Identification of functionally relevant residues of the rat ileal apical sodium-dependent bile acid cotransporter.
The mechanisms underlying the transport of bile acids by apical sodium-dependent bile acid transporter (Asbt) are not well defined. To further identify the functionally relevant residues, thirteen conserved negatively (Asp and Glu) and positively (Lys and Arg) charged residues plus Cys-270 of rat Asbt were replaced with Ala or Gln by site-directed mutagenesis. Seven of the fourteen residues of rat Asbt were identified as functionally important by taurocholate transport studies, substrate inhibition assays, confocal microscopy, and electrophysiological methods. The results showed that Asp-122, Lys-191, Lys-225, Lys-256, Glu-261, and Lys-312,Lys-313 residues of rat Asbt are critical for transport function and may determine substrate specificity. Arg-64 may be located at a different binding site to assist in interaction with non-bile acid organic anions. For bile acid transport by Asbt, Na(+) ion movement is a voltage-dependent process that tightly companied with taurocholate movement. Asp-122 and Glu-261 play a critical role in the interaction of a Na(+) ion and ligand with Asbt. Cys-270 is not essential for the transport process. These studies provide new details about the amino acid residues of Asbt involved in binding and transport of bile acids and Na(+).
Sun
An-Qiang
AQ
Department of Pediatrics, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA. An-Qiang.Sun@mssm.edu
Balasubramaniyan
Natarajan
N
Chen
Haijun
H
Shahid
Mohammad
M
Suchy
Frederick J
FJ
D000818:Animals; D001647:Bile Acids and Salts / Q000378:metabolism*; D019556:COS Cells; D002352:Carrier Proteins / Q000378:metabolism / Q000502:physiology*; D002522:Chlorocebus aethiops; D004594:Electrophysiology; D007082:Ileum / Q000473:pathology; D008562:Membrane Glycoproteins / Q000378:metabolism / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D051381:Rats; D012964:Sodium / Q000378:metabolism; D013656:Taurocholic Acid / Q000494:pharmacology; D014982:Xenopus laevis
D001647:Bile Acids and Salts; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; C037643:bile acid binding proteins; D013656:Taurocholic Acid; D012964:Sodium