Aquaporin 9 is the major pathway for glycerol uptake by mouse erythrocytes, with implications for malarial virulence

Abstract

Human and rodent erythrocytes are known to be highly permeable to glycerol. Aquaglyceroporin aquaporin (AQP)3 is the major glycerol channel in human and rat erythrocytes. However, AQP3 expression has not been observed in mouse erythrocytes. Here we report the presence of an aquaglyceroporin, AQP9, in mouse erythrocytes. AQP9 levels rise as reticulocytes mature into erythrocytes and as neonatal pups develop into adult mice. Mice bearing targeted disruption of both alleles encoding AQP9 have erythrocytes that appear morphologically normal. Compared with WT cells, erythrocytes from AQP9-null mice are defective in rapid glycerol transport across the cell membrane when measured by osmotic lysis, [(14)C]glycerol uptake, or stopped-flow light scattering. In contrast, the water and urea permeabilities are intact. Although the physiological role of glycerol in the normal function of erythrocytes is not clear, plasma glycerol is an important substrate for lipid biosynthesis of intraerythrocytic malarial parasites. AQP9-null mice at the age of 4 months infected with Plasmodium berghei survive longer during the initial phase of infection compared with WT mice. We conclude that AQP9 is the major glycerol channel in mouse erythrocytes and suggest that this transport pathway may contribute to the virulence of intraerythrocytic stages of malarial infection.

Fig. 1.

Expression of AQP9 in mouse erythrocytes. Membrane protein samples (20 μg per lane) from mouse, rat, and human erythrocytes with (+) and without (−) PNGase F digestion analyzed by immunoblotting. (A) Mouse (mRBC), rat (rRBC), and human (hRBC) erythrocyte membrane proteins analyzed by immunoblot using anti-AQP3. Mouse kidney medulla membrane proteins (mKd) were analyzed as positive controls. (B) Mouse, rat, and human erythrocyte membrane proteins analyzed by immunoblot by using anti-AQP9. Rat liver membrane proteins (rLv) were analyzed as positive controls. (C) Erythrocyte and liver membrane proteins from WT and AQP9-null (KO) mice analyzed by immunoblot by using anti-AQP9. (D) The morphology of Wright–Giemsa-stained WT and AQP9-null erythrocytes. (E) Erythrocyte membranes from WT and AQP9-null mice analyzed by SDS/PAGE stained with Coomassie blue. (Scale bars: 10 μm.)

Fig. 2.

Expression of AQP9 and AQP1 in various developmental stages of mouse erythrocytes. (Upper) Membrane protein samples from mouse erythrocytes after PNGase F digestion were analyzed by immunoblotting. (Lower) Quantitation of the density of the bands on the immunoblots are shown. Three mice were studied for each age group. Erythrocyte membrane proteins from mice of various ages analyzed by immunoblot using anti-AQP9 (20 μg per lane) (A) or anti-AQP1 (0.1 μg per lane) (B); mature erythrocyte (RBC) and reticulocyte (Retic) membrane proteins analyzed by immunoblot using anti-AQP9 (20 μg per lane) (C) or anti-AQP1 (0.1 μg per lane) (D).

Fig. 3.

Solute permeability of mouse erythrocytes. (A) The glycerol permeability of WT (filled circles; n = 3), AQP9 heterozygous (Het) (open squares; n = 3), and AQP9-null (open triangles; n = 3) erythrocytes assayed by osmotic lysis in a 0.3 M glycerol solution. (B) The glycerol permeability of WT (triangles) and AQP9-null (squares) erythrocytes assayed by [¹⁴C]glycerol uptake. Representative data of five independent experiments are shown. (C–E) The solute permeabilities of erythrocytes from WT and AQP9-null mice measured by monitoring light scattering with a stopped-flow spectrophotometer after rapid mixing of solute-loaded erythrocytes with an equal volume of sucrose solution. Inhibition of solute permeability was achieved by incubating erythrocytes with 0.1 mM HgCl₂ for 5 min before measurement. The kinetics of 10 measurements were averaged and fitted to a first-order exponential equation. (C) Glycerol permeability. (D) Water permeability. (E) Urea permeability.

Fig. 4.

Effect of AQP9 on malarial virulence. Both WT (bold line; n = 6) and AQP9-null (thin line; n = 7) female mice were infected with equal numbers of P. berghei (10⁵) on day 0. The survival of the mice was examined daily for 23 days.