Thiel
Plant Membrane Biophysics
Prof. Dr. Gerhard Thiel
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Research
Structure function relations of potassium-channels
The main focus of the lab is to investigate structure/function correlates in simple K+-channel proteins. Together with the group of Anna Moroni (Milano, Italy) and Jim Van Etten (Lincoln, USA) we have cloned and functionally expressed a K+-channel, Kcv, from the Chlorella virus PBVC-1. This channel, Kcv, has the advantage of being with only 94 amino acids truly minimal. That is it consists of the two transmembrane domains a pore loop with minimal N and C termini and small extracellular loops; like other K+-channels it forms functional teteramers (see Fig. 1). This miniature viral K+-channel serves as ideal model system to examine basic architectural principles of K+-channel proteins because:
- Kcv has many functional properties, which are similar to other more complex K+-channels. This includes cation selectivity, sensitivity to voltage and susceptibility to blockers
- other than crystallized bacterial channels Kcv and its mutants are easy to express in heterologous systems such Xenopus oocytes, mammalian cell lines and bilayers
- A large collection of channel orthologs with interesting structural and functional deviations from the reference channel Kcv is available
The current work on structure/function correlates in Kcv relies on a combination of complementary experimental and computational methods. Figure 1. Molecular dynamic model of Kcv (Sascha Tayefeh, AG Kast)Biochemical and structural aspects of the protein are investigated in our lab and the Moroni lab. In the center of this work is the electrophysiological and biochemical characterization of channel mutants and orthologs from other viruses. Molecular dynamic simulations complement interpretation of these data on the atomic level of the protein structure. The group of Stefan Kast has generated a robust model of the Kcv protein for computational studies (Fig. 1).
The function of channels in viral infection and replication
With the discovery of viral K+ channels we started to examine the role of these proteins in the life cycle of the virus. This work combines our background on classical plant physiology with the excellent virology in the lab of our partner Jim Van Etten at ULN. A bulk of data supports the Figure2 Presense of Bloggercurrent hypothesis that the presence of the K+-channel in the virus particle is obligatory for infection. One of the first steps of infection is a membrane depolarization of the host, which is generated by the insertion of the viral channel into the electrical continuum of the host plasma membrane. The depolarization results in a loss of K+ from the host; this generates a driving force for water efflux. Altogether this lowers the turgor pressure in the host and decreases the energetic barrier for the ejection of the large viral dsDNA genome into the host. If the viral K+-channel is blocked for example Ba2+ stops the infection at an early state and DNA cannot be ejected from the particle (Fig. 2).
Radiation biology
A new entry to the lab is the work on cellular responses to heavy ion radiation. The group of Gerhard Kraft at the GSI (Gesellschaft für Schwerionenforschung) close to Darmstadt has established a successful tumor therapy on the basis of heavy ion radiation. In this context there are still many open questions on the cellular responses to this form of radiation. In collaboration with our partners at the GSI we use our experience in fluorescence imaging to examine radiation triggered signal transduction cascades.
Elementary mechanism of exo- and endocytosis in plants
These processes are studied in single plant cell protoplasts by electrophysiological techniques of patch-clamping, where the exo- and endocytotic activity is monitored by changes in membrane capacitance. The regulation of these processes is examined by altering key cellular parameters such as the mechanic forces in the plasma membrane, protein synthesis and the cytosolic calcium activity. The latter is accomplished by flash photolysis of caged compounds and measured ratiometrically using fura-2 as Ca2+-sensor.
Methods
Patch-Clamp Methods
Single channel recording
Whole-cell patch-clamp recording
Cell membrane capacitance measurements with two-phase lock-in amplifier
Reconstruction of macroscopic electrical cell parameters (membrane capacitance and conductance)
High resolution measurements of unitary capacitance changes
Conventional electrophysiological methods
Voltage recordings
Two electerode voltage-clamp
Action-potential-clamp
Fluorescence-based measurements of intracellular ion (Ca2+, pH etc.) activities
Flash photolysis
Microinjection methods
Fluorescence Microscopy
Confocal Microscopy