Rapid Binding and Transmembrane Diffusion of Pepducins in Phospholipid Bilayers

Abstract

Pepducins are GPCR-targeted lipopeptides designed to anchor in the cell membrane lipid bilayer and modulate the receptor/G protein signal transduction pathway via an allosteric mechanism. It is thus presumed that pepducins cross the plasma membrane by some mechanism, possibly passive diffusion. The goal of this research is to study the biophysical transport properties of pepducins in model membranes. We utilized fluorescent probes that measure the binding (fluorescein phosphatidylethanolamine - FPE) and diffusion (pH probe - pyranine) of charged ligands across the lipid bilayer of large unilamellar vesicles (LUV) comprised of egg-phosphatidylcholine. We tested pepducins with a palmitate or myristate linked to the N-terminal of the peptide sequence (KKSRALF). The GPCR target for these pepducins is the protease activator receptor 1 (PAR1). Addition of pepducins (0.16-5.0 mol%) to LUVs labeled in the outer leaflet with FPE or containing entrapped pyranine produced a fast (<2s) and dose-dependent increase in the fluorescence of both probes. The fast response of FPE, resulting from the insertion of positive charges (lysine and arginines residues) into the outer leaflet, demonstrated rapid partitioning into the membrane. The increase in pyranine fluorescence indicated alkalinization of the intravesicular compartment, probably due to protonation of the lysine residues. In order for this to be detected, the pepducin must cross the membrane. The peptide alone (not acylated) did not cause any change in the fluorescence of either FPE or pyranine. These data are consistent with favorable partitioning of pepducins into the membrane and rapid passive diffusion to the sites of their action at the cytosolic leaflet of the plasma membrane.