Role of proline 1150 in functional interactions between the membrane spanning domains and nucleotide binding domains of the MRP1 (ABCC1) transporter

The ATP-binding cassette multidrug resistance protein 1 (MRP1) mediates ATP-dependent cellular efflux of drugs and organic anions. We previously described a mutant, MRP1-Pro1150Ala, which exhibits selectively increased estradiol glucuronide (E217betaG) and methotrexate transport as well as altered interactions with ATP. We have now further explored the functional importance of MRP1-Pro1150 at the interface of transmembrane helix 15 and cytoplasmic loop 7 (CL7) by replacing it with Gly, Ile, Leu and Val. All four mutants exhibited a phenotype similar to MRP1-Pro1150Ala with respect to organic anion transport and [gamma32P]8N3ATP photolabeling. They also displayed very low levels of substrate-independent vanadate-induced trapping of [alpha32P]8N3ADP. To better understand the relationship between the altered nucleotide interactions and transport activity of these mutants, [alpha32P]8N3ADP trapping experiments were performed under different conditions. Unlike leukotriene C4, E217betaG decreased [alpha32P]8N3ADP trapping by both wild-type and mutant MRP1. [alpha32P]8N3ADP trapping by MRP1-Pro1150Ala could be increased by using Ni2+ instead of Mg2+, and by decreasing temperature; however, the transport properties of the mutant remained unchanged. We conclude that the reduced [alpha32P]8N3ADP trapping associated with loss of Pro1150, or the presence of E217betaG, is due to enhanced ADP release following ATP hydrolysis rather than a reduction in ATP hydrolysis itself. We hypothesize that loss of Pro1150 alters the role of CL7 as a coupling helix that mediates signaling between the nucleotide binding domains and some substrate binding sites in the membrane spanning domains of MRP1.