Identification of a protein-protein interaction between KCNE1 and the activation gate machinery of KCNQ1.
KCNQ1 channels assemble with KCNE1 transmembrane (TM) peptides to form voltage-gated K(+) channel complexes with slow activation gate opening. The cytoplasmic C-terminal domain that abuts the KCNE1 TM segment has been implicated in regulating KCNQ1 gating, yet its interaction with KCNQ1 has not been described. Here, we identified a protein-protein interaction between the KCNE1 C-terminal domain and the KCNQ1 S6 activation gate and S4-S5 linker. Using cysteine cross-linking, we biochemically screened over 300 cysteine pairs in the KCNQ1-KCNE1 complex and identified three residues in KCNQ1 (H363C, P369C, and I257C) that formed disulfide bonds with cysteine residues in the KCNE1 C-terminal domain. Statistical analysis of cross-link efficiency showed that H363C preferentially reacted with KCNE1 residues H73C, S74C, and D76C, whereas P369C showed preference for only D76C. Electrophysiological investigation of the mutant K(+) channel complexes revealed that the KCNQ1 residue, H363C, formed cross-links not only with KCNE1 subunits, but also with neighboring KCNQ1 subunits in the complex. Cross-link formation involving the H363C residue was state dependent, primarily occurring when the KCNQ1-KCNE1 complex was closed. Based on these biochemical and electrophysiological data, we generated a closed-state model of the KCNQ1-KCNE1 cytoplasmic region where these protein-protein interactions are poised to slow activation gate opening.
Lvov
Anatoli
A
Department of Biochemistry and Molecular Pharmacology, Programs in Neuroscience and Chemical Biology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Gage
Steven D
SD
Berrios
Virla M
VM
Kobertz
William R
WR
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003545:Cysteine; D004220:Disulfides / Q000378:metabolism; D006801:Humans; D015640:Ion Channel Gating; D051657:KCNQ1 Potassium Channel / Q000235:genetics / Q000378:metabolism*; D008564:Membrane Potentials; D008958:Models, Molecular; D009154:Mutation; D011188:Potassium / Q000378:metabolism*; D024642:Potassium Channels, Voltage-Gated / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D013997:Time Factors; D014162:Transfection
D004220:Disulfides; C489622:KCNE1 protein, human; D051657:KCNQ1 Potassium Channel; C496909:KCNQ1 protein, human; D024642:Potassium Channels, Voltage-Gated; D003545:Cysteine; D011188:Potassium