scholarly journals Reaction Dynamics of ATP Hydrolysis Catalyzed by P-Glycoprotein

Biochemistry ◽  
2014 ◽  
Vol 53 (6) ◽  
pp. 991-1000 ◽  
Author(s):  
Michele Scian ◽  
Mauro Acchione ◽  
Mavis Li ◽  
William M. Atkins
Keyword(s):  
Atp Hydrolysis ◽  
Reaction Dynamics ◽  
P Glycoprotein

scholarly journals ATP-dependent thermostabilization of human P-glycoprotein (ABCB1) is blocked by modulators

2019 ◽  
Vol 476 (24) ◽  
pp. 3737-3750 ◽  
Author(s):  
Sabrina Lusvarghi ◽  
Suresh V. Ambudkar
Keyword(s):  
Conformational Changes ◽  
Drug Transport ◽  
Atp Hydrolysis ◽  
Atp Binding ◽  
Dependent Manner ◽  
Deficient Mutant ◽  
Concentration Dependent Manner ◽  
Glycoprotein P ◽  
Binding Domains ◽  
P Glycoprotein

P-glycoprotein (P-gp), an ATP-binding cassette transporter associated with multidrug resistance in cancer cells, is capable of effluxing a number of xenobiotics as well as anticancer drugs. The transport of molecules through the transmembrane (TM) region of P-gp involves orchestrated conformational changes between inward-open and inward-closed forms, the details of which are still being worked out. Here, we assessed how the binding of transport substrates or modulators in the TM region and the binding of ATP to the nucleotide-binding domains (NBDs) affect the thermostability of P-gp in a membrane environment. P-gp stability after exposure at high temperatures (37–80°C) was assessed by measuring ATPase activity and loss of monomeric P-gp. Our results show that P-gp is significantly thermostabilized (>22°C higher IT50) by the binding of ATP under non-hydrolyzing conditions (in the absence of Mg2+). By using an ATP-binding-deficient mutant (Y401A) and a hydrolysis-deficient mutant (E556Q/E1201Q), we show that thermostabilization of P-gp requires binding of ATP to both NBDs and their dimerization. Additionally, we found that transport substrates do not affect the thermal stability of P-gp either in the absence or presence of ATP; in contrast, inhibitors of P-gp including tariquidar and zosuquidar prevent ATP-dependent thermostabilization in a concentration-dependent manner, by stabilizing the inward-open conformation. Altogether, our data suggest that modulators, which bind in the TM regions, inhibit ATP hydrolysis and drug transport by preventing the ATP-dependent dimerization of the NBDs of P-gp.

scholarly journals Transport of Alzheimer’s associated amyloid-β catalyzed by P-glycoprotein

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0250371
Author(s):  
James W. McCormick ◽  
Lauren Ammerman ◽  
Gang Chen ◽  
Pia D. Vogel ◽  
John G. Wise
Keyword(s):  
Cell Culture ◽  
Atp Hydrolysis ◽  
Amyloid Β ◽  
Md Simulations ◽  
Membrane Transporter ◽  
Biochemical Activity ◽  
Glycoprotein P ◽  
P Glycoprotein ◽  
Hydrolysis Activity

P-glycoprotein (P-gp) is a critical membrane transporter in the blood brain barrier (BBB) and is implicated in Alzheimer’s disease (AD). However, previous studies on the ability of P-gp to directly transport the Alzheimer’s associated amyloid-β (Aβ) protein have produced contradictory results. Here we use molecular dynamics (MD) simulations, transport substrate accumulation studies in cell culture, and biochemical activity assays to show that P-gp actively transports Aβ. We observed transport of Aβ40 and Aβ42 monomers by P-gp in explicit MD simulations of a putative catalytic cycle. In in vitro assays with P-gp overexpressing cells, we observed enhanced accumulation of fluorescently labeled Aβ42 in the presence of Tariquidar, a potent P-gp inhibitor. We also showed that Aβ42 stimulated the ATP hydrolysis activity of isolated P-gp in nanodiscs. Our findings expand the substrate profile of P-gp, and suggest that P-gp may contribute to the onset and progression of AD.

Drug efflux mediated by the human multidrug resistance P-glycoprotein is inhibited by cell swelling

1994 ◽  
Vol 107 (12) ◽  
pp. 3281-3290
Author(s):  
A. Sardini ◽  
G.M. Mintenig ◽  
M.A. Valverde ◽  
F.V. Sepulveda ◽  
D.R. Gill ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Chloride Channels ◽  
Drug Transport ◽  
Atp Hydrolysis ◽  
Cell Swelling ◽  
Drug Efflux ◽  
Fluorescent Substrate ◽  
Membrane Stretch ◽  
P Glycoprotein ◽  
In Cells

P-glycoprotein (P-gp), the product of the human multidrug resistance (MDR1) gene, confers multidrug resistance on cells by acting as an ATP-dependent drug transporter. A method using confocal microscopy was developed to measure the transport activity of P-gp from the rate of movement of doxorubicin, a fluorescent substrate of P-gp, across the membrane of a single cell. Recent work has shown that expression of P-gp enhances the activation of chloride channels in response to cell swelling, suggesting that membrane stretch might switch P-gp from a drug-transporting mode to a mode in which it activates chloride channels. In agreement with this idea, we find that cell swelling inhibits drug efflux in cells expressing P-gp but is without effect on the slower background efflux in cells not expressing P-gp and in cells transiently transfected with a mutated MDR1 in which the ATP hydrolysis sites had been inactivated. The identification of a novel means for inhibiting P-gp-mediated drug transport may have implications for the reversal of multidrug resistance during chemotherapy.

scholarly journals Reversing the direction of drug transport mediated by the human multidrug transporter P-glycoprotein

2020 ◽  
Vol 117 (47) ◽  
pp. 29609-29617
Author(s):  
Andaleeb Sajid ◽  
Sabrina Lusvarghi ◽  
Megumi Murakami ◽  
Eduardo E. Chufan ◽  
Biebele Abel ◽  
...  
Keyword(s):  
Drug Transport ◽  
Atp Hydrolysis ◽  
Binding Pocket ◽  
Drug Binding ◽  
Drug Uptake ◽  
Cancer Drug ◽  
Binding Domains ◽  
Cancer Drug Resistance ◽  
P Glycoprotein ◽  
Cell Transforming

P-glycoprotein (P-gp), also known as ABCB1, is a cell membrane transporter that mediates the efflux of chemically dissimilar amphipathic drugs and confers resistance to chemotherapy in most cancers. Homologous transmembrane helices (TMHs) 6 and 12 of human P-gp connect the transmembrane domains with its nucleotide-binding domains, and several residues in these TMHs contribute to the drug-binding pocket. To investigate the role of these helices in the transport function of P-gp, we substituted a group of 14 conserved residues (seven in both TMHs 6 and 12) with alanine and generated a mutant termed 14A. Although the 14A mutant lost the ability to pump most of the substrates tested out of cancer cells, surprisingly, it acquired a new function. It was able to import four substrates, including rhodamine 123 (Rh123) and the taxol derivative flutax-1. Similar to the efflux function of wild-type P-gp, we found that uptake by the 14A mutant is ATP hydrolysis-, substrate concentration-, and time-dependent. Consistent with the uptake function, the mutant P-gp also hypersensitizes HeLa cells to Rh123 by 2- to 2.5-fold. Further mutagenesis identified residues from both TMHs 6 and 12 that synergistically form a switch in the central region of the two helices that governs whether a given substrate is pumped out of or into the cell. Transforming P-gp or an ABC drug exporter from an efflux transporter into a drug uptake pump would constitute a paradigm shift in efforts to overcome cancer drug resistance.

scholarly journals Characterization of the Catalytic Cycle of ATP Hydrolysis by Human P-glycoprotein

2001 ◽  
Vol 276 (15) ◽  
pp. 11653-11661 ◽  
Author(s):  
Zuben E. Sauna ◽  
Suresh V. Ambudkar
Keyword(s):  
Multidrug Resistance ◽  
Functional Outcomes ◽  
Atp Hydrolysis ◽  
Substrate Binding ◽  
Nucleotide Binding ◽  
Catalytic Cycle ◽  
Nucleotide Binding Sites ◽  
P Glycoprotein ◽  
Random Manner

P-glycoprotein (Pgp) is a plasma membrane protein whose overexpression confers multidrug resistance to tumor cells by extruding amphipathic natural product cytotoxic drugs using the energy of ATP. An elucidation of the catalytic cycle of Pgp would help design rational strategies to combat multidrug resistance and to further our understanding of the mechanism of ATP-binding cassette transporters. We have recently reported (Sauna, Z. E., and Ambudkar, S. V. (2000)Proc. Natl. Acad. Sci. U. S. A.97, 2515–2520) that there are two independent ATP hydrolysis events in a single catalytic cycle of Pgp. In this study we exploit the vanadate (Vi)-induced transition state conformation of Pgp (Pgp·ADP·Vi) to address the question of what are the effects of ATP hydrolysis on the nucleotide-binding site. We find that at the end of the first hydrolysis event there is a drastic decrease in the affinity of nucleotide for Pgp coincident with decreased substrate binding. Release of occluded dinucleotide is adequate for the next hydrolysis event to occur but is not sufficient for the recovery of substrate binding. Whereas the two hydrolysis events have different functional outcomesvis à visthe substrate, they show comparablet12for both incorporation and release of nucleotide, and the affinities for [α-32P]8-azido-ATP during Vi-induced trapping are identical. In addition, the incorporation of [α-32P]8-azido-ADP in two ATP sites during both hydrolysis events is also similar. These data demonstrate that during individual hydrolysis events, the ATP sites are recruited in a random manner, and only one site is utilized at any given time because of the conformational change in the catalytic site that drastically reduces the affinity of the second ATP site for nucleotide binding. In aggregate, these findings provide an explanation for the alternate catalysis of ATP hydrolysis and offer a mechanistic framework to elucidate events at both the substrate- and nucleotide-binding sites in the catalytic cycle of Pgp.

scholarly journals In vivo FRET analyses reveal a role of ATP hydrolysis–associated conformational changes in human P-glycoprotein

2020 ◽  
Vol 295 (15) ◽  
pp. 5002-5011 ◽  
Author(s):  
Ryota Futamata ◽  
Fumihiko Ogasawara ◽  
Takafumi Ichikawa ◽  
Atsushi Kodan ◽  
Yasuhisa Kimura ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Drug Transport ◽  
Atp Hydrolysis ◽  
Hek293 Cells ◽  
Atp Binding ◽  
Binding Domains ◽  
P Glycoprotein ◽  
Atp Binding And Hydrolysis

P-glycoprotein (P-gp; also known as MDR1 or ABCB1) is an ATP-driven multidrug transporter that extrudes various hydrophobic toxic compounds to the extracellular space. P-gp consists of two transmembrane domains (TMDs) that form the substrate translocation pathway and two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. At least two P-gp states are required for transport. In the inward-facing (pre-drug transport) conformation, the two NBDs are separated, and the two TMDs are open to the intracellular side; in the outward-facing (post-drug transport) conformation, the NBDs are dimerized, and the TMDs are slightly open to the extracellular side. ATP binding and hydrolysis cause conformational changes between the inward-facing and the outward-facing conformations, and these changes help translocate substrates across the membrane. However, how ATP hydrolysis is coupled to these conformational changes remains unclear. In this study, we used a new FRET sensor that detects conformational changes in P-gp to investigate the role of ATP binding and hydrolysis during the conformational changes of human P-gp in living HEK293 cells. We show that ATP binding causes the conformational change to the outward-facing state and that ATP hydrolysis and subsequent release of γ-phosphate from both NBDs allow the outward-facing state to return to the original inward-facing state. The findings of our study underscore the utility of using FRET analysis in living cells to elucidate the function of membrane proteins such as multidrug transporters.

Structural Flexibility of the Linker Region of Human P-Glycoprotein Permits ATP Hydrolysis and Drug Transport†

Biochemistry ◽  
1998 ◽  
Vol 37 (39) ◽  
pp. 13660-13673 ◽  
Author(s):  
Christine A. Hrycyna ◽  
Lisa E. Airan ◽  
Ursula A. Germann ◽  
Suresh V. Ambudkar ◽  
Ira Pastan ◽  
...  
Keyword(s):  
Drug Transport ◽  
Atp Hydrolysis ◽  
Structural Flexibility ◽  
Linker Region ◽  
P Glycoprotein
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