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J. Biol. Chem., Vol. 282, Issue 8, 5296-5301, February 23, 2007

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Fast and Selective Ammonia Transport by Aquaporin-8^*

Sapar M. Saparov, Kun Liu, Peter Agre, and Peter Pohl¹

From the Institut für Biophysik, Kepler Universität Linz, Altenbergerstrasse 69, A-4040 Linz, Austria and Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710

Received for publication, October 3, 2006 , and in revised form, November 29, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The transport of ammonia/ammonium is fundamental to nitrogen metabolism in all forms of life. So far, no clear picture has emerged as to whether a protein channel is capable of transporting exclusively neutral NH₃ while excluding H⁺ and . Our research is the first stoichiometric study to show the selective transport of NH₃ by a membrane channel. The purified water channel protein aquaporin-8 was reconstituted into planar bilayers, and the exclusion of or H⁺ was established by ensuring a lack of current under voltage clamp conditions. The single channel water permeability coefficient of 1.2 x 10^–14 cm³/subunit/s was established by imposing an osmotic gradient across reconstituted planar bilayers, and resulting minute changes in ionic concentration close to the membrane surface were detected. It is more than 2-fold smaller than the single channel ammonia permeability (2.7 x 10^–14 cm³/subunit/s) that was derived by establishing a transmembrane ammonium concentration gradient and measuring the resulting concentration increases adjacent to the membrane. This permeability ratio suggests that electrically silent ammonia transport may be the main function of AQP8.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Aquaporins (AQPs)² are commonly believed to mediate fast and selective water transport (1). However, some members of the protein family may have other functions. The intracellular acid-sensing aquaporin-6 (AQP6), for example, serves as an anion channel (2). The function of aquaporin-8 (AQP8) is under dispute. First, it was suggested that AQP8-mediated water transport may be particularly important for the rapid expansion of mitochondrial volume (3). In a contrasting study, it was concluded that the rapid volume equilibration in mitochondria in response to an osmotic gradient was due to its small size (high surface-to-volume ratio) rather than to AQP-mediated high membrane water permeability (4). Moreover, only mild phenotype differences between wild-type and AQP8-deficient mice were found (5).

In addition, AQP8 was anticipated to participate in ammonia transport. AQP8 was able to rescue the growth of yeast defective in ammonium uptake, suggesting that the protein is involved in NH₃ transport in humans. Increased acidification of the oocyte medium containing was in accordance with NH₃ diffusion through the protein (6). Voltage clamp experiments suggested that AQP8 conducts as well (7). Because growth complementation could be an indirect effect of AQP8 expression, light-scattering experiments with reconstituted vesicles were conducted. They revealed AQP8 permeability to formamide, suggesting that the protein may transport ammonium in vivo and physiologically contribute to the acid-base equilibrium (8). However, comparative phenotype studies in wild-type versus AQP8 null mice revealed no significant or only very small differences in serum ammonia, colonic ammonia absorption, renal ammonia clearance, and liver ammonia accumulation (9). It is difficult to interpret these results as evidence against physiologically significant AQP8-facilitated NH₃ transport in mice. Because of the importance of ammonium homeostasis, several ammonium transport pathways are likely to exist. This is crucial for the urine pH adjustment and the acid-base equilibrium of body fluid. One of the alternative NH₃ transport pathways was identified in terms of RhBG and RhCG, the non-erythroid members of the Rh family (10). These proteins were also knocked out in mice, and neither distal tubular acidosis nor hyperammonemia was detected (11).

The assumption that a double knock out of both AQP8 and Rh proteins leads to a detectable phenotype remains to be tested. This expectation is based on the observation that the apical membranes of AQP8-expressing cells must maintain large chemical and osmotic gradients and therefore be effectively impermeable to small molecules, including NH₃ and water. Tightening of the lipid matrix and, thus, a reduced permeability is achieved by high concentrations of glycosphingolipids and sphingomyelin in the outer membrane leaflet (12). It is therefore not surprising that, for example, the expression of hepatic AQP8 was associated with apical microdomain fractions enriched in cholesterol and sphingolipids (13). With respect to the low basal NH₃ permeability of the epithelial membrane, the requirement for controllable proteinaceous NH₃ transport machinery becomes obvious. AQP8 is the ideal candidate because it is largely localized in intracellular vesicles and can be redistributed to plasma membranes via a microtubule-dependent, cAMP-stimulated mechanism (14).

The molecular mechanism for ammonia transport by AQP8 has not yet been resolved. Evidence was reported showing both NH₃ and transport (7), although it was not possible to differentiate whether transport occurred through the aquaporin itself or whether secondary effects related to rapid NH₃ transport took place. We have addressed this question by reconstituting purified AQP8 into planar lipid bilayers mimicking the lipid composition of epithelial plasma membranes. Functional reconstitution was confirmed by water flux measurements. Simultaneous ion and ammonia flux measurements revealed perfect NH₃ selectivity, i.e. ammonia transport by AQP8 is electrically silent. We found that ammonia permeability exceeds water permeability 2-fold, suggesting that ammonia transport may be the main function of AQP8.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Expression and Purification of AQP8 from Yeast—Rat AQP8 was expressed in pep4 Saccharomyces cerevisiae and purified as previously described (8). In brief, after induction with 2% (w/w) galactose harvested by centrifugation, and three French press cycles, the membrane fraction was recovered from the supernatant by ultracentrifugation. AQP8 protein was solubilized in n-octyl--D-glucoside. The His₁₀-tagged protein was absorbed by a nickel column. Its purity was better than 90% as revealed by the comparison of Coomassie-stained SDS gels with bands obtained by immunoblotting.

Protein Reconstitution into Planar Bilayers—Purified AQP8 proteins were first reconstituted into proteoliposomes by dialysis (15). In brief, the reconstitution mixture was prepared at room temperature by sequentially adding 100 mM MOPS-sodium, pH 7.5 (Fluka, Buchs, Switzerland), 1.25% (w/v) octyl glucoside, purified AQP8 (final concentration 0.5–1 mg/ml), and 20–50 mg/ml of preformed lipid vesicles. The latter consisted of a 3:2:1 molar mixture of cholesterol, E. coli lipid extract (Avanti%20Polar%20Lipids">Avanti Polar Lipids, Alabaster, AL), and sphingomyelin. It was loaded into SPECTRA/POR 2.1 dialysis tubing, molecular weight cut-off 15,000 (Spectrum Laboratories, Laguna Hills, CA), and dialyzed against 100 volumes of assay buffer for 48 h at 4 °C. Proteoliposomes were harvested by ultracentrifugation (60 min at 100,000 x g) and were resuspended into assay buffer (compare Fig. 1) at a concentration of 5–10 mg/ml.

At the air-water interface of vesicle suspensions, monolayers were formed spontaneously (16). Two such monolayers were combined to form a planar bilayer in the 150-µm-diameter aperture of a 25-µm-thick polytetrafluoroethylene septum separating the two aqueous phases of the chamber (15, 17). The septum was pretreated with a hexadecane-hexane mixture (volume ratio of 1:200).

Water Flux Measurements—Transmembrane osmotic water flow was derived from solute dilution in the immediate membrane vicinity (18). The solute concentration at the interface, C_s, increases with the distance, x, to the membrane: C(x) = C_s exp(–vx/D + bx³/3D), where –v and b are – the linear drift velocity of the osmotic volume flow and the stirring parameter, respectively. In the steady state, v was obtained by fitting the concentration distribution of Na⁺ ions to this equation. v is related to P_f by P_f = v/(C_osm V_w) (19). J_w is the product of water concentration and v. Na⁺ concentrations were measured by microelectrodes made of glass capillaries, the tips (1–2 µm in diameter) of which were filled with mixture A of Sodium Ionophore I (Fluka, Dreisenhofen, Germany). Movement of the electrodes relative to the membrane was realized by a hydraulic stepdrive (Narishige).

Ion Flux Measurements—Ag/AgCl reference electrodes were immersed into the buffer solutions at both sides of the planar bilayers. Under voltage clamp conditions, the transmembrane current was measured by a patch clamp amplifier (model EPC9; HEKA Electronics). The recording filter was a 4-pole Bessel with 3-dB corner frequency of 0.1 kHz. The acquired raw data were analyzed with the help of the TAC software package (Bruxton Corp., Seattle, WA). A Gaussian filter of 0.3 Hz was applied to reduce noise.

Ammonia Flux Measurements—Ammonium chloride was added to the buffer solutions surrounding the bilayer. Its concentration at the cis side of the membrane was higher than at the trans side. The resulting transmembrane NH₃ flux gave rise to a pH and a gradient in the immediate membrane vicinity. The latter was measured in terms of a potential difference between a selective microelectrode and a reference electrode, both placed in the buffer solution at the same side of the membrane (20). Movement of the electrodes relative to the membrane was realized by a hydraulic stepdrive (Narishige).

Theoretical Model for NH₃ Transport—In the model, the membrane flux of is neglected. This assumption is justified by the lack of any incremental conductivity after AQP8 reconstitution (Fig. 2). For acidic pH the experimental results are analyzed precisely by solving the complete system of differential equations that takes into account all relevant chemical reactions in the immediate membrane vicinity (20, 21) as shown in Equations 1 and 2.

(Eq. 1)

(Eq. 2)

J_i, D_i, c_i(x) denote, respectively, flux, diffusion coefficient, and concentration of the ith species, where 1 = H⁺, 2 = NH₃, , 4 = OH^–, 5 = MES^–, 6 = H-MES. R_i(c) is the specific local rate of expenditure of the ith species in chemical reactions 1 and 2.

REACTION 1

REACTION 2

At the membrane-water interface, the fluxes of all species are required to be equal to zero, except for J₂, as shown in Equation 3.

(Eq. 3)

For other boundary conditions, please compare Antonenko et al. (21). The numerical solutions are derived, assuming that the rates of chemical reactions (like dissociation/recombination of water, buffer, and NH₃) are very high compared with the rate of diffusion through the USL, so that the local chemical equilibrium is maintained.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
To demonstrate functional reconstitution, we measured osmotic water flow through purified AQP8 channels by imposing an osmotic gradient across reconstituted planar bilayers and detected resulting minute changes in ionic concentration close to the membrane surface (15, 17). We formed planar membranes that mimicked the composition of epithelial cells (cholesterol:E. coli lipid extract:sphingomyelin = 3:2:1). In line with previous experiments (12), these membranes exhibited a very low osmotic water permeability, P_f, of only (11.0 ± 1.5) µm/s. Reconstitution of AQP8 at lipid:protein mass ratio, r_m = 100, resulted in a 3-fold increase in P_f (Fig. 1). The incremental water permeability, P_f.c, allowed calculation of the hydraulic permeability coefficient of a single channel, p_f, from the absolute hydraulic conductivity of all channels P_f.c and the number of channels, n (19). n is anticipated to be equal to the total number of lipid molecules, L, in the bilayer divided by the molar lipid to protein ratio, r, where L is derived from two times (for both leaflets) the membrane area, A, divided by the area, b, per lipid molecule as shown in Equation 4

(Eq. 4)

where M_P and M_L are the molecular masses of the protein (30 kDa) and the lipid (700 Da), respectively. For b = 70 A², p_f of AQP8 is found to be (2.4 ± 0.2) x 10^–14 cm³ subunit^–1 s^–1. The result was obtained assuming that protein incorporation is 100% efficient. The real efficiency is, most probably, smaller. Reconstitution of AQP0, for example, was 50% efficient (22). If our value is in the same range, p_f adopts a value of (1.2 ± 0.1) x 10^–14 cm³ subunit^–1 s^–1. That is in reasonable agreement with results reported previously using a Xenopus oocyte expression system (23). p_f allows calculation of the AQP8 turnover numbers for water, T_W as shown in Equation 5

(Eq. 5)

where N_A and V_W are the Avogadro number and the molecular volume of water, respectively.

View larger version (18K): [in this window] [in a new window]   FIGURE 1. Water transport by AQP8. Reconstitution of AQP8 (protein:lipid = 1:100) augmented water permeability of bare lipid bilayers (cholesterol:E. coli lipid extract:sphingomyelin = 3:2:1) from 11 to 27 µm/s. Water permeability was calculated from the dilution of Na+ ions shown as a function of the distance to the membrane. Osmotic water flux was induced by 1 M urea. The buffer contained 20 mM MES, 100 mM NaCl, 1 mM NH4Cl, pH 6.0.

(Eq. 6)

Consequently, the ion:water selectivity was better than 1:10⁹ (compare Refs. 17, 24).

View larger version (49K): [in this window] [in a new window]   FIGURE 2. Exclusion of NH+4 transport by AQP8. The current voltage characteristics of bare lipid bilayers and bilayers reconstituted with AQP8 are not altered by the hundredfold augmentation of the NH4Cl bulk concentration. A voltage ramp (duration 3 min) was applied in the interval from –90 to +90 mV. The current was measured with a frequency of 100 Hz and than filtered at 0.3 Hz (compare "Experimental Procedures"). The resulting data cloud is plotted. The spline lines were obtained by applying a local smoothing technique (100 intervals) using polynomial regression and weights computed from the Gaussian density function (SigmaPlot). The similarity of their slopes indicates that AQP8 excludes other ions as well. Membrane and buffer composition were as in Fig. 1.

(Eq. 7)

(Eq. 8)

D_NH3, , and x are the aqueous diffusion coefficients of NH₃, , and the distance to the membrane, respectively. is equal to 10^{(pH – p K)}. A concentration gradient was imposed across the planar bilayer, and the resulting small changes in concentration close to the membrane were detected by scanning ammonia-selective microelectrodes. The difference in polarization adjacent to bare (Fig. 3A) and reconstituted planar bilayers (Fig. 3B) indicated NH₃ transport by aquaporins. Plotting versus the transmembrane concentration gradient reveals a linear dependence (Fig. 3C). Calculation of was performed taking into account the accompanying chemical reactions of ammonia and buffer. The set of differential equations (see "Experimental Procedures") was solved numerically to fit the experimental profiles for 0 < x <50 µm (20, 21). NH₃ permeabilities of 16 and 105 µm/s were computed for bare and AQP8-containing bilayers, respectively.

View larger version (24K): [in this window] [in a new window]   FIGURE 3. NH3 flux J as a function of the transmembrane NH4Cl concentration gradient. The concentration in the trans compartment was measured by scanning microelectrodes as a function of the distance to the membrane. A, representative concentration profiles visualizing NH3 diffusion through a bare lipid bilayer made of a lipid mixture (cholesterol:E. coli lipid extract:sphingomyelin = 3:2:1). B, representative concentration profiles showing NH3 diffusion through reconstituted AQP8. For membrane formation the same lipid composition as in panel A was used. The protein:lipid ratio was 1:50. C, NH3 flux at different bulk concentrations in the cis compartment. JNH3 and PNH3 were calculated using the analytical model of weak base diffusion (Equations 1, 2, 3 and Reactions 1 and 2). The regression lines correspond to NH3 permeabilities of 16 and 105 µm/s for bare and AQP8-containing bilayers, respectively. The buffer solution contained 20 mM MES, 100 mM NaCl, pH 6.0. The trans compartment contained 1 mM NH4Cl.

View larger version (13K): [in this window] [in a new window]   FIGURE 4. Inhibition of AQP8 mediated NH3 transport by Hg2+. Reconstitution of AQP8 in a protein:lipid ratio of 1:120 resulted in a NH3 permeability of 50 µm/s. 1 mM Hg2+ reduced the permeability to that of a bare bilayer (here to 15 µm/s). The corresponding transmembrane NH3 fluxes were 0.2 and 0.06 nmol cm–2 s–1 in the absence and presence of Hg2+, respectively. The trans and cis NH4Cl concentrations in the bulk were equal to 1 and 80 mM, respectively. Buffer composition was as in Fig. 1.

For further proof of NH₃ conductance by AQP8, flux dependence on membrane protein abundance was measured. Reconstitution of increasing amounts of AQP8 was accompanied by an increasing concentration polarization in the immediate membrane vicinity (Fig. 5). As before, the set of differential equations (see "Experimental Procedures") was solved numerically for different . For each AQP8 concentration the parameter was changed iteratively until the deviation of the theoretical profile from the experimental one was minimal. Plotting as a function of 1/r_m allowed calculation of single channel ammonium permeability (Fig. 5, inset) as shown in Equation 9

(Eq. 9)

where P_NH3,c is the incremental NH₃ permeability introduced by AQP8 reconstitution. Assuming 50% reconstitution efficiency p_NH3 was equal to (2.7 ± 0.2) x 10^–14 cm³ subunit^–1 s^–1 and, thus, 2-fold higher than the respective single channel coefficient for water. The number of neutral ammonia molecules, T_NH3, transported by AQP8 per second can be assessed by calculation of the turnover number per channel as shown in Equation 10

(Eq. 10)

where N_A and V_NH3 are the Avogadro number and the molecular volume of NH₃, respectively.

View larger version (22K): [in this window] [in a new window]   FIGURE 5. NH3 membrane permeablity as a function of AQP8 membrane abundance. The trans and cis NH4Cl concentrations in the bulk were equal to 1 and 80 mM, respectively. In the absence of the protein (spline line), a modest increase in the cis concentration was observed in the immediate membrane vicinity. Upon reconstitution of AQP8, the augmentation of concentration adjacent to the membrane became more pronounced. The more protein was reconstituted, the higher was the concentration polarization. The gray lines represent concentration profiles generated by the theoretical model of weak base diffusion (Equations 1, 2, 3 and Reactions 1 and 2). The values used to calculate the theoretical curves are plotted in the inset as a function of the protein:lipid ratio. Buffer composition was as in Fig. 1.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

AQP8 transports ammonia very efficiently. With 8 x 10⁵ substrate molecules/s/channel (Equation 10), T_NH3 of AQP8 is comparable with the respective number of 2 x 10⁶ estimated for the human rhesus-associated glycoprotein (28). Because the physiological NH₃ concentration is orders of magnitude smaller than the concentration of H₂O, the NH₃ conduction rate need not be as high as that of H₂O to match diffusion limited rates for arriving at the pore (29). In agreement with this prediction, we have obtained a 500-fold higher T_W (Equation 5).

Comparison of the actual single channel permeability coefficients reveals, in contrast, a preference for NH₃ over H₂O as shown in Equation 11.

(Eq. 11)

Because of their larger molecular volume (V_NH3 >> V_W), the channel accommodates less gas than water molecules. Thus, albeit their higher transport velocity, AQP8 transports less NH₃ molecules than water molecules. It should be noted that the actual velocity of water transport may be underestimated because it was derived assuming that the pores are densely packed with water. In single file transport, the density of water inside the channel may be lower than in bulk (30, 31). Molecular dynamics simulations support the view that liquid-vapor oscillations occur in the channel (32, 33).

Equation 11 reveals a preference for ammonia over water. This result conflicts with a cell culture study in which the fluorescence of the pH-sensing yellow fluorescent protein was used to assess rat or mouse AQP8 ammonia permeability. The reason for the reported extremely low AQP8 single channel NH₃-to-water permeability of only 0.03 (9) is not clear. However, it is likely that the kinetics of pH changes are, at least in part, determined by compensatory transport events aimed to maintain cellular pH. Calculating P_NH3 from the time course of pH changes alone (9) should be hampered (i) by passive fluxes of CO₂ and H⁺ through the plasma membrane as well as (ii) by different types of pH-regulating transporters, like cation-H⁺ exchangers, transporters, and H⁺-ATPases, and (iii) proton exchange with intracellular compartments such as mitochondria and lysosomes. Lacking the entire list of uncertainties, reconstituted bilayers offer the opportunity to measure NH₃ flux directly. The only uncertainty of the bilayer system is that the reconstituted protein may adopt a slightly different quaternary structure. At least for the aqueous pore of the AQP8 monomer, which is the channel path for water and ammonia, this can be ruled out by the perfect match between the p_f values of the reconstituted protein and the one in an expression system (23). Theoretically, there is still a possibility that transport of charged species occurs through a putative fifth pore in the center of the aquaporin tetramer. It is rather unlikely in AQP8 because (i) so far ion channel activity has been ascribed only to AQP1 (34), (ii) nonphysiologically high cGMP concentrations (1 mM) are required (35), and (iii) ion channel activity was not reproduced by a variety of laboratories (36, 37).

AQP8 is the first ammonia-transporting channel for which the exclusion of has been shown. The net transport of NH₃ across the plasma membrane by kidney Rh glycoproteins RhBG and RhCG results from an exchange of for H⁺ (38). A similar mechanism for AQP8 can be ruled out because the estimated upper limit of H⁺ fluxes of 1 x 10^–15 mol cm^–2 s^–1 is negligible compared with the ammonia flux of 0.4 nmol cm^–2 s^–1 (Equation 6). NH₃/H⁺ cotransport or transport as proposed for AMT-1 from the hyperthermophilic archaeon Archaeglobus fulgidus (39) or the plant ammonium transporters LeAMT1 (40) and LeAMT2 (41) can be excluded for the same reason. Exclusion of NH₃ as well as saturable transport kinetics found for LeAMT1; 1 (42) indicate a transporting mechanism that contrasts with AQP8.

A transport mechanism much closer to AQP8 has been proposed for AmtB from E. coli. Mainly based on structural considerations (43, 44) and on molecular dynamics simulations (45), NH₃ selectivity was suggested. However, occasional passage of or H⁺ was not excluded. It would be stabilized by ring currents of the rich aromatic environment at the constriction zone using the acid/base properties of the imidazole nitrogens to assist in proton transfer (43). The authors concluded that a stoichiometric measure of conductance of neutral and charged species is required to establish whether ion conductance takes place, a study we now have carried out for AQP8.

Summarizing, AQP8 exhibits a preference for neutral NH₃ molecules over water, suggesting a physiological role in maintenance of acid-base equilibrium. In physiological concentrations AQP8 may augment the basal NH₃ conductivity 3- to 5-fold.

	FOOTNOTES

 
^* This work was supported by the Austrian Science Fund (FWF W1201-N13). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. peter.pohl{at}jku.at.

² The abbreviations used are: AQP, aquaporin; MES, 4-morpholineethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid.

	ACKNOWLEDGMENTS

 
We thank Quentina Beatty for critically reading the manuscript.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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