Calmodulin interaction with hEAG1 visualized by FRET microscopy.
BACKGROUND
Ca(2+)-mediated regulation of ion channels provides a link between intracellular signaling pathways and membrane electrical activity. Intracellular Ca(2+) inhibits the voltage-gated potassium channel EAG1 through the direct binding of calmodulin (CaM). Three CaM binding sites (BD-C1: 674-683, BD-C2: 711-721, BD-N: 151-165) have been identified in a peptide screen and were proposed to mediate binding. The participation of the three sites in CaM binding to the native channel, however, remains unclear.


RESULTS
Here we studied the binding of Ca(2+)/CaM to the EAG channel by visualizing the interaction between YFP-labeled CaM and Cerulean-labeled hEAG1 in mammalian cells by FRET. The results of our cellular approach substantiate that two CaM binding sites are predominantly involved; the high-affinity 1-8-14 based CaM binding domain in the N-terminus and the second C-terminal binding domain BD-C2. Mutations at these sites completely abolished CaM binding to hEAG1.


CONCLUSIONS
We demonstrated that the BD-N and BD-C2 binding domains are sufficient for CaM binding to the native channel, and, therefore, that BD-C1 is unable to bind CaM independently.
Gonçalves
J Tiago
JT
Molecular Biology of Neuronal Signals, Max-Planck Institute for Experimental Medicine, Göttingen, Germany. goncalves@ucla.edu
Stühmer
Walter
W
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001681:Biological Assay; D002147:Calmodulin / Q000378:metabolism*; D051638:Ether-A-Go-Go Potassium Channels / Q000737:chemistry / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008853:Microscopy / Q000379:methods*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D017434:Protein Structure, Tertiary; D051381:Rats
D002147:Calmodulin; D051638:Ether-A-Go-Go Potassium Channels; C496884:KCNH1 protein, human