Biophysical Society Thematic Meeting | Ascona, Switzerland

Liposomes, Exosomes, and Virosomes: From Modeling Complex Membrane Processes to Medical Diagnostics and Drug Delivery

Poster Abstracts

14-POS Board 7 Pepducins Rapidly Bind and Diffuse Across Membranes Kellen Brunaldi 1 , James Hamilton 2 . 1 State University of Maringá, Maringá, Paraná, Brazil, 2 Boston University School of Medicine, Boston, MA, USA. The objective of this study was to evaluate the transmembrane transport of pepducins containing a palmitate or myristate linked to peptides corresponding to the third loop of PAR1 (protease activator receptor 1) and to the first loop of CXCR1/CXCR2 (chemokine receptors). Large unilamellar vesicles (LUV) of egg-phosphatidylcholine (egg-PC) were either encapsulated with the pH sensitive fluorescent probe pyranin or labeled in the outer leaflet of the lipid bilayer with the charge sensitive fluorescent probe FPE (fluorescein phosphatidylethanolamine). HEK293 cells were also labeled with FPE. Pepducins at final concentrations from 0.3 to 1.25 μM (< 1 mol% with respect to egg-PC) produced less than 2% of calcein leakage. Therefore, the transport assays were carried within these concentrations. Pepducins added to LUVs or HEK293 cells labeled with FPE or LUVs with pyranine produced a fast (t1/2 < 2 s) and dose-dependent increase in the fluorescence. The peptide alone (not acylated) did not affect the fluorescence, confirming the importance of acyl chain in anchoring the pepducins in the lipid bilayer. The fast FPE response reflected a net positive change in the interface of the zwitterionic egg-PC (partitioning) and as expected, PAR1 pepducins (3+ positive net charges) produced higher levels of FPE fluorescence than CXCR pepducins (1+ positive net charge). The increase in pyranine fluorescence indicated alkalinization of the intravesicular compartment, probably induced by the basic residues that had reached the inner leaflet of the lipid bilayer. We concluded that pepducins were able to bind rapidly to the external leaflet of lipid bilayers and flip to the inner leaflet by simple diffusion. The fatty acid anchor was mandatory for these processes to take place. These data give support to the biological effects of pepducins explained by their interaction with the intracellular loops of GPCRs.

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