Transporters are vital for numerous cellular functions, from nutrient uptake and waste removal to maintaining ion balance and facilitating signal transduction. They are indispensable for cell survival and overall organismal health and are implicated in numerous diseases. Despite their importance, the functional characterization of transporters has been hampered by technical challenges and high costs associated with traditional assays.

In a recent study, researchers from Irina Borodina’s lab introduced a robust and efficient workflow combining continuous-exchange cell-free protein synthesis (CECF) and solid supported membrane-based electrophysiology (SSME). This approach provides a direct, real-time, label-free method for transporter characterization, significantly enhancing throughput and reliability.

The novel workflow leverages the strengths of CECF to produce high-quality membrane proteins in the presence of nanodiscs (NDs). NDs facilitate the co-translational insertion and folding of membrane proteins into a native-like lipid environment, ensuring their functional integrity. The synthesized proteins are then incorporated into proteoliposomes and analyzed using SSME.

The study validated this approach by successfully expressing and characterizing five diverse transporters: EmrE and SugE (drug/H+-coupled antiporters from E. coli), LacY (lactose permease from E. coli), NhaA (Na+/H+ antiporter from E. coli), and AAC2 (mitochondrial ADP/ATP carrier from S. cerevisiae).

For each transporter, kinetic parameters such as KM (Michaelis constant), IMAX (maximum current), and pH dependency were evaluated.

Key findings included:

• EmrE: Characterized with tetrapropylammonium (TPA+), revealing pH-dependent transport kinetics.
• SugE: Demonstrated functionality even after precipitation and refolding, with specific substrate interactions causing pre-steady state currents.
• LacY: Showed distinct transport kinetics for lactose, lactulose, and melibiose.
• NhaA: Thermostabilized variant showed improved stability and activity.
• AAC2: Both steady-state and pre-steady state currents were measured, providing detailed mechanistic insights.

The developed workflow provides a robust, efficient, and cost-effective method for the direct functional assessment of transporter proteins. This novel approach can be executed within five days and offers significant advantages for applications in medical and biotechnological research, facilitating the study of transporters that are otherwise difficult to characterize using traditional methods.

Clearly, the integration of cell-free protein synthesis and solid supported membrane-based electrophysiology represents a significant advancement in the field of transporter research, providing a powerful tool for future studies.

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Find the original article here: https://www.sciencedirect.com/science/article/pii/S156753942400094X

Learn more about Solid Supported Membrane-Based Electrophysiology and SURFE²R devices here: https://www.nanion.de/products/surfe2r-n1/