Arrhythmogenic calmodulin mutations impede activation of small-conductance calcium-activated potassium current

Heart Rhythm. 2016 Aug;13(8):1716-23. doi: 10.1016/j.hrthm.2016.05.009. Epub 2016 May 7.

Abstract

Background: Apamin-sensitive small-conductance calcium-activated potassium (SK) channels are gated by intracellular Ca(2+) through a constitutive interaction with calmodulin.

Objective: We hypothesize that arrhythmogenic human calmodulin mutations impede activation of SK channels.

Methods: We studied 5 previously published calmodulin mutations (N54I, N98S, D96V, D130G, and F90L). Plasmids encoding either wild-type or mutant calmodulin were transiently transfected into human embryonic kidney 293 cells that stably express subtype 2 of SK protein channels (SK2 cells). Whole-cell voltage-clamp recording was used to determine apamin-sensitive current densities. We also performed optical mapping studies in normal murine hearts to determine the effects of apamin in hearts with (n=7) or without (n=3) pretreatment with sea anemone toxin.

Results: SK2 cells transfected with wild-type calmodulin exhibited an apamin-sensitive current density of 33.6 pA/pF (31.4-36.5 pA/pF) (median and confidence interval 25th-75th percentile), which was significantly higher than that observed for cells transfected with N54I (17.0 pA/pF [14.0-27.7 pA/pF]; P = .016), F90L (22.6 pA/pF [20.3-24.3 pA/pF]; P = .011), D96V (13.0 pA/pF [10.9-15.8 pA/pF]; P = .003), N98S (13.7 pA/pF [8.8-20.4 pA/pF]; P = .005), and D130G (17.6 pA/pF [13.8-24.6 pA/pF]; P = .003). The decrease in SK2 current densities was not associated with a decrease in membrane protein expression or intracellular distribution of the channel protein. Apamin increased the ventricular action potential duration at 80% repolarization (from 79.6 ms [63.4-93.3 ms] to 121.8 ms [97.9-127.2 ms]; P = .010) in hearts pretreated with anemone toxin but not in control hearts.

Conclusion: Human arrhythmogenic calmodulin mutations impede the activation of SK2 channels in human embryonic kidney 293 cells.

Keywords: Arrhythmias; Catecholaminergic polymorphic ventricular tachycardia; Ion channels; Long QT syndrome; Patch clamp.

Publication types

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

MeSH terms

  • Action Potentials
  • Animals
  • Arrhythmias, Cardiac
  • Blotting, Western
  • Calmodulin / genetics*
  • Calmodulin / metabolism
  • DNA / genetics*
  • DNA Mutational Analysis
  • Disease Models, Animal
  • HEK293 Cells
  • Humans
  • Ion Transport
  • Mice
  • Mutation*
  • Myocytes, Cardiac / metabolism*
  • Myocytes, Cardiac / pathology
  • Patch-Clamp Techniques
  • Small-Conductance Calcium-Activated Potassium Channels / genetics*
  • Small-Conductance Calcium-Activated Potassium Channels / metabolism
  • Tachycardia, Ventricular / genetics*
  • Tachycardia, Ventricular / metabolism
  • Tachycardia, Ventricular / pathology

Substances

  • Calmodulin
  • Small-Conductance Calcium-Activated Potassium Channels
  • DNA