The enterohepatic circulation of bile acids comprises several transmembraneous transport steps. The proposed research will focus on two of them: the uptake from blood into the hepatocyte across the sinusoidal plasma membrane, and the secretion into bile across the canalicular membrane. The latter process is rate-limiting for the entire enterohepatic circulation. Its deficiency could have pathological consequences: cholestasis on one hand, gallstone formation due to a decreased bile acid/cholesterol ratio on the other. It is proposed to investigate the molecular mechanism of both hepatic bile acid transporters with respect to three major properties: (i) Specificity towards different bile acids, their conjugates, and their sulfates and glucuronides (the latter are enhanced in cholestasis). A corollary question is the possibility of several distinct transporters specific for the above classes of compounds. (ii) Driving force. The coupling of bile acid transport to ion (e.g. Na+) grandients and to the electrical membrane potential will be studied. (iii) Subunit composition. Published data suggest that the same protein might participate in both the sinusoidal and the canalicular transport. Since the mode of action of these transporters is different, the existence of additional subunits conferring the special properties to a common channel is possible and will be checked. Initial experiments will be carried out on purified canalicular and sinusoidal membranes. However, the simultaneous presence of other ion permeabilities will interfere with many experiments. Therefore, the transporters will be solubilized and reconstituted. Employing reconstitution as the assay, the purification of the transporters will be carried out. The purification in conjunction with reconstitution will form an integral part of the search for regulatory subunits; in addition, the reconstituted system (crude or purified) will be used for the remaining experiments listed above.
