Amino Acid Residues in Transmembrane Domain 10 of Organic Anion Transporting Polypeptide 1B3 Are Critical for Cholecystokinin Octapeptide Transport
详细信息 查看全文
- 作者：Chunshan Gui ; Bruno Hagenbuch
- 刊名：Biochemistry
- 出版年：2008
- 出版时间：September 2, 2008
- 年：2008
- 卷：47
- 期：35
- 页码：9090-9097
- 全文大小：389K
- 年卷期：v.47,no.35(September 2, 2008)
- ISSN：1520-4995
文摘
Human organic anion transporting polypeptides (OATP) 1B1 and 1B3 are multispecific transporters that mediate uptake of amphipathic organic compounds into hepatocytes. The two OATPs contain 12 transmembrane domains (TMs) and share 80% amino acid sequence identity. Besides common substrates with OATP1B1, OATP1B3 specifically transports cholecystokinin octapeptide (CCK-8). To determine which structural domains and/or residues are important for the substrate selectivity of OATP1B3, we constructed a series of chimeric proteins between OATP1B3 and 1B1, expressed them in HEK293 cells, and determined rates of uptake of CCK-8 along with surface expression of the proteins. Replacing TM10 in OATP1B3 with TM10 of OATP1B1 resulted in a dramatically reduced degree of CCK-8 transport, indicating that TM10 is crucial for recognition and/or translocation of CCK-8. Using site-directed mutagenesis, we identified three key residues within TM10, namely, Y537, S545, and T550. When we replaced these residues with the corresponding amino acid residues found in OATP1B1, the level of CCK-8 transport was similarly low as for the replacement of the whole TM10. Kinetic experiments showed that the Km values for CCK-8 transport in the TM10 replacement and triple mutant were only 1.3 and 1.1 μM, respectively, as compared to 16.3 μM for wild-type OATP1B3. Similarly, the Vmax values dropped from 495.5 pmol (normalized mg)−1 min−1 for wild-type OATP1B3 to 13.3 and 19.0 pmol (normalized mg)−1 min−1 for the TM10 replacement and triple mutant, respectively. Molecular modeling indicated that two of the three identified residues might form hydrogen bonds with CCK-8. In conclusion, we have identified three amino acid residues (Y537, S545, and T550) in TM10 of OATP1B3 that are important for CCK-8 transport.