Abstract
The goal of this study was to compare single channel water and glycerol permeabilities of mammalian aquaporins (AQP) 1-5 and the major intrinsic protein of lens fiber (MIP). Each of the six cloned cDNAs from rat was left untagged or was epitope-tagged with c-Myc or FLAG at either the N or C terminus so that results would not depend on epitope identity or location. The constructs were expressed in Xenopus oocytes for measurement of osmotic water permeability (Pf), [3H]glycerol uptake, and protein expression. Each of the 30 epitope-tagged constructs was expressed strongly at the oocyte plasma membrane. The 10-min uptake of [3H]glycerol was increased significantly (range of 4.5-8-fold over control) in oocytes expressing untagged AQP3 (GLIP) and each of the four tagged AQP3 constructs; [3H]glycerol uptake was not increased in oocytes expressing AQP1, AQP2, AQP4, AQP5, or MIP. In oocytes microinjected with 5 ng of cRNA, average Pf values (in cm/s x 10(-3)) were 0.67 +/- 0.06 (control), 19 +/- 2 (AQP1), 10 +/- 1 (AQP2), 8 +/- 2 (AQP3), 29 +/- 1 (AQP4), 10 +/- 1 (AQP5), and 1.3 +/- 0.2 (MIP), and they were relatively insensitive to the presence, identity, or location of the epitope tag. Pf values were not affected by protein kinase A or C activation. After normalization for plasma membrane expression by immunoprecipitation of microdissected plasma membranes, single channel water permeabilities (pf, referenced to the AQP1 pf of 6 x 10(-14) cm3/s) were (in cm3/s x 10(-14)) 3.3 +/- 0.2 (AQP2), 2.1 +/- 0.3 (AQP3), 24 +/- 0.6 (AQP4), 5.0 +/- 0.4 (AQP5), and 0.25 +/- 0.05 (MIP); pf values were insensitive to epitope identity and location. These results indicate very different intrinsic water permeabilities for the mammalian aquaporin homologs, with the pf value for AQP4 remarkably higher than those for the others. The pf values establish limits on aquaporin tissue densities required for physiological function and suggest significant structural and functional differences among the aquaporins.
Highlights
Five proteins with homology to the major intrinsic protein of lens fiber (MIP) (1) have been cloned in mammals and given the name aquaporins
Most studies of aquaporin function have concluded that AQP1, AQP2, AQP4, and AQP5 are selective for transport of water without passage of ions, protons, urea, and glycerol, whereas Abrami et al (29) reported that AQP1 may transport small polar non-electrolytes in addition to water
The cDNAs encoding rat AQP1–5 and MIP were epitopetagged at the N or C terminus with the c-Myc or FLAG sequence, and the untagged and tagged constructs were subcloned into an oocyte expression vector
Summary
Five proteins with homology to the major intrinsic protein of lens fiber (MIP) (1) have been cloned in mammals and given the name aquaporins. AQP3 (alternate name GLIP (glycerol-transporting intrinsic protein)) was cloned by three laboratories (4 – 6) and is expressed at the basolateral membrane of kidney collecting duct and in multiple epithelia (7). In the case of AQP1, where purification to homogeneity has been possible, measurement of “per channel” or “single channel” water permeability (pf) indicates that each AQP1 monomer has a relatively low pf of ϳ6 ϫ 10Ϫ14 cm3/s (27, 28) This relatively low pf requires the presence of very high densities of AQP1 water channels in membranes (Ͼ103/m2, compared with generally Ͻ1 ion channel/m2) to confer significantly increased water permeability. The principal goal of this study was to determine quantitatively the single channel water and glycerol permeabilities of mammalian AQP1–5 and MIP. Single channel water permeabilities were determined from ratios of measured oocyte permeabilities to plasma membrane expression levels. The considerable heterogeneity in water channel function of the aquaporin family proteins was an unexpected finding with interesting implications for water channel structure and physiology
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