SSM-based Electrophysiology: We are investigating a number of membrane proteins that perform active transport of charged substrates over the cell membrane. For this purpose we investigate the charge translocation by these proteins using solid supported membrane based electrophysiology (SSM-based electrophysiology). The electrophysiological techniques are supplemented by spectroscopic methods. In order to assess the contribution of single amino acid residues to the transport process, functional analysis of mutants of the transport proteins is performed.
The systems currently under investigation are: Na+/melibiose -cotransporter (melB), lactose permease LacY, bacterial Na+/H+ exchanger (NhaA), human Na+/H+ exchanger hsNHA2, mammalian Cl-/H+ exchanger ClC7, bacterial Cl-/H+ exchanger ecClC. These are transporters from the bacterial plasma membrane or from the membranes of intracellular compartments which are difficult to investigate using conventional electrophysiology (patch-clamp or voltage-clamp techniques).
For the investigation we use reconstituted systems (protein reconstituted in liposomes) or membrane fragment preparations. The advantage of a purified preparation is obvious: no interaction with intracellular components or other membrane proteins, a well controlled lipid environment and a well defined aqueous phase. Membrane fragments are more easily prepared and allow the investigation of the transport protein in its natural environment.
The solid supported membrane consists of an alkanethiol monolayer (Thiol) with a lipid monolayer (PC) on top. Proteoliposomes containing the transport protein under investigation are adsorbed to this surface. The transport protein (in the figure the Na+/proline cotransporter putP) is activated via a rapid solution exchange at the solid supported membrane. Transient currents corresponding to the electrogenic translocation of the substrates are measured. In the figure proline concentration jumps of 0.5 to 150 mM in the presence of 100 mM Na+ are shown.
Drug screening. The SSM is a robust platform which allows parallelization and automation of functional transporter testing. It is, therefore, well suited for the screening of pharmacologically interesting compounds. In a project supported by the German Ministry for Education and Research (BMBF) we develop and apply assays for rapid screening of compounds that modulate the function of the glucose transporter SGLT.
Gaiko, O., Janausch, I., Geibel, S., Vollert, H., Arndt, P., Gonski, S., and Fendler, K. (2011) Robust Electrophysiological Assays using Solid Supported Membranes: the Organic Cation Transporter OCT2, Australian Journal of Chemistry 64, 31-35.
Ganea, C., Meyer-Lipp, K., Lemonnier, R., Krah, A., Leblanc, G., and Fendler, K. (2011) G117C MelB, a mutant melibiose permease with a changed conformational equilibrium, Biochim Biophys Acta 1808, 2508-2516.
Mager, T., Rimon, A., Padan, E., and Fendler, K. (2011) Transport mechanism and pH regulation of the Na+/H+ antiporter NhaA from Escherichia coli: An electrophysiological study, J Biol Chem 286, 23570-23581.
Schulz, P., Werner, J., Stauber, T., Henriksen, K., and Fendler, K. (2010) The G215R mutation in the Cl-/H+-antiporter ClC-7 found in ADO II osteopetrosis does not abolish function but causes a severe trafficking defect, PLoS One 5, e12585.
PhD and Diploma thesis:
Garcia-Celma, J. J. (2009) Electrophysiological Characterization of Cation Coupled Symporters and Investigation of Physicochemical Surface Processes with a Solid-Supported Membrane, Johann Wolfgang Goethe-University, Frankfurt/Main, Germany.
Prof. Dr. Klaus Fendler
Abteilung für Biophysikalische Chemie
Tel.: +49 (0) 69 6303-2035
Fax: +49 (0) 69 6303-2002