P-glycoprotein retains function when reconstituted into a sphingolipid and cholesterol rich environment

Szabolcs Modok, Catherine Heyward, and Richard Callaghan

Nuffield Department of Clinical Laboratory Sciences, Oxford University, Oxford OX3 9DU

Corresponding Author: richard.callaghan{at}ndcls.ox.ac.uk

Numerous reports have proposed a functional association of the multidrug resistance P-glycoprotein (P-gp) within rigid detergent resistant membrane microdomains (e.g. rafts or caveolae). Such microdomains are highly specialised with respect to function and lipid composition. The activity of P-gp has been well documented to display sensitivity to its lipid environment, and a functional association in membrane microdomains will require that the multidrug transporter retains activity in the “raft” microenvironment. To address this issue, purified hamster P-gp was reconstituted in liposomes comprising sphingomyelin and cholesterol, both highly enriched in membrane microdomains and known to impart a liquid-ordered phase to bilayers. The activity of P-gp was compared to proteoliposomes composed of crude egg-phosphatidylcholine (unsaturated lipids) or dipalmitoyl phosphatidylcholine (saturated lipids), in the presence or absence of cholesterol. P-gp function, as measured by maximal ATP hydrolysis, was not significantly altered by the nature of the lipid species in the proteoliposomes. However, the potencies of nicardipine and XR9576 to modulate the ATPase activity of P-gp were significantly increased in the sphingolipid based proteoliposomes. By implication, this effect may arise by either altered drug-Pgp interaction or through transmission of drug binding events to the ATP catalytic machinery. The drug-Pgp interaction was investigated by measurement of the rates of [3H]-XR9576 association and dissociation from the transporter. The lipid environment of P-gp did not affect kinetic parameters characterising drug binding. In summary, P-gp retains function in liquid ordered cholesterol and sphingolipid model membranes where the communication between the transmembrane and the nucleotide binding domains following drug binding to the protein is more efficient.

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