Genetic, cellular, and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore

Abstract

Cell-penetrating peptides (CPPs) allow intracellular delivery of bioactive cargo molecules. The mechanisms allowing CPPs to enter cells are ill-defined. Using a CRISPR/Cas9-based screening, we discovered that KCNQ5, KCNN4, and KCNK5 potassium channels positively modulate cationic CPP direct translocation into cells by decreasing the transmembrane potential (Vm). These findings provide the first unbiased genetic validation of the role of Vm in CPP translocation in cells. In silico modeling and live cell experiments indicate that CPPs, by bringing positive charges on the outer surface of the plasma membrane, decrease the Vm to very low values (–150 mV or less), a situation we have coined megapolarization that then triggers formation of water pores used by CPPs to enter cells. Megapolarization lowers the free energy barrier associated with CPP membrane translocation. Using dyes of varying dimensions in CPP co-entry experiments, the diameter of the water pores in living cells was estimated to be 2 (–5) nm, in accordance with the structural characteristics of the pores predicted by in silico modeling. Pharmacological manipulation to lower transmembrane potential boosted CPP cellular internalization in zebrafish and mouse models. Besides identifying the first proteins that regulate CPP translocation, this work characterized key mechanistic steps used by CPPs to cross cellular membranes. This opens the ground for strategies aimed at improving the ability of cells to capture CPP-linked cargos in vitro and in vivo.

Ionic Strength and Solution Composition Dictate the Adsorption of Cell-Penetrating Peptides onto Phosphatidylcholine Membranes

“…Still, the membrane potential seems to play a role in cells, as demonstrated by a recent experimental study highlighting the role of ion channels in generating permeable pores. 22 Moreover, very recent experiments with giant unilamellar vesicles (GUVs) composed of neutral PC lipids at very low ARCPP concentrations have actually shown pore-free translocation of ARCPPs, 35 , 36 in contrast to previous literature.…”

Dose-dependent nuclear delivery and transcriptional repression with a cell-penetrant MeCP2

“…This result suggests that Tat- t MeCP2 gains entry into cells, albeit less efficiently ( Extended Data Fig. 6b ), via a different subpopulation of endosomes 48 or one or more non-endosomal pathways 49 .…”

“…This result suggests that Tat-tMeCP2 gains entry into cells, albeit less efficiently (Extended Data Fig. 6b), via a different subpopulation of endosomes 48 or one or more non-endosomal pathways 49 . Delivered ZF-tMeCP2 interacts with partner proteins in the NCoR/SMRT complex Next we explored the function of tMeCP2 proteins delivered to the nucleus of CHO-K1 cells, which express low levels of endogenous MeCP2 (Extended Data Fig.…”

Cell-Penetrating Botulinum Neurotoxin Type A With Improved Cellular Uptake and Therapeutic Index

“…It has been shown that ZFP penetrates cells through macropinocytosis and, to a lesser extent, caveolindependent endocytosis (Gaj et al, 2014). In addition, TAT or other CPPs may penetrate cells through water pore-mediated direct translocation (Trofimenko et al, 2021). Therefore, one may envision that fusion of CPPs can compromise the cell-targeting specificity of BoNTA proteins.…”