Visualization of the Ca2+-dependent regulation of voltage-gated Ether-à-go-go channels by FRET microscopy

Goncalves, Jose Tiago

dc.contributor.advisor	Stühmer, Walter Prof. Dr.	de
dc.contributor.author	Goncalves, Jose Tiago	de
dc.date.accessioned	2006-08-22T06:48:28Z	de
dc.date.accessioned	2013-01-18T14:25:00Z	de
dc.date.available	2013-01-30T23:51:13Z	de
dc.date.issued	2006-08-22	de
dc.identifier.uri	http://hdl.handle.net/11858/00-1735-0000-0006-B362-1	de
dc.identifier.uri	http://dx.doi.org/10.53846/goediss-3227
dc.format.mimetype	application/pdf	de
dc.language.iso	eng	de
dc.rights.uri	http://webdoc.sub.gwdg.de/diss/copyr_diss.html	de
dc.title	Visualization of the Ca2+-dependent regulation of voltage-gated Ether-à-go-go channels by FRET microscopy	de
dc.type	doctoralThesis	de
dc.title.translated		de
dc.contributor.referee	Neher, Erwin Prof. Dr.	de
dc.date.examination	2006-07-03	de
dc.subject.dnb	570 Biowissenschaften	de
dc.subject.dnb	Biologie	de
dc.description.abstracteng	Several 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.	de
dc.contributor.coReferee	Wouters, Fred Prof. Dr.	de
dc.subject.topic	Molecular Biology & Neurosciences Program	de
dc.subject.ger		de
dc.subject.eng	EAG	de
dc.subject.eng	Ether-à-go-go	de
dc.subject.eng	Calcium	de
dc.subject.eng	voltage-gated	de
dc.subject.eng	FRET	de
dc.subject.eng	photoconversion	de
dc.subject.eng	YFP	de
dc.subject.eng	synapse	de
dc.subject.bk	42.63	de
dc.identifier.urn	urn:nbn:de:gbv:7-webdoc-1275-6	de
dc.identifier.purl	webdoc-1275	de
dc.affiliation.institute	Göttinger Graduiertenschule für Neurowissenschaften und molekulare Biowissenschaften (GGNB)	de
dc.subject.gokfull	MED 311: Physiologie {Medizin}	de
dc.identifier.ppn	610580892	de

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Fakultät für Biologie und Psychologie (inkl. GAUSS) [1621]

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