Efficient entry of cell-penetrating peptide nona-arginine into adherent cells involves a transient increase in intracellular calcium.
Bottom Line: 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.Intriguingly, we found that R9 at 10-20 μM and 37°C induces repetitive spikes in intracellular Ca(2+) concentration.To conclude, we uncovered a novel mechanistic link between calcium signalling and entry of cationic peptides.
Affiliation: 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. email@example.com.Show MeSH
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Mentions: Mechanistic dependence of the R9 entry on calcium signalling suggested that drugs known to release calcium from intracellular stores may lower concentration of the R9-TAMRA required for its efficient entry at 37°C. We treated the cells with flufenamic acid, a member of fenamate class of non-steroidal anti-inflammatory drugs, which have been shown to release calcium form mitochondria [32,33]. Since, in our experiments, we consistently observed a several minute delay between addition of 10 μM R9-TAMRA and the onset of calcium spikes (Figure 5B; Supplementary Video S2), we applied flufenamic acid to cells either together with 2 μM R9-TAMRA or 5 min after the peptide addition. We observed no effect on the entry of R9-TAMRA when flufenamic acid was added together with the peptide (Figure 10A). In contrast, application of flufenamic acid 5 min after peptide addition resulted in efficient entry of R9-TAMRA into the cytosol and nucleus of many cells (Figure 10A). Interestingly, flufenamic acid applied either with or before the peptide induced an increase in intracellular calcium concentration (Figure 10B). However, whereas addition of drug 5 min after peptide application resulted in a sustained increase in calcium concentration (Figure 10B, top trace), in the case of simultaneous application of flufenamic acid and the peptide, the calcium level rapidly returned to background levels (Figure 10B, bottom trace). We conclude that efficient entry of R9-TAMRA can be promoted by a drug-induced increase in intracellular calcium concentration.
Affiliation: 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. firstname.lastname@example.org.