No Evidence for Plasma Membrane Potential‐Independent Cell Penetrating Peptide Direct Translocation

ABSTRACT Cell‐penetrating peptides (CPPs) are small peptides that can carry bioactive cargoes into cells. CPPs access the cell's cytosol via direct translocation across the plasma membrane. We and others have shown that direct translocation of CPPs occurs through water pores that are formed upon hyperpolarization of the cell's membrane. Direct translocation through water pores can therefore be blocked by depolarizing the plasma membrane. Other direct translocation mechanisms have been proposed that would not rely on membrane hyperpolarization. It has been reported, for example, that in HEK cells, CPP translocation occurs in a plasma membrane potential‐independent manner, in contrast to HeLa cells, where CPP access to the cytosol required plasma membrane hyperpolarization. To address these apparent discrepant data, we have tested the requirement of plasma membrane hyperpolarization in a series of cell lines, including HEK and HeLa cells, for CPP direct translocation. Our data, obtained from a wide range of CPP concentrations, show that efficient direct translocation always requires plasma membrane hyperpolarization. We discuss the possible reasons why earlier studies have not evidenced the importance of the plasma membrane potential in the cytosolic uptake of CPPs in some cell lines.