scholarly journals Studies of Human MDR1-MDR2 Chimeras Demonstrate the Functional Exchangeability of a Major Transmembrane Segment of the Multidrug Transporter and Phosphatidylcholine Flippase

1999 ◽  
Vol 19 (2) ◽  
pp. 1450-1459 ◽  
Author(s):  
Yi Zhou ◽  
Michael M. Gottesman ◽  
Ira Pastan
Keyword(s):  
Functional Relationship ◽  
Transmembrane Domain ◽  
Site Directed Mutagenesis ◽  
Multidrug Transporter ◽  
Transmembrane Segment ◽  
Glycoprotein P ◽  
Hydrophobic Compounds ◽  
Functional Differences ◽  
P Glycoprotein ◽  
Human Mdr1

ABSTRACT P-glycoprotein (P-gp), encoded by the MDR1 gene, is a plasma membrane transporter which effluxes a large number of structurally nonrelated hydrophobic compounds. The molecular basis of the broad substrate recognition of P-gp is not well understood. Despite the 78% amino acid sequence identity of the MDR1 andMDR2 transporter, MDR2, which has been identified as a phosphatidylcholine transporter, does not transport most MDR1 substrates. The structural and functional differences between MDR1 and MDR2 provide an opportunity to identify the residues essential for the broad substrate spectrum of MDR1. Using an approach involving exchanging homologous segments of MDR1 and MDR2 and site-directed mutagenesis, we have demonstrated that MDR1 residues Q330, V331, and L332 in transmembrane domain 6 are sufficient to allow an MDR2 backbone in the N-terminal half of P-gp to transport several MDR1 substrates, including bisantrene, colchicine, vinblastine, and rhodamine-123. These studies help define some residues important for multidrug transport and indicate the close functional relationship between the multidrug transporter (MDR1) and phosphatidylcholine flippase (MDR2).

scholarly journals Characterization and tissue localization of zebrafish homologs of the human ABCB1 multidrug transporter

2021 ◽  
Author(s):  
Robert W. Robey ◽  
Andrea N. Robinson ◽  
Fatima Ali-Rahmani ◽  
Lyn M. Huff ◽  
Sabrina Lusvarghi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Multidrug Transporter ◽  
Glycoprotein P ◽  
Tissue Localization ◽  
Brain Vasculature ◽  
P Glycoprotein ◽  
Capillary Endothelial Cells ◽  
The Brain ◽  
Viable Model

ABSTRACTGiven its similarities with mammalian systems, the zebrafish has emerged as a potential model to study the blood-brain barrier (BBB). Capillary endothelial cells at the human BBB express high levels of P-glycoprotein (P-gp, encoded by the ABCB1 gene) and ABCG2 (encoded by the ABCG2 gene). However, little information has been available about ATP-binding cassette transporters expressed at the zebrafish BBB. In this study, we focus on the characterization and tissue localization of two genes that are similar to human ABCB1, zebrafish abcb4 and abcb5. Cytotoxicity assays with stably-transfected cell lines revealed that zebrafish Abcb5 cannot efficiently transport the substrates doxorubicin and mitoxantrone compared to human P-gp and zebrafish Abcb4. Additionally, zebrafish Abcb5 did not transport the fluorescent probes BODIPY-ethylenediamine or LDS 751, while they were readily transported by Abcb4 and P-gp. A high-throughput screen conducted with 90 human P-gp substrates confirmed that zebrafish Abcb4 has overlapping substrate specificity with P-gp. Basal ATPase activity of zebrafish Abcb4 and Abcb5 was comparable to that of human P-gp. In the brain vasculature, RNAscope probes to detect abcb4 colocalized with staining by the P-gp antibody C219, while abcb5 was not detected. Zebrafish abcb4 also colocalized with claudin-5 expression in brain endothelial cells. Abcb4 and Abcb5 had different tissue localizations in multiple zebrafish tissues, consistent with different functions. The data suggest that zebrafish Abcb4 most closely phenocopies P-gp and that the zebrafish may be a viable model to study the role of the multidrug transporter P-gp at the BBB.

scholarly journals Modulation of drug-stimulated ATPase activity of human MDR1/P-glycoprotein by cholesterol

2006 ◽  
Vol 401 (2) ◽  
pp. 597-605 ◽  
Author(s):  
Yasuhisa Kimura ◽  
Noriyuki Kioka ◽  
Hiroaki Kato ◽  
Michinori Matsuo ◽  
Kazumitsu Ueda
Keyword(s):  
Atpase Activity ◽  
Molecular Mass ◽  
Binding Site ◽  
Drug Binding ◽  
Soluble Form ◽  
Strongly Correlated ◽  
Drug Binding Site ◽  
Hydrophobic Compounds ◽  
P Glycoprotein ◽  
Human Mdr1

MDR1 (multidrug resistance 1)/P-glycoprotein is an ATP-driven transporter which excretes a wide variety of structurally unrelated hydrophobic compounds from cells. It is suggested that drugs bind to MDR1 directly from the lipid bilayer and that cholesterol in the bilayer also interacts with MDR1. However, the effects of cholesterol on drug–MDR1 interactions are still unclear. To examine these effects, human MDR1 was expressed in insect cells and purified. The purified MDR1 protein was reconstituted in proteoliposomes containing various concentrations of cholesterol and enzymatic parameters of drug-stimulated ATPase were compared. Cholesterol directly binds to purified MDR1 in a detergent soluble form and the effects of cholesterol on drug-stimulated ATPase activity differ from one drug to another. The effects of cholesterol on Km values of drug-stimulated ATPase activity were strongly correlated with the molecular mass of that drug. Cholesterol increases the binding affinity of small drugs (molecular mass <500 Da), but does not affect that of drugs with a molecular mass of between 800 and 900 Da, and suppresses that of valinomycin (molecular mass >1000 Da). Vmax values for rhodamine B and paclitaxel are also increased by cholesterol, suggesting that cholesterol affects turnover as well as drug binding. Paclitaxel-stimulated ATPase activity of MDR1 is enhanced in the presence of stigmasterol, sitosterol and campesterol, as well as cholesterol, but not ergosterol. These results suggest that the drug-binding site of MDR1 may best fit drugs with a molecular mass of between 800 and 900 Da, and that cholesterol may support the recognition of smaller drugs by adjusting the drug-binding site and play an important role in the function of MDR1.

scholarly journals MEMANTINE, AS A P-GLYCOPROTEIN EXPRESSION MODULATOR, ENHANCES LEVETIRACETAM THERAPEUTIC RESPONSE IN EPILEPTIC PATIENTS

2020 ◽  
pp. 104-108
Author(s):  
SAHAR AHMED HARBY ◽  
RASHA A NASSRA ◽  
JAIDAA F MEKKY ◽  
SAMIA M ALI ◽  
CHERINE A ISMAIL
Keyword(s):  
Therapeutic Response ◽  
Seizure Control ◽  
Multidrug Transporter ◽  
Potential Therapeutic Target ◽  
Glycoprotein P ◽  
P Glycoprotein ◽  
Epileptic Patients ◽  
Patients With Epilepsy ◽  
Transporter Expression

Objective: The principal aim of the present study was to assess the importance of multidrug transporter; P-glycoprotein (P-gp) as a potential therapeutic target in patients with epilepsy. Can P-gp transporter expression modulation by memantine add to the standard antiepileptic drugs (AEDs) response? Methods: A cohort of 56 epilepsy patients was included in a 4 monthly visits prospective study. Patients were on levetiracetam (LEV) 1000 mg/ day alone or combined with other AEDs. They were randomly assigned into two groups; LEV only group including LEV-treated patients and LEV + memantine group including patients on LEV with add-on oral memantine 10 mg/day until the end of the study. During monthly follow-up visits, therapeutic responses were evaluated for each patient by recording the monthly seizures frequency. Blood samples were drawn from every patient twice (on the first and last visits) for assessment of P-gp mRNA expression level. Results: Fifty patients completed the study. At the end of 4th month, LEV only group showed a non-significant decrease in P-gp expression and seizure frequency compared to the 1st month, whereas, in LEV + memantine group, P-gp expression was significantly reduced and associated with significant seizure control. Conclusion: Memantine by hindering P-gp overexpression was apt to enhance LEV efficacy and exhibit a better seizure control.

Alteration of Substrate Specificity by Mutations at the His61Position in Predicted Transmembrane Domain 1 of Human MDR1/P-Glycoprotein†

Biochemistry ◽  
1997 ◽  
Vol 36 (29) ◽  
pp. 8883-8889 ◽  
Author(s):  
Yoshitomo Taguchi ◽  
Kouichi Kino ◽  
Masaki Morishima ◽  
Tohru Komano ◽  
Susan E. Kane ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Transmembrane Domain ◽  
P Glycoprotein ◽  
Human Mdr1

scholarly journals Active participation of membrane lipids in inhibition of multidrug transporter P-glycoprotein

2021 ◽  
Author(s):  
Karan Kapoor ◽  
Shashank Pant ◽  
Emad Tajkhorshid
Keyword(s):  
Membrane Lipids ◽  
Active Participation ◽  
Multidrug Transporter ◽  
Glycoprotein P ◽  
P Glycoprotein

Lipid invasion of P-glycoprotein, enhanced by binding of an inhibitor.

scholarly journals The reconstituted P-glycoprotein multidrug transporter is a flippase for glucosylceramide and other simple glycosphingolipids

2005 ◽  
Vol 389 (2) ◽  
pp. 517-526 ◽  
Author(s):  
Paul D. W. Eckford ◽  
Frances J. Sharom
Keyword(s):  
Efflux Pump ◽  
Drug Binding ◽  
Sugar Residue ◽  
Multidrug Transporter ◽  
Membrane Phospholipids ◽  
Binding Domains ◽  
Hydrophobic Compounds ◽  
P Glycoprotein ◽  
Fluorescence Quenching Technique ◽  
Hydrolysis Of

The Pgp (P-glycoprotein) multidrug transporter, which is linked to multidrug resistance in human cancers, functions as an efflux pump for non-polar drugs, powered by the hydrolysis of ATP at its nucleotide binding domains. The drug binding sites of Pgp appear to be located within the cytoplasmic leaflet of the membrane bilayer, suggesting that Pgp may function as a ‘flippase’ for hydrophobic compounds. Pgp has been shown to translocate fluorescent phospholipids, and it has been suggested that it may also interact with GlcCer (glucosylceramide). Here we use a dithionite fluorescence quenching technique to show that reconstituted Pgp can flip several NBD (nitrobenzo-2-oxa-1,3-diazole)-labelled simple glycosphingolipids, including NBD–GlcCer, from one leaflet of the bilayer to the other in an ATP-dependent, vanadate-sensitive fashion. The rate of NBD–GlcCer flipping was similar to that observed for NBD-labelled PC (phosphatidylcholine). NBD–GlcCer flipping was inhibited in a concentration-dependent, saturable fashion by various Pgp substrates and modulators, and inhibition correlated well with the Kd for binding to the protein. The addition of a second sugar to the headgroup of the glycolipid to form NBD–lactosylceramide drastically reduced the rate of flipping compared with NBD–PC, probably because of the increased size and polarity contributed by the additional sugar residue. We conclude that Pgp functions as a broad-specificity outwardly-directed flippase for simple glycosphingolipids and membrane phospholipids.

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