ATPase activity of nucleotide binding domains of human MDR3 in the context of MDR1

Although human MDR1 and MDR3 share 86% similarity in their amino acid sequences and are predicted to share conserved domains for drug recognition, their physiological transport substrates are quite different: MDR1 transports xenobiotics and confers multidrug resistance, while MDR3 exports phosphatidylcholine into bile. Although MDR1 shows high ATPase activity, attempts to demonstrate the ATPase activity of human MDR3 have not succeeded. Therefore, it is possible that the difference in the functions of these proteins is caused by their different ATPase activities. To test this hypothesis, a chimera protein containing the transmembrane domains (TMDs) of MDR1 and the nucleotide binding domains (NBDs) of MDR3 was constructed and analyzed. The chimera protein was expressed on the plasma membrane and conferred resistance against vinblastine and paclitaxel, indicating that MDR3 NBDs can support drug transport. Vanadate-induced ADP trapping of MDR3 NBDs in the chimera protein was stimulated by verapamil as was MDR1 NBDs. The purified chimera protein showed drug-stimulated ATPase activity like MDR1, while its Vmax was more than 10-times lower than MDR1. These results demonstrate that the low ATPase activity of human MDR3 cannot account for the difference in the functions of these proteins, and furthermore, that TMDs determine the features of NBDs. To our knowledge, this is the first study analyzing the features of human MDR3 NBDs.

Graphical abstractMDR3 NBD, which hydrolyzes ATP more than 10-times slower than MDR1 NBD, can support drug transport by MDR1 TMD.Figure optionsDownload full-size imageDownload high-quality image (208 K)Download as PowerPoint slideHighlights► MDR1 and MDR3 transport different substrates, xenobiotic and phosphatidylcholine, respectively. ► MDR3 NBDs hydrolyze ATP more than 10-times slower than MDR1 NBDs. ► ATPase activity cannot account for the difference in the functions of MDR1 and MDR3.