Sorting nexin 27 interacts with multidrug resistance-associated protein 4 (MRP4) and mediates internalization of MRP4.

Multidrug resistance-associated protein 4 (MRP4/ABCC4) makes a vital contribution to the bodily distribution of drugs and endogenous compounds because of its cellular efflux abilities. However, little is known about the mechanism regulating its cell surface expression. MRP4 has a PDZ-binding motif, which is a potential sequence that modulates the membrane expression of MRP4 via interaction with PDZ adaptor proteins. To investigate this possible relationship, we performed GST pull-down assays and subsequent analysis with matrix-assisted laser desorption/ionization-time of flight mass spectrometry. This method identified sorting nexin 27 (SNX27) as the interacting PDZ adaptor protein with a PDZ-binding motif of MRP4. Its interaction was confirmed by a coimmunoprecipitation study using HEK293 cells. Knockdown of SNX27 by siRNA in HEK293 cells raised MRP4 expression on the plasma membrane, increased the extrusion of 6-[(14)C]mercaptopurine, an MRP4 substrate, and conferred resistance against 6-[(14)C]mercaptopurine. Cell surface biotinylation studies indicated that the inhibition of MRP4 internalization was responsible for these results. Immunocytochemistry and cell surface biotinylation studies using COS-1 cells showed that SNX27 localized to both the early endosome and the plasma membrane. These data suggest that SNX27 interacts with MRP4 near the plasma membrane and promotes endocytosis of MRP4 and thereby negatively regulates its cell surface expression and transport function.

AP2 adaptor complex mediates bile salt export pump internalization and modulates its hepatocanalicular expression and transport function.

UNLABELLED: The bile salt export pump (BSEP) mediates the biliary excretion of bile salts and its dysfunction induces intrahepatic cholestasis. Reduced canalicular expression of BSEP resulting from the promotion of its internalization is one of the causes of this disease state. However, the molecular mechanism underlying BSEP internalization from the canalicular membrane (CM) remains unknown. We have shown previously that 4-phenylbutyrate (4PBA), a drug used for ornithine transcarbamylase deficiency (OTCD), inhibited internalization and subsequent degradation of cell-surface-resident BSEP. The current study found that 4PBA treatment decreased significantly the expression of α- and µ2-adaptin, both of which are subunits of the AP2 adaptor complex (AP2) that mediates clathrin-dependent endocytosis, in liver specimens from rats and patients with OTCD, and that BSEP has potential AP2 recognition motifs in its cytosolic region. Based on this, the role of AP2 in BSEP internalization was explored further. In vitro analysis with 3×FLAG-human BSEP-expressing HeLa cells and human sandwich-culture hepatocytes indicates that the impairment of AP2 function by RNA interference targeting of α-adaptin inhibits BSEP internalization from the plasma membrane and increases its cell-surface expression and transport function. Studies using immunostaining, coimmunoprecipitation, glutathione S-transferase pulldown assay, and time-lapse imaging show that AP2 interacts with BSEP at the CM through a tyrosine motif at the carboxyl terminus of BSEP and mediates BSEP internalization from the CM of hepatocytes. CONCLUSION: AP2 mediates the internalization and subsequent degradation of CM-resident BSEP through direct interaction with BSEP and thereby modulates the canalicular expression and transport function of BSEP. This information should be useful for understanding the pathogenesis of severe liver diseases associated with intrahepatic cholestasis.

Involvement of the pyrilamine transporter, a putative organic cation transporter, in blood-brain barrier transport of oxycodone.

The purpose of this study was to characterize blood-brain barrier (BBB) transport of oxycodone, a cationic opioid agonist, via the pyrilamine transporter, a putative organic cation transporter, using conditionally immortalized rat brain capillary endothelial cells (TR-BBB13). Oxycodone and [3H]pyrilamine were both transported into TR-BBB13 cells in a temperature- and concentration-dependent manner with Km values of 89 and 28 microM, respectively. The initial uptake of oxycodone was significantly enhanced by preloading with pyrilamine and vice versa. Furthermore, mutual uptake inhibition by oxycodone and pyrilamine suggests that a common mechanism is involved in their transport. Transport of both substrates was inhibited by type II cations (quinidine, verapamil, and amantadine), but not by classic organic cation transporter (OCT) substrates and/or inhibitors (tetraethylammonium, 1-methyl-4-phenylpyridinium, and corticosterone), substrates of OCTN1 (ergothioneine) and OCTN2 (L-carnitine), or organic anions. The transport was inhibited by metabolic inhibitors (rotenone and sodium azide) but was insensitive to extracellular sodium and membrane potential for both substrates. Furthermore, the transport of both substrates was increased at alkaline extracellular pH and decreased in the presence of a protonophore (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone). Intracellular acidification induced with ammonium chloride enhanced the uptakes, suggesting that the transport is driven by an oppositely directed proton gradient. The brain uptake of oxycodone measured by in situ rat brain perfusion was increased in alkaline perfusate and was significantly inhibited by pyrilamine. These results suggest that blood-brain barrier transport of oxycodone is at least partly mediated by a common transporter with pyrilamine, and this transporter is an energy-dependent, proton-coupled antiporter.