The First Cytoplasmic Loop in the Core Structure of the ABCC1 (Multidrug Resistance Protein 1; MRP1) Transporter Contains Multiple Amino Acids Essential for Its Expression.

ABCC1 (human multidrug resistance protein 1 (hMRP1)) is an ATP-binding cassette transporter which effluxes xeno- and endobiotic organic anions and confers multidrug resistance through active drug efflux. The 17 transmembrane α-helices of hMRP1 are distributed among three membrane spanning domains (MSD0, 1, 2) with MSD1,2 each followed by a nucleotide binding domain to form the 4-domain core structure. Eight conserved residues in the first cytoplasmic loop (CL4) of MSD1 in the descending α-helix (Gly ³⁹² , Tyr ⁴⁰⁴ , Arg ⁴⁰⁵ ), the perpendicular coupling helix (Asn ⁴¹² , Arg ⁴¹⁵ , Lys ⁴¹⁶ ), and the ascending α-helix (Glu ⁴²² , Phe ⁴³⁴ ) were targeted for mutagenesis. Mutants with both alanine and same charge substitutions of the coupling helix residues were expressed in HEK cells at wild-type hMRP1 levels and their transport activity was only moderately compromised. In contrast, mutants of the flanking amino acids (G392I, Y404A, R405A/K, E422A/D, and F434Y) were very poorly expressed although Y404F, E422D, and F434A were readily expressed and transport competent. Modeling analyses indicated that Glu ⁴²² and Arg ⁶¹⁵ could form an ion pair that might stabilize transporter expression. However, this was not supported by exchange mutations E422R/R615E which failed to improve hMRP1 levels. Additional structures accompanied by rigorous biochemical validations are needed to better understand the bonding interactions crucial for stable hMRP1 expression.