NOVEL TECHNIQUES FOR IN VIVO CHARACTERIZATION OF SHORT PEPTIDES AND PROTEINS IN MEMBRANE PERMEABILIZATION AND SIGNAL TRANSDUCTION

My scientific interest is focused on the field of cellular electrical activity, ranging from the study of intracellular enzymatic processes to the characterization of new generation of drugs. For this purpose I also used the most powerful techniques of investigation, including patch-clamp technique, fluorescence imaging, and surface plasmon resonance (SPR) spectroscopy. Moreover, to shed light on complex molecular mechanisms, unconventional strategies were employed, requiring sometimes the realization of specific devices not commercially available. In particular my PhD thesis includes two different scientific projects: the biophysical characterization of antimicrobial peptides and the modulation of visual phototransduction in vertebrate cones. In the first project, the patch-clamp technique was employed to study the pore forming properties of synthetic cecropin-melittin hybrid peptide (CM15), alamethicin F50/5 and its synthetic analog [L-Glu(OMe)7,18,19] under strict physiological conditions. These short peptides selectively permeabilize the bacteria plasma membrane leading to their lyses and death: they are therefore a source of antibacterial molecules, and inspiration for novel and more selective drugs. I pursued this study by recording the ion current through the channels formed by these peptides, once inserted in the membrane of photoreceptor rod outer segment membrane (OS) isolated from frog retinae. The peptides were applied to (and removed from) the extracellular OS side in ~50 ms with a computer-controlled microperfusion system, so that the ion channel characteristics (as its selectivity, blockade and gating) and the dynamics of pore formation could be precisely assessed. On the basis of the electrophysiological recordings obtained, it was demonstrated that, different than alamethicins, CM15 produced voltage-independent membrane permeabilisation, repetitive peptide application caused a progressive permeabilisation increase, and no single-channel events were detected at low peptide concentrations. Collectively, these results indicate that CM15 form pores according to a toroidal model. Moreover, in order to understand the divalent-cation dependency of [L-Glu(OMe)7,18,19] binding to the lipid bilayer at the molecular level, the electrophysiological experiments were paralleled with experiments employing SPR spectroscopy. Results indicate the presence of Ca2+ in the external solution increases the probability of formation of smaller and more stable [L-Glu(OMe)7,18,19] pores. The second project of this thesis concerns the investigation of the physiological role of the neuronal calcium sensor zGCAP3 in the photrasduction cascade. This study was pursued through the simulation of an over expression and a knock-down of this protein, by delivering it, or its monoclonal antibody, into zebrafish cone cytosol, while recording their photoresponses with the patch-clamp technique. The intracellular protein delivery was attained via the patch pipette, by ejecting the proteins out of a tube inserted into the pipette lumen. A microperfusion system was employed to apply the desired exogenous molecules with a precise timing. However, the long tapered shape of the pipette shank make it very difficult to perfuse efficiently the cell with this strategy. For this reason a pressure polishing setup was assembled to enlarge the patch pipette shank, through the calibrated combination of heat and air pressure. This allowed to insert quartz or plastic tubes in the pipette lumen very close to its tip. In order to obtain a substantial and specific silencing of the zGCAP3s in zebrafish cones, surface plasmon resonance experiments were performed to allow the selection of a monoclonal antibody with strong affinity for zGCAP3 and low cross interaction with other components of the phototransduction cascade. Results showed that the perfusion with GCAP3 did not altered significantly the light response, while he anti-zGCAP3 incorporation in the cytosol caused the progressive photoresponse fall, followed by the progressive fall of saturating flash response amplitude, probably due to the progressive GC inhibition. The unexpected lack of an effect of zGCAP3 incorporation in the cone cytosol, suggests that the endogenous number of zGCAP3 is saturating, i.e. their number is equal or above the number of their target molecules (guanylate cyclase), therefore any further increase of zGAP3 in the cytosol is uneffective.