**3. Roles of Additional TMD0 and L0 Domains**

The functional role of TMD0 in other proteins like MRP1, MRP2, SUR1 is well defined, which may be involved in stabilization and retention of the transporter in the plasma membrane or in regulation of channel activities. By topological modeling of TMD0 of MRP6, we demonstrated that it contains five transmembrane domains with the N- and C-termini on the external and cytoplasmic side, respectively. These TMs are inserted into the membrane individually on the basis of hydrophobicity and without affecting each other. In addition, we have also found that disease-causing mutations did not affect the membrane insertion of these TMs [25]. In a further study, we have done the structural and functional characterization of L0 loop of ABCC6. We have found that L0 loop of ABCC6 is well structured (Figure 3) as it contains aromatic residues, and three α-helical regions; it resembles the homologous L0 loops of other MRPs and is responsible for plasma membrane localization of TMD0 [13]. However, to understand the exact role of TMD0 and L0 loop (N-terminal Region) of ABCC6, we have constructed two variants of N-terminal lacking TMD0 (∆TMD0) and a variant lacking both TMD0 and L0 (∆TMD0L0). Interestingly, we have found that ∆TMD0L0 not only failed to exhibit transport activity, but it was also not able to localize at the basolateral side of the plasma membrane, which reflects that L0 loop is important for both activities. Thus, these findings suggest that L0 not only contains

a basolateral sorting signal but that L0 also contributes to folding ABCC6 into a cellular sorting-competent state, which is necessary to pass the endoplasmic reticulum (ER) quality control system and continue through the secretory pathway. In addition, we also found that L0 loop of ABCC6 interacts with the ion channels like other members of ABCC family and might be involved in the modulation of Ca2+ channels of plasma membrane [20].

**Figure 3.** Homology model of L0 loop. The loop includes the region between residues E205 and A262. The PDB structure 5UJ9 corresponding to MRP1 was used as template.
