Efficient entry of cell-penetrating peptide nona-arginine into adherent cells involves a transient increase in intracellular calcium

Understanding the mechanism of entry of cationic peptides such as nona-arginine (R-9) into cells remains an important challenge to their use as efficient drug-delivery vehicles. At nanomolar to low micromolar R-9 concentrations and at physiological temperature, peptide entry involves endocytosis. In contrast, at a concentration >= 10 mu M, R-9 induces a very effective non-endocytic entry pathway specific for cationic peptides. We found that a similar entry pathway is induced at 1-2 mu M concentrations of R-9 if peptide application is accompanied by a rapid temperature drop to 15 degrees C. Both at physiological and at sub-physiological temperatures, this entry mechanism was inhibited by depletion of the intracellular ATP pool. Intriguingly, we found that R-9 at 10-20 mu M and 37 degrees C induces repetitive spikes in intracellular Ca2+ concentration. This Ca2+ signalling correlated with the efficiency of the peptide entry. Pre-loading cells with the Ca2+ chelator BAPTA(1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid) inhibited both Ca2+ spikes and peptide entry, suggesting that an increase in intracellular Ca2+ precedes and is required for peptide entry. One of the hallmarks of Ca2+ signalling is a transient cell-surface exposure of phosphatidylserine (PS), a lipid normally residing only in the inner leaflet of the plasma membrane. Blocking the accessible PS with the PS-binding domain of lactadherin strongly inhibited non-endocytic R-9 entry, suggesting the importance of PS externalization in this process. To conclude, we uncovered a novel mechanistic link between calcium signalling and entry of cationic peptides. This finding will enhance our understanding of the properties of plasma membrane and guide development of future drug-delivery vehicles.