Bile salt excretion in skate liver is mediated by a functional analog of Bsep/Spgp, the bile salt export pump

Biliary secretion of bile salts in mammals is mediated in part by the liver -specific ATP-dependent canalicular membrane protein Bsep/Spgp, a member of the ATP-binding cassette superfamily. We examined whether a similar transp ort activity exists in the liver of the evolutionarily primitive marine fis h Raja erinacea, the little skate, which synthesizes mainly sulfated bile a lcohols rather than bile salts. Western blot analysis of skate liver plasma membranes using antiserum raised against rat liver Bsep/Spgp demonstrated a dominant protein band with an apparent molecular mass of 210 kDa, a size larger than that in rat liver canalicular membranes, similar to 160 kDa. Im munofluorescent localization with anti-Bsep/Spgp in isolated, polarized ska te hepatocyte clusters revealed positive staining of the bile canaliculi, c onsistent with its selective apical localization in mammalian liver. Functi onal characterization of putative ATP-dependent canalicular bile salt trans port activity was assessed in skate liver plasma membrane vesicles, with [H -3]taurocholate as the substrate. [H-3]taurocholate uptake into the vesicle s was mediated by ATP-dependent and -independent mechanisms. The ATP-depend ent component was saturable, with a Michaelis-Menten constant (K-m) for tau rocholate of 40 +/- 7 mu M and a K-m for ATP of 0.6 +/- 0.1 mM, and was com petitively inhibited by scymnol sulfate (inhibition constant of 23 mu M), t he major bile salt in skate bile. ATP-dependent uptake of taurocholate into vesicles was inhibited by known substrates and inhibitors of Bsep/Spgp, in cluding other bile salts and bile salt derivatives, but not by inhibitors o f the multidrug resistance protein-1 or the canalicular multidrug resistanc e-associated protein, indicating a distinct transport mechanism. These find ings provide functional and structural evidence for a Bsep/Spgp-like protei n in the canalicular membrane of the skate liver. This transporter is expre ssed early in vertebrate evolution and transports both bile salts and bile alcohols.