Urea Transporter UT3 Functions as an Efficient Water Channel

Abstract

A family of molecular urea transporters (UTs) has been identified whose members appear to have an exceptionally high transport turnover rate. To test the hypothesis that urea transport involves passage through an aqueous channel, osmotic water permeability was measured in Xenopus oocytes expressing UTs. The UT3 class of urea transporters functioned as efficient water channels. Quantitative measurement of single channel water permeability ( p f ) using epitopetagged rat UTs gave p f (in cm 3 /s ؋ 10 ؊14 ) of 0.14 ؎ 0.11 (UT2) and 1.4 ؎ 0.2 (UT3), compared with 6.0 and 2.3 for water channels AQP1 and AQP3, respectively. Relative single channel urea permeabilities ( p urea ) were 1.0 (UT2), 0.44 (UT3), and 0.0 (AQP1). UT3-mediated water and urea transport were weakly temperature-dependent (activation energy <4 kcal/mol), inhibited > 75% by the urea transport inhibitor 1,3-dimethylthiourea, but not inhibited by the water transport inhibitor HgCl 2 . To test for a common water/urea pore, the urea reflection coefficient ( urea ) was measured by independent induced osmosis and solvent drag methods. In UT3-expressing oocytes, the time course of oocyte volume in response to different urea gradients (induced osmosis) gave urea ϳ0.3 for the UT3 pathway, in agreement with urea determined by the increase in uptake of [ 14 C]urea during osmotic gradient-induced oocyte swelling (solvent drag). In oocytes of comparable water and urea permeability coexpressing AQP1 (permeable to water, not urea) and UT2 (permeable to urea, not water), urea ‫؍‬ 1. These results indicate that UT3 functions as a urea/ water channel utilizing a common aqueous pathway. The water transporting function and low urea reflection coefficient of UT3 in vasa recta may be important for the formation of a concentrated urine by countercurrent exchange in the kidney. Several related urea transporters (UTs)1 have been cloned recently. The UT2 transporter (rat form referred to as rUT2, Ref. 1) was first identified in rabbit by expression cloning (2). The UT2 cDNA encodes a 397-amino acid glycoprotein that is expressed in the medullary portion of descending limbs of Henle in kidney (3, 4). UT1 is larger protein (929 amino acids) that contains the UT2 sequence at its C terminus fused to a 67% identical amino acid sequence at its N terminus (5). UT1 functions a cAMP-regulated urea transporter that is expressed at the apical membrane of inner medullary collecting duct cells. UT1 and UT2 are thought to be derived from a single gene by alternative splicing. A 391-amino acid urea transporter expressed in erythrocytes was initially cloned from human bone marrow (HUT11, Ref. 6) and subsequently from rat kidney (named UT3, Ref. 7). Rat UT3 has 62% amino acid identity to UT2 and is expressed strongly in kidney in descending vasa recta in the inner stripe of outer medulla, as well as in testis and brain (7,8). It is thought that these urea transporters have an important role in the urinary concentrating mechanism to establish a hypertonic renal medullary interstitium (9).UT3 ...

Triazolothienopyrimidine Inhibitors of Urea Transporter UT-B Reduce Urine Concentration

“…However, UTB inh -14 inhibited water transport significantly in AQP1-null erythrocytes (Figure 5B, right), supporting our prior conclusion that erythrocyte water transport is mediated, in part, by UT-B. 22,27 Figure 5C shows no significant inhibition of CFTR or TMEM16A chloride conductance by 10 mM UTB inh -14, as measured by short-circuit current. Last, by MTT assay, incubation of MDCK cells with UTB inh -14 up to 10 mM for 24 hours (where it remains soluble) showed no cytotoxicity ( Figure 5D).…”

Urea-selective Concentrating Defect in Transgenic Mice Lacking Urea Transporter UT-B

“…Urea permeability in erythrocytes from null mice was 45-fold lower than that from wild-type mice, indicating that UT-B was functionally deleted. We found previously that rat UT-B transported some water along with urea when heterologously expressed in Xenopus oocytes (21). Mouse UT-B also functioned as a weak water channel and an efficient urea transporter in Xenopus oocytes.…”

“…5Ј and 3Ј end cDNA sequences were obtained by 5Ј-and 3Ј-RACE (CLONTECH). For functional measurements of urea and water permeability, the mouse UT-B cDNA (full-length coding sequence) was subcloned into oocyte expression plasmid pSP64T, and in vitro transcribed cRNA (5 ng) was expressed in Xenopus laevis oocytes as described previously (21). The structure of the mouse UT-B gene was analyzed by PCR amplification of exon-intron-exon fragments by using C57/bl6 mouse liver genomic DNA as template.…”

“…UT-B has similar membrane topology to UT-A2 based on hydropathy analysis and has functional characteristics similar to UT-A1 and UT-A2. We reported that rat UT-B heterologously expressed at a relatively high level in Xenopus oocytes functioned as a urea/water channel utilizing a common aqueous pathway (21), however, the relevance of water transport by UT-B in mammalian physiology is unresolved (22).…”

Channels for water efflux and influx involved in volume regulation of murine spermatozoa

“…Two-way ANOVA revealed that there were no Loo et al (1996Loo et al ( , 1999Loo et al ( , 2002, Yang & Verkman (1998 Fig. 1).…”