Genetic, cellular and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore
- University of Lausanne, Switzerland
- Università della Svizzera italiana, Scuola Universitaria Professionale della Svizzera Italiana, Switzerland
- Politecnico di Torino, Italy
- Université de Lausanne, Switzerland
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 CPPs 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 membrane. This opens the ground for strategies aimed at improving the ability of cells to capture CPP-linked cargos in vitro and in vivo.
Data availability
DNA sequencing data from the CRISPR/Cas9-based screens are available through the following link: https://www.ncbi.nlm.nih.gov/sra/SRP161445.
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CRISPR/Cas9 screen to identify genes involved in uptake of cell-penetrating peptidesNCBI Gene Expression Omnibus, SRP161445.
Article and author information
Author details
Evgeniya Trofimenko
Funding
Swiss National Science Foundation (CRSII3_154420,IZCSZ0-174639)
- Christian Widmann
Swiss National Science Foundation (158116)
- Nadja Chevalier
Swiss National Science Foundation (320030_170062)
- Francesca Amati
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Patricia Bassereau, Institut Curie, France
Ethics
Animal experimentation: All experiments were performed according to the principles of laboratory animal care and Swiss legislation under ethical approval (Swiss Animal Protection Ordinance; permit number VD3374.a).
Version history
- Preprint posted: February 26, 2020 (view preprint)
- Received: April 27, 2021
- Accepted: October 28, 2021
- Accepted Manuscript published: October 29, 2021 (version 1)
- Version of Record published: December 2, 2021 (version 2)
Copyright
© 2021, Trofimenko et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Genetic, cellular and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore
eLife 10:e69832.
https://doi.org/10.7554/eLife.69832
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