Energetic and Molecular Water Permeation Mechanisms of the Human Red Blood Cell Urea Transporter B

Slim Azouzi,Catherine Etchebest,Caroline Le Van Kim,Yves Colin Aronovicz,Pierre Ripoche,Marc Gueroult,Isabelle Mouro-Chanteloup,Sandrine Genetet,Michael B Butterworth

doi:10.1371/journal.pone.0082338

Abstract

Urea transporter B (UT-B) is a passive membrane channel that facilitates highly efficient permeation of urea. In red blood cells (RBC), while the major function of UT-B is to transport urea, it is assumed that this protein is able to conduct water. Here, we have revisited this last issue by studying RBCs and ghosts from human variants with defects of aquaporin 1 (AQP1) or UT-B. We found that UT-B's osmotic water unit permeability (pfunit) is similar to that of AQP1. The determination of diffusional permeability coefficient (Pd) allowed the calculation of the Pf/Pd ratio, which is consistent with a single-file water transport. Molecular dynamic simulations of water conduction through human UT-B confirmed the experimental finding. From these results, we propose an atomistic description of water–protein interactions involved in this permeation. Inside the UT-B pore, five water molecules were found to form a single-file and move rapidly along a channel by hydrogen bond exchange involving two critical threonines. We further show that the energy barrier for water located in the central region coincides with a water dipole reorientation, which can be related to the proton exclusion observed experimentally. In conclusion, our results indicate that UT-B should be considered as a new member of the water channel family.

Highlights

Aquaporin 1 (AQP1) and urea transporter (UT-B) are integral membrane proteins, both expressed in erythrocytes, allowing a rapid transport of water and urea across red blood cell (RBC) membranes, respectively [1,2]
The osmotic water permeability coefficients Pf were determined from a stopped-flow light-scattering analysis of RBCs exhibiting the Colton-null [2] and Kidd-null [49] phenotypes which are characterized by a total deficiency of AQP1 and Urea transporter B (UT-B), respectively (Table 1)
[2] Addition of 0.5 mM HgCl2 to all RBC samples significantly decreased the Pf values and resulted in very similar residual water permeabilities regardless the phenotype of the erythrocytes (Figure 1A). These results indicate that AQP1 is not the only pathway for water transport in human RBCs and that at least a second mercury-sensitive protein might assume the residual water permeability in AQP1null RBCs, in agreement with previous works [2].This suggests that UT-B protein and/or AQP3 proteins could be, like AQP1, a mercury-sensitive water channel

Summary

Introduction

Aquaporin 1 (AQP1) and urea transporter (UT-B) are integral membrane proteins, both expressed in erythrocytes, allowing a rapid transport of water and urea across red blood cell (RBC) membranes, respectively [1,2]. These functions protect RBCs from osmotic stress when they pass through the vasa recta in the medulla where urea accumulates in high concentration [3]. By generating double knockout mice lacking both UT-B and AQP1, Yang and Verkman proposed the presence of an aqueous pore through UT-B involved in water transport [8]

Methods

Results

Conclusion