Fast and selective ammonia transport by aquaporin-8

Abstract

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 NH(3) while excluding H(+) and NH(4)(+). Our research is the first stoichiometric study to show the selective transport of NH(3) by a membrane channel. The purified water channel protein aquaporin-8 was reconstituted into planar bilayers, and the exclusion of NH(4)(+) 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) cm(3)/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) cm(3)/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.

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.

FIGURE 2. Exclusion of NH4+ 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.

FIGURE 3. NH₃ flux J as a function of the transmembrane NH₄Cl concentration gradient

The ${\mathrm{NH}}_4^{+}$ 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 ${\mathrm{NH}}_4^{+}$ bulk concentrations in the cis compartment. J_NH3 and P_NH3 were calculated using the analytical model of weak base diffusion (Equations 1-3 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.

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 50μm/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.

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 ${\mathrm{NH}}_4^{+}$ concentration was observed in the immediate membrane vicinity. Upon reconstitution of AQP8, the augmentation of ${\mathrm{NH}}_4^{+}$ 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 1-3 and Reactions 1 and 2). The $P_{{\mathit{NH}}_3}^M$ 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.