Analysis of the interactions between the C-terminal cytoplasmic domains of KCNQ1 and KCNE1 channel subunits

Renjian Zheng, Keith Thompson, Edmond Obeng-Gyimah, Dana Alessi, Jerri Chen, Huiyong Cheng, Thomas V. McDonald

Research output: Contribution to journalArticle

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Abstract

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 α-subunit and the KCNE1 β-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) contributedmost to the bimolecular interactionwith a dissociation constant of ∼4 μM. 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.

Original languageEnglish (US)
Pages (from-to)75-84
Number of pages10
JournalBiochemical Journal
Volume428
Issue number1
DOIs
StatePublished - May 15 2010

Fingerprint

Association reactions
Mutation
Long QT Syndrome
Kinetics
Surface Plasmon Resonance
Surface plasmon resonance
Ion Channels
Cultured Cells
Chemical activation
Cells
Membranes
Amino Acids
Experiments

Keywords

  • Ion channel
  • KCNE1
  • KCNQ1
  • Long QT syndrome (LQTS)
  • Voltage-gated potassium channel

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology
  • Medicine(all)

Cite this

Zheng, R., Thompson, K., Obeng-Gyimah, E., Alessi, D., Chen, J., Cheng, H., & McDonald, T. V. (2010). Analysis of the interactions between the C-terminal cytoplasmic domains of KCNQ1 and KCNE1 channel subunits. Biochemical Journal, 428(1), 75-84. https://doi.org/10.1042/BJ20090977

Analysis of the interactions between the C-terminal cytoplasmic domains of KCNQ1 and KCNE1 channel subunits. / Zheng, Renjian; Thompson, Keith; Obeng-Gyimah, Edmond; Alessi, Dana; Chen, Jerri; Cheng, Huiyong; McDonald, Thomas V.

In: Biochemical Journal, Vol. 428, No. 1, 15.05.2010, p. 75-84.

Research output: Contribution to journalArticle

Zheng, R, Thompson, K, Obeng-Gyimah, E, Alessi, D, Chen, J, Cheng, H & McDonald, TV 2010, 'Analysis of the interactions between the C-terminal cytoplasmic domains of KCNQ1 and KCNE1 channel subunits', Biochemical Journal, vol. 428, no. 1, pp. 75-84. https://doi.org/10.1042/BJ20090977
Zheng, Renjian ; Thompson, Keith ; Obeng-Gyimah, Edmond ; Alessi, Dana ; Chen, Jerri ; Cheng, Huiyong ; McDonald, Thomas V. / Analysis of the interactions between the C-terminal cytoplasmic domains of KCNQ1 and KCNE1 channel subunits. In: Biochemical Journal. 2010 ; Vol. 428, No. 1. pp. 75-84.
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AB - 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 α-subunit and the KCNE1 β-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) contributedmost to the bimolecular interactionwith a dissociation constant of ∼4 μM. 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.

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