Analysis of double knockout mice lacking aquaporin-1 and urea transporter UT-B. Evidence for UT-B-facilitated water transport in erythrocytes.
We reported increased water permeability and a low urea reflection coefficient in Xenopus oocytes expressing urea transporter UT-B (former name UT3), suggesting that water and urea share a common aqueous pathway (Yang, B., and Verkman, A. S. (1998) J. Biol. Chem. 273, 9369-9372). Although increased water permeability was confirmed in the Xenopus oocyte expression system, it has been argued (Sidoux-Walter, F., Lucien, N., Olives, B., Gobin, R., Rousselet, G., Kamsteeg, E. J., Ripoche, P., Deen, P. M., Cartron, J. P., and Bailly, P. (1999) J. Biol. Chem. 274, 30228-30235) that UT-B does not transport water when expressed at normal levels in mammalian cells such as erythrocytes. To quantify UT-B-mediated water transport, we generated double knockout mice lacking UT-B and the major erythrocyte water channel, aquaporin-1 (AQP1). The mice had reduced survival, retarded growth, and defective urinary concentrating ability. However, erythrocyte size and morphology were not affected. Stopped-flow light scattering measurements indicated erythrocyte osmotic water permeabilities (in cm/s x 0.01, 10 degrees C): 2.1 +/- 0.2 (wild-type mice), 2.1 +/- 0.05 (UT-B null), 0.19 +/- 0.02 (AQP1 null), and 0.045 +/- 0.009 (AQP1/UT-B null). The low water permeability found in AQP1/UT-B null erythrocytes was also seen after HgCl(2) treatment of UT-B null erythrocytes or phloretin treatment of AQP1 null erythrocytes. The apparent activation energy for UT-B-mediated water transport was low, <2 kcal/mol. Estimating 14,000 UT-B molecules per mouse erythrocyte, the UT-B-dependent P(f) of 0.15 x 10(-4) cm/s indicated a substantial single channel water permeability of UT-B of 7.5 x 10(-14) cm(3)/s, similar to that of AQP1. These results provide direct functional evidence for UT-B-facilitated water transport in erythrocytes and suggest that urea traverses an aqueous pore in the UT-B protein.
Department of Medicine, Cardiovascular Research Institute, University of California, San Francisco, California 94143-0521, USA. byang@itsa.ucsf.edu
Baoxue
B
Yang
A S
AS
Verkman
D000818:Animals; D051398:Aquaporin 1; D020346:Aquaporins / Q000235:genetics* / Q000502:physiology*; D004912:Erythrocytes / Q000378:metabolism; D015151:Immunoblotting; D007671:Kidney Concentrating Ability; D007700:Kinetics; D008027:Light; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009995:Osmosis; D012542:Scattering, Radiation; D013997:Time Factors; D014867:Water / Q000378:metabolism; D014981:Xenopus
C496021:Aqp1 protein, mouse; D020346:Aquaporins; D026901:Membrane Transport Proteins; C452857:urea transporter B, mouse; D014867:Water; D051398:Aquaporin 1