Functional Assay For Characterizing Human P-Glycoprotein Transport Using The Pore Forming Peptide Gramicidin A

P-glycoprotein (P-gp), a member of the ATP-Binding Cassette (ABC) transporter family, actively transports chemotherapeutics out of cancer cells, leading to reduced efficacy of these drugs. Thus the development of functional assays for better characterization of P-gp is important in finding ways to combat multidrug resistance (MDR) in cancer treatment. We establish a novel approach that takes advantage of the single molecule sensitivity of the patch clamp technique and provides access to the intracellular environment as well as direct control of the cellular transmembrane potential. Briefly, the assay measures whole cell current in the presence of ion channel forming peptide gramicidin A (gA). Since gA is a substrate of P-gp, changes in gA current report the efflux of gA by P-gp. For instance, gA current in chemosensitive parental cells that did not express P-gp, increased with time in a dose-dependent manner. In contrast, P-gp expressing resistant cells exhibited significantly reduced gA current due to its efflux by P-gp. Consistently, P-gp inhibitors restored gA current in P-gp expressing cells to a level comparable to that in parental cells. HeLa cells transduced with non-functional mutant (E556Q/E1201Q) P-gp displayed large gA currents comparable to control HeLa cells. This can be attributed to the loss of transport activity due to defective ATP hydrolysis by this mutant P-gp. Additionally fitting the gA currents data to a model that quantifies the kinetic constants showed that P-gp efflux activity was correlated with the intracellular ATP concentration. Moreover, to our surprise, membrane depolarization enhanced P-gp activity whereas hyperpolarization reduced its activity. Collectively our findings demonstrate that whole cell gA current can be used to monitor P-gp function.