Functional Reconstitution of ATP‐Dependent Transporters from the Solubilized Hepatocyte Canalicular Membrane

Abstract

The hepatocyte canalicular membrane contains several primary‐active ATP‐dependent export carriers including one for bile salts and one for leukotriene C₄ and related conjugates. The molecular identity of both transporters has not been fully elucidated. To establish a transport assay that allows the purification and identification of the proteins involved in ATP‐dependent bile salt transport and in leukotriene C₄ transport, we reconstituted solubilized hepatocyte canalicular membranes into phospholipid bilayers using a rapid dilution method. The proteoliposomes formed exhibited both [³H]taurocholate and [³H]leukotriene C₄ uptake, which was much higher in the presence of ATP than in the presence of the non‐hydrolyzable ATP‐analog AdoPP [CH₂]P or in the absence of nucleotides. Nucleotide requirement and osmotic sensitivity of [³H]taurocholate transport indicates true transport into the vesicle lumen. Optimized conditions for reconstitution included the addition of a high concentration of an osmolyte (glycerol) and the presence of exogenous phospholipids (0.3%) during solubilization. Highest transport rates were obtained by reconstitution into acetone/ether‐precipitated Escherichia coli phospholipid supplemented with 20% cholesterol and by use of octylglucoside concentrations between 30 mM and 50 mM. Taurocholate transport was non‐competitively inhibited by vanadate (K_i= 39 μM). The kinetic parameters of cyclosporin A inhibition (K_i= 2.6 μM for taurocholate and 4.3 μM for leukotriene C₄ transport) as well as the affinities of taurocholate (K_m= 12 μM) and leukotriene C₄ (K_m= 0.5 μM) in the proteoliposome system indicate that the reconstitution resulted in functionally active transport systems, which are representative of ATP‐dependent transport in the intact plasma membrane.