N-glycosylation of ABC transporters is associated with functional activity in sandwich-cultured rat hepatocytes.

Hepatobiliary elimination via canalicular efflux transport proteins plays a key role in the clearance of endo- and xenobiotics. Correct membrane localization and coordinated action of the transport systems are essential for vectorial transport of drugs from blood into the bile. While basolaterally localized uptake transporters are responsible for the inward transport of substances from the blood into the hepatocyte, apically expressed ATP-dependent transport proteins such as P-glycoprotein (P-gp), multidrug resistance-associated protein (Mrp2) and breast cancer resistance protein (Bcrp) mediate the outward efflux into the bile canaliculus. Using sandwich-cultured rat hepatocytes we have characterized the expression and maturation of P-gp, Mrp2 and Bcrp transport proteins as well as their transport function over several days. The re-differentiation of the hepatocytes, which only occurs in sandwich configuration involves de novo synthesis and subsequent posttranslational N-glycosylation of all three transport proteins. Only fully N-glycosylated isoforms of the transporters were associated with functional activity as visualized by excretion of specific fluorescent substrates into the canalicular network. However, in what way N-glycosylation affects the functional activity of the ABC transporters investigated remains to be determined.