In this project, two strategies will be employed in order to precisely assess the biophysical characteristics of the pore-forming peptides. The first strategy consists in inserting the peptides in cells where all the endogenous sources of membrane current are blocked. By using whole-cell patch-clamp recordings, it will be investigated whether these peptides can insert in the membrane and generate an exogenous current. This will allow us to study the peptide in strict physiological conditions. The voltage dependency of the exogenous current will be studied under various extracellular and intracellular ionic conditions that can be controlled rapidly and precisely, so that to study, besides the current-to-voltage characteristics, the ion selectivity of the pore. Another preparation that we will employ in this project is the whole-cell, inside-out or outside-out excised-patch recording from giant unilamellar vesicles (GUV). These have a diameter larger than 10 μm and are comparable to the cells. This will allow us to assess whether the membrane lipid composition affects the kinetics of peptide insertion into the lipid bilayer. Also it provides information on the biophysical properties of the peptidic pore, by precisely controlling the lipid composition of the GUV.
MRP project (Medical relevant peptides: biophysical properties and dynamics of pore formation in natural membranes). Progetto finanziato dal CNISM (Consorzio Nazionale Interuniversitario per le Scienze fisiche della Materia).
RISPOLI, Giorgio
2007
Abstract
In this project, two strategies will be employed in order to precisely assess the biophysical characteristics of the pore-forming peptides. The first strategy consists in inserting the peptides in cells where all the endogenous sources of membrane current are blocked. By using whole-cell patch-clamp recordings, it will be investigated whether these peptides can insert in the membrane and generate an exogenous current. This will allow us to study the peptide in strict physiological conditions. The voltage dependency of the exogenous current will be studied under various extracellular and intracellular ionic conditions that can be controlled rapidly and precisely, so that to study, besides the current-to-voltage characteristics, the ion selectivity of the pore. Another preparation that we will employ in this project is the whole-cell, inside-out or outside-out excised-patch recording from giant unilamellar vesicles (GUV). These have a diameter larger than 10 μm and are comparable to the cells. This will allow us to assess whether the membrane lipid composition affects the kinetics of peptide insertion into the lipid bilayer. Also it provides information on the biophysical properties of the peptidic pore, by precisely controlling the lipid composition of the GUV.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
