Conserved amino acids in the region connecting membrane spanning domain 1 to nucleotide binding domain 1 are essential for expression of the MRP1 (ABCC1) transporter
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.