KCNJ2 mutation causes an adrenergic-dependent rectification abnormality with calcium sensitivity and ventricular arrhythmia

Abstract

Background: KCNJ2 mutations are associated with a variety of inherited arrhythmia syndromes including catecholaminergic polymorphic ventricular tachycardia 3.

Objective: To characterize the detailed cellular mechanisms of the clinically recognized KCNJ2 mutation R67Q.

Methods: Kir2.1 current density was measured from COS-1 cells transiently transfected with wild-type human Kir-2.1 (WT-Kir2.1) and/or a heterozygous missense mutation in KCNJ2 (R67Q-Kir2.1) by using the whole-cell voltage clamp technique. Catecholamine activity was simulated with protein kinase A-stimulating cocktail exposure. Phosphorylation-deficient mutants, S425N-Kir2.1 and S425N-Kir2.1/R67Q-S425N-Kir2.1, were used in a separate set of experiments. HA- or Myc-Tag-WT-Kir2.1 and HA-Tag-R67Q-Kir2.1 were used for confocal imaging.

Results: A 33-year-old woman presented with a catecholaminergic polymorphic ventricular tachycardia-like clinical phenotype and was found to have KCNJ2 missense mutation R67Q. Treatment with nadolol and flecainide resulted in the complete suppression of arrhythmias and symptom resolution. Under baseline conditions, R67Q-Kir2.1 expressed alone did not produce inward rectifier current while cells coexpressing WT-Kir2.1 and R67Q-Kir2.1 demonstrated the rectification index (RI) similar to that of WT-Kir2.1. After PKA stimulation, R67Q-Kir2.1/WT-Kir2.1 failed to increase peak outward current density; WT-Kir2.1 increased by 46% (n = 5), while R67Q-Kir2.1/WT-Kir2.1 decreased by 6% (n = 6) (P = .002). Rectification properties in R67Q-Kir2.1/WT-Kir2.1 demonstrated sensitivity to calcium with a decreased RI in the high-calcium pipette solution (RI 20.3% ± 4.1%) than in the low-calcium pipette solution (RI 36.5% ± 5.7%) (P < .05). Immunostaining of WT-Kir2.1 and R67Q-Kir2.1 individually and together showed a normal membrane expression pattern and colocalization by using the Pearson correlation coefficient.

Conclusions: R67Q-Kir2.1 is associated with an adrenergic-dependent clinical and cellular phenotype with rectification abnormality enhanced by increased calcium. These findings are a significant advancement of our knowledge and understanding of the phenotype-genotype relationship of arrhythmia syndromes related to KCNJ2 mutations.

Keywords: Arrhythmia syndrome; CPVT3; Inherited arrhythmia; KCNJ2; Kir2.1; Potassium inward-rectifier channel; Ventricular arrhythmia.

Figure 1. ECG and Holter recordings from Proband

A. Baseline ECG from a 33-year-old female harboring KCNJ2 R67Q mutation. The ECG shows a normal QTc (430 msec). There are prominent U waves, seen best in lead V2, with a QUc 620 msec. B. Holter monitor from the same patient demonstrates frequent polymorphic ventricular ectopy (top panel) and ventricular ectopy with bi-directional and polymorphic qualities (lower panel) both occurring during exercise.

Figure 2. R67Q-Kir2.1 lacks dominant negative effect when co-expressed with WT-Kir2.1

Schematic of protocol used to measure currents and representative Kir2.1 current density traces are illustrated in the left panel: WT-Kir2.1 (top), R67Q-Kir2.1 (middle), R67Q/WT-Kir2.1 (bottom). Dotted line indicates 0 pA, scale bar represents 50 ms and 10 pA/pF. The right panel shows baseline current voltage (I-V) relationship for WT-Kir2.1 (filled squares; n=9), R67Q-Kir2.1 alone (filled triangles, n=3), and co-expressed R67Q/WT-Kir2.1 (filled triangles, n=10). Homomeric R67Q-Kir2.1 channels do not produce current while co-expressed WT and R67Q-Kir2.1 channels produce typical I_K1. The current reduction is as expected as the total WT-Kir2.1 DNA is 50% less in the co-expression experiments. * denotes p < 0.05 by Student’s t-test.

Figure 3. Response of R67Q/WT-Kir2.1 to PKA-cocktail differs from WT-Kir2.1

A. Acute application of PKA-CT to WT-Kir2.1. For each cell, I_K1 was recorded after 8 minutes of perfusion with control bath solution (filled squares, n=5). PKA-CT was then perfused and Kir2.1 was recorded again after another 8 minutes (open squares, n=5). B. Contemporaneously, this experiment was repeated with co-expressed R67Q/WT-Kir2.1 (control filled triangles, n=6; PKA-CT open triangles, n=6). After application of PKA-CT, outward currents in the physiologically significant range (see inset) increased in WT cells (p=0.06 at −50 mV) but decreased in cells with co-expressed Kir2.1. Insets highlight the current at physiologically significant potentials. C. Example current density traces in all experiment conditions are shown below. Dotted line indicates 0 pA, scale bar represents 50 ms and 10 pA/pF.

Figure 4

A,B. R67Q-Kir2.1/WT-Kir2.1 fails to have a typical WT-Kir2.1 outward current increase following incubation with PKA-CT. I–V plots obtained under control conditions or after 2 hour incubation with PKA-CT. A. WT-Kir2.1 (control filled squares, n=9; PKA-CT open squares, n=6). B. R67Q/WT-Kir2.1 (control filled triangles, n=10; PKA-CT open triangles, n=6). C and D. The increase in outward currents with PKA-CT is phosphorylation dependent. I–V plots obtained under control conditions or after 2 hours of incubation with PKA-CT using phosphorylation deficient constructs (S425N mutants). C. S425N-Kir2.1 (control filled squares, n=7; PKA-CT open squares, n=5). D. R67Q-S425N-Kir2.1/S425N-Kir2.1 (control filled triangles, n=7; PKA-CT open triangles, n=5). Insets highlight the current at physiologically significant potentials.

Figure 5. R67Q-Kir2.1 affects channel rectification index and may alter channel sensitivity to calcium

COS-1 cells expressing either WT-Kir2.1 (left hand portion of figure) or R67Q/WT-Kir2.1 (right hand portion) were incubated for 2 hours in control bath solution or PKA-CT containing solution. I_K1 was then measured using a pipette solution with no added Ca²⁺ (low Ca²⁺) or a pipette solution containing 4.2 mmol Ca²⁺(high Ca²⁺). A rectification index (RI) was then calculated by dividing the value of the outward current at −60 mV by the absolute value of the current at −100 mV then multiplying by 100(14). * denotes p<0.05 by Student’s t-test.

Figure 6. Pattern of localization of Myc-WT-Kir2.1 (red), HA-R67Q-Kir2.1 (green) and HA-R67W-Kir2.1 (green) transiently transfected in COS-1 cells

Wild-type (a, b and c) and mutant (R67Q (d, e and f); R67W (g, h and i)) Kir2.1 both show very similar pattern of localization 1) punctuate pattern and 2) edge of the cell. DAPI staining was used to identify the nucleus. Scale bar = 25 µm.

Fig 7. Pattern of co-localization of Myc-WT-Kir2.1 (red) with HA-WT-Kir2.1 or HA-R67Q-Kir2.1 (green) or HA-R67W-Kir2.1 (green) in COS-1 cells

COS-1 cells show very similar pattern of co-localization of Myc-WT-Kir2.1 (c, g and h) with that of HA-WT-Kir2.1 (b) or HA-R67Q-Kir2.1 (f) or HA-R67W-Kir2.1 (j) in the staining pattern. Co-localization was confirmed by Pearson’s correlation coefficient of >0.5 indicating significant co-localization, also evident from the merged panels (d, h and l). DAPI staining was used to identify the nucleus. Scale bar = 25 µm.