Inward Rectifier Potassium Channels (Kir2.x) and Caveolin-3 Domain-Specific Interaction: Implications for Purkinje Cell-Dependent Ventricular Arrhythmias

Circ Arrhythm Electrophysiol. 2018 Jan;11(1):e005800. doi: 10.1161/CIRCEP.117.005800.

Abstract

Background: In human cardiac ventricle, IK1 is mainly comprised Kir2.1, but Kir2.2 and Kir2.3 heterotetramers occur and modulate IK1. Long-QT syndrome-9-associated CAV3 mutations cause decreased Kir2.1 current density, but Kir2.x heterotetramers have not been studied. Here, we determine the effect of long-QT syndrome-9-CAV3 mutation F97C on Kir2.x homo- and heterotetramers and model-associated arrhythmia mechanisms.

Methods and results: Super-resolution microscopy, co-immunoprecipitation, cellular electrophysiology, on-cell Western blotting, and simulation of Purkinje and ventricular myocyte mathematical models were used. Kir2.x isoforms have unique subcellular colocalization in human cardiomyocytes and coimmunoprecipitate with Cav3. F97C-Cav3 decreased peak inward Kir2.2 current density by 50% (-120 mV; P=0.019) and peak outward by 75% (-40 mV; P<0.05) but did not affect Kir2.3 current density. FRET (Förster resonance energy transfer) efficiency for Kir2.2 with Cav3 is high, and on-cell Western blotting demonstrates decreased Kir2.2 membrane expression with F97C-Cav3. Cav3-F97C reduced peak inward and outward current density of Kir2.2/Kir2.1 or Kir2.2/Kir2.3 heterotetramers (P<0.05). Only Cav3 scaffolding and membrane domains co-immunoprecipitation with Kir2.1 and Kir2.2 and Kir2.x-N-terminal Cav3 binding motifs are required for interaction. Mathematical Purkinje, but not ventricular, myocyte model incorporating simulated current reductions, predicts spontaneous delayed after-depolarization-mediated triggered activity.

Conclusions: Kir2.x isoforms have a unique intracellular pattern of distribution in association with specific Cav3 domains and that critically depends on interaction with N-terminal Kir2.x Cav3-binding motifs. Long-QT syndrome-9-CAV3 mutation differentially regulates current density and cell surface expression of Kir2.x homomeric and heteromeric channels. Mathematical Purkinje cell model incorporating experimental findings suggests delayed after-depolarization-type triggered activity as a possible arrhythmia mechanism.

Keywords: Purkinje cells; blotting, Western; immunoprecipitation; mutation; potassium channels.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Caveolin 3 / genetics*
  • Caveolin 3 / metabolism
  • Cells, Cultured
  • DNA / genetics*
  • DNA Mutational Analysis
  • Heart Ventricles / metabolism
  • Heart Ventricles / pathology
  • Humans
  • Membrane Potentials
  • Mutation*
  • Myocytes, Cardiac / metabolism*
  • Myocytes, Cardiac / pathology
  • Patch-Clamp Techniques
  • Polymerase Chain Reaction
  • Potassium Channels, Inwardly Rectifying / genetics*
  • Potassium Channels, Inwardly Rectifying / metabolism
  • Purkinje Cells / metabolism*
  • Purkinje Cells / pathology
  • Tachycardia, Ventricular / genetics*
  • Tachycardia, Ventricular / metabolism
  • Tachycardia, Ventricular / pathology

Substances

  • Caveolin 3
  • Potassium Channels, Inwardly Rectifying
  • DNA