The N‐terminal of the mouse urea transporter UT‐A3 contains an 8 residue segment crucial for normal function

Mouse UT‐A3 is expressed in the inner medullary collecting duct, where it localises to the basolateral region of the cell and plays an important role in urine concentration. Currently we have investigated the role of the N‐terminal in the function of mUT‐A3. Xenopus laevis oocytes were injected with 1.5ng cRNA encoding mUTA3, its mutants or 50nl of H2O and function was assessed by measuring urea uptake as described previously (Fenton, 2000). mUT‐A3 and its mutants were N‐terminally tagged with eGFP. The eGFP tag did not alter mUT‐A3 function. The N‐terminal of mUT‐A3 was truncated by 55 and 111 residues (M55‐start and M111‐start). When expressed in Xenopus oocytes the M55‐start truncation was functional. However, no urea uptake was observed in oocytes expressing M111‐start, indicating that the region between M55 and M111 is required to produce a functional urea transporter. Further N‐terminal truncation mutants were synthesised, with the clones beginning with residues S68, G94 and A103. When expressed in Xenopus oocytes these three truncation mutants were functional. Deleting residues 103–113 produced a non‐functional clone. These results indicate that an 8 amino acid stretch between residues 103 and 111 is crucial for mUT‐A3 to function normally. It is unclear whether the lack of function observed in the M111‐ start clone or the 103–113 deletion mutant is due to a defect in trafficking or disruption of urea movement through the pore. Funded by Kidney Research UK.