Biophysical mechanisms of membrane perturbation and signal transduction produced by proteins and peptides

My primary research interest is focused on the field of cellular electrical activity, ranging from the ion channels that generates it, up to the study of intracellular processes regulating it, and new generation of drugs. For this purpose, during my Ph.D. I have learnt and improved different cutting-edge techniques, i.e. the patch-clamp technique, the fluorescence imaging, and the synthesis and use of model membranes. Moreover, to explore particular aspects of these molecular mechanisms and to overcome the issues raised during the investigations, non-conventional strategies were employed, even requiring the development of specific devices not commercially available. In summary, my Ph.D. thesis is focused on two projects: the biophysical characterization of a particular class of membrane active peptides, and the modulation of visual phototransduction in vertebrate cones. In the first project, I investigated the mechanism of membrane perturbation of cell-penetrating and antimicrobial peptides using the patch-clamp technique. Cell-penetrating peptides (CPPs) are short peptides that are able to cross the cell membrane via energy-dependent and/or independent mechanisms, with low toxicity and without the use of specific receptors. This ability is preserved even when CPPs are conjugated with a large cargo, thus representing an innovative pharmacological tool for the diffusion of large and hydrophilic drugs into the cells. Despite the mechanism of cellular uptake is still debated in literature, it has been proved that it can occur by either direct translocation or endocytosis. In the latter case, though, the cargo-peptide complex often remains trapped inside the endocytic vesicles and is not able to reach its therapeutic target. A possible solution to this problem could be found in another class of small peptides, similar to CPPs, i.e. the antimicrobial peptides (AMPs). AMPs are 12-50 amino acids long peptides, which represent an essential part in the innate immune system in most organisms. Indeed, they are among the first defensive molecules released during infections and their activity is direct thorough the membrane of bacteria, causing its destruction and consequently the death of the pathogen. Therefore, the ability of AMPs to disrupt biological membranes could be exploited to improve the CPPs escape from the endocytic vesicles in addition to, of course, their application as a novel class of antibiotics. The idea is to conjugate the CPP with a molecule that possess an antimicrobial activity, which can destroy the vesicle membrane, and help the complex to reach its target once it has been internalized in the cell. On this ground, the first project I carried out regards the study of a novel chimeric peptide, CM18-Tat11, composed of the antimicrobial peptide CM18 (a cecropin-mellitin hybrid peptide) linked to the cell-penetrating peptide Tat11 (derived from the basic domain of HIV-1 Tat protein). In particular, I investigated the membrane perturbing activity of this peptide (and of its elements) using the patch-clamp technique and operating under strictly physiological conditions. This study has been carried out by recording the ion current flowing through the channels formed by these peptides (if any), once inserted in the membrane of Chinese hamster ovary (CHO) cells. In these experiments, the peptides were applied to (and removed from) the extracellular CHO membrane in ~50 ms with a computer-controlled microperfusion system. Therefore, besides assessing ion channel characteristics, the dynamics of pore formation and disaggregation could be precisely evaluated as well. I found that CM18-Tat11 produces a large and irreversible plasma membrane lysis, at concentration where CM18 and Tat11 give instead a nearly reversible membrane permeabilization and no perturbation, respectively. Furthermore, using the same method, I studied the biophysical characteristic of another antimicrobial peptide, called CM12, which sequence was obtained from the optimization of CM18. When applied on CHO, CM12 and CM18 produce voltage-independent membrane permeabilization, and no single-channel events were detected at low peptides concentration. These results indicate that both peptides form pores according to a toroidal model, in which the lipid layer bends continuously through the pore so that the core is formed by both lipid head groups and the peptides. Finally, I have studied the single-channels properties generated by the pore-forming peptide alamethicin (Alm) F50/5 and its [L-Glu(OMe)7,18,19] analog inserted in a natural membrane and in giant unilamellar vesicles (GUVs). The possibility to compare the channel activity in the precisely controlled lipid environment of GUVs, with the one recorded in a natural membrane, will open new possibilities in the biophysical characterization of the pores. The second project of this thesis is focused on the study of the physiological role of the calcium sensor GCAP3 (guanylate cyclase activated protein 3) in the phototransduction cascade in zebrafish. I pursued this study simulating the over expressions and the knockdown of this protein, through the delivery of zGCAP3, or of its monoclonal antibody, into zebrafish cone cytoplasm, while recording their photorensponses with the patch-clamp technique. The proteins were administered inside the cone via the patch pipette thanks to an intracellular perfusion system developed in this thesis. This system allows the delivery of exogenous molecules inside the cell with a controlled timing, by expelling them with a small teflon tube inserted into the pipette lumen controlled by a microperfusion apparatus. Results indicated that the increase in the concentration in zGCAP3 did not altered significantly the light response, while the perfusion with the antibody anti-zGCAP3 caused the progressive fall of the dark current, together with the progressive slowing down of the flash response kinetics. The surprising lack of an effect of zGCAP3 incorporation, suggests that the endogenous number of zGCAP3 is saturating, therefore any further increase of this sensor is ineffective. However, the effects of the antibody can be explained as an inhibition of the target enzyme of zGCAP3, which is the guanylate cyclase (GC). Finally, no experiments mentioned above would have been accomplished without the development of a “pressure-polishing” system, which makes it possible to modify the geometry of the patch-clamp pipette. The pipette shank (the final part of the pipette) is, in fact, very long and tapered, thus generating a high resistance to the passage of ions and molecules, and making very difficult to perfuse efficiently the cell with the internal perfusion. The pressure polishing setup I developed enlarged the patch pipette shank, using a calibrated combination of heat and air pressure. These pipettes minimized errors in membrane potential control and allowed the insertion of teflon tubes in the pipette lumen very close to its tip.