Energetic and molecular water permeation mechanisms of the human red blood cell urea transporter B.

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

Cα fluctuations computed from each simulation.

<p>(a) Root Mean Square Fluctuation (RMSF), RMSF of simulations on human model (blue and green) and bovine structure in red. (b) Same information projected on the 3D structure. In red, residues exhibiting low fluctuations, and in blue, residues with high fluctuations.</p

UT-B and AQP1 unit permeabilities deduced from rate constants and permeabilities to water (pf and pd), to proton and to ammonia of human RBC variants.

†<p>UT-B unit permeabilities calculated from the number of UT-B copy per RBC which is described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082338#pone-0082338-t001" target="_blank">table 1</a>.</p>††<p>Means and ranges of AQP1 unit permeabilities calculated from the published values of AQP1 copy number (150 000–200 000) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082338#pone.0082338-Yang2" target="_blank">[8]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082338#pone.0082338-Agre1" target="_blank">[51]</a>. Experiments performed at 15°C.</p

Comparison of Proton and ammonia transports in ghost from RBC variants.

<p>(A) Time course of intracellular pH decrease in ghosts subjected to pH gradient from 7.1 to 7.6. (B) Time course of fluorescence changes of ghosts derived from control (green), UT-B_null (blue), AQP1_null (red), RhAG_null (black) RBCs and subjected to 10 mM ammonium inwardly-directed gradient followed by stopped-flow analysis.</p

Hydrogen bonds and potential of mean force of water in the pore.

<p>(A) The average number of hydrogen bonds over the MD simulation between water and, in blue, the protein and, in red, the water molecules. (B) Snapshot of water molecules' organization in the lumen of the pore, with the hydrogen bond network. Thr177 and Thr339 are displayed in blue. (C) Potential of Mean Force of a water molecule along the pore calculated from the water density. Green lines indicate the average z-coordinate of CG atoms for Thr177 and 339, over the three subunits. Residues in stick define sub-site along pore axis.</p

Electrostatic potential on the surface of UT-B (a) and AQP1 (b).

<p>View along the pore with water molecules located in the pore. Electrostatic potential defined between −10 (red) and 10 (blue) KT/e.</p

Alignment and superposition between bUT-B and hUT-B.

<p>Alignment between bovine and human UT-B shows 80% of sequence identity. Superimposition between human model (red) and bovine structure (blue) on Cα is less than 1 Å.</p

Antigen and protein expression of human RBCs.

<p>Values indicate the copy number of membrane proteins per red cell (×10³), except for AQP1 (anti-Colton antigen), which was measured as mean of fluorescence intensity. n.t.: not tested. MFI: Mean of Fluorescence Intensity.</p