Molecular characterization of the mercurial sensitivity of a frog urea transporter (fUT).
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 5-min preincubation in 0.5 mM mercury chloride (HgCl2) also significantly reduced urea uptake. The transport of urea by fUT was rendered mercury (Hg2+) 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, Hg2+ sensitivity was reestablished. The transport of urea by the mouse UTs mUT-A2 and mUT-A3 was not sensitive to Hg2+. Introducing cysteine residues analogous to that mutated in fUT into mUT-A2 or mUT-A3 did not induce Hg2+ sensitivity. Additionally, introducing the double cysteine, histidine mutations into mUT-A2 or mUT-A3 still did not induce Hg2+ sensitivity, indicating that a region outside of the M-I region also contributes to the Hg2+-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 COOH terminal of fUT determine Hg2+ sensitivity, and we propose that differences in the folding of this region between fUT and mUT-A2/mUT-A3 allow access of Hg2+ to the fUT channel pore.
Stewart
Gavin S
GS
Faculty of Life Sciences, University of Manchester, United Kingdom.
Smith
Craig P
CP
Cooper
Gordon J
GJ
D000595:Amino Acid Sequence; D000818:Animals; D003545:Cysteine; D005260:Female; D015870:Gene Expression; D006639:Histidine; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics; D008627:Mercuric Chloride / Q000494:pharmacology; D017669:Mercury Compounds / Q000494:pharmacology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D017354:Point Mutation; D018075:RNA, Complementary / Q000235:genetics; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D014162:Transfection; D014508:Urea / Q000378:metabolism; D014982:Xenopus laevis; D000096982:Urea Transporters
D003545:Cysteine; D006639:Histidine; D026901:Membrane Transport Proteins; D008627:Mercuric Chloride; D017669:Mercury Compounds; D018075:RNA, Complementary; D011994:Recombinant Proteins; D014508:Urea; D000096982:Urea Transporters