Functional activity of membrane channels and transporters by electrophysiology and fluorescence.

The different functions of the eukaryotic cell are made possible by the plasma membrane and the complex system of membranes that are present within it. The membrane is not an insurmountable barrier, rather an internal-external communication system controlled by ion channels and transporters that allow the movement of ions, peptides, sugars and molecules of all kinds. Through the techniques of electrophysiology (patch-clamp, voltage-clamp) and the heterologous expression in membranes of known models (oocytes, cell lines and vacuoles of plant cells) we can study in real time the functional activity of membrane channels and transporters. I participated in different projects aimed at studying the functional activity and modulation of chloride channels (ClC-K) involved in Bartter syndrome, at characterizing VRAC channels (volume-regulated anion channels) essential for the regulation of cell volume, and at investigating the localization and characterization of TMEM16E / ANO5 protein associated with GDD, genetic bone disease and with various forms of muscular dystrophy.
Currently my activity is focused on the study of ionic channels and transporters, for example NHX, present in the tonoplast (membrane of the vacuole). The vacuole is a compartment of the plant cell with various functions: from the maintenance of cellular turgidity, to the accumulation of ions and essential elements up to the detoxification of the cell. The study of the membrane of an organelle so important for the plant cell is interesting for the consequences that this research can have at an agronomic level. In addition, the vacuole represents an ideal system for the heterologous expression of mammalian (also human) membrane proteins. Recently, together with prof. Armando Carpaneto (DISTAV of the University of Genoa) we have developed a technique that combines electrophysiology and fluorescence measurements (FLEP: fluorescence combined with excised patch) to monitor the activity of membrane transporters. We used fluorescent probes (e.g. Fura and BCECF) sensitive to variations in calcium or proton concentrations to highlight the functional mechanism of non-electrogenic transporters not detectable with the usual electrophysiological techniques.

Members:
Antonella Gradogna (Principal investigator)
Joachim Scholz - Starke