N-terminal arginines modulate plasma-membrane localization of Kv7.1/KCNE1 channel complexes

PLoS One. 2011;6(11):e26967. doi: 10.1371/journal.pone.0026967. Epub 2011 Nov 4.

Abstract

Background and objective: The slow delayed rectifier current (I(Ks)) is important for cardiac action potential termination. The underlying channel is composed of Kv7.1 α-subunits and KCNE1 β-subunits. While most evidence suggests a role of KCNE1 transmembrane domain and C-terminus for the interaction, the N-terminal KCNE1 polymorphism 38G is associated with reduced I(Ks) and atrial fibrillation (a human arrhythmia). Structure-function relationship of the KCNE1 N-terminus for I(Ks) modulation is poorly understood and was subject of this study.

Methods: We studied N-terminal KCNE1 constructs disrupting structurally important positively charged amino-acids (arginines) at positions 32, 33, 36 as well as KCNE1 constructs that modify position 38 including an N-terminal truncation mutation. Experimental procedures included molecular cloning, patch-clamp recording, protein biochemistry, real-time-PCR and confocal microscopy.

Results: All KCNE1 constructs physically interacted with Kv7.1. I(Ks) resulting from co-expression of Kv7.1 with non-atrial fibrillation '38S' was greater than with any other construct. Ionic currents resulting from co-transfection of a KCNE1 mutant with arginine substitutions ('38G-3xA') were comparable to currents evoked from cells transfected with an N-terminally truncated KCNE1-construct ('Δ1-38'). Western-blots from plasma-membrane preparations and confocal images consistently showed a greater amount of Kv7.1 protein at the plasma-membrane in cells co-transfected with the non-atrial fibrillation KCNE1-38S than with any other construct.

Conclusions: The results of our study indicate that N-terminal arginines in positions 32, 33, 36 of KCNE1 are important for reconstitution of I(Ks). Furthermore, our results hint towards a role of these N-terminal amino-acids in membrane representation of the delayed rectifier channel complex.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Arginine / physiology*
  • Cell Membrane / metabolism
  • Fluorescent Antibody Technique
  • Humans
  • KCNQ1 Potassium Channel / chemistry
  • KCNQ1 Potassium Channel / metabolism*
  • Microscopy, Confocal
  • Molecular Sequence Data
  • RNA, Messenger / genetics
  • Real-Time Polymerase Chain Reaction
  • Sequence Homology, Amino Acid
  • Transcription, Genetic

Substances

  • KCNQ1 Potassium Channel
  • KCNQ1 protein, human
  • RNA, Messenger
  • Arginine