(190n) Transport of Amyloid-? across the Blood Brain Barrier By P-Glycoprotein: A Novel Therapeutic Target in Alzheimer’s Disease
An ATPase activity assay was used to quantify AÎ² binding by P-gp. When a ligand binds P-gp, ATP is hydrolyzed to release inorganic phosphate (Pi). The concentration of Pi, measured via absorbance, correlates with binding activity. Inverted vesicles, which allow Ab to bind P-gp on the outer vesicle surface, are incubated alone (negative control), in the presence of verapamil (positive control), or in the presence of AÎ² prepared to optimize different assembly states. Results demonstrate that AÎ² oligomers preferentially bind P-gp compared to AÎ² monomer or larger aggregates, including fibril and sonicated fibril.
Uniquely, P-gp contains two distinct binding sites. To determine the P-gp binding site with which oligomeric AÎ² interacts, a competition binding assay was performed. Rhodamine 123 (R123) and Hoechst 33342 (H33342) are fluorescent compounds with well-described alternate binding sites on P-gp. The transport of each R123 and H33342 by P-gp into a vesicle is evidenced by a quenching of fluorescence allowing for the calculation of each compoundâs transport rate; this transport is impeded by the presence of any compound that binds to the same site on P-gp. Here, inverted vesicles and MgATP are incubated in the presence of both oligomeric AÎ² and either R123 or H33342 for identification of the binding site. The rate of fluorescence quenching shows oligomeric AÎ²âs competitive binding relationship to a specific binding site on P-gp.
Together, this study demonstrates the size of the AÎ² aggregate species plays a crucial role in the binding of AÎ² to P-gp for transport. In addition, through the competitive binding assay, the probable P-gp binding site of AÎ² has been identified. Future studies will confirm these relationships between oligomeric AÎ² and P-gp in a cellular transport assay.