Point mutations in the aromatic/arginine region in aquaporin 1 allow passage of urea, glycerol, ammonia, and protons

Abstract

Water-specific aquaporins (AQP), such as the prototypical mammalian AQP1, stringently exclude the passage of solutes, ions, and even protons. Supposedly, this is accomplished by two conserved regions within the pore, a pair of canonical asparagine-proline-alanine (NPA) motifs, the central constriction, and an aromatic/arginine (ar/R) constriction, the outer constriction. Here, we analyzed the function of three residues in the ar/R constriction (Phe-56, His-180, and Arg-195) in rat AQP1. Individual or joint replacement of His-180 and Arg-195 by alanine and valine residues, respectively (AQP1-H180A, AQP1-R195V, and AQP1-H180A/R195V), did not affect water permeability. The double mutant AQP1-H180A/R195V allowed urea to pass. In line with the predicted solute discrimination by size, replacement of both Phe-56 and His-180 (AQP1-F56A/H180A) enlarged the maximal diameter of the ar/R constriction 3-fold and enabled glycerol and urea to pass. We further show that ammonia passes through all four AQP1 mutants, as determined (i) by growth complementation of yeast deletion strains with ammonia, (ii) by ammonia uptake from the external solution into oocytes, and (iii) by direct recordings of ammonia induced proton currents in oocytes. Unexpectedly, removal of the positive charge in the ar/R constriction in AQP1-R195V and AQP1-H180A/R195V appeared to allow the passage of protons through AQP1. The data indicate that the ar/R constriction is a major checkpoint for solute permeability, and that the exquisite electrostatic proton barrier in AQPs comprises both the NPA constriction as well as the ar/R constriction.

Fig. 1.

Shape of the ar/R constriction of AQP1 and mutants. For orientation, Top Right depicts a side view of the crystal structure of AQP1 (1J4N) with F56, H180, C189, and R195 as sticks (numbering according to the rat sequence; pymol software, DeLano Scientific, San Francisco). The red bar indicates the plane shown in the cross sections A–E.(A) Wild-type ar/R constriction. Amino acid residues that directly contribute to the constriction are drawn in full color. The black bands denote 1-Å-thick sections of the Connolly surface at this site (sybyl software, Tripos Associates, St. Louis). (B–E) Mutant outlines of ar/R constriction with individual mutations printed red. Calculations were based on the bovine AQP1 crystal structure (1J4N) and energy-optimized by using the sybyl threading algorithms. For additional description, see text.

Fig. 2.

Water and solute permeability of AQP1 mutants. (A) Expression of AQP1 wild-type and mutants in Xenopus oocytes determined by Western blotting. (B) Water permeability of AQP1 wild-type and mutants. (C) Urea and glycerol permeability. (n = 3 for controls [con], n = 5–12 for AQP1 and mutants; error bars denote SEMs.)

Fig. 3.

Yeast growth complementation on and methylammonium plates by AQP1 mutants. (A) Expression control of AQP1 and mutants in 31019bΔmep1-3 (endogenous transporters deleted) by Western blotting. (B) 31019bΔmep1-3 expressing wild-type or mutant AQP1 were spotted at none, 1:10², and 1:10⁴ dilutions (left to right) on agar plates containing 3 mM (NH₄)₂SO₄ as a nitrogen source at the indicated pH values. Shown is the cell growth after 5 days. Here, cell growth indicates NH₃ uptake. (B) BY4742Δfps1 yeast (deletion of the endogenous aquaglyceroporin Fps1) expressing wild-type or mutant AQP1 spotted at none, 1:10, and 1:100 dilutions (left to right) on selective medium containing 0.1% proline as the nitrogen source and 100 mM cytotoxic methylammonium at the indicated pH values. Cell growth was monitored after 5 days. The isogenic parent strain BY4742 (top row) as well as sham-transformed BY4742Δfps1 yeast (second row) were used as controls. Cell growth indicates passage of methylamine through AQP1 mutants (see text for further explanations).

Fig. 4.

Ammonia- and pH-induced proton currents in Xenopus oocytes expressing AQP1 wild-type and mutants. (A) Sample trace of clamp current (-50 mV) induced in an AQP1-H180A/R195V-expressing oocyte by changes in pH_e and of the bathing solution. The baseline current was -100 nA. pH_e was intermittently changed from 7.4 (control) to a range of values from 5.6 to 8.6 ± a concurrent change in (5 mM Na⁺ was replaced by 5 mM ), as indicated by the bars on top. In the acidic range, pH_e changes resulted in inward currents that were independent of the presence of . In the alkaline range, changes in pH_e resulted in small outward currents, whereas simultaneous addition of induced large inward currents. The L_P of the oocyte was 9.6·10^-3 cm·s^-1. (B) Filled symbols: total clamp currents (-50 mV) induced by 5 mM at various pH_e values in uninjected oocytes (control) and oocytes expressing AQP1 wild-type and mutants. The open symbols represent the currents induced by AQP-facilitated ammonia passage, i.e., we subtracted the background current induced by ammonia diffusion (control, Top Left) and the current produced by ammonia-independent proton permeation of certain AQP1 mutants (depicted in C). (C) Clamp currents elicited exclusively by changes in pH_e. For clarity, we show only the currents for AQP1-R195V and AQP1-H180A/R195V. The currents for AQP1 wild-type and the AQP1-H180A mutant were not significantly different from the miniscule currents already observed in uninjected control oocytes. The current for AQP1-F56A/H180A (not shown) was only significantly different at pH_e 5.6, i.e., 17 ± 2 nA (n > 5 for AQPs and n = 3 for uninjected controls).