Human hepatic bile acid transporter Na+/taurocholate co-transporting polypeptide (NTCP) represents the liver-specific entry receptor for the Hepatitis B and D Viruses (HBV/HDV). Chronic hepatitis B and D affect several million people worldwide, but treatment options are limited. Recently, HBV/HDV entry inhibitors targeting NTCP have emerged as promising novel drug candidates. Nevertheless, the exact molecular mechanism that NTCP uses to mediate virus binding and entry into hepatocytes is still not completely understood. It is already known that human NTCP mRNA expression is down-regulated under cholestasis. Furthermore, incubation of rat hepatocytes with the secondary bile acid taurolithocholic acid (TLC) triggers internalization of the rat Ntcp protein from the plasma membrane. In the present study, the long-term inhibitory effect of TLC on transport function, HBV/HDV receptor function and membrane expression of human NTCP were analyzed in HepG2 and HEK293 cells stably overexpressing NTCP. Even after short pulse preincubation, TLC had a significant long-lasting inhibitory effect on the transport function of NTCP, but the NTCP protein was still present at the plasma membrane. Furthermore, binding of the HBV/HDV myr-preS1 peptide and susceptibility for in vitro HDV infection were significantly reduced by TLC preincubation. We hypothesize that TLC rapidly accumulates in hepatocytes and mediates long-lasting trans-inhibition of the transport and receptor function of NTCP via a particular TLC binding site at an intracellularly accessible domain of NTCP. Physiologically, this trans-inhibition might protect hepatocytes from toxic overload of bile acids. Pharmacologically, it provides an interesting novel NTCP target site for potential long-acting HBV/HDV entry inhibitors.

Overexpression of single genes in mammalian cells is widely used to investigate protein function in basic and applied biosciences and in drug research. A better understanding of interactions of two proteins is an important next step in the advancement of our understanding of complex biological systems. However, simultaneous and robust overexpression of two or more genes is challenging. The Flp-In system integrates a vector into cell lines at a specific genomic locus, but has not been used for integration of more than one gene. Here we present a modification of the Flp-In system that enables the simultaneous targeted integration of two genes. We describe the modification and generation of the vectors required and give the complete protocol for transfection and validation of correct genomic integration and expression. We also provide results on the stability and reproducibility, and we functionally validated this approach with a pharmacologically relevant combination of a membrane transporter facilitating drug uptake and an enzyme mediating drug metabolism.