TAILIEUCHUNG - Báo cáo khoa học: Transport of taurocholate by mutants of negatively charged amino acids, cysteines, and threonines of the rat liver sodium-dependent taurocholate cotransporting polypeptide Ntcp

The relevance of functional amino acids for taurocholate transport by the sodium-dependent taurocholate cotrans-porting polypeptide Ntcp was determined by site-directed mutagenesis. cRNAfrom28 single-pointsmutants of the rat liver Ntcp clone was expressed in Xenopus laevisoocytes. Mutations were generated in five conserved negatively chargedaminoacids (aspartates andglutamates)whichwere present in nine members of the SBAT-family, in two non-conserved negatively charged amino acids, in all eight Ntcp-cysteines, and in two threonines from a protein kinase C consensus region of theNtcpC-terminus. . | Eur. J. Biochem. 270 1117-1127 2003 FEBS 2003 doi Transport of taurocholate by mutants of negatively charged amino acids cysteines and threonines of the rat liver sodium-dependent taurocholate cotransporting polypeptide Ntcp Daniel Zahner Uta Eckhardt and Ernst Petzinger Institute of Pharmacology and Toxicology Justus-Liebig-University Giessen Germany The relevance of functional amino acids for taurocholate transport by the sodium-dependent taurocholate cotransporting polypeptide Ntcp was determined by site-directed mutagenesis. cRNA from 28 single-points mutants of the rat liver Ntcp clone was expressed in Xenopus laevis oocytes. Mutations were generated in five conserved negatively charged amino acids aspartates and glutamates which were present in nine members of the SBAT-family in two nonconserved negatively charged amino acids in all eight Ntcp-cysteines and in two threonines from a protein kinase C consensus region of the Ntcp C-terminus. Functional amino acids were Asp115 Glu257 and Cys266 which were found to be essential for the maintenance of taurocholic acid transport. Asp115 is located in the large intracellular loop III whereas Glu257 and Cys266 are located in the large extracellular loop VI. Four mutations of threonines from the C-terminus of the Ntcp by alanines or tyrosines showed no effects on sodium-dependent taurocholate transport. Introduction of the FLAG motif into several transport negative point mutations demonstrated that all mutated proteins besides one were present within the cell membrane of the oocytes and provided proof that an insertion defect has not caused transport deficiency by these Ntcp mutants. The latter was observed only with the transport negative mutant Asp24Asn. In conclusion loop amino acids are required for sodium-dependent substrate translocation by the Ntcp. Keywords bile acids P-loop glutamate aspartate membrane protein. The sodium-dependent taurocholate cotransporting polypeptide Ntcp .

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