Studies On The Essential Amino Acid Residues In Transmembrane Domains 1 And N-terminus Of Organic Anion Transporting Polypeptide 1B1 (OATP1B1)

Organic anion transporting polypeptides?OATPs,gene symbol SLCO?are involved in the transport of various endogenous substrates within human body and play crucial roles in the absorption of a broad spectrum of drugs and xenobiotics.OATP1B1 is an important member of the OATP family and is specifically expressed at the basolateral membrane of human hepatocytes,mediating the absorption and elimination of a wide variety of negatively charged and/or amphiphilic compounds.Therefore,functional alternation of OATP1B1 would affect the pharmacokinetics,pharmacodynamics,and drug-drug interactions of many drugs.The structure-function relationship studies of OATP1B1 hence may help us better understand the molecular mechanisms of hepatic drug transport and provide a guideline for clinical therapeutic applications.Membrane proteins are involved in various physiological activities within cells such as cellular material exchange,cell recognition,immune responses,cell signaling regulation.In addition,they are binding targets for many drugs,Transmembrane domains?TMs?are key components of membrane proteins and homologous modeling had found that OATP1B3and OATP2B1,two members of the OATP family,have their transmembrane regions symmetrically distributed to form a positively charged pore that adapts to the function of transporting negatively charged compounds.Our previous study of OATP2B1 revealed several important amino acid residues located in TM1 of the transporter.OATP1B1 and OATP2B1,although both are OATPs,belong to different subfamilies.Whether TM1 of OATP1B1 is involved in substrate transport is unclear.In addition,previous studies of OATP1B3,which belongs to the same subfamily of OATP1B1 and shows high homology with OATP1B1,demonstrated that the N-terminus is essential for targeting OATP1B3 to the plasma membrane.Whether N-terminus of OATP1B1 exerts a similar function is unknown.In the current study,site-directed mutagenesis,uptake function measurement and cell surface biotin labeling coupled with Western blotting were applied to study the TM1 and N-terminus of OATP1B1.The experimental results obtained are as follows:1.Alanine-scanning was performed to study the TM1 of OATP1B1.Most of the alanine mutants retained transport function of prototypic substrate estrone-3-sulfate?ES??>50%function of wild-type OATP1B1?.However,alanine substitution of two positively charged amino acid residues,K41 and K49,resulted in dramatic reduction of transport function,showing about 20%and 30%ES uptake,respectively,compared to the wild type.K41A and K49A also exhibited significant decrease in transport of taurocholate,another prototypic substrate of OATP1B1.These date suggested that lysine residues at these two positions may participate in the substrate transport process of OATP1B1;2.Membrane protein analysis of K41A and K49A revealed that the cell surface protein level of K41A decreased about 60%,while that of K49A was similar to wild-type OATP1B1.Total protein level of the two mutants correlated well with the cell surface protein expression,indicating that the reduced expression of K41A on cell surface is due to the change of total protein expression;3.When K41 was replaced with the positively charged arginine,transport function and protein expression of K41R were significantly increased compared to K41A.On the other hand,when K41 was substituted with the negatively charged aspartic acid,the transport function of K41E was similar to K41A;while its protein expression was even lower than K41A.These results suggested that a positive charge at position 41 is important for protein stability and uptake function of OATP1B1.On the other hand,K49R showed a similar uptake function to that of K49A,implicating that the specific lysine at position 49 is irreplaceable;4.Kinetic analysis showed that K41A and K49A exhibited an altered Km at the high-affinity and low-affinity binding site,respectively,for ES uptake,suggesting that these two lysine residues may be involved in the formation of different substrate interaction channels.V_maxax of both mutants was reduced significantly,which indicated that alanine substitution also affects turnover rate of ES by the transporter;5.Our studies of the N-terminal residues of OATP1B1 revealed that when amino acids2-18 was truncated,the mutant retained comparable function to that of the wild-type transporter.However,when amino acids 2-27 were removed,protein expression as well as uptake function were reduced to almost undetectable level.In addition,protease inhibitor MG132 partially restored the protein level and transport function,suggesting that amino acid residues 19-27 of OATP1B1 may be important for proper function of the transporter protein;6.Further analysis of amino acid residues 19-27 revealed two conservative motifs-a cluster of positively charged residues with the sequence of KKTR?19-22?and a sequence YGNCL at positions 23-27 that shows similarity with the conserved sorting motif YXX?37??where X is any amino acid,?37?is a hydrophobic amino acid?.Studies of these two motifs found that KKTR plays a role in maintaining protein stability,while YCNGL is important not only for protein stability,but also for the interaction of OATP1B1 and its substrates.