Sensitivity of P-glycoprotein tryptophan residues to benzodiazepines and ATP interaction.
Plasma membrane P-glycoprotein is a member of the ATP-binding cassette family of membrane transporters. In the present study tryptophan intrinsic fluorescence was used to understand the P-glycoprotein response to three benzodiazepines (bromazepam, chlordiazepoxide and flurazepam) in the presence and absence of ATP. Fluorescence emission spectra showed a red shift on the maximal emission wavelength upon interaction of P-glycoprotein with all benzodiazepines. Benzodiazepine association with nucleotide-bound P-glycoprotein also showed this trend and the quenching profile was attributed to a sphere-of-action model, for static fluorescence. Furthermore, quenching data of benzodiazepine-bound P-glycoprotein with ATP were concentration dependent and saturable, indicating that nucleotide binds to P-glycoprotein whether drug is present or not. These results seems in agreement with the proposal of the ATP-switch model by Higgins and Linton, where substrate binding to the transporters initiates the transport cycle by increasing the ATP binding affinity.
Lima
Sofia A C
SA
REQUIMTE, Departamento de Química da Faculdade Ciências da Universidade do Porto, Porto, Portugal.
Cordeiro-da-Silva
Anabela
A
de Castro
Baltazar
B
Gameiro
Paula
P
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry*; D000818:Animals; D001665:Binding Sites; D001960:Bromazepam / Q000737:chemistry*; D020134:Catalytic Domain; D002707:Chlordiazepoxide / Q000737:chemistry*; D005453:Fluorescence; D005479:Flurazepam / Q000737:chemistry*; D051379:Mice; D008956:Models, Chemical; D010843:Pichia / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002707:Chlordiazepoxide; D014364:Tryptophan; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D005479:Flurazepam; D001960:Bromazepam