Inhibition of human ether a go-go potassium channels by Ca2+/calmodulin

Abstract

Intracellular Ca2+ inhibits voltage-gated potassium channels of the ether a go-go (EAG) family. To identify the underlying molecular mechanism, we expressed the human version hEAG1 in Xenopus oocytes. The channels lost Ca2+ sensitivity when measured in cell-free membrane patches. However, Ca2+ sensitivity could be restored by application of recombinant calmodulin (CaM). In the presence of CaM, half inhibition of hEAG1 channels was obtained in 100 nM Ca2+. Overlay assays using labelled CaM and glutathione S-transferase (GST) fusion fragments of hEAG1 demonstrated direct binding of CaM to a C-terminal domain (hEAG1 amino acids 673-770). Point mutations within this section revealed a novel CaM-binding domain putatively forming an amphipathic helix with both sides being important for binding. The binding of CaM to hEAG1 is, in contrast to Ca2+-activated potassium channels, Ca2+ dependent, with an apparent K(D) of 480 nM. Co-expression experiments of wild-type and mutant channels revealed that the binding of one CaM molecule per channel complex is sufficient for channel inhibition.