4 YEAR PhD IN NEUROSCIENCE
Laboratory of Molecular Pharmacology
Molecular pharmacology and physiology of potassium channels
We study several aspects of potassium channel molecular physiology, combining molecular biology with electrophysiology, histochemistry and biochemistry. In particular, we investigate the mechanisms responsible for targeting, clustering and regulation of calcium-activated potassium channels to understand how these channels influence the signal processing of neurons in the central nervous system. A second line of research concerns the structural and functional properties of the modulatory potassium channel subunits, which comprise one-fourth of all voltage-gated K+ channels. The aim is to understand the physiological relevance of these ion channels in the heart and the central nervous system. Finally, we are screening and characterizing novel drugs and toxins acting as highly selective blockers or openers of potassium channels. These molecules are invaluable tools to define the function of different potassium channels.
The following projects are starting points and can be expanded and developed into a PhD.
Project A: Studying ion channels by suppression strategies.
In this project you will generate potassium channel constructs, which can be used
to study their function by suppression strategies. The structure of potassium channels
makes it difficult to generate antibodies, which bind to the extracellular part
of the channel. To overcome this problem, we plan to modify a calcium-activated
potassium channel subunit in such a way that it can be recognised by commercially
available antibodies, without changing the functionality of the channel. Furthermore,
we intend to fuse SK channel subunits with the green fluorescent protein, which
is a well known strategy to visualise proteins without antibodies. After fusing
the two proteins together, we will express the construct in HEK293 cells and compare
its distribution with untagged SK channels. You will learn various techniques of
molecular biology (PCRs, cloning, sequencing, etc.). To analyse the generated constructs,
you will learn how to culture cells and to introduce your constructs into these
cells, perform immunocytochemistry, and use conventional and confocal microscopy.
Project B: Stoichiometry of potassium channel analysed by fluorescence
resonance energy transfer (FRET).
Potassium channels are formed by the assembly of up to four a-subunits which can
be identical or different. Sometimes accessory proteins, which regulate channel
properties, assemble with the channel core. Fluorescence resonance energy transfer
(FRET) is one method to analyse the composition of these molecular complexes. You
will use FRET to study the subunit composition of an ion channel. Therefore you
will learn the necessary molecular biology to generate fluorescently labelled constructs
and their transfection into cells in culture. Using conventional immunofluorescence
you will control whether the generated constructs work. FRET analysis will be performed
at a confocal microscope. This project can evolve into a PhD project where these
constructs or similar ones will be used to address the question of how ion channels
assembly is regulated.
D'hoedt, D., K. Hirzel, P. Pedarzani & M. Stocker (2004)
Domain analysis of the calcium-activated potassium channel SK1 from rat brain: Functional expression and toxin sensitivity.
J. Biol. Chem. 279: 12088-12092.
M. Stocker (2004)
Ca2+-activated K+ channels: Molecular determinants and function of the SK-family
Nature Reviews Neuroscience 5: 758-770.
Stocker M., M. Krause and P. Pedarzani (1999)
An apamin-sensitive Ca2+-activated K+ current in hippocampal pyramidal neurons.
Proc. Natl. Acad. Sci. USA 96:4662-4667