A series of cyclic peptides, [(DipR)(WR)4], [(DipR)2(WR)3], [(DipR)3(WR)2], [(DipR)4(WR)], and [DipR]5, and their linear counterparts containing arginine (R) as positively charged residues and tryptophan (W) or diphenylalanine (Dip) as hydrophobic residues, were synthesized and evaluated for their molecular transporter efficiency. The in vitro cytotoxicity of the synthesized peptides was determined in human epithelial ovary adenocarcinoma cells (SK-OV-3), human lymphoblast peripheral blood cells (CCRF-CEM), human embryonic epithelial kidney healthy cells (HEK-293), human epithelial mammary gland adenocarcinoma cells (MDA-MB-468), pig epithelial kidney normal cells (LLC-PK1), and human epithelial fibroblast uterine sarcoma cells (MES-SA). A concentration of 5–10 µM and 3 h incubation were selected in uptake studies. The cellular uptake of a fluorescent-labeled phosphopeptide, stavudine, lamivudine, emtricitabine, and siRNA was determined in the presence of peptides via flow cytometry. Among the peptides, [DipR]5 (10 µM) was found to be the most efficient transporter and significantly improved the uptake of F’-GpYEEI, i.e., by approximately 130-fold after 3 h incubation in CCRF-CEM cells. Confocal microscopy further confirmed the improved delivery of fluorescent-labeled [DipR]5 (F’-[K(DipR)5]) alone and F’-GpYEEI in the presence of [DipR]5 in MDA-MB-231 cells. The uptake of fluorescent-labeled siRNA (F’-siRNA) in the presence of [DipR]5 with N/P ratios of 10 and 20 was found to be 30- and 50-fold higher, respectively, compared with the cells exposed to F’-siRNA alone. The presence of endocytosis inhibitors, i.e., nystatin, chlorpromazine, chloroquine, and methyl β-cyclodextrin, did not completely inhibit the cellular uptake of F’-[K(DipR)5] alone or F’-GpYEEI in the presence of [DipR]5, suggesting that a combination of mechanisms contributes to uptake. Circular dichroism was utilized to determine the secondary structure, while transmission electron microscopy was used to evaluate the particle sizes and morphology of the peptides. The data suggest the remarkable membrane transporter property of [DipR]5 for improving the delivery of various small molecules and cell-impermeable negatively charged molecules (e.g., siRNA and phosphopeptide).

Aim Small‐conductance Ca2+‐activated potassium (SK) channels are activated exclusively by increases in intracellular Ca2+ that binds to calmodulin constitutively associated with the channel. Wild‐type SK2 channels are activated by Ca2+ with an EC50 value of ~0.3 μmol/L. Here, we investigate hydrophobic interactions between the HA helix and the S4‐S5 linker as a major determinant of channel apparent Ca2+ sensitivity. Methods Site‐directed mutagenesis, electrophysiological recordings and molecular dynamic (MD) simulations were utilized. Results Mutations that decrease hydrophobicity at the HA‐S4‐S5 interface lead to Ca2+ hyposensitivity of SK2 channels. Mutations that increase hydrophobicity result in hypersensitivity to Ca2+. The Ca2+ hypersensitivity of the V407F mutant relies on the interaction of the cognate phenylalanine with the S4‐S5 linker in the SK2 channel. Replacing the S4‐S5 linker of the SK2 channel with the S4‐S5 linker of the SK4 channel results in loss of the hypersensitivity caused by V407F. This difference between the S4‐S5 linkers of SK2 and SK4 channels can be partially attributed to I295 equivalent to a valine in the SK4 channel. A N293A mutation in the S4‐S5 linker also increases hydrophobicity at the HA‐S4‐S5 interface and elevates the channel apparent Ca2+ sensitivity. The double N293A/V407F mutations generate a highly Ca2+ sensitive channel, with an EC50 of 0.02 μmol/L. The MD simulations of this double‐mutant channel revealed a larger channel cytoplasmic gate. Conclusion The electrophysiological data and MD simulations collectively suggest a crucial role of the interactions between the HA helix and S4‐S5 linker in the apparent Ca2+ sensitivity of SK2 channels.

The cell membrane properties create a significant obstacle in intracellular delivery of cell-impermeable and negatively charged molecules. Herein, we report the synthesis and biological evaluation of a novel series of hybrid cyclic-linear peptides containing alternative positive and hydrophobic amino acids on the ring and side chain [(RW) 5 ]K(RW) X (X = 1−5) to compare their molecular transporter efficiency. The peptides were synthesized through Fmoc solid-phase peptide synthesis. In vitro cytotoxicity of the peptides showed that the peptides did not exhibit any significant cytotoxicity at the concentration of 10 μM in human leukemia carcinoma cell line (CCRF-CEM), human ovarian adenocarcinoma cells (SK-OV-3), human epithelial embryonic kidney healthy (HEK-293), and human epithelial mammary gland adenocarcinoma cells (MDA-MB-231) after 3 h incubation. The cellular uptake of a fluorescence-labeled phosphopeptide (F′-GpYEEI) and anti-human immunodeficiency virus (HIV) drugs (lamivudine (F′-3TC), emtricitabine (F′-FTC), Stavudine (F′-d4T)), where F′ is carboxyfluorescein, was measured in the presence of the peptides in CCRF-CEM and SK-OV-3 cells. Among all peptides, [(RW) 5 K](RW) 5 (10 μM) was the most efficient transporter that improved the cellular uptake of F′-GpYEEI (2 μM) by 18-and 11-fold in CCRF-CEM and SK-OV-3, respectively, compared with F′-GpYEEI alone. Fluorescence-activated cell sorting (FACS) analysis results indicated that the cellular uptake of fluorescencelabeled peptide (F′-[(RW) 5 K](RW) 5 ) was only partially inhibited by chlorpromazine as an endocytosis inhibitor after 3 h incubation in MDA-MB-231 cells. These data suggest the potential of this series of hybrid cyclic-linear peptides as cell-penetrating peptides and molecular transporters.