inhibit aquaporins in the plasma membrane of internodes of Chara corallina. Compared to conventional agents such as HgCl₂, *OH proved to be more effective in blocking water channels and was less toxic to the cell. When internodes were treated for 30 min, cell hydraulic conductivity (Lp) decreased by 90% or even more. This effect was reversed within a few minutes after removing the radicals from the medium. In contrast to HgCl₂, radical treatment reduced membrane permeability of small lipophilic organic solutes (ethanol, acetone, 1‐propanol, and 2‐propanol) by only 24 to 52%, indicating some continued limited movement of these solutes across aquaporins. The biggest effect of *OH treatment on solute permeability was found for isotopic water (HDO), which largely used water channels to cross the membrane. Inhibition of aquaporins reduced the diffusional water permeability (P_d) by about 70%. For the organic test solutes, which mainly use the bilayer to cross the membrane, channel closure caused anomalous (negative) osmosis; that is, cells had negative reflection coefficients (σ_s) and were transiently swelling in a hypertonic medium. From the ratio of bulk (Lp or osmotic permeability coefficient, P_f) to diffusional (P_d) permeability of water, the number (N) of water molecules that align in water channels was estimated to be N = P_f/P_d = 46 (on average). Radical treatment decreased N from 46 to 11, a value still larger than unity, which would be expected for a membrane lacking pores. The gating of aquaporins by *OH radicals is discussed in terms of a direct action of the radicals when passing the pores or by an indirect action via the bilayer. The rapid recovery of inhibited channels may indicate an easy access of cytoplasmic antioxidants to closed water channels. As hydrogen peroxide is a major signalling substance during different biotic and abiotic stresses, the reversible closure of water channels by *OH (as produced from H₂O₂ in the apoplast in the presence of transition metals such as Fe²⁺ or Cu⁺) may be downstream of the H₂O₂ signalling. This may provide appropriate adjustments in water relations (hydraulic conductivity), and a common response to different kinds of stresses.     

Gating of aquaporins by heavy metals in <Emphasis Type="Italic">Allium cepa</Emphasis> L. epidermal cells

Changes in the water permeability, aquaporin (AQP) activity, of leaf cells were investigated in response to different heavy metals (Zn²⁺, Pb²⁺, Cd²⁺, Hg²⁺). The cell pressure probe experiments were performed on onion epidermal cells as a model system. Heavy metal solutions at different concentrations (0.05 μM–2 mM) were used in our experiments. We showed that the investigated metal ions can be arranged in order of decreasing toxicity (expressed as a decrease in water permeability) as follows: Hg>Cd>Pb>Zn. Our results showed that β-mercaptoethanol treatment (10 mM solution) partially reverses the effect of AQP gating. The magnitude of this reverse differed depending on the metal and its concentration. The time course studies of the process showed that the gating of AQPs occurred within the first 10 min after the application of a metal. We also showed that after 20–40 min from the onset of metal treatment, the water flow through AQPs stabilized and remained constant. We observed that irrespective of the metal applied, the effect of AQP gating can be recorded within the first 10 min after the administration of metal ions. More generally, our results indicate that the toxic effects of investigated metal ions on the cellular level may involve AQP gating.     

Mean residence time of molecules diffusing in a cell bounded by a semi-permeable membrane: Monte Carlo simulations and an expression relating membrane transition probability to permeability

 The rapid exchange of water across erythrocyte membranes is readily measured using an NMR method that entails doping a suspension of cells with a moderately high concentration of Mn²⁺ and measuring the rate of transverse relaxation of the nuclear magnetisation. Analysis of the data yields an estimate of the rate constant for membrane transport, from which the membrane permeability can be determined. It is assumed in the analysis that the efflux rate of the water is solely a function of the rate of membrane permeation and that the time it takes for intracellular water molecules to diffuse to the membrane is relatively insignificant. The limits of this assumption were explored by using random-walk simulations of diffusion in cells modelled as parallel planes, spheres, and biconcave discs. The rate of membrane transport was specified in terms of a transition probability but it was not initially clear what the relationship should be between this parameter and the diffusional membrane permeability P _d. This relationship was derived and used to show that the mean residence time for a water molecule is determined by P _d when the diffusion coefficient is above a certain threshold value; it is determined by the distance to the membrane below that value. Received: 7 January 2000 / Revised version: 4 April 2000 / Accepted: 4 April 2000     

Comparative NMR studies of diffusional water permeability of red blood cells from different species: XVIII platypus (Ornithorhynchus anatinus) and saltwater crocodile (Crocodylus porosus)

As part of a programme of comparative measurements of P _d (diffusional water permeability) the RBCs (red blood cells) from an aquatic monotreme, platypus (Ornithorhynchus anatinus), and an aquatic reptile, saltwater crocodile (Crocodylus porosus) were studied. The mean diameter of platypus RBCs was estimated by light microscopy and found to be ～6.3 μm. P _d was measured by using an Mn²⁺‐doping 1H NMR (nuclear magnetic resonance) technique. The P _d (cm/s) values were relatively low: ～2.1×10^?3 at 25°C, 2.5×10^?3 at 30°C, 3.4×10^?3 at 37°C and 4.5 at 42°C for the platypus RBCs and ～2.8×10^?3 at 25°C, 3.2×10^?3 at 30°C, 4.5×10^?3 at 37°C and 5.7×10^?3 at 42°C for the crocodile RBCs. In parallel with the low water permeability, the E _a,d (activation energy of water diffusion) was relatively high, ～35 kJ/mol. These results suggest that “conventional” WCPs (water channel proteins), or AQPs (aquaporins), are probably absent from the plasma membranes of RBCs from both the platypus and the saltwater crocodile.     

Stimulating Effect of External Myo-Inositol on the Expression of Mutant Forms of Aquaporin 2

Myo-inositol (MI; hexahydroxycyclohexane, C₆H₆O₁₂) is a small neutral molecule used as a compatible osmolyte in the kidney medulla. At high concentrations, MI appears to act as a chemical chaperone and was shown to promote plasma membrane expression of the impaired cystic fibrosis chloride channel (Δ508-CFTR). In the present study, we measured whether MI could increase expression of two human aquaporin 2 (AQP2) mutants which were recently identified as causing nephrogenic diabetes insipidus (NDI). Both proteins (D150E and G196D) were expressed in Xenopus laevis oocytes, but only D150E displayed an increase in oocyte water permeability (P _f). Adding 5 mM MI to the bathing solution for 24 h produced a 50% increase in the D150E-associated P _f, while it had no effect on noninjected oocytes or on oocytes expressing wt-AQP2 or G196D. Western blots performed on purified plasma membrane preparations confirmed that MI increased the amount of D150E present at the plasma membrane, while G196D was always undetectable. X. laevis oocytes are remarkably impermeable to MI, and the effect of MI on D150E expression does not require the presence of intracellular MI. The effect of external MI was dose-dependent (K _0.5 was 130 μM) and specific with respect to other forms of inositols. Further studies on a second group of AQP2 mutants causing NDI showed that K228E activity was similarly stimulated by MI, while V71M, A70D and S256L were not. It is concluded that physiological concentrations of extracellular MI can stimulate the expression of a specific subgroup of AQP2 mutants.     

Reconstitution of a Regulated Transepithelial Water Pathway in Cells Transfected with AQP2 and an AQP1/AQP2 Hybrid Containing the AQP2-C Terminus

Transepithelial water permeability was measured in LLC-PK1 cells stably transfected with aquaporins (AQPs): AQP1, AQP2, and a chimera of AQP1 and AQP2 containing 41 amino acids of the C-terminus of AQP2. Transepithelial water fluxes (Jw) were not previously reported in cells transfected with aquaporins. Jw were now recorded each minute using a specially developed experimental device. A significant increase in Posm after forskolin (FK) plus vasopressin (VP) was found in AQP2 transfected cells (39.9 ± 8.2 vs. 12.5 ± 3.3 cm · sec⁻¹· 10⁻³), but not in cells transfected with AQP1 (15.3 ± 3.6 vs. 13.4 ± 3.6 cm · sec⁻¹· 10⁻³). In the case of the AQP1/2 cells (chimera) the FK plus VP induced Posm was smaller than in AQP2 cells but significantly higher than in mock cells at rest (18.1 ± 4.8 vs. 6.7 ± 1.0 cm · sec⁻¹· 10⁻³). The increases in Posm values were not paralleled by increases in ¹⁴C-Mannitol permeability. HgCl₂ inhibited the hydrosmotic response to FK plus VP in AQP2 transfected epithelia. Results were comparable to those observed, in parallel experiments, in a native ADH-sensitive water channel containing epithelial barrier (the toad urinary bladder). Electron microscopy showed confluent LLC-PK1 cells with microvilli at the mucosal border. The presence of spherical or elongated intracellular vacuoles was observed in AQP2 transfected cells, specially after FK plus VP stimulus and under an osmotic gradient. These results demonstrate regulated transepithelial water permeability in epithelial cells transfected with AQP2. Received: 24 June 1997/Revised: 16 September 1997     

Enantiodifferent Proton Exchange in Alanine and Asparagine in the Presence of H 2 17 O

Using Time Domain ¹H Nuclear Magnetic Resonance with H₂¹⁷O (H₂¹⁷O-TD-¹HNMR), we found [H₂¹⁷O]- and pH-controlled chiral differences in proton exchange properties in alanine (Ala) and asparagine (Asn). To minimize and equalize chemical impurities, Asn enantiomers were purified by crystallization from racemic solution. At <0.1 M H₂¹⁷O, a shift in isoelectric pH (pI) occurred, ~1.14 kJ mol⁻¹ l-d-Asn ΔΔG ^o′ in the 5.91–6.42 pH range. One potential source for this asymmetry is the enantio-different magnetic moments (lμ↑ ≠ dμ↓) produced by neutral ring currents in the chiral center, leading to enantio-different nuclear spin organization and charge distribution in the amino group. At ≥pI, dissimilar interactions may occur in the hydration of the amino group with H₂¹⁷O (NH₂/H₂¹⁷O ≠ NH₂/H₂¹⁶O; NH₃ ⁺/H₂¹⁷O ≠ NH₂/H₂¹⁷O; l-*C-NH₂/H₂¹⁷O ≠ d-*C-NH₂/H₂¹⁷O). As lμ↑ ≠ dμ↓, the l-*C-amino and the d-*C-amino groups are diastereo spin-isomers. The nuclear spin of ¹⁷O may be parallel or antiparallel with the ortho-¹H¹H pair; hence two ortho-H₂¹⁷O molecules exist, also diastereo spin-isomers. As the pK of H₂¹⁷O is different from H₂¹⁶O, dissimilarities between l-*C- and d-*C-amino groups are converted into proton exchange differences. During H₂¹⁷O-TD-¹HNMR, the H₂¹⁷O molecule is a “probe” of the state of the amino group. Regarding prebiotic evolution: prebiotic chirality may not require stochastic symmetry breaking or preexisting chiral conditions; chemical chiral effects due to lμ↑ ≠ dμ↓ are small and need chiral amplification to generate an enantiomeric excess significant for prebiotic evolution; and prebiotic symmetry breaking was homochiral because the effect of lμ↑ and dμ↓ on the amino group should be similar in all alpha amino acids.     

Diffusional water permeability of mammalian red blood cells

An extensive programme of comparative nuclear magnetic resonance measurements of the membrane diffusional permeability for water (P_d) and of the activation energy (E_a,d) of this process in red blood cells (RBCs) from 21 mammalian species was carried out. On the basis of P_d, these species could be divided into three groups. First, the RBC's from humans, cow, sheep and “large” kangaroos (Macropus giganteus and Macropus rufus) had P_d values 5 × 10⁻³ cm/s at 25°C and 7 × 10⁻³ cm/s at 37°C. The RBCs from other marsupial species, mouse, rat, guinea pig and rabbit, had P_d values roughly twice higher, whereas echidna RBCs were twice lower than human RBCs. The value of E_a,d was in most cases correlated with the values of P_d. A value of E_a,d -26 kJ/mol was found for the RBCs from humans and the species having similar P_d values. Low values of E_a,d (ranging from 15 to 22 kJ/mol) appeared to be associated with relatively high values of P_d. The highest value of E_a,d (33 kJ/mol) was found in echidna RBCs. This points to specialized channels for water diffusion incorporated in membrane proteins; a relatively high water permeability of the RBC membrane could be due to a greater number of channel proteins. There are, however, situations where a very high water permeability of RBCs is associated with a high value of E_a,d (above 25 kJ/mol) as in the case of RBCs from mouse, rat and tree kangaroo. Moreover, it was found that P_d in different species was positively correlated to the RBC membrane phosphatidylcholine and negatively correlated to the sphingomyelin content. This suggests that in addition to the number of channel proteins, other factors are involved in the water permeability of the RBC membrane.     

Biological significance and topological basis of aquaporin-partnering protein-protein interactions

Aquaporins (AQPs) are intramolecular channels essential for transport of H₂O, CO₂, and other small substrates across membranes. Through this function, AQPs can modulate CO₂ uptake and assimilation in plants and regulate water relations and many other physiological processes in all living organisms. To execute their physiological roles, AQPs may experience 3 types of hetero-molecular interaction, between AQPs and their kinases; between AQP isoforms; and between AQPs and other proteins that are neither AQPs nor kinases. Interacting with non-AQP non-kinase proteins may enable AQPs to extend their functions beyond substrate transport, and most fascinatingly, to serve as a gateway control for translocation of virulence effectors from pathogenic bacteria into the cytosol of eukaryotic cells. In this mini review, we will summarize the latter 2 types of interaction and discuss the physiological and/or pathological significance. We will also discuss a research angle to elucidate the structural basis of AQP-partnering protein interactions.     

Effect of PCMBS on CO2 permeability of Xenopus oocytes expressing aquaporin 1or its C189S mutant

A recent study onXenopus oocytes [N. L. Nakhoul,M. F. Romero, B. A. Davis, and W. F. Boron. Am. J. Physiol. 274 (CellPhysiol. 43): C543-548, 1998] injected withcarbonic anhydrase showed that expressing aquaporin 1 (AQP1) increasesby ~40% the rate at which exposing the cell toCO₂ causes intracellular pH tofall. This observation is consistent with several interpretations.Overexpressing AQP1 might increase apparentCO₂ permeability by1) allowingCO₂ to pass through AQP1,2) stimulating injected carbonicanhydrase, 3) enhancing theCO₂ solubility of the membrane'slipid, or 4) increasing theexpression of a native "gas channel." The purpose of the presentstudy was to distinguish among these possibilities. We found thatexpressing the H₂O channel AQP1 inXenopus oocytes increases theCO₂ permeability of oocytes in anexpression-dependent fashion, whereas expressing theK⁺ channel ROMK1 has no effect.The mercury derivativep-chloromercuriphenylsulfonic acid(PCMBS), which inhibits the H₂Omovement through AQP1, also blocks the AQP1-dependent increase inCO₂ permeability. Themercury-insensitive C189S mutant of AQP1 increases theCO₂ permeability of the oocyte tothe same extent as does the wild-type channel. However, the C189S-dependent increase in CO₂permeability is unaffected by treatment with PCMBS. These data rule outoptions 2-4 listed above. Thusour results suggest that CO₂passes through the pore of AQP1 and are the first data to demonstratethat a gas can enter a cell by a means other than diffusing through themembrane lipid.