Biophysical Society Thematic Meeting

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

, James Hamilton

State University of Maringá, Maringá, Paraná, Brazil,

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.