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P-Glycoprotein is a member of the ABC superfamily of membrane transporters, and functions as an ATP-driven active efflux pump for natural products and chemotherapeutic drugs. Overexpression of P-glycoprotein is a major cause of multidrug resistance in human cancers. Sulfhydryl modification agents are known to inactivate both P-glycoprotein ATPase activity and transport function. In the present study, P-glycoprotein purified from CHRB30 cells was covalently labeled at two conserved Cys residues, one within each of the nucleotide binding domains, using 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid (MIANS). MIANS modification inactivated P-glycoprotein ATPase function, in a concentration-dependent fashion. Increasing concentrations of ATP blocked MIANS labeling with an IC50 of 0.37 mM (similar to the KM for ATP hydrolysis), which suggests that the label is located close to the site of ATP binding within the nucleotide binding domain. A blue shift in the fluorescence spectrum of MIANS bound to P-glycoprotein indicated that the labeled Cys residues are situated in a nonpolar environment. MIANS-labeled P-glycoprotein was still able to bind ATP, as demonstrated by quenching of the fluorescence, with a Kd of 0.46 mM. Addition of a variety of drugs and chemosensitizers to MIANS-labeled P-glycoprotein led to substantial quenching of the probe fluorescence within the nucleotide binding domains. Dissociation constants for drug binding measured by fluorescence quenching were in the range of 0.77 microM for vinblastine to 158 microM for colchicine. Quenching by ATP and drugs was independent and additive, suggesting that each produces a defined change in the protein. The rate of MIANS labeling of Pgp was reduced in the presence of drugs and chemosensitizers, implying that a long-range conformational change arises from drug binding which alters the accessibility of the nucleotide binding domains to MIANS. These results suggest that there is conformational communication between the drug binding site(s) of P-glycoprotein and the ATPase catalytic sites within the nucleotide binding domains.

A large body of evidence suggests that an increase in the brain b-amyloid (Ab) burden contributes to the etiology of Alzheimer's disease (AD). Much is now known about the intracellular processes regulating the production of Ab, however, less is known regarding its secretion from cells. We now report that p-glycoprotein (p-gp), an ATP-binding cassette (ABC) transporter, is an Ab ef¯ux pump. Pharmacological blockade of p-gp rapidly decrease extracellular levels of Ab secretion. In vitro binding studies showed that addition of synthetic human Ab1±40 and Ab1±42 peptides to hamster mdr1-enriched vesicles labeled with the¯uorophore MIANS resulted in saturable quenching, suggesting that both peptides interact directly with the transporter. Finally, we were able to directly measure transport of Ab peptides across the plasma membranes of p-gp enriched vesicles, and showed that this phenomenon was both ATP-and p-gp-dependent. Taken together, our study suggests a novel mechanism of Ab detachment from cellular membranes, and represents an obvious route towards identi®cation of such a mechanism in the brain.

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