Inhibition of human ether a go-go potassium channels by Ca super(2+)-calmodulin binding to the cytosolic N- and C-termini

• Published in: The FEBS journal 2006-03, Vol.273 (5), p.1074-1086
• Main Authors: Ziechner, Ulrike, Schoenherr, Roland, Born, Anne-Kathrin, Gavrilova-Ruch, Oxana, Glaser, Ralf W, Malesevic, Miroslav, Kuellertz, Gerhard, Heinemann, Stefan H
• Format: Article
• Language: English
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/j.1742-4658.2006.05134.x

Human ether a go-go potassium channels (hEAG1) open in response to membrane depolarization and they are inhibited by Ca super(2+)-calmodulin (CaM), presumably binding to the C-terminal domain of the channel subunits. Deletion of the cytosolic N-terminal domain resulted in complete abolition of Ca super(2+)-CaM sensitivity suggesting the existence of further CaM binding sites. A peptide array-based screen of the entire cytosolic protein of hEAG1 identified three putative CaM-binding domains, two in the C-terminus (BD-C1: 674-683, BD-C2: 711-721) and one in the N-terminus (BD-N: 151-165). Binding of GST-fusion proteins to Ca super(2+)-CaM was assayed with fluorescence correlation spectroscopy, surface plasmon resonance spectroscopy and precipitation assays. In the presence of Ca super(2+), BD-N and BD-C2 provided dissociation constants in the nanomolar range, BD-C1 bound with lower affinity. Mutations in the binding domains reduced inhibition of the functional channels by Ca super(2+)-CaM. Employment of CaM-EF-hand mutants showed that CaM binding to the N- and C-terminus are primarily dependent on EF-hand motifs 3 and 4. Hence, closure of EAG channels presumably requires the binding of multiple CaM molecules in a manner more complex than previously assumed.