Multiple Membrane-associated Tryptophan Residues Contribute to the Transport Activity and Substrate Specificity of the Human Multidrug Resistance Protein, MRP1

Identification of Proline Residues in the Core Cytoplasmic and Transmembrane Regions of Multidrug Resistance Protein 1 (MRP1/ABCC1) Important for Transport Function, Substrate Specificity, and Nucleotide Interactions

“…Finally, the functional importance of the four MSD2 TM Pro residues at positions 343, 448, 557, and 595 is consistent with their location in the same TM helices (TM6, TM8, TM10, and TM11, respectively) that contain other amino acids shown in previous studies to be important for MRP1 drug binding and/or transport activity (24,26,50,53,54). However, of these four MSD2 TM Pro mutants that display a global and substantial loss of transport activity, only one of them (P595A) showed a complete loss of photolabeling by LTC 4 and a major decrease in uptake affinity for this substrate.…”

“…Our current model of MRP1 based on the crystal structures of the bacterial lipid transporter MsbA predicts that the analogous Pro 448 in TM8 also lines the substrate translocation pathway of MRP1 (50) and probably is also water-accessible, although this needs to be confirmed experimentally. Finally, it is worth noting that a conserved Trp residue is located within one helical turn of TM8 MRP1-Pro 448 and TM3 CFTR-Pro 205 (MRP1-Trp 445 and CFTR-Trp 202 ) and has been shown to be important for the function of these two ABCC proteins (24,59). Previous biophysical and modeling studies of channel and transport proteins indicate that the solvent accessibility of a Trp residue in a TM ␣-helix would be diminished by an Ala substitution of a Pro residue located at either position i ϩ 3 or i ϩ 4 (60).…”

“…The template for mutagenesis of Pro residues in MSD2 was prepared by cloning a 1.9-kb BamHI/SphI fragment (containing nucleotides 841-2701 encoding amino acids 281-900) from pcDNA3.1(Ϫ)-MRP1 K into pBluescriptSK(ϩ) (Stratagene, La Jolla, CA) into which a SphI site had been introduced into the multiple cloning site (24). The template for mutagenesis of Pro residues in MSD3 was prepared by cloning a 2-kb XmaI fragment (containing nucleotides 2337-4322 encoding amino acids 780 -1440) from pcDNA3.1(Ϫ)-MRP1 K into pGEM-3Z (Promega, Madison, WI) (23).…”

“…For example, previous studies have identified a number of aromatic, polar, and charged amino acids in or proximal to the TM helices in both MSD2 (amino acids 323-601) and MSD3 (amino acids 972-1248) that play an important role in determining MRP1 transport activity and substrate specificity (23)(24)(25)(26)(27)(28)(29)(30). Other studies have shown that Ala substitution of Cys residues in these domains as well as the cytoplasmic and extracytoplasmic regions of MRP1 do not appear to have major effects on its transport function (31).…”

Role of the NH2-terminal Membrane Spanning Domain of Multidrug Resistance Protein 1/ABCC1 in Protein Processing and Trafficking

“…Those limited elements identified, such as the cluster of basic amino acids in CL1 and conserved aromatic and Pro residues (Figure 9), seem likely to contribute to determining the orientation and positions of TMs such as TM1 and TM4, respectively (Ito et al, 2003;Koike et al, 2004). Furthermore, conserved tryptophans in MSD0 have been mutated singly, without affecting the transport activity of the protein (Koike et al, 2002). Nevertheless, studies of three ABCC proteins, SUR1A, MRP2, and Ycf1, indicate that MSD0 has at least a partially conserved role in protein trafficking (Fernandez et al, 2002;Mason and Michaelis, 2002;Babenko and Bryan, 2003).…”