The P-glycoprotein multidrug transporter

Pgp (P-glycoprotein) (ABCB1) is an ATP-powered efflux pump which can transport hundreds of structurally unrelated hydrophobic amphipathic compounds, including therapeutic drugs, peptides and lipid-like compounds. This 170 kDa polypeptide plays a crucial physiological role in protecting tissues from toxic xenobiotics and endogenous metabolites, and also affects the uptake and distribution of many clinically important drugs. It forms a major component of the blood–brain barrier and restricts the uptake of drugs from the intestine. The protein is also expressed in many human cancers, where it probably contributes to resistance to chemotherapy treatment. Many chemical modulators have been identified that block the action of Pgp, and may have clinical applications in improving drug delivery and treating cancer. Pgp substrates are generally lipid-soluble, and partition into the membrane before the transporter expels them into the aqueous phase, much like a ‘hydrophobic vacuum cleaner’. The transporter may also act as a ‘flippase’, moving its substrates from the inner to the outer membrane leaflet. An X-ray crystal structure shows that drugs interact with Pgp within the transmembrane regions by fitting into a large flexible binding pocket, which can accommodate several substrate molecules simultaneously. The nucleotide-binding domains of Pgp appear to hydrolyse ATP in an alternating manner; however, it is still not clear whether transport is driven by ATP hydrolysis or ATP binding. Details of the steps involved in the drug-transport process, and how it is coupled to ATP hydrolysis, remain the object of intensive study.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2016270117 ◽

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.

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Shedding light on drug transport: structure and function of the P-glycoprotein multidrug transporter (ABCB1)This paper is one of a selection of papers published in this Special Issue, entitled CSBMCB — Membrane Proteins in Health and Disease.

Biochemistry and Cell Biology ◽

10.1139/o06-199 ◽

2006 ◽

Vol 84 (6) ◽

pp. 979-992 ◽

Cited By ~ 70

Author(s):

Frances J. Sharom

Keyword(s):

Lipid Bilayer ◽

Drug Transport ◽

Atp Hydrolysis ◽

Physiological Role ◽

Structure And Function ◽

Drug Uptake ◽

Spectroscopic Techniques ◽

Binding Domains ◽

P Glycoprotein ◽

And Function

P-glycoprotein (Pgp; ABCB1), a member of the ATP-binding cassette (ABC) superfamily, exports structurally diverse hydrophobic compounds from the cell, driven by ATP hydrolysis. Pgp expression has been linked to the efflux of chemotherapeutic drugs in human cancers, leading to multidrug resistance (MDR). The protein also plays an important physiological role in limiting drug uptake in the gut and entry into the brain. Substrates partition into the lipid bilayer before interacting with Pgp, which has been proposed to function as a hydrophobic vacuum cleaner. Low- and medium-resolution structural models of Pgp suggest that the 2 nucleotide-binding domains are closely associated to form a nucleotide sandwich dimer. Pgp is an outwardly directed flippase for fluorescent phospholipid and glycosphingolipid derivatives, which suggests that it may also translocate drug molecules from the inner to the outer membrane leaflet. The ATPase catalytic cycle of the protein is thought to proceed via an alternating site mechanism, although the details are not understood. The lipid bilayer plays an important role in Pgp function, and may regulate both the binding and transport of drugs. This review focuses on the structure and function of Pgp, and highlights the importance of fluorescence spectroscopic techniques in exploring the molecular details of this enigmatic transporter.

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

Biochemical Journal ◽

10.1042/bcj20190736 ◽

2019 ◽

Vol 476 (24) ◽

pp. 3737-3750 ◽

Cited By ~ 5

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.

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

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.012042 ◽

2020 ◽

Vol 295 (15) ◽

pp. 5002-5011 ◽

Cited By ~ 4

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.

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Acute pain alters P-glycoprotein-containing protein complexes in rat cerebral microvessels: Implications for P-glycoprotein trafficking

Journal of Cerebral Blood Flow & Metabolism ◽

10.1177/0271678x18803623 ◽

2018 ◽

Vol 38 (12) ◽

pp. 2209-2222 ◽

Cited By ~ 6

Author(s):

Margaret E Tome ◽

Chelsea K Jarvis ◽

Charles P Schaefer ◽

Leigh M Jacobs ◽

Joseph M Herndon ◽

...

Keyword(s):

Efflux Pump ◽

Protein Complexes ◽

Activity Increase ◽

Cerebral Microvessels ◽

Cns Drug Delivery ◽

Therapeutic Drugs ◽

P Glycoprotein ◽

Cell Plasma ◽

Rat Paw ◽

Major Drug

P-glycoprotein (PgP) is the major drug efflux pump in human cerebral microvessels. PgP prevents pathogens, toxins and therapeutic drugs from entering the CNS. Understanding the molecular regulation of PgP activity will suggest novel mechanisms to improve CNS drug delivery. Previously, we found that during peripheral inflammatory pain (PIP) (3 h after λ carrageenan injection in the rat paw), PgP traffics to the cortical microvessel endothelial cell plasma membrane concomitant with increased PgP activity. In the current study, we measured the changes in composition of PgP-containing protein complexes after PIP in rat microvessel isolates. We found that a portion of the PgP is contained in a multi-protein complex that also contains the caveolar proteins CAV1, SDPR, PTRF and PRKCDBP. With PIP, total CAV1 bound to PgP was unchanged; however, phosphorylated CAV1 (Y14P-CAV1) in the complex increased. There were few PgP/CAV1 complexes relative to total PgP and CAV1 in the microvessels suggesting CAV1 bound to PgP is unlikely to affect total PgP activity. However, both PgP and CAV1 trafficked away from the nucleus in response to PIP. These data suggest that P-CAV1 bound to PgP potentially regulates PgP trafficking and contributes to the acute PgP activity increase after a PIP stimulus.

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Mg2+-dependent ATP occlusion at the first nucleotide-binding domain (NBD1) of CFTR does not require the second (NBD2)

Biochemical Journal ◽

10.1042/bj20081068 ◽

2008 ◽

Vol 416 (1) ◽

pp. 129-136 ◽

Cited By ~ 14

Author(s):

Luba Aleksandrov ◽

Andrei Aleksandrov ◽

John R. Riordan

Keyword(s):

Atp Hydrolysis ◽

Nucleotide Binding ◽

Binding Pocket ◽

Atp Binding ◽

Transmembrane Conductance Regulator ◽

Transmembrane Conductance ◽

Bivalent Cation ◽

Binding Domains ◽

Rapid Hydrolysis ◽

Cystic Fibrosis Transmembrane

ATP binding to the first and second NBDs (nucleotide-binding domains) of CFTR (cystic fibrosis transmembrane conductance regulator) are bivalent-cation-independent and -dependent steps respectively [Aleksandrov, Aleksandrov, Chang and Riordan (2002) J. Biol. Chem. 277, 15419–15425]. Subsequent to the initial binding, Mg2+ drives rapid hydrolysis at the second site, while promoting non-exchangeable trapping of the nucleotide at the first site. This occlusion at the first site of functional wild-type CFTR is somewhat similar to that which occurs when the catalytic glutamate residues in both of the hydrolytic sites of P-glycoprotein are mutated, which has been proposed to be the result of dimerization of the two NBDs and represents a transient intermediate formed during ATP hydrolysis [Tombline and Senior (2005) J. Bioenerg. Biomembr. 37, 497–500]. To test the possible relevance of this interpretation to CFTR, we have now characterized the process by which NBD1 occludes [32P]N3ATP (8-azido-ATP) and [32P]N3ADP (8-azido-ADP). Only N3ATP, but not N3ADP, can be bound initially at NBD1 in the absence of Mg2+. Despite the lack of a requirement for Mg2+ for ATP binding, retention of the NTP at 37 °C was dependent on the cation. However, at reduced temperature (4 °C), N3ATP remains locked in the binding pocket with virtually no reduction over a 1 h period, even in the absence of Mg2+. Occlusion occurred identically in a ΔNBD2 construct, but not in purified recombinant NBD1, indicating that the process is dependent on the influence of regions of CFTR in addition to NBD1, but not NBD2.

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Structure of a zosuquidar and UIC2-bound human-mouse chimeric ABCB1

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1717044115 ◽

2018 ◽

Vol 115 (9) ◽

pp. E1973-E1982 ◽

Cited By ~ 67

Author(s):

Amer Alam ◽

Raphael Küng ◽

Julia Kowal ◽

Robert A. McLeod ◽

Nina Tremp ◽

...

Keyword(s):

Lipid Membrane ◽

Binding Pocket ◽

Conformational Epitope ◽

Multidrug Transporter ◽

Inhibitory Antibody ◽

Binding Domains ◽

Atp Binding Cassette Transporter ◽

P Glycoprotein ◽

Inhibitory Agents ◽

Human Specific

The multidrug transporter ABCB1 (P-glycoprotein) is an ATP-binding cassette transporter that has a key role in protecting tissues from toxic insult and contributes to multidrug extrusion from cancer cells. Here, we report the near-atomic resolution cryo-EM structure of nucleotide-free ABCB1 trapped by an engineered disulfide cross-link between the nucleotide-binding domains (NBDs) and bound to the antigen-binding fragment of the human-specific inhibitory antibody UIC2 and to the third-generation ABCB1 inhibitor zosuquidar. Our structure reveals the transporter in an occluded conformation with a central, enclosed, inhibitor-binding pocket lined by residues from all transmembrane (TM) helices of ABCB1. The pocket spans almost the entire width of the lipid membrane and is occupied exclusively by two closely interacting zosuquidar molecules. The external, conformational epitope facilitating UIC2 binding is also visualized, providing a basis for its inhibition of substrate efflux. Additional cryo-EM structures suggest concerted movement of TM helices from both halves of the transporters associated with closing the NBD gap, as well as zosuquidar binding. Our results define distinct recognition interfaces of ABCB1 inhibitory agents, which may be exploited for therapeutic purposes.

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

Journal of Cell Science ◽

10.1242/jcs.107.12.3281 ◽

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.

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Modeling of P-glycoprotein-involved epithelial drug transport in MDCK cells

AJP Renal Physiology ◽

10.1152/ajprenal.1999.277.1.f84 ◽

1999 ◽

Vol 277 (1) ◽

pp. F84-F96 ◽

Cited By ~ 6

Author(s):

Shinya Ito ◽

Cindy Woodland ◽

Balázs Sarkadi ◽

Guido Hockmann ◽

Scott E. Walker ◽

...

Keyword(s):

Drug Transport ◽

Diffusion Processes ◽

Efflux Pump ◽

Mdck Cells ◽

Basolateral Membrane ◽

Drug Efflux ◽

Drug Transfer ◽

P Glycoprotein ◽

Drug Efflux Pump ◽

Apical Membranes

P-glycoprotein (P-gp) on the apical membranes of epithelial cells is known as a drug efflux pump. However, unclear is its integral quantitative role in the overall epithelial drug transfer, which also involves distinct diffusion processes in parallel and sequence. We used a simple three-compartment model to obtain kinetic parameters of each drug transfer mechanism, which can quantitatively describe the transport time courses of P-gp substrates, digoxin and vinblastine, across P-gp-expressing MDCK cell monolayers grown on permeable filters. Our results show that the model, which assumes a functionally single drug efflux pump in the apical membrane with diffusion across two membranes and intercellular junctions, is the least complex model with which to quantitatively reproduce the characteristics of the data. Interestingly, the model predicts that the MDCK apical membranes are less diffusion permeable than the basolateral membrane for both drugs and that the distribution volume of vinblastine is 10-fold higher than that of digoxin. Additional experiments verified these model predictions. The modeling approach is feasible to quantitatively describe overall kinetic picture of epithelial drug transport. Further model refinement is necessary to incorporate other modes of drug transport such as transcytosis. Also, whether P-gp solely accounts for the pump function in this model awaits more studies.

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