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Research Profile Prof. Dr. Klaus Fendler

Prof. Dr. Klaus Fendler
  • Dr. rer. nat. (Solid State Physics) University of Konstanz, 1983
  • Habilitation (Biophysical Chemistry) University of Frankfurt, 1995
  • Adjunct Professor, University of Frankfurt, 2003
  • At the Institute since 1983

 

 

 

Transport mechanism of ion translocating membrane proteins

The solid supported membrane consists of an alkanethiol monolayer (Thiol) with a lipid monolayer (PC) on top.

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.

 

SSM cuvette designed for rapid solution exchange.

Rapid solution exchange: Time-resolved information is important for the investigation of the transport mechanism. We have, therefore, developed a rapid solution exchange SSM system which allows solution exchange at the surface of the SSM within 2 ms.

96-well plate for SSM-based electrophysiology

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.

The team

Selected Publications:

Garcia-Celma, J. J., Smirnova, I. N., Kaback, H. R., and Fendler, K. (2009) Electrophysiological characterization of LacY, Proc Natl Acad Sci U S A 106, 7373-7378.

Krause, R., Watzke, N., Kelety, B., Dorner, W., and Fendler, K. (2009) An automatic electrophysiological assay for the neuronal glutamate transporter mEAAC1, J Neurosci Methods 177, 131-141.

Schulz, P., Dueck, B., Mourot, A., Hatahet, L., and Fendler, K. (2009) Measuring ion channels on solid supported membranes, Biophys J 97, 388-396.

Garcia-Celma, J. J., Ploch, J., Smirnova, I., Kaback, H. R., and Fendler, K. (2010) Delineating electrogenic reactions during lactose/H+ symport, Biochemistry 49, 6115-6121.

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.

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, Biochimica et Biophysica Acta (BBA) - Biomembranes In Press, Uncorrected Proof.

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.

PhD and Diploma thesis:

Al-Tamari, H. (2009) Cloning, expression and Electrophysiological Characterization of the Human Na+/H+ Exchanger (HsNha2), Justus Liebig Universität, Giessen.

Diekmann, M. (2009) Funktionelle Chrarakterisierung des Natrium/Protonen-Austauschers NHA2 des Menschen, Universität Osnabrück, Osnabrück.

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.

Moritz, A. (2009) Transportmessungen am humanen Natrium/Protonen Austauscher NHA2, Fachhochschule Bingen, Bingen.

Werner, J. (2009) Molekularbiologische Untersuchungen am elektrogenen ClC-7-Transporter, Hochschule Mannheim, Mannheim.

Kleinschmidt, U. (2010) Transporter-Assays auf festkörperunterstützten Membranen - Etablierung des Saccharomyces-Expressionssystems Hochschule Darmstadt, Darmstadt.

Krautwurst, E. (2010) Elektrophysiologische Untersuchungen zum Transportmechanismus der Lactose Permease LacY aus Escherischia coli, Fachhochschule Frankfurt, Frankfurt/Main.

Mamaeva, N. (2010) Elektrophysiologische Charakterisierung des bakteriellen Nitrittransporters NirC, Technische Universität Darmstadt, Darmstadt.

Schulz, P. (2010) Elektrophysiologische Charakterisierung des lysosomalen Cl-/H+-Antiporters ClC-7 mit Hilfe der SSM-Technik, Johann Wolfgang Goethe Universität, Frankfurt/Maín.

Bazzone, A. (2011) Transportmechanismen der Laktose Permease: Messungen unter asymmetrischen pH-Bedingungen, Johann Wolfgang Goethe Universität, Frankfurt/Main.

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Contact:

Max Planck Institute of Biophysics

Prof. Dr. Klaus Fendler
Department of Biophysical Chemistry

Phone: +49 (0) 69 6303-2035
Fax: +49 (0) 69 6303-2002

E-mail: fendler(at)biophys.mpg.de

Research Group:

Postdoc:

  • Christine Keipert
  • Slavoj Kresak
  • Patrick Schulz

Graduate students:

  • Thomas Mager
  • Octavian Calinescu

Diploma student:

  • Andre Bazzone

Technical assistant:

  • Lina Hatahet
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