**6. Regulation of the Transport Activity of Canalicular ABC Transporters**

At the canalicular membrane, ABCB1, ABCB4, ABCB11, ABCC2 and ABCG5 have been proposed to mostly reside within glycosphingolipids-, cholesterol- and caveolin-1 (Cav-1)-enriched raft microdomains [27,79–81]. These domains could provide a favorable environment for the regulation of the activity of canalicular ABC transporters. Indeed, the shift of ABCB1 and ABCB11 from cholesterol-enriched microdomains to low-cholesterol domains lowered their transport activity [82–85]. Moreover, accumulating evidence indicates that phospholipids and cholesterol are required for the proper function of ABCB1, ABCB4, ABCB11 and ABCC2 [86–93]. In purified membrane vesicles, delipidation due to detergent action inactivates ABCB1, whereas phospholipid addition fully restores the ATPase activity of the transporter [94,95]. Phosphoinositides, lipid products from PI3K-mediated activities, are required for ABCB11 and ABCC2 activation because their addition reverses the negative effect of PI3K inhibitors on the activity of these transporters [40,96].

The importance of membrane cholesterol content has been highlighted in Atp8b1 deficient mice [92]. Indeed, in these mice, the normal phospholipid asymmetry of the canalicular membrane is lost, thereby enhancing sensitivity to cholesterol extraction by hydrophobic BA and subsequent loss of ABCB11 and ABCC2 activity [92]. How exactly membrane cholesterol influences the transport activity of these transporters is not known, but this may involve allosteric modulations and/or indirect means such as changes in membrane fluidity. Several studies have proposed that cholesterol directly interacts with the ABCB1 substrate binding site and thereby facilitates the recognition of small drugs (<500 Da) [89,97,98]. Cyclodextrin treatment or Cav-1 overexpression leads to cholesterol depletion from the plasma membrane, which inhibits ABCB1 transport activity by increasing membrane fluidity and loosening lipid packing density [99]. However, Moreno

and colleagues showed that Cav-1 overexpression in mice increases both bile flow and the biliary secretion of phospholipids, BA and cholesterol, suggesting a positive role of Cav-1 in ABCB11 transport activity [100]. Considering the role of Cav-1 in intracellular cholesterol trafficking [101,102], some of the Cav-1 effects may be indirect and mediated through cholesterol homeostasis. Alternatively, Cav-1 may also directly bind ABCB1 and inhibit its transport activity [99,103–105]. It has been reported that the binding capacity of Cav-1 to ABCB1 is negatively modulated by Src kinase-mediated Cav-1 phosphorylation, a process facilitated by RACK1, which interacts with both Src and ABCB1 [105–107].

Several studies suggest a role for phosphorylation in the regulation of canalicular ABC transporters. Phosphorylation sites in the linker domain of ABCB1 have been well documented at Ser661, Ser667, Ser671, Ser675, and Ser683 [108–112]. Likewise, six potential phosphorylation sites have been found in the linker region of ABCC2 at Ser904, Ser912, Ser916, Ser917, Ser922 and Ser926 [113] and in the N-terminal domain of ABCB4 at Thr34, Thr44 and Ser49 [114]. An analysis of ABCB11 amino acid sequence also predicted multiple potential serine/threonine phosphorylation sites [115]. Overwhelming evidence indicates that PKC is a major player in ABC transporter phosphorylation and activity regulation. Phosphorylation within the linker domain of ABCB1 is specific for PKCα in purified vesicles from Sf9 cells [116]. The coexpression of PKCα and ABCB1 increases the ATPase activity of the transporter in insect and ovarian cells [116,117], while PKC inhibitor treatment did not alter ATPase activity in MCF-7 cells [118]. It has been shown that PKCα also mediates ABCB11 phosphorylation [115], as well as ABCC2 phosphorylation, resulting in stimulation of the intrinsic transport activity of ABCC2 [113]. PKC-dependent phosphorylation has also been shown to regulate ABCB4-mediated PC secretion [114]. The variation-induced impairment of ABCB4 N-terminal phosphorylation may also be related to a decrease in ABCB4-mediated PC secretion [114]. The substitution of all conserved serines in the linker domain of ABCC2 by non-phosphorylatable alanines significantly reduces the basal transport activity of ABCC2, while substitution into aspartates (mimicking constitutive phosphorylation of the residues) increases it [113]. Conversely, the role of ABCB1 phosphorylation in the regulation of its transport activity is less obvious because the substitution of potentially phosphorylatable residues by aspartates or non-phosphorylatable residues has no effect [119,120].
