Benzodiazepine-mediated structural changes in the multidrug transporter P-glycoprotein: an intrinsic fluorescence quenching analysis.
P-glycoprotein expressed in Pichia pastoris was used to study the drug binding sites of different benzodiazepines. The effect of bromazepam, chlordiazepoxide, diazepam and flurazepam on P-glycoprotein structure was investigated by measuring the intrinsic fluorescence of the transporter tryptophan residues. Purified mouse mdr1a transporter in mixed micelles of 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid and 1,2-dimiristoyl-sn-glycerol-3-phosphocholine emitted fluorescence at 340 nm indicative of the fluorophores in a relatively apolar environment. Acrylamide and iodide ion were used as collisional quenchers toward distinct regions of the transporter, the protein and the interface protein-surface, respectively. Binding of ATP induced conformational changes at the protein surface level in accordance with the location of the nucleotide binding sites. Bromazepam interaction with the transporter was located at the protein-surface interface, diazepam at the membrane region and chlordiazepoxide at the protein surface. Only the flurazepam interaction site was not detected by the quenchers used. All benzodiazepines were able to elicit reorientation of the protein fluorophores on the P-glycoprotein-ATP complex.
Lima
Sofia A C
SA
Rede de Química e Tecnologia (REQUIMTE), Departamento de Química, Faculdade Ciências, Universidade do Porto, Porto, Portugal.
Cordeiro-da-Silva
Anabela
A
de Castro
Baltazar
B
Gameiro
Paula
P
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000737:chemistry* / Q000235:genetics / Q000493:pharmacokinetics*; D020106:Acrylamide / Q000493:pharmacokinetics; D000255:Adenosine Triphosphate / Q000493:pharmacokinetics; D000818:Animals; D014151:Anti-Anxiety Agents / Q000493:pharmacokinetics / Q000494:pharmacology; D001569:Benzodiazepines / Q000493:pharmacokinetics / Q000494:pharmacology*; D005479:Flurazepam / Q000031:analogs & derivatives / Q000493:pharmacokinetics; D007454:Iodides / Q000493:pharmacokinetics; D051379:Mice; D008823:Micelles; D010843:Pichia / Q000235:genetics; D011487:Protein Conformation / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000493:pharmacokinetics
D018435:ATP Binding Cassette Transporter, Subfamily B; D014151:Anti-Anxiety Agents; D007454:Iodides; D008823:Micelles; D001569:Benzodiazepines; D020106:Acrylamide; C037324:7-chloro-5-(2'-fluorophenyl)-2,3-dihydro-2-oxo-1H-1,4-benzodiazepine-1-acetaldehyde; D014364:Tryptophan; D000255:Adenosine Triphosphate; C095122:multidrug resistance protein 3; D005479:Flurazepam
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