Reduction of SK current in rat CPVT model with heterozygous calmodulin 3 mutation D132E is caused by impaired intracellular Ca2+ homeostasis.

Abstract

Small conductance Ca2+-activated K+ (SK) channels are voltage independent and their gating is governed by Ca2+ via calmodulin (CaM) constitutively bound to the C-terminal CaM-binding domain (CaMBD). Recently, several CaM mutations have been linked to a hereditary arrhythmia syndrome, Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT). Although in vitro studies demonstrated that CaM mutations cause loss-of-SK-channel complex function, the exact mechanisms remain elusive. The main objective of this work is to delineate potential roles and mechanisms of SK channels dysregulation in CPVT-linked calmodulinopathy. To achieve this goal we generated a novel rat model with CPVT-associated calmodulin 3 (Cam3) point mutation D132E. In vivo ECG recordings of heterozygous CPVT CaM3D132E rats demonstrated increased incidence of epinephrine+caffeine induced ventricular tachycardia. Using whole-cell patch-clamp and confocal Ca2+-imaging we recorded ISK in isolated littermate control (LMC) and D132E myocytes challenged with β-adrenergic agonist isoproterenol under preserved intracellular Ca2+ cycling. Patch-clamp recordings showed reduced ISK in CaMD132E myocytes vs LMCs. However, the amplitude of Ca2+ transients that evoke ISK was also significantly lower in mutant myocytes. To test directly whether CaM3D132E causes SK channels dysfunction we expressed rat SK2 tethered with CaMD132E or WT CaM in rat myocytes. Simultaneous ISK/Ca2+ recordings revealed that this mutation causes two-fold reduction in Ca2+-dependent activation of the channels. In vitro stopped-flow experiments confirmed that CaMD132E mutation impairs CaM interaction with SK2 CaMBD. RNA fluorescence imaging experiments demonstrated lack of CaM3 and SK2/3 mRNAs spatial overlap eliminating possibility of co-translation. These data suggest that CaM3 D132E mutation causes ISK downregulation primarily due to Ca2+ transients amplitude reduction, while direct loss-of-function effects on plasmalemmal SK channels play minimal role given reduced ability of mutant CaM to bind to the channel.