ABCMdb

PMID: 18058211

Sauna ZE, Kim IW, Ambudkar SV
Genomics and the mechanism of P-glycoprotein (ABCB1).
J Bioenerg Biomembr. 2007 Dec;39(5-6):481-7., [PubMed]

Sentences

No. Mutations Sentence Comment

77 ABCB1 p.Tyr401Trp ABCB1 p.Tyr401Trp ABCB1 p.Tyr1044Trp ABCB1 p.Tyr1044Phe ABCB1 p.Tyr401Phe Although the mutants Y401F, Y401W, Y1044F and Y1044W transported fluorescent substrates similar to wild-type protein, there was a significant decrease (2-2.5 fold) in the affinity of the nucleotide for the Y401W mutant P-gp. Login to comment 78 ABCB1 p.Tyr401Leu ABCB1 p.Tyr401Cys Y401C and Y401L mutant P-gps showed partial (30-50%) transport function. Login to comment 79 ABCB1 p.Tyr1044Ala ABCB1 p.Tyr1044Ala ABCB1 p.Tyr401Ala However, transport was completely abolished in Y401A, Y1044A, and Y401/Y1044A mutant P-gps and there was no detectable binding of 8-azido[α-32 P]ATP or TNP-ATP, Vi-induced trapping of nucleotide, and ATP hydrolysis in these mutant P-gps. Login to comment 108 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln In addition, we characterized the double mutant (E556Q/E1201Q) where the glutamates in both NBDs were simultaneously mutated (Sauna et al. 2002). Login to comment 110 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln Additional studies from our laboratory (using the E556Q/E1201Q mutant of human P-gp) demonstrated that the occlusion of ATP is strongly temperature-dependent and that the occluded nucleotide conformation shows reduced binding of the transport-substrate [125 I]iodoarylazidoprazosin ([125 I]IAAP) (Sauna et al. 2006). Login to comment 115 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln Double mutants of the conserved glutamate, however, are hydrolysis deficient and cannot generate ADP and occlude ATP in a pre-hydrolysis intermediate, P-gp(E556Q/E1201Q)·MgATP (see Fig. 2) and represent the enzyme-substrate (E·S) state. Login to comment 117 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln Both the Senior group and we have demonstrated that unlike the ATP-driven dimers obtained with isolated NBDs, which exhibit a stoichiometry of 2 mol ATP per dimer (see for example, Smith et al. 2002), P-gp shows a maximal P-gp + MgATP P-gp•MgATP P-gp•MgADP•Pi P-gp•MgADP•Vi Vi Pi Wild-type Mutant E556/1201Q P-gp + MgATP P-gp•MgATP P-gp•MgADP•Pi E•P E•S ATP in exchangeable form Trapped ADP in non-exchangeable form "Vi-trapped state" Occluded ATP in non-exchangeable form "Occluded-nucleotide conformation" Fig. 2 Schematic showing the ATPase reaction in wild-type and mutant (E556Q/E1201Q) P-gps. Login to comment 119 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln The E556Q/E1201Q mutant P-gp shows minimal ATP hydrolysis and "occludes" MgATP in a non-exchangeable form. Login to comment 132 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln Our studies with the P-gp mutant (E556Q/E1201Q) also allow us to address the question of what role, if any, the occluded nucleotide conformation plays in the transport pathway. Login to comment 136 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln The formation of the occluded nucleotide conformation of the mutant human P-gp, E556Q/E1201Q, has an activation energy of ~60 kJ/mol and the activation energies for nucleotide trapping and decrease in drug binding are equivalent (Sauna et al. 2006). Login to comment 139 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln Conclusions A substantive body of work now exists that suggests that SNPs in the human MDR1 gene (both synonymous and non-synonymous) can affect protein levels as well as 0 5 10 15 20 25 30 35 40 0 50 100 150 200 250 0.00 0.05 0.10 0.15 0.20 0.25 0.30 [125I]IAAPincorporated (arbitraryunits) [α-32P]ATPincorporated (arbitraryunits) ATP ATP NC NC ATP ATP Temperature, oC Fig. 3 Occlusion of ATP in the mutant P-gp (E556Q/E1201Q) is accompanied by a high-affinity to low affinity switch at the TMDs. Login to comment 140 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln The E556Q/E1201Q mutant P-gp occludes [α-32 P]ATP in a temperature-dependent manner (black squares). Login to comment