25.07.2021 | European Biophysical Societies' Association (EBSA 2021)
Dr. Andre Bazzone (Nanion Technologies) will present the following poster:
Title: SSM-based Electrophysiology meets SGLT1 and GAT1
Subtitle: Impact of different driving forces on Na+/substrate co-transport and binding events on a gold-coated sensor. The unconventional alternative to electrophysiology.
Authors: Andre Bazzone, Rocco Zerlotti, Maria Barthmes, Niels Fertig
We developed functional assays for the human Na+/GABA and Na+/glucose co-transporters GAT1 and SGLT1, using solid supported membrane -based electrophysiology (SSME). In conventional electrophysiology voltage steps are used to trigger pre steady-state (PSS) and transport currents, which are commonly recorded in whole cells at a defined holding potential. Transport and PSS electrogenicity in SGLT1 and GAT1 triggered by voltage steps is postulated to be a result from transitions within the substrate-free carriers, e.g. the alternating access of the charged sodium binding sites within the empty carrier. In contrast, SSME utilizes membrane vesicles at 0 mV and the transport cycle is triggered by applying a substrate concentration gradient as the main driving force. Using SSME, we observed substrate-induced PSS currents, likely representing conformational transitions within the substrate-loaded carrier, which are not observed with conventional electrophysiology. We examined the impact of different driving forces on influx, efflux, and PSS currents, focusing on sodium gradients and membrane voltage. We found that internal accumulation of sodium reduces Vmax, rendering sodium release rate limiting at 0 mV. Application of membrane voltage affected the apparent KM in SGLT1, but Vmax in GAT1. We also found that transport properties in influx and efflux modes are highly asymmetric for GAT1, while being more similar for SGLT1.