Research Interests

 

Structural Movements in Potassium Channel Gating

Methods

Electrophysiological Measurements of Ion Channels in Giant Membrane Patches

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As ion flow through channel proteins in a membrane gives rise to electrical currents, ion channel function can be studied by amplifying the tiny currents flowing between an electrode in a glass pipette, representing one side of the membrane, and a second electrode in the bath, constituting the other side of the membrane. 

 

 

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In order to generate such measurable currents, we need to achieve high expression levels of ion channels at the plasma membrane. Therefore, ion channels are heterologously expressed in Xenopus oocytes by microinjection of in vitro transcribed cRNA. The Xenopus oocyte expression system has a high protein synthesis capacity and is easily manageable because of the large oocyte diameter.
 

 

 

 

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After an incubation period, the cleared oocyte plasma membrane is approached with a polished wide-bore glass pipette, forming an electric gigaseal between membrane lipid and glass. With the lipid bilayer as insulator, current now can only flow from the pipette to the bath electrode through our channels. Giant membrane patches can be excised, exposing to the bath the intracellular face of the channels („inside-out“ configuration) or reforming at the pipette tip to the „outside-out“ configuration.
 

 

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In so-called voltage-clamp mode, the membrane potential can be controlled and varied according to specific protocols, enabling us to monitor the current response generated by the opening or closing of ion channels according to voltage or other stimuli like activators or blockers.
 

 

 

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These isolated patches are examined by rapid solution exchanges in a piezo-driven application system, which allows us to resolve kinetics on a millisecond timescale, determine on- and off-rates and measure binding affinities of ligands or pharmacological substances. We are also capable of protein modification with silver and cysteine-reactive reagents to analyze state-dependent structural changes. The patch clamp technique can also be carried out with cultured cells transfected with the ion channel DNA of interest to study channels in a native mammalian membrane environment.
 


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For measurements of extracellular modulators or for channel proteins generating only small currents, patch-clamp recordings using whole Xenopus oocytes are carried out in a two-electrode voltage clamp setup (TEVC). Two sharp glass pipettes with electrodes are inserted into the oocyte, one to control plasma membrane potential according to the experimental protocol, the second to record the currents in response to the membrane voltage steps. Extracellular solutions can be exchanged via a gravity-flow application system. As manipulation of whole oocytes in this setup is generally faster to manage, the setup is a good choice for screening or bachelor and master projects.
 

 

 

Zwei Studenten und eine Tasse KaffeePatch-clamp Fluorimetry

The expansion of the genetic code can be utilized for site-specific incorporation of an non-natural fluorescent amino acid by translating the ‚amber‘ stop codon with an orthogonal tRNA(CUA)/ tRNA-synthetase pair. This makes it possible to insert a fluorescent marker at certain positions in the tertiary structure of an ion channel, where we e.g. expect structural changes in case of gating events. The fluorescence readout of a very fast camera correlated with simultaneously recorded current traces provides information about alterations in the chemical environment of the fluorophore when a specific voltage protocol is applied, showing movements and distance changes in protein structure.
 

 

Zwei Studenten und eine Tasse KaffeePlanar patch-clamp for whole-cell measurements & artificial bilayer environments

In the planar patch-clamp setup, the glass pipette is replaced by a planar glass chip with a small aperture, on which the gigaseal is formed either by a cell membrane or by an artificial lipid bilayer. After seal formation, the cell membrane is disrupted to gain access to the intracellular space; alternatively, proteins are reconstituted in the artificial bilayer. As in the pipette setup, intra- or extracellular solutions can be exchanged by a gravity flow application system. These setups are capable of single-channel measurements. 
 


Molecular Biology, Cell culture and Protein Biochemistry Lab

Besides the electrophysiology labs, our group operates a molecular biology standard lab for procedures like cloning and site-directed mutagenesis, a cell culture lab for cell transfection and fluorescence microscopy, and a protein biochemistry lab for cell-free expression of ion channels and small-scale protein purification. In addition, we have access to a confocal laser scanning microscope to visualize fluorescently labeled cells or fluorescent fusion proteins.

Pharmacological Modulation of K2P Channel Gating

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Synthetic Modification of Potassium Channels

Potassium Channels: K2P, Kv, Kir Channels
Structure-Function Relationship in K+-Channels
Pharmacological Regulation of Ion Channels
Regulation of Ion Channels by Membrane Lipids
Artificially Modified Ion Channels

Electrophysiological Measurements of Ion Channels in Giant Membrane Patches

As ion flow through channel proteins in a membrane gives rise to electrical currents, ion channel function can be studied by amplifying the tiny currents flowing between an electrode in a glass pipette, representing one side of the membrane, and a second electrode in the bath, constituting the other side of the membrane. Membrane potential can be controlled during the experiments by injecting current through this pipette.
To achieve high channel expression corresponding to high currents at the plasma membrane, ion channels are heterologously expressed in Xenopus oocytes by microinjection of in-vitro transcribed cRNA. After an incubation period, the cleared oocyte plasma membrane is approached with a polished wide-bore glass pipette, forming an electric gigaseal between membrane lipid and glass. Giant membrane patches can be excised, exposing to the bath the intracellular face of the channels (inside-out configuration) or reforming at the pipette tip in outside-out configuration. 
These patches can now be examined by rapid solution exchanges in a piezo-driven application system, which allows us to resolve kinetics on a millisecond timescale, determine on- and off-rates and precisely measure binding affinities of blockers, ligands or pharmacological substances. We are also capable of fast protein modification with silver and cysteine-reactive reagents to analyze state-dependent structural changes.
The patch clamp technique can also be carried out with whole cultured cells transfected with the ion channel of interest to study channels in a mammalian membrane environment.

TEVC

For measurements of extracellular modulators or for channel proteins generating only small currents, patch-clamp recordings using whole Xenopus oocytes are carried out in a two-electrode voltage clamp setup. Two sharp glass pipettes with electrodes are inserted into the oocyte, one to control plasma membrane potential according to the experimental protocol, the second to record the currents in response to the membrane voltage steps. Extracellular solution is exchanged via a gravity-flow application system. As manipulation of whole oocytes in this setup is generally faster to handle, the setup is a good choice for screening or bachelor and master projects.


Patch-clamp fluorimetry

The expansion of the genetic code can be utilized for site-specific incorporation of an non-natural fluorescent amino acid by translating the ‚amber‘ stop codon with an orthogonal tRNA(CUA)/ tRNA-synthetase pair. This enables us to insert a fluorescent marker at certain positions in the tertiary structure of an ion channel, where we suspect structural changes in case of gating events. The fluorescence readout of a very fast camera correlated with simultaneously recorded current traces provides information about alterations in the chemical environment of the fluorophore when e.g. a specific voltage protocol is applied, showing movements and distance changes in protein structure.


Planar patch-clamp for whole-cell measurements & artificial bilayer environments

In the planar patch-clamp setup, the glass pipette is replaced by a planar glass chip with a small aperture, on which the gigaseal is formed either by a cell membrane or by an artificial lipid bilayer. After seal formation, the cell membrane is disrupted to gain access to the intracellular space; alternatively, purified proteins is reconstituted in the artificial bilayer. As in the pipette setup, intra- or extracellular solutions can be exchanged by a gravity flow application system. As noise levels are quite low, these setups are capable of single-channel measurements. 

Molecular Biology, Cell culture and Protein Biochemistry Lab

Besides the electrophysiology labs, our group operates a molecular biology standard lab for procedures like cloning and site-directed mutagenesis, a cell culture lab for cell transfection and fluorescence microscopy, and a protein biochemistry lab for cell-free expression of ion channels and small-scale protein purification. In addition, we have access to confocal laser scanning microscope to visualize fluorescently labeled cells or fluorescent fusion proteins.