Membrane permeation of arginine-rich cell-penetrating peptides independent of transmembrane potential as a function of lipid composition and membrane fluidity

Abstract

Cell-penetrating peptides (CPPs) are prominent delivery vehicles to confer cellular entry of (bio-) macromolecules. Internalization efficiency and uptake mechanism depend, next to the type of CPP and cargo, also on cell type. Direct penetration of the plasma membrane is the preferred route of entry as this circumvents endolysosomal sequestration. However, the molecular parameters underlying this import mechanism are still poorly defined. Here, we make use of the frequently used HeLa and HEK cell lines to address the role of lipid composition and membrane potential. In HeLa cells, at low concentrations, the CPP nona-arginine (R9) enters cells by endocytosis. Direct membrane penetration occurs only at high peptide concentrations through a mechanism involving activation of sphingomyelinase which converts sphingomyelin into ceramide. In HEK cells, by comparison, R9 enters the cytoplasm through direct membrane permeation already at low concentrations. This direct permeation is strongly reduced at room temperature and upon cholesterol depletion, indicating a complex dependence on membrane fluidity and microdomain organisation. Lipidomic analyses show that in comparison to HeLa cells HEK cells have an endogenously low sphingomyelin content. Interestingly, direct permeation in HEK cells and also in HeLa cells treated with exogenous sphingomyelinase is independent of membrane potential. Membrane potential is only required for induction of sphingomyelinase-dependent uptake which is then associated with a strong hyperpolarization of membrane potential as shown by whole-cell patch clamp recordings. Next to providing new insights into the interplay of membrane composition and direct permeation, these results also refute the long-standing paradigm that transmembrane potential is a driving force for CPP uptake.

Internalization mechanisms of cell-penetrating peptides

“…It has been shown that at low concentration, arginine-rich CPPs are mainly endocytosed, whereas rapid cytoplasmic entry occurred at higher concentration [41]. The latter is associated with the accumulation of the peptide at certain membrane areas called nucleation zones [42]. New findings of these type could further broaden what is already a very wide horizon in the field of CPP research -their internalization mechanism.…”

Synthetic molecular evolution of hybrid cell penetrating peptides

“…Toward solving the delivery problem inherent to peptide, PNA, and PMO cargoes, cell penetrating peptides (CPPs) have shown promise as vehicles capable of transporting such cell-impermeant cargo to cytosolic or nuclear targets. However, there remains a need to identify CPPs with higher efficiencies, lower effective treatment concentrations, decreased cytotoxicity, and alternative mechanisms of action 2 , 6 – 8 .…”

“…At low concentrations (<10 µM), the cationic guanidinium-rich tat and its analogs, including nona-arginine (Arg9), enter cells mostly by endocytosis 31 . At higher concentrations, a mostly energy-independent mechanism of entry dominates as the peptide enters cells directly, perhaps after accumulation at ceramide-rich nucleation zones on the plasma membrane 8 . Penetratin is an amphipathic CPP that is capable of either direct translocation through the plasma membrane or translocation via the formation of a transient membrane structure 32 .…”

Action of antimicrobial peptides and cell-penetrating peptides on membrane potential revealed by the single GUV method

“…The effects of the increase in K + concentration in aqueous solution outside eukaryotic cells on the entry of CPPs into their cytosol have been studied, and the results suggest that the efficiency of entry of CPPs decreases with a decrease in negative membrane potential (Rothbard et al 2004(Rothbard et al , 2005Henriques et al 2005). An opposite result was also reported, namely, that entry of a CPP, nona-arginine (R 9 ) into cells, does not depend on membrane potential (Wallbrecher et al 2017). Similar experiments have been performed for AMPs, and the results suggest that activities of some AMPs decrease with a decrease in negative membrane potential (Wu et al 1999).…”