calmodulin interaction with heag1 visualized by fret microscopy钙调蛋白与heag1交互可视化的显微镜.pdfVIP

Calmodulin Interaction with hEAG1 Visualized by FRET Microscopy 1 ¤ ¨ 1,2 J. Tiago Gonc¸alves * , Walter Stuhmer ¨ 1 Molecular Biology of Neuronal Signals, Max-Planck Institute for Experimental Medicine, Gottingen, Germany, 2 DFG Research Center for Molecular Physiology of the ¨ Brain, Gottingen, Germany Abstract Background: Ca2+-mediated regulation of ion channels provides a link between intracellular signaling pathways and membrane electrical activity. Intracellular Ca2+ 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. Methodology/Principal Findings: Here we studied the binding of Ca2+/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/Significance: 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. ¨ Citation: Gonc¸alves JT, Stuhmer W (2010) Calmodulin Interaction with hEAG1 Visual