Fig 1.
(A) Shown is a two-dimensional illustration of the domain structure of MRP1, a so-called ‘long’ ABCC transporter [58]. MSD0 (beige) is present only in a subset of ABCC/MRP subfamily members and is connected to MSD1 by cytoplasmic loop 3 (often referred to as Lo). The core ABC domain structure is comprised of MSD1/NBD1 (green), CR1 (orange), MSD2/NBD2 (blue) and CR2 (red). The TMs of MRP1 are numbered 1–17, and the three N-glycosylation sites at Asn19, Asn23 and Asn1006 are indicated by small branches. (B) Shown are views of a homology model of the core human MRP1 structure [43] based on the cryo-EM structure of apo bovine Mrp1 (PDB ID: 5UJ9) at 0° and 180° rotation on a vertical axis so that CR1 (orange) and CR2 (red) are clearly visible. The colours of the other domains are the same as in panel (A) with a portion of cytoplasmic loop 3 between MSD0 and MSD1 coloured pink. MSD, membrane spanning domain; NBD, nucleotide binding domain; CR, connecting region.
Fig 2.
Predicted location and sequence alignments of targeted CR1-MRP1 residues.
(A) Shown is a cartoon of the secondary structure of MRP1-CR1 (amino acids 600–642) obtained from the atomic homology model of human MRP1 based on the cryo-EM structure of apo bovine Mrp1 (PDB: 5UJ9) [43]. The side chains of the initial four targeted CR1 residues [Ser612, Arg615, His622, Glu624] are shown in stick form and coloured by element where oxygen is red and nitrogen is blue. Sequence alignments of human MRP1-CR1 with (B) bovine Mrp1; (C) human ABCC 5-domain and 4-domain transporter homologs; and (D) human ABCC non-transporter homologs were generated using Clustal Omega [60]. The initial four and subsequent single CR1 amino acids targeted for mutagenesis in this study (Ser612, Arg615, His622, Glu624, and Phe619, respectively) are highlighted in dark orange and purple, respectively. In (B), the diamond symbols (♦) above the sequence denotes the seven amino acids in this region (and the COOH-proximal end of TM11) investigated previously (Pro600, Met601, Val602, Ile603, Ser604, Ser605, Arg633) [57, 72, 73] (Cole laboratory, unpublished).
Fig 3.
CR1 mutations S612A, R615A, H622A and E624A have different effects on total and membrane MRP1 levels in transfected HEK cells.
(A) Shown are representative immunoblots of whole cell extracts (10 μg protein loaded per lane) prepared from HEK cells transfected with wild-type (WT) and mutant (S612A, R615A, H622A, E624A) MRP1 pcDNA expression vectors as well as untransfected cells (HEK) (negative control). MRP1 was detected with mAb QCRL-1 [54], and anti-Na+/K+-ATPase served as a protein loading control. A vertical black line indicates where irrelevant lanes from the immunoblot were removed by cropping images; the vertical space between lanes indicates independent immunoblots. The images in each panel are from a single blot with the region between MRP1 and the loading control cropped out. (B) WT and S612A, R615A, H622A and E624A mutant MRP1 levels, normalized for protein loading based on the Na+/K+-ATPase signal, were estimated using densitometry and plotted as a percent of wild-type MRP1 levels. Bars represent the mean values (± SD) of results obtained from three independent transfections. * P<0.05, unpaired t-test. (C) Shown are non-quantitative confocal microscopy images of HEK cells 24 h after transfection with WT and CR1 mutant (S612A, R615A, H622A, E624A) pcDNA expression vectors. MRP1 (green) was detected using rat mAb MRPr1 [56] and Alexa Fluor 488-conjugated goat anti-rat as the primary and secondary antibodies, respectively. Untransfected cells (HEK) were used as a negative control. Nuclei are stained blue. Signals from the two channels were acquired independently and the merged images are presented. White calibration bars (bottom right) represent 10 μm.
Fig 4.
Ala substitution of CR1-His622 has no effect on levels or organic anion transport activity of MRP1.
(A) Shown is a representative immunoblot of inside-out membrane vesicles (1 μg protein per lane) prepared from HEK cells transfected with wild-type (WT) and H622A MRP1 pcDNA expression vectors as well as untransfected cells (HEK) (negative control). The blot was probed for MRP1 (Mab QCRL-1) [54] and Na+/K+-ATPase as a loading control. The vertical black line indicates where irrelevant lanes from the immunoblot were removed by cropping the image. The images in each panel are from a single blot with the region between MRP1 and the loading control (Na+/K+-ATPase) cropped out. (B & C) Transport activity was measured as ATP-dependent uptake of (B) [3H]E217βG and (C) [3H]LTC4 into inside-out membrane vesicles and expressed as a percent of wild-type MRP1 uptake. The values shown have been adjusted to take into account minor differences in mutant MRP1 levels in the membrane vesicles relative to wild-type MRP1. Bars represent the mean values (± SD) of results obtained from three independent experiments. n.s., not significant P>0.05, unpaired t-test.
Fig 5.
Potential bonding interactions of CR1 Arg615 and Glu624 and effect of structure-guided double exchange mutations on MRP1 levels.
Upper panels, Shown are potential electrostatic bonding interactions between (A) Lys406 and Glu624; and (B) Arg615 and Asp430, and (C) Arg615 and Phe619 derived from an atomic homology model of human MRP1 [43] based on the apo bovine Mrp1 cryo-EM structure (PDB ID: 5UJ9). While several bonding interactions are theoretically possible, for clarity, only the potential bond (dotted line) with the shortest predicted distance between each pair of amino acids is shown. Side chains are shown as sticks and coloured by element where oxygen is red and nitrogen is blue. Lower panels, Shown are representative immunoblots of cell extracts (10 μg protein per lane) prepared from HEK cells transfected with (A) wild-type (WT) and mutant (E624A, E624K, K406E, K406E/E624K) MRP1 and (B) WT and mutant (R615A, R615D, D430R, D430R/R615D) MRP1; and (C) WT and mutant (R615A, R615F, F619R, R615F/F619R) pcDNA expression vectors. Extracts from untransfected cells (HEK) served as negative controls. MRP1 was detected with mAb QCRL-1 [54], and anti-Na+/K+-ATPase was used as a protein loading control. The images in each panel are from a single blot with the region between MRP1 and the loading control cropped out. The vertical black lines in (A) indicate where irrelevant lanes from the immunoblot were removed by cropping images. Similar results were obtained with extracts from two independent transfections.
Fig 6.
Levels of mutant MRP1 proteins in transfected HEK cells.
Shown are immunoblots of cell extracts (10 μg protein per lane) prepared from HEK cells transfected with pcDNA3.1(-) expression vectors encoding wild-type MRP1 (WT) and (A) single K406A/R/E mutants; (B) single E624A/D mutants; (C) single R615A/K mutants; and (D) single F619A/R/Y mutants. Untransfected cells (HEK) served as a negative control. Relative MRP1 levels were quantified by densitometry and adjusted as needed to take into account differences in gel loading by probing blots with antibodies against α-tubulin (loading control). Blots shown are from single experiments; comparable results were obtained in at least one additional independent transfection and immunoblot. For conservative mutants R615K, E624D, and F619Y, levels were comparable to wild-type hMRP1 (1.3, 0.8, and 1.0-fold, respectively; means of 2 independent observations). The images in each panel are from a single blot with the region between MRP1 and the loading control cropped out.
Fig 7.
Location of amino acids found essential for stable expression of human MRP1 in mammalian HEK cells.
Shown are the locations of amino acids identified in this study and in previously published studies [48, 59, 63, 88] or in unpublished work (Cole laboratory) to be essential for stable MRP1 expression in HEK cells. Amino acids are considered essential if their mutation to a non-conservative or conservative amino acid reduced MRP1 levels by >80% as measured by immunoblotting. Critical amino acids identified in the present study are colored to match their domains (green, orange). Those from previous studies are uncolored in regular font while those from unpublished work are uncolored in italics. MSD, membrane spanning domain; CL, cytoplasmic loop; NBD, nucleotide binding domain; CR, connecting region.