Transient pores account for cell-penetrating peptide and homeoprotein translocation

Homeoproteins (HPs) and cell-penetrating peptides (CPPs) enter cells by endocytosis and direct membrane crossing (translocation). However, unlike endocytosis, translocation remains globally unknown. Here, we developed an electrophysiological approach to assess the internalization of CPPs (Tat, R₉, penetratin and R₆W₃) and the HPs Otx2 and En2 though single-cell unitary transient currents in mammalian cells. At resting membrane potential, CPPs or HPs lead to submillisecond transient pores, faster than any endocytosis event, which reveal the rapid passage of the peptide across the membrane i.e., by translocation. We evidenced that expression of specific membrane glycosaminoglycans is mandatory for translocation-induced transient pores. Associated transient currents are supralinearly enhanced by hyperpolarization and poorly affected by depolarization. Moreover, a CPP-conjugated bioactive cargo similarly translocates into cytosol. Finally, we show similar HPs-evoked transient pores in brain cortical pyramidal cells, showing the physiological relevance of translocation, with crucial biotechnological and therapeutical consequences for cell delivery purposes.