Visualization of the Ca2+-dependent regulation of voltage-gated Ether-à-go-go channels by FRET microscopy
von Jose Tiago Goncalves
Datum der mündl. Prüfung:2006-07-03
Betreuer:Prof. Dr. Walter Stühmer
Gutachter:Prof. Dr. Erwin Neher
Gutachter:Prof. Dr. Fred Wouters
EnglischSeveral studies have suggested a role for EAG channels in the regulation of neuronal excitability, possibly by contributing to the maintenance of the resting membrane potential. EAG channels mediate inward-rectifying currents with activation close to the membrane resting potential which would be compatible with such a physiological function. Knowledge on the regulation of the activity of this kind of channels is therefore potentially important for the study of neuronal signaling.EAG1 has been known to be inhibited by intracellular Ca2+ with a half maximal concentration of 100 nM and this inhibition has been shown to be mediated by direct binding of calmodulin (CaM) to the C-terminal domain (aa. 707-726) of the channel. The regulation of EAG activity was studied by visualizing its interaction with CaM, using an intermolecular FRET approach. FRET was measured between YFP-labeled CaM and several Cerulean-labeled EAG constructs. The fusion-proteins were expressed in HEK cells and the interaction was found to be Ca2+-dependent as the FRET efficiency decreases with increasing Ca2+ concentrations and is abolished when using a Ca2+-insensitive CaM mutant.Mutations in the previously described CaM binding site at aa. 707-726 were found to prevent the binding of CaM to the C-terminus domain and consequently to prevent the inhibition of hEAG1 by CaM. The FRET assay described in the present study found that the interaction of the C-term-rEAG1 with CaM could be fully inhibited by Ca2+ depletion, whereas the same treatment merely reduced the interaction of CaM with the full-length hEAG1. This interaction was found to be accounted for by the presence of a further, high affinity CaM binding domain in the N-terminus of the channel at aa. 151-164.In conclusion the present results show that two CaM binding sites are present in hEAG1 subunits - a high-afinity CaM binding domain of the form 1-8-14 in the N-terminus and the previously described C-terminal binding domain at aa. 707- 726. Mutations at these sites lead to the complete disruption of CaM binding to the hEAG1, these two binding domains are therefore responsible for CaM binding. These findings indicate that the regulation of hEAG1 by Ca2+ /CaM is more complex than previously thought.
Keywords: EAG; Ether-à-go-go; Calcium; voltage-gated; FRET; photoconversion; YFP; synapse