Concerted action of two cation filters in the aquaporin water channel

Abstract

Aquaporin (AQP) facilitated water transport is common to virtually all cell membranes and is marked by almost perfect specificity and high flux rates. Simultaneously, protons and cations are strictly excluded to maintain ionic transmembrane gradients. Yet, the AQP cation filters have not been identified experimentally. We report that three point mutations turned the water-specific AQP1 into a proton/alkali cation channel with reduced water permeability and the permeability sequence: H(+) >>K(+) >Rb(+) >Na(+) >Cs(+) >Li(+). Contrary to theoretical models, we found that electrostatic repulsion at the central asn-pro-ala (NPA) region does not suffice to exclude protons. Full proton exclusion is reached only in conjunction with the aromatic/arginine (ar/R) constriction at the pore mouth. In contrast, alkali cations are blocked by the NPA region but leak through the ar/R constriction. Expression of alkali-leaking AQPs depolarized membrane potentials and compromised cell survival. Our results hint at the alkali-tight but solute-unselective NPA region as a feature of primordial channels and the proton-tight and solute-selective ar/R constriction variants as later adaptations within the AQP superfamily.

Figure 1

Models of wild-type rat AQP1 (A) and AQP1-N76D/H180A/R195V (B). The channel path depictions are based on the crystal structure of bovine AQP1 (PDB #1j4n). Shape and diameters of the aromatic/arginine region (His180, Arg195) and the NPA region (Asn76, Asn192) are indicated by grey ellipses. Distances between the backbone carbonyl oxygens along the channel are given in Å. The figure was generated using Pymol.

Figure 2

Water and solute permeability of AQP1 mutants. (A) Water permeability of rat wild-type AQP1 and the indicated AQP1 mutants was determined from the oocyte shrinkage obtained by adding 20 mosm mannitol to the bathing solution (Beitz et al, 2006). (B) Urea permeability was calculated from the oocyte swelling rate in a 130 mM isotonic inward gradient (Beitz et al, 2006). n=5–14, ±s.e.m, ^**P<0.01 versus wild-type AQP1 or AQP1-H180A/R195V. (C) Expression levels of wild-type AQP1 and AQP1 mutants were tested by western blotting with total oocyte membrane protein (protein from one oocyte per lane) and a specific anti-AQP1 antiserum. Non-glycosylated (lower band) as well as glycosylated forms of AQP1 (upper bands) were detected. (D) For detection of AQP1 in the plasma membrane, intact oocytes were treated with Ludox and polyacrylic acid to increase the specific weight of the plasma membrane (Leduc-Nadeau et al, 2007). After homogenization, the plasma membrane leaflets were collected by low-speed centrifugations and plasma membrane protein from 25 oocytes was loaded per lane. The numbers above the blots in C and D indicate expression levels relative to wild-type AQP1.

Figure 3

Ion permeability of AQP1 mutants. Current–voltage relationships were obtained by the two-electrode voltage-clamp technique (Beitz et al, 2006). A series of voltage steps were applied and the resulting steady state currents recorded. The examples are from an oocyte expressing AQP1-N76D/H180A/R195V bathed in 100 mM NaCl (A) or in 100 mM ChCl (B), the latter also served as control bathing solution. All solutions contained an additional (mM) 2 KCl, 1 CaCl₂, 1 MgCl₂, 10 HEPES or MES, and pH was adjusted using Tris base. (C) The voltage was changed from a holding potential of −50 mV to a value between +40 and −140 mV in steps of 20 mV and the current was read after 100 ms. The test solutions were applied for about 30 s, at which time the voltage steps were applied. (D) Steady state currents obtained in 100 mM ChCl at an external pH of 7.4, 6.5, 6.0, or 5.5. (E) Steady state currents plotted as a function of voltage in 100 mM choline chloride (ChCl), in 25 mM KCl + 75 mM ChCl (25 K⁺), in 50 mM NaCl + 50 mM ChCl (50 Na⁺), and in 100 mM NaCl (100 Na⁺). (F) Permeability (P) for H⁺, Na⁺, and K⁺, please note the different scales. The P-values were calculated from the clamp currents (I_C) obtained at a clamp voltage V of −120 mV from the equation P=I_CRT/(VF²C), an approximation to the Goldman–Hodgkin–Katz equation applicable at high negative potentials. C is the concentration, F is the Faraday's constant, and R is the gas constant. Values for the wild-type AQP1 (rAQP1 wt) and the mutants AQP1-N76D, AQP1-H180A/R195V, AQP1-N76D/H180A/R195V, AQP1-N192D, and AQP1-N192D/H180A/R195V are shown. Currents observed with uninjected oocytes were not significantly different from oocytes expressing wild-type AQP1. n=5–14, ±s.e.m.

Figure 4

Phenotypic yeast assays for ion permeability of AQP1 mutants. (A) Expression control of AQP1 mutants in the CY162 and MA5 yeast strains by western blot. (B) Yeast expressing rat wild-type AQP1 or the indicated mutants were spotted in serial 1:10 dilutions on low-salt arginine phosphate medium (Anderson et al, 1992) of different pH (H⁺) or supplemented with sodium (Na⁺) or potassium (K⁺) chloride at pH 5.5. Mock transformed yeast (−) or yeast transformed with an Arabidopsis potassium channel, AtKAT1, served as controls. For the H⁺ stress assay and the K⁺ growth complementation assay, the CY162 strain (Anderson et al, 1992) lacking both endogenous potassium channels Trk1 and 2 was used. The Na⁺ stress test was done with the sodium sensitive MA5 strain (Benito et al, 2004).

Figure 5

Model of the AQP joint cation filters. (A) The primordial AQPs already selected against inorganic cations, such as Na⁺ and K⁺, because of a positive electrostatic field from the helix dipols and the lack of cation coordination sites in the NPA region (filter I); yet, protons leaked through. Later, a second cation filter evolved in the ar/R region, which fully excluded protons (filter II) and provided individual selectivity properties for water, glycerol, urea, and ammonia. (B) Disturbance of the filter regions by point mutations leads to Na⁺ leakage as was the case for the AQP1-N192D mutant (ND; exchange of asparagine for aspartate in the NPA region), enables low proton permeability (HA/RV; removal of positive charges in the ar/R region) or switches the AQP into a cation channel (ND/HA/RV; combination of the NPA and ar/R mutations).