Abstract
Canalicular secretion of bile salts is a vital function of the vertebrate liver, yet the molecular identity of the involved ATP-dependent carrier protein has not been elucidated. We cloned the full-length cDNA of the sister of P-glycoprotein (spgp; Mr approximately 160,000) of rat liver and demonstrated that it functions as an ATP-dependent bile salt transporter in cRNA injected Xenopus laevis oocytes and in vesicles isolated from transfected Sf9 cells. The latter demonstrated a 5-fold stimulation of ATP-dependent taurocholate transport as compared with controls. This spgp-mediated taurocholate transport was stimulated solely by ATP, was inhibited by vanadate, and exhibited saturability with increasing concentrations of taurocholate (Km approximately 5 microM). Furthermore, spgp-mediated transport rates of various bile salts followed the same order of magnitude as ATP-dependent transport in canalicular rat liver plasma membrane vesicles, i.e. taurochenodeoxycholate > tauroursodeoxycholate = taurocholate > glycocholate = cholate. Tissue distribution assessed by Northern blotting revealed predominant, if not exclusive, expression of spgp in the liver, where it was further localized to the canalicular microvilli and to subcanalicular vesicles of the hepatocytes by in situ immunofluorescence and immunogold labeling studies. These results indicate that the sister of P-glycoprotein is the major canalicular bile salt export pump of mammalian liver.
Highlights
Bile formation is an important function of vertebrate liver [1]
Membrane vesicles isolated from spgp cDNA-infected Sf9 cells demonstrated marked ATP-dependent uptake of taurocholate, whereas control vesicles from wild type cells did not (Fig. 3A)
This conclusion is derived from the functional expression of the full-length bile salt export pump (BSEP)/spgp cDNA in X. laevis oocytes (Fig. 2) and in Sf9 insect cells (Figs. 3 and 4A)
Summary
Materials—[3H]Taurocholic acid (2.1– 4.6 Ci/mmol), [3H]cholic acid (13.2 Ci/mmol), and [4C]glycocholic acid (44.6 mCi/mmol) were obtained from NEN Life Science Products. 3H-Labeled taurochenodeoxycholic and tauroursodeoxycholic acids of high specific activity (2– 60 Ci/mmol) were prepared as described previously (20 –22). After cloning and sequencing a single polymerase chain reaction product of 430 bp was identified that revealed an 88% identity to the published portion of the pig liver spgp cDNA [19]. Functional Expression of spgp in X. laevis Oocytes—Oocytes were prepared, injected with cRNAs, cultured, and preloaded with 28.5 pmol of [3H]taurocholate (60 nl of 475 M [3H]taurocholate (2.1 Ci/mmol)) as described elsewhere [23, 24]. [3H]Taurocholate efflux from individual oocytes was determined at 25 °C as described previously [18, 24] using a Naϩ-free incubation medium of 100 mM choline chloride, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES/Tris, pH 7.5. A multiple tissue Northern blot (CLONTECH) (ϳ2 g of poly(Aϩ) RNA per lane) was hybridized with the full-length spgp cDNA clone or with a -actin probe labeled with [␣-32P]dCTP by random priming. Immunofluorescence and Immunogold Electron Microscopy—Immunofluorescence studies [27] and immunogold electron microscopy studies on ultrathin frozen-thawed sections of perfusion-fixed (3% formaldehyde, 0.1% glutaraldehyde) liver were performed as described previously [28, 29]
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