Inhibition of human ether à go-go potassium channels by Ca(2+)/calmodulin.
Intracellular Ca(2+) inhibits voltage-gated potassium channels of the ether à go-go (EAG) family. To identify the underlying molecular mechanism, we expressed the human version hEAG1 in Xenopus oocytes. The channels lost Ca(2+) sensitivity when measured in cell-free membrane patches. However, Ca(2+) 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 Ca(2+). 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 Ca(2+)-activated potassium channels, Ca(2+) 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.
Schönherr
R
R
Arbeitsgruppe Molekulare und zelluläre Biophysik, am Klinikum der Friedrich-Schiller-Universität Jena, Drackendorfer Strabetae 1, D-07747 Jena, Germany.
Löber
K
K
Heinemann
S H
SH
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D002147:Calmodulin / Q000378:metabolism / Q000494:pharmacology*; D027682:Cation Transport Proteins; D004268:DNA-Binding Proteins; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; D006801:Humans; D015640:Ion Channel Gating; D008969:Molecular Sequence Data; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000737:chemistry / Q000378:metabolism; D024642:Potassium Channels, Voltage-Gated; D011485:Protein Binding; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators; D000071230:Transcriptional Regulator ERG; D014982:Xenopus laevis
D002147:Calmodulin; D027682:Cation Transport Proteins; D004268:DNA-Binding Proteins; C072312:ERG protein, human; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; C496884:KCNH1 protein, human; C092509:KCNH6 protein, human; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D011994:Recombinant Proteins; D015534:Trans-Activators; D000071230:Transcriptional Regulator ERG; D002118:Calcium