The regions connecting the membrane spanning and nucleotide binding domains of MRP1 are functionally distinct
Multidrug resistance protein 1 (MRP1) is a 190 kDa ATP-binding cassette transporter which effluxes xeno- and endobiotic organic anions including estradiol glucuronide (E217βG) and the pro-inflammatory leukotriene C4 (LTC4). MRP1 also confers multidrug resistance by reducing intracellular drug accumulation through active efflux. MRP1 has three membrane spanning domains (MSD), two of which form the solute translocation pathway, and two functionally asymmetric nucleotide binding domains (NBD) which bind and hydrolyze ATP. MSD1/2 are linked to NBD1/2 by connecting regions (CR) 1 (aa 600-642) and CR2 (aa 1249-1291), respectively. To test the hypothesis that the CRs are functionally distinct, eight CR residues (four in CR1: S612A, R615A, H622A, E624A and four in CR2: T1270A, P1275A, W1287A, G1291A), were targeted for Ala substitution based on sequence conservation and structural location of the residue, and their phenotypes investigated. Cellular levels of three of four CR1 mutants (S612A, R615A, E624A) were substantially lower than wild-type MRP1 (by 60%, 95%, 95%, respectively; p<0.05). Of the four CR2 mutants, only W1287A levels were markedly reduced (by 80%; p<0.05) and more conservatively substituted mutants (W1287F, W1287Y) remained <65% of wild-type MRP1levels (p<0.05). Analyses of apo bMrp1/MRP1 cryo-EM structures and models suggested that some of these mutation-sensitive residues might participate in stabilizing interactions. However, this idea was not supported by double exchange mutations which all failed to improve MRP1 levels. Immunofluorescence showed that while CR1-H622A localized to the plasma membrane like wild-type MRP1, the poorly expressing CR1 mutants (S612A, R615A, E624A) were retained intracellularly. In contrast, all four CR2 mutants, including the poorly expressing W1287A, localized exclusively to the plasma membrane. For CR mutants expressed at levels comparable to wild-type MRP1 (H622A, T1270A, P1275A, G1291A), only CR2-G1291A exhibited a substrate selective change in [3H]LTC4 transport (reduced by 40%; p<0.05) whereas [3H]E217βG and estrone sulphate transport were largely unaffected. Together these results support the hypothesis that CR1 and CR2 play important, but distinct, roles. CR1 may be more important for the plasma membrane expression of MRP1 whereas CR2 may play a larger role in transport activity. The mechanisms underlying these functional differences should be investigated further.