P-Glycoprotein Activity Is Non-Monotonically Modulated by Transmembrane Voltage

Abstract

P-glycoprotein (Pgp) is a broadly polyspecific exporter of hydrophobic and amphiphilic compounds that plays an important role in the blood-brain barrier and in multidrug resistance in cancer. Several research groups have recently made significant strides toward elucidating the mechanism of Pgp's catalytic cycle, but the biophysical properties of the cellular microenvironment can have strong effects on the function of Pgp and other ATP-binding cassette (ABC) transporters in as-yet poorly defined ways. Here we report that Pgp, MRP1, and BCRP, three of the most clinically relevant multidrug resistance-linked ABC transporters, are strongly and non-monotonically regulated by the transmembrane electrical potential difference. We confirmed this result with several assays, which used fluorescence and electrophysiological methods to measure the efflux of transporter substrates from two human cell lines and giant unilamellar vesicles and controlled the voltage using electrolyte gradients or the patch clamp technique. At hyperpolarized (highly inside-negative) potentials, each transporter displayed a pronounced reduction in efflux activity compared to the activity at the resting potential; Pgp and MRP1 also showed reduced activity at inside-positive potentials. The transport data is well-described by a model in which the transmembrane potential biases the conformational distribution of the proteins in a manner similar to the mechanism of action of voltage-gated ion channels. In this model, which is compatible with the “alternating access” framing of Pgp function, each catalytic cycle contains two transitions with an opposite dependence on the transmembrane voltage due to the movement of an equivalent of 3-4 elementary charges back and forth across the membrane. This finding, which points to an additional axis for modulating transporter activity, underscores the importance of electric field effects on the function of all membrane proteins, particularly those that undergo large conformational changes in the transmembrane region.