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Case Reports
. 2009 Oct;2(5):540-7.
doi: 10.1161/CIRCEP.109.872309. Epub 2009 Aug 25.

Protein kinase A-dependent biophysical phenotype for V227F-KCNJ2 mutation in catecholaminergic polymorphic ventricular tachycardia

Amanda L Vega  1 , David J Tester, Michael J Ackerman, Jonathan C Makielski
Affiliations
Case Reports

Protein kinase A-dependent biophysical phenotype for V227F-KCNJ2 mutation in catecholaminergic polymorphic ventricular tachycardia

Amanda L Vega et al. Circ Arrhythm Electrophysiol. 2009 Oct.

Abstract

Background: KCNJ2 encodes Kir2.1, a pore-forming subunit of the cardiac inward rectifier current, I(K1). KCNJ2 mutations are associated with Andersen-Tawil syndrome and catecholaminergic polymorphic ventricular tachycardia. The aim of this study was to characterize the biophysical and cellular phenotype of a KCNJ2 missense mutation, V227F, found in a patient with catecholaminergic polymorphic ventricular tachycardia.

Methods and results: Kir2.1-wild-type (WT) and V227F channels were expressed individually and together in Cos-1 cells to measure I(K1) by voltage clamp. Unlike typical Andersen-Tawil syndrome-associated KCNJ2 mutations, which show dominant negative loss of function, Kir2.1WT+V227F coexpression yielded I(K1) indistinguishable from Kir2.1-WT under basal conditions. To simulate catecholamine activity, a protein kinase A (PKA)-stimulating cocktail composed of forskolin and 3-isobutyl-1-methylxanthine was used to increase PKA activity. This PKA-simulated catecholaminergic stimulation caused marked reduction of outward I(K1) compared with Kir2.1-WT. PKA-induced reduction in I(K1) was eliminated by mutating the phosphorylation site at serine 425 (S425N).

Conclusions: Heteromeric Kir2.1-V227F and WT channels showed an unusual latent loss of function biophysical phenotype that depended on PKA-dependent Kir2.1 phosphorylation. This biophysical phenotype, distinct from typical Andersen-Tawil syndrome mutations, suggests a specific mechanism for PKA-dependent I(K1) dysfunction for this KCNJ2 mutation, which correlates with adrenergic conditions underlying the clinical arrhythmia.

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Figures

Figure 1
Figure 1
Twelve lead electrocardiograms (A) and selected tracings from a treadmill stress test (B) for the CPVT patient with V227F-KCNJ2 mutation. For further description see text.
Figure 2
Figure 2
Kir2.1-V227F has distinct molecular phenotype for IK1. (A) Representative IK1 traces for the Kir2.1-WT, Kir2.1-V227F, and co-expressed Kir2.1-WT+Kir2.1-V227F (B) Current-voltage (IV) plots for Kir2.1-WT (closed squares; n=14), Kir2.1-V227F (closed circles; n=16) and co-expressed Kir2.1-WT+V227F (closed triangles; n=18) expressed in Cos-1 cells. The inset shows detail of outward currents. V227F significantly reduces current amplitudes of homomeric channels but heteromeric channels are the same as WT. (* p<.05 by ANOVA for V227F versus both WT and WT+V227F)
Figure 3
Figure 3
IK1 measured from Cos-1 cells transfected with the dominant negative mutation Kir2.1-AAA. (A) Representative traces from KiR2.1-AAA, co-expressed Kir2.1-WT and Kir2.1-AAA, and co-expressed Kir2.1-V227F and Ki2.1-AAA. (B) Peak current-relationships for KiR2.1-AAA (open circles), co-expressed Kir2.1-WT and Kir2.1-AAA (open squares), co-expressed Kir2.1-V227F (open triangles). For comparison the relationships for Kir2.1WT (closed squares) and Kir2.1V227F + Kir2.1WT (closed triangles) are shown. These results show that Kir2.1-AAA shows dominant negative suppression of Kir2.1-V227F suggesting that the V227F mutant channel co-assemble with other Kir2.1 subunits.
Figure 4
Figure 4
PKA activation reduced outward IK1 for heteromeric Kir2.1 WT+V227F channels. From each cell, a complete IV plot was obtained before (closed symbols) and after (open symbols) five minutes of PKA activation. (A) Representative current after (bottom row) 5 minutes of PKA activation. Peak IV plots for Kir2.1-WT (squares, n=6) (B), Kir2.1-V227F (circles, n=9) (C) and co-expressed Kir2.1-WT+V227F (triangles, n=7) (D). PKA activity significantly decreased inward IK1 for all groups. PKA tended (not significant) to increase outward IK1 for WT (B inset), did not affect outward IK1 for V227F, but significantly (*p<.05) decreased outward IK1 for Kir2.1-WT+V227F (D inset).
Figure 5
Figure 5
PKA activation reduced outward IK1 for heteromeric Kir2.1 WT+V227F channels after 2 hours of PKA activation. Cells were incubated in control solutions or PKA activations solutions for 2 hours then patch-clamped to measure IK1 density. (N numbers in parentheses, *p<.05 PKA vs non-PKA).
Figure 6
Figure 6
PKA consensus site S425 required for PKA-mediated effects on Kir2.1 channels. (A) Representative IK1 traces of Kir2.1-S425N, Kir2.1-S425N+Kir2.1- V227F/S425N and Kir2.1-WT+Kir2.1-V227F/S425N in the presence or absence of the PKA activation (B) IV plot for Kir2.1-S425N with PKA activation (open squares; n=5) or without PKA activation (closed squares; n=8) shows nearly complete abrogation of inhibition seen in WT (Figure 4B). (c) IV plot for Kir2.1-S425N+Kir2.1-V227F/S4245N with PKA activation (open triangle; n=5) and without PKA activation (closed triangle; n=11) shows abrogation of the inhibitory effect seen in heteromeric channels (Figure 4C). (D) IV plot for Kir2.1-WT+Kir2.1-V227F/S425N with PKA activation (open diamond; n=8) and without PKA activation (closed diamond; n=11) shows that the inhibitory effect remains with a WT subunit that can be phosphorylated. (* p<.05)
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