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 (R9) into cells remains an important challenge to their use as efficient drug-delivery vehicles. At nanomolar to low micromolar R9 concentrations and at physiological temperature, peptide entry involves endocytosis. In contrast, at a concentration ≥10 μM, R9 induces a very effective non-endocytic entry pathway specific for cationic peptides. We found that a similar entry pathway is induced at 1-2 μM concentrations of R9 if peptide application is accompanied by a rapid temperature drop to 15°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 R9 at 10-20 μM and 37°C induces repetitive spikes in intracellular Ca(2+) concentration. This Ca(2+) signalling correlated with the efficiency of the peptide entry. Pre-loading cells with the Ca(2+) chelator BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) inhibited both Ca(2+) spikes and peptide entry, suggesting that an increase in intracellular Ca(2+) precedes and is required for peptide entry. One of the hallmarks of Ca(2+) 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 R9 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.
Section on Membrane Biology, Program of Physical Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Building 10/Room 10D05, 10 Center Drive, Bethesda, MD 20892-1855, U.S.A. melikovk@mail.nih.gov.
Kamran
K
Melikov
Section on Membrane Biology, Program of Physical Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Building 10/Room 10D05, 10 Center Drive, Bethesda, MD 20892-1855, U.S.A.
Ann
A
Hara
Section on Membrane Biology, Program of Physical Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Building 10/Room 10D05, 10 Center Drive, Bethesda, MD 20892-1855, U.S.A.
Kwabena
K
Yamoah
Section on Membrane Biology, Program of Physical Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Building 10/Room 10D05, 10 Center Drive, Bethesda, MD 20892-1855, U.S.A.
Elena
E
Zaitseva
Section on Membrane Biology, Program of Physical Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Building 10/Room 10D05, 10 Center Drive, Bethesda, MD 20892-1855, U.S.A.
Eugene
E
Zaitsev
Section on Membrane Biology, Program of Physical Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Building 10/Room 10D05, 10 Center Drive, Bethesda, MD 20892-1855, U.S.A.
Leonid V
LV
Chernomordik
D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects*; D002448:Cell Adhesion / Q000187:drug effects; D057846:Cell-Penetrating Peptides / Q000493:pharmacokinetics* / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D006367:HeLa Cells; D006801:Humans; D009842:Oligopeptides / Q000493:pharmacokinetics* / Q000494:pharmacology
D057846:Cell-Penetrating Peptides; D009842:Oligopeptides; C491427:nonaarginine; D002118:Calcium
calcium imaging; calcium intracellular release; cell-penetrating peptide (CPP); microscopy; phosphatidylserine; transient receptor potential channels (TRP channels)