Abstract 417: Increased Protein Kinase-A Activity Reduces Current Expression of Co-Expressed Wild Type and Novel V227F-KCNJ2 Mutant Kir2.1 Channels -A Novel Mechanism for the Pathogenesis of Catecholaminergic Polymorphic Ventricular Tachycardia
KCNJ2 encodes the alpha subunit of the inward rectifying potassium channel, Kir2.1, and causes Andersen-Tawil syndrome (ATS1), but has only recently been implicated in cat-echolaminergic polymorphic ventricular tachycardia (CPVT). Presently, mutations in the RyR2-encoded cardiac ryanodine receptor and CASQ2-endcoded calsequestrin 2 account for about two-thirds of CPVT cases, causing dysfunctional calcium handling in cardiac myocytes. We previously discovered a KCNJ2 missense mutation, V227F, in a RyR2/CASQ2-genotype negative patient diagnosed with CPVT. Whole cell voltage clamp technique revealed markedly decreased current expression of mutant channels expressed alone compared to wild type (WT). However, when co-expressed with WT subunits as would be found in the patient, the currents were indistinguishable from WT channels alone. Because the clinical phenotype manifests with catecholaminergic stimulation, we hypothesized that increased Protein Kinase-A (PKA) activity in Cos-1 cells may alter current expression of co-expressed WT and mutant channels. Cells transiently expressing either or both mutant and WT cDNA for 24 hours were incubated with a PKA stimulating cocktail (100μM forskolin and 10μM IBMX) for 2 hours at 37°C before electrophysiology study. Incubation of PKA cocktail dramatically reduced inward current density at −140mV by 52% of co-expressed WT and mutant channels when compared to those not incubated with the cocktail (−300±20 pA/pF n=18 vs. −143±18 pA/pF n=7 p<0.001). A marked decrease in outward current density over the voltage range of terminal repolarization (70% reduction at −60mV 11.9±1.9 pA/pF n=18 vs. 3.6±0.5 pA/pF n=7 p<0.05; 68% reduction at −40mV 9.1±1.6 pA/pF n=18 vs. 3.0±0.9 pA/pF n=7 p<0.05) was observed in cells incubated with PKA cocktail. Incubation of WT or mutant channels alone with PKA cocktail had no significant effect on currents over the range of terminal repolarization. This is the first observation of a PKA-sensitive KCNJ2 mutation. These findings may have implications for novel expression defect mechanisms and potentially a novel CPVT susceptibility gene.