Analysis of the interactions between the C-terminal cytoplasmic domains of KCNQ1 and KCNE1 channel subunits.
Ion channel subunits encoded by KCNQ1 and KCNE1 produce the slowly activating K+ current (IKs) that plays a central role in myocardial repolarization. The KCNQ1 alpha-subunit and the KCNE1 beta-subunit assemble with their membrane-spanning segments interacting, resulting in transformation of channel activation kinetics. We recently reported a functional interaction involving C-terminal portions of the two subunits with ensuing regulation of channel deactivation. In the present study, we provide evidence characterizing a physical interaction between the KCNQ1-CT (KCNE1 C-terminus) and the KCNE1-CT (KCNE1 C-terminus). When expressed in cultured cells, the KCNE1-CT co-localized with KCNQ1, co-immunoprecipitated with KCNQ1 and perturbed deactivation kinetics of the KCNQ1 currents. Purified KCNQ1-CT and KCNE1-CT physically interacted in pull-down experiments, indicating a direct association. Deletion analysis of KCNQ1-CT indicated that the KCNE1-CT binds to a KCNQ1 region just after the last transmembrane segment, but N-terminal to the tetramerization domain. SPR (surface plasmon resonance) corroborated the pull-down results, showing that the most proximal region (KCNQ1 amino acids 349-438) contributed most to the bimolecular interaction with a dissociation constant of approximately 4 microM. LQT (long QT) mutants of the KCNE1-CT, D76N and W87F, retained binding to the KCNQ1-CT with comparable affinity, indicating that these disease-causing mutations do not alter channel behaviour by disruption of the association. Several LQT mutations involving the KCNQ1-CT, however, showed various effects on KCNQ1/KCNE1 association. Our results indicate that the KCNQ1-CT and the KCNE1-CT comprise an independent interaction domain that may play a role in IKs channel regulation that is potentially affected in some LQTS (LQT syndrome) mutations.
Zheng
Renjian
R
Department of Medicine, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Thompson
Keith
K
Obeng-Gyimah
Edmond
E
Alessi
Dana
D
Chen
Jerri
J
Cheng
Huiyong
H
McDonald
Thomas V
TV
D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D015640:Ion Channel Gating; D051657:KCNQ1 Potassium Channel / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D024642:Potassium Channels, Voltage-Gated / Q000737:chemistry* / Q000378:metabolism*; D014162:Transfection
C489622:KCNE1 protein, human; D051657:KCNQ1 Potassium Channel; C496909:KCNQ1 protein, human; D024642:Potassium Channels, Voltage-Gated