Short-term regulation of bile acid uptake by microfilament-dependent translocation of rat ntcp to the plasma membrane

The Naf-taurocholate cotransport polypeptide (ntcp) is the primary transpor ter for the uptake of bile acids in the liver. The second messenger adenosi ne 3':5'-cyclic monophosphate (cAMP) rapidly increases ntcp protein concent ration in the plasma membrane, yet the mechanism is unknown. To investigate this, HepG2 cells were transiently transfected with a carboxy-terminal-tag ged green fluorescence protein (GFP) conjugate of ntcp, and then examined b y confocal video microscopy. Transporter activity was directly assayed with H-3-taurocholic acid (TC) scintigraphy. ntcp-GFP targeted to the plasma me mbrane in transfected cells, and the conjugate protein transported H-3-TC a s effectively as unmodified rat ntcp, Stimulation of ntcp-GFP cells with cA MP increased GFP fluorescence in the plasma membrane by 40% (P < .0001) wit hin 2.5 minutes and by 55% within 10 minutes. Similarly, cAMP increased tra nsport of bile acids by 30%, Cytochalasin D, an inhibitor of microfilaments , did not prevent ntcp-GFP from targeting to the plasma membrane, but compl etely abolished the increase in GFP fluorescence seen in response to cAMP. In contrast, the microtubule inhibitor, nocodazole, prevented development o f membrane fluorescence in 48 (96%) of 50 cells. Cells regained plasma memb rane fluorescence within 2 hours after nocodazole removal. These findings s uggest that targeting of ntcp to the plasma membrane consists of 2 steps: 1 ) delivery of ntcp to the region of the plasma membrane via microtubules; a nd 2) insertion of ntcp into the plasma membrane, in a microfilament- and c AMP-sensitive fashion.