The outer membrane of Gram-negative bacteria contains a variety of pore-forming structures collectively referred to as porins. Some of these are voltage-dependent, but weakly so, closing at high voltages. Triplin, a novel bacterial pore-former, is a three-pore structure, highly voltage dependent, with a complex gating process. The three pores close sequentially: pore 1 at positive potentials, 2 at negative and 3 at positive. A positive domain containing 14 positive charges (the voltage sensor) translocates through the membrane during the closing process and the translocation is proposed to take place by the domain entering the pore and thus blocking it resulting in the closed conformation. This mechanism of pore closure is supported by kinetic measurements that show that in the closing process the voltage sensor travels through most of the transmembrane voltage before reaching the energy barrier. Voltage-dependent blockage of the pores by polyarginine but not by a 500 fold higher concentrations of polylysine, is consistent with the model of pore closure, with the sensor consisting mainly of arginine residues, and with the presence, in each pore, of a complementary surface that serves as a binding site for the sensor.

Given the pathological role of hyperphosphorylated Tau aggregation in Alzheimer’s disease (AD) our laboratory previously developed the novel Tau aggregation inhibitor peptide, RI-AG03. As Tau aggregates accumulate intracellularly, it is essential that Tau-targeting peptides efficiently traverse the cell membrane. In addition, these peptides need to avoid sequestration into cell organelles, particularly those involved in degradation, to ensure interaction with Tau in the cytoplasm. Here, we examine the cellular uptake and intracellular trafficking of RI-AG03 when the peptide is unconjugated or linked to nanoliposome carrier particles. The impact of adding the cell penetrating peptide (CPP) sequences, polyarginine (polyR) and transactivator of transcription (TAT), to RI-AG03 was also characterised. Our data show that the conjugation of CPP-containing RI-AG03 to liposomes dramatically increases cellular liposomes uptake. The majority of this uptake occurs via direct cell penetration. However, we find that macropinocytosis is also an important mechanism for unconjugated and RI-AG03-polyR-conjugated, but not RI-AG03-TAT-conjugated, liposome uptake. In agreement with this macropinocytosis-mediated internalisation, liposome lipids localise to lysosomes and macropinosomes following cellular uptake. However, while unconjugated RI-AG03 peptides also localise to lysosomes, liposome conjugation prevents this localisation, with the peptide detaching from the liposomes at the cell surface and evading organelle entrapment. Overall, this study demonstrates that CPP sequences and nanoliposomes carriers enhance the intracellular delivery of RI-AG03, furthering the possibility of the peptide’s use in targeting Tau pathology in AD.