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Department of Molecular Physiology

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patch clamp
Caged compounds: fast phototriggers for patch-clamp

 

Kinetic experiments with high time resolution can reveal much about ion channels. To do such experiments with a chemically gated channel (e.g. calcium- , cGMP-, or transmitter-gated channels) it is necessary to generate rapid jumps of the ligand concentration. The way how the channel responds to this jump can yield information about the gating mechanism and other aspects of channel function. How do you trigger concentration jumps of cGMP or calcium inside living cells? A solution is the use of caged compounds (caged cGMP or caged Ca). In a caged compound, the biologically active molecule is coupled to a "cage", a residue that prevents bioaction but can be cleaved off by a flash of UV light. Cells can be loaded with such a caged compound through the patch-clamp micropipette (upper left). If for example caged cGMP is loaded into a cell that expresses cGMP-gated channels, a kinetic experiment can be started by directing a UV flash onto the loaded cell. This releases cGMP within less than a microsecond and opens the channel. The electrode within the micropipette then records the resulting current (lower left) which can be analyzed at high time resolution. For experiments with caged compounds, light from a UV-flash lamp is fed into the microscope through a light guide and focussed on the test cell through the objective lens (right).

  


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