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Inhibition of human ether à go‐go potassium channels by Ca 2+ /calmodulin binding to the cytosolic N‐ and C‐termini
Author(s) -
Ziechner Ulrike,
Schönherr Roland,
Born AnneKathrin,
GavrilovaRuch Oxana,
Glaser Ralf W,
Malesevic Miroslav,
Küllertz Gerhard,
Heinemann Stefan H
Publication year - 2006
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2006.05134.x
Subject(s) - calmodulin , cytosol , chemistry , potassium channel , biophysics , c terminus , binding site , binding domain , depolarization , n terminus , biochemistry , biology , peptide sequence , amino acid , enzyme , gene
Human ether à go‐go potassium channels (hEAG1) open in response to membrane depolarization and they are inhibited by Ca 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 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 2+ /CaM was assayed with fluorescence correlation spectroscopy, surface plasmon resonance spectroscopy and precipitation assays. In the presence of Ca 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 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.