Aquaporin 4 as a NH3 Channel

Abstract

Ammonia is a biologically potent molecule, and the regulation of ammonia levels in the mammalian body is, therefore, strictly controlled. The molecular paths of ammonia permeation across plasma membranes remain ill-defined, but the structural similarity of water and NH3 has pointed to the aquaporins as putative NH3-permeable pores. Accordingly, a range of aquaporins from mammals, plants, fungi, and protozoans demonstrates ammonia permeability. Aquaporin 4 (AQP4) is highly expressed at perivascular glia end-feet in the mammalian brain and may, with this prominent localization at the blood-brain-interface, participate in the exchange of ammonia, which is required to sustain the glutamate-glutamine cycle. Here we observe that AQP4-expressing Xenopus oocytes display a reflection coefficient <1 for NH4Cl at pH 8.0, at which pH an increased amount of the ammonia occurs in the form of NH3 Taken together with an NH4Cl-mediated intracellular alkalization (or lesser acidification) of AQP4-expressing oocytes, these data suggest that NH3 is able to permeate the pore of AQP4. Exposure to NH4Cl increased the membrane currents to a similar extent in uninjected oocytes and in oocytes expressing AQP4, indicating that the ionic NH4 (+) did not permeate AQP4. Molecular dynamics simulations revealed partial pore permeation events of NH3 but not of NH4 (+) and a reduced energy barrier for NH3 permeation through AQP4 compared with that of a cholesterol-containing lipid bilayer, suggesting AQP4 as a favored transmembrane route for NH3 Our data propose that AQP4 belongs to the growing list of NH3-permeable water channels.

Keywords: ammonia; aquaporin 4 (AQP4); membrane protein; molecular dynamics; permeability.

FIGURE 1.

The Lewis structure of NH₃ (left) and H₂O (right). NH₃ and H₂O have several similarities including dipole moment (1.47 D for NH₃ and 1.85 for H₂O), tetrahedral electronic structure, bond angle (106.7° for NH₃ and 104.5° for H₂O), and bond length (101.7 pm for NH₃ and 95.8 pm for H₂O).

FIGURE 2.

The reflection coefficient is reduced for ammonia in AQP4- and AQP8-expressing oocytes. A and B, volume traces from an AQP4-expressing, AQP8-expressing, or uninjected oocyte challenged with a hyperosmotic gradient of 20 mosm (marked with a black bar) of either 10 mm NaCl (black trace) or 10 mm NH₄Cl (red trace) at pH_o 7.4 (A) or pH_o 8.0 (B). C, A summary of the reflection coefficients for ammonia for AQP4-expressing (left panel), AQP8-expressing (middle panel), and uninjected oocytes (right panel) at pH_o 7.4 and pH_o 8.0. The reflection coefficient is calculated from two control measurements (10 mm NaCl as the osmolyte) and two measurements using 10 mm NH₄Cl as the osmolyte for each oocyte; n = 7–12. Statistical significance was determined with paired Student's t test. **, p < 0.01; ***, p < 0.001; ns, not significant.

FIGURE 3.

pH_i changes in response to exposure to ammonia in uninjected and in AQP4-expressing oocytes. A, transport of ammonia as either NH₄⁺ or NH₃. If NH₄⁺ crosses the cell membrane, it will cause an intracellular acidification, whereas influx of NH₃ will cause an intracellular alkalization. B and D, pH_i traces from an uninjected oocyte (B) and from two AQP4-expressing oocytes (D) exposed to 10 mm NH₄Cl (marked with a black bar). C, overview of the individual pH_i changes in uninjected oocytes after 15 min of exposure to 10 mm NH₄Cl, summarized in the inset, n = 15. E, overview of the individual pH_i changes in AQP4-expressing oocytes after 15 min of exposure to 10 mm NH₄Cl, summarized in the inset, n = 33. F, overview of the NH₄Cl-induced ΔpH_i in uninjected (Uninj.) oocytes (n = 15) and AQP4-expressing oocytes (n = 33). Statistical significance was determined with paired Student's t test (unpaired Student's t test in panel F). ***, p < 0.001; ns, not significant.

FIGURE 4.

No aquaporin-mediated NH₄⁺ permeation. A and D, representative current traces in uninjected (left panel), AQP4 (middle panel)-, and AQP8 (right panel)-expressing oocytes at pH_o 7.4 (A) and pH_o 8.0 (D) before and in the presence of 5 mm NH₄Cl, marked with a black bar. The currents were recorded from single oocytes at a holding potential of −50 mV. B and E, representative I/V relationships of uninjected (left panels), AQP4 (middle panels)-, or AQP8 (right panels)-expressing oocytes at pH_o 7.4 (B) and pH_o 8.0 (E) before and after 5 min of treatment with 5 mm NH₄Cl. C and F, summarized I/V relationships of uninjected (Uninj.) oocytes (white) and oocytes expressing AQP4 (gray) or AQP8 (black) before and after treatment with 5 mm NH₄Cl at pH_o 7.4 (C) and pH_o 8.0 (F), n = 9 of each, with the currents obtained at V_m = −60 mV summarized in the insets. Statistical significance was determined with two-way analysis of variance with Šídák's multiple comparison post hoc test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.

FIGURE 5.

Accumulation of NH₄⁺ close to the channel surface. In free simulations the NH₄⁺ density (illustrated as blue mesh) is placed closely to the glutamate and aspartate residues on the protein surface, shown in red licorice representation.

FIGURE 6.

Potential of mean force for NH₃ permeation (A). Shown is a comparison of the PMF for NH₃ through the AQP4 channel with the membrane. The PMF of NH₃ through AQP4 is shown in blue. The PMF for NH₃ through a pure POPC lipid bilayer is shown in cyan. The PMF for NH₃ through a lipid bilayer with 20% cholesterol is shown in purple. The uncertainty measured via bootstrapping is shown in the shaded region around the curves. The broken violet curve is the PMF for NH₃ through AQP4 extracted from the free simulations. B, radius profile of the AQP4 pore along the channel axis. The shaded region represents the standard deviation in the profile over the simulation trajectory. C, population histogram of the important pore lining residues along the channel axis.

FIGURE 7.

Comparison of water and NH₃ permeation in AQP4. A, NH₃ has a significantly larger (∼5 kJ/mol) free energy barrier over water in AQP4 calculated from free simulations. B, as NH₃ loses hydration via hydrogen bonding on its entry into the channel, the average number of hydrogen bonds to the channel axis. C, hydrogen bonding energy (HBE) difference for water and NH₃ along the channel axis (HBE_NH3 − HBE_water). The positive difference indicates that hydrogen bonding with water is favored in the channel.