MRP1 belongs to subfamily "C" of the ABC transporter superfamily. The nucleotide-binding domains (NBDs) of the C family members are relatively divergent compared with many ABC proteins. They also differ in their ability to bind and hydrolyze ATP. In MRP1, NBD1 binds ATP with high affinity, whereas NBD2 is hydrolytically more active. Furthermore, ATP binding and/or hydrolysis by NBD2 of MRP1, but not NBD1, is required for MRP1 to shift from a high to low affinity substrate binding state. Little is known of the structural basis for these functional differences. One minor structural difference between NBDs is the presence of Asp COOH-terminal to the conserved core Walker B motif in NBD1, rather than the more commonly found Glu present in NBD2. We show that the presence of Asp or Glu following the Walker B motif profoundly affects the ability of the NBDs to bind, hydrolyze, and release nucleotide. An Asp to Glu mutation in NBD1 enhances its hydrolytic capacity and affinity for ADP but markedly decreases transport activity. In contrast, mutations that eliminate the negative charge of the Asp side chain have little effect. The decrease in transport caused by the Asp to Glu mutation in NBD1 is associated with an inability of MRP1 to shift from high to low affinity substrate binding states. In contrast, mutation of Glu to Asp markedly increases the affinity of NBD2 for ATP while decreasing its ability to hydrolyze ATP and to release ADP. This mutation eliminates transport activity but potentiates the conversion from a high to low affinity binding state in the presence of nucleotide. These observations are discussed in the context of catalytic models proposed for MRP1 and other ABC drug transport proteins.ATP-binding cassette (ABC) 1 transporters are ubiquitous transmembrane proteins that couple ATP hydrolysis to the energy-dependent transport of a wide variety of endogenous and exogenous molecules across biological membranes. Multidrug resistance protein (MRP) 1 (ABCC1) belongs to the "C" subfamily of the ABC superfamily and was discovered by virtue of its ability to cause multidrug resistance when overexpressed in a human small cell lung cancer cell line (1, 2). The MRP1 multidrug resistance phenotype is similar to that resulting from overexpression of P-glycoprotein (P-GP), and involves resistance to many relatively hydrophobic, natural product type, cytotoxic agents. However, unlike P-GP, MRP1 can also transport various structurally unrelated organic anionic conjugates, including: glutathione, glucuronide, and sulfate conjugates, such as the potent mediator of inflammation, cysteinyl leukotriene LTC 4 , the cholestatic glucuronide-conjugated estrogen E 2 17␤G, the sulfate conjugate estrone 3-sulfate, and the glutathione epoxide conjugate of the highly mutagenic aflatoxin B1 (3-10).Typically, the functional form of ABC proteins consists of two hydrophilic nucleotide-binding domains (NBDs) located at the cytoplasmic surface of the membrane and two hydrophobic transmembrane spanning domains (MSD) that are thought to for...

Cellular senescence is induced by stresses and results in a stable proliferation arrest accompanied by a pro-inflammatory secretome. Senescent cells accumulate during aging, promoting various age-related pathologies and limiting lifespan. The endoplasmic reticulum (ER) inositol 1,4,5-trisphosphate receptor, type 2 (ITPR2) calcium-release channel and calcium fluxes from the ER to the mitochondria are drivers of senescence in human cells. Here we show that Itpr2 knockout (KO) mice display improved aging such as increased lifespan, a better response to metabolic stress, less immunosenescence, as well as less liver steatosis and fibrosis. Cellular senescence, which is known to promote these alterations, is decreased in Itpr2 KO mice and Itpr2 KO embryo-derived cells. Interestingly, ablation of ITPR2 in vivo and in vitro decreases the number of contacts between the mitochondria and the ER and their forced contacts induce premature senescence. These findings shed light on the role of contacts and facilitated exchanges between the ER and the mitochondria through ITPR2 in regulating senescence and aging.