Characterisation of a novel urea transporter from human colon

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University of Manchester (2003) J Physiol 552P, P117

Communications: Characterisation of a novel urea transporter from human colon

Elizabeth A. Potter, Craig P. Smith, Robert A. Fenton* and Gavin S. Stewart.

School of Biological Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK and *Laboratory of Kidney and Electrolyte Metabolism, NHLBI, National Institutes of Health, Bethesda, MD 20892-1603, USA

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Urea movement across plasma membranes is modulated by facilitative transport proteins that are the product of UT-A and UT-B genes. UT-A proteins have been detected in the mammalian colon (Ritzhaupt et al. 1998, Stewart et al. 2002), where they are proposed to participate in urea nitrogen recycling. We have characterised a 1789-bp cDNA isolated from human colon mucosa encoding a new UT-A isoform (hUT-A6; GenBank Acc. no. AK074236).

hUT-A6 cDNA has a putative open reading frame encoding a 235-amino-acid protein, making it the smallest member of the UT-A subfamily. hUT-A6 is the result of alternative splicing of the UT-A1 gene and contains a novel 127 bp exon between exons 5 and 6. hUT-A6 contains putative consensus PKC phosphorylation sites (Ser15, Ser71, Ser197), but is devoid of putative consensus sequences for PKA phosphorylation. hUT-A6 also contains a putative consensus glycosylation site which lies within the novel exon. The Kyte-Doolittle plot of hUT-A6 suggests this protein shares essentially the same structural configuration as the N-terminal 216-amino acids of hUT-A1, with a hydrophobic core flanked by hydrophilic N-and C-termini.

Xenopus oocyte expression studies were performed as described previously (Smith et al. 1995). Expression of hUT-A6 cRNA in Xenopus oocytes mediated a significant increase in urea flux, compared to water-injected controls, that was significantly inhibited by 15 min pre-incubation with 0.5 mM phloretin (n = 8, P < 0.05, ANOVA). hUT-A6-mediated urea flux was significantly increased by 10 min pre-incubated with a cAMP cocktail (0.5 mM cAMP-0.5 mM IBMX-0.05 mM forskolin) compared to unstimulated levels (n = 8, P < 0.05, ANOVA).

hUT-A6 represents a novel human urea transporter orthalogue and despite its small size, encodes a functional, phloretin-inhibitable facilitative urea transporter similar to other characterised UT-A proteins. Furthermore, this UT-A isoform is positively regulated by a cAMP cocktail despite containing no cAMP-dependent phosphorylation consensus sites, suggesting an alternative intracellular signalling pathway possibly distinct from direct PKA phosphorylation.

This work was supported by the BBSRC and the Royal Society.

Where applicable, experiments conform with Society ethical requirements.

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