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1 Faculty of Life Sciences, Stopford Building, University of Manchester, Manchester, United Kingdom
2 Department of Biomedical Science, Alfred Denny Building, University of Sheffield, Sheffield, S.Yorkshire, United Kingdom
* To whom correspondence should be addressed. E-mail: g.j.cooper{at}shef.ac.uk.
The amphibian urea transporter (fUT) shares many properties with the mammalian urea transporters (UT) derived from UT-A and UT-B genes. The transport of urea by fUT is inhibited by the mercurial agent p-chloromercuribenzenesulfonic acid (pCMBS). We found that in oocytes expressing cRNA encoding fUT, a five minute pre-incubation in 0.5mM mercury chloride (HgCl2) also significantly reduced urea uptake. The transport of urea by fUT was rendered mercury (Hg++) insensitive by mutating either of the residues C185 or H187, both of which lie within the M-I region (close to the hypothetical UT pore). In oocytes expressing a mixture of the C185 and H187 mutants, Hg++-sensitivity was re-established. The transport of urea by the mouse UTs, mUT-A2 and mUT-A3 was not sensitive to Hg++. Introducing cysteine residues analogous to that mutated in fUT into mUT-A2 or mUT-A3 did not induce Hg++ sensitivity. Additionally introducing the double cysteine, histidine mutations into mUT-A2 or mUT-A3 still did not induce Hg++ sensitivity, indicating that a region outside of the M-I region also contributes to the Hg++ induced block of fUT. Using a series of chimeras formed between UT-A3 and fUT we found that as well as C185 and H187, residues within the C-terminal of fUT determine Hg++ sensitivity and we propose that differences in the folding of this region between fUT and mUT-A2/mUT-A3 allow access of Hg++ to the fUT channel pore.
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