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FOR2143


Institute of Physiology
University of Freiburg
Hermann-Herder-Str. 7
79104 Freiburg
Germany
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Physiology I
N.N.

Physiology II
Prof. Bernd Fakler
Tel. +49 761 203-5176
Fax +49 761 203-5191
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  • We apply various configurations of the patch-clamp technique (whole-cell, cell-attached, excised inside-out, outside-out) to native and genetically manipulated cells and subcellular compartments. They enable us to monitor protein function and protein-protein interactions at high-resolution.
  • We use a large spectrum of biochemical techniques to detect and quantify membrane proteins (mainly ion channels and receptors), their post-translational modifications and association with other proteins in complexes and protein networks.
  • Modern mass spectrometers coupled with liquid chromatography enable us to identify several hundreds of proteins from complex samples with high confidence and sequence coverage. In addition, they provide quantitative data that let us determine stability, specificity and stoichiometry of protein-protein interactions as well as absolute protein abundance.
  • Nuclear magnetic resonance spectroscopy (NMR) provides information on structure and dynamics of biological macromolecules at atomic resolution under near-physiological conditions. We use it to examine proteins participating in the nano-environment of membrane proteins with regard to their 3D structure, mobility and interactions.
  • Using innovative microsystems, we work to enhance resolution and throughput of electrical recording of ionic currents. We develop biohybrid sensing devices based on single biological nanopores in membrane microarrays and study the interaction of natural and synthetic polymers with pore-forming membrane proteins.
  • To understand how neurons collectively process information, we develop optogenetic tools as well as new technologies for recordings from neurons in vivo and imaging of cell activity using photon Ca2+ and functional approaches. With computational network models we gain information on the principles underlying information processing in complex neuronal circuits.

Electrophysiology

Neuron Pip
AP-EPSC Trace
Postsynaptic current (EPSC) evoked by a presynaptic AP (paired recording)

We employ a variety of electrophysiological techniques to

  • characterize the molecular and biophysical properties of membrane proteins, ion channels, transporters and GPCRs (including conformational changes ("gating"), permeation and binding of ions and/or ligands),
  • analyze their interaction with auxiliary subunits and other associated proteins and
  • precisely monitor the time-course of protein-protein interactions and their significance for the signal transduction at the plasma membrane.

For these purposes, we use the following techniques and recording configurations:

  • patch-clamp recordings in whole-cell, and excised-patch (inside-out, outsice-out) configuration,
  • giant patch-clamp recordings (patch-pipettes with Ø of up to 20 µm)
  • two-electrode voltage- and current-clamp,
  • rapid piezo-driven application of agonists (ligand-controlled channels and receptors)
  • lock-in capacitance measurements (changes in membrane surface associated with exo- and endocytosis).

These techniques are applied on membrane proteins and protein complexes that are

  • heterologously expressed in culture-cells (CHO, HEK, COS) and Xenopus oocytes,
  • present in native cells, mostly CNS neurons in brain slices and/or neuronal cultures, before and after molecular and or genetic manipulations (eg. virally-driven overexpression of mutant protein, protein knock-down via sh-RNAs).
Fast Application
Fast Application
Fast Application Trace
Current through AMPA-type glutamate receptors evoked by 1-ms application of the agonist
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