Properties of WT and mutant hERG K(+) channels expressed in neonatal mouse cardiomyocytes

Abstract

Mutations in human ether-a-go-go-related gene 1 (hERG) are linked to long QT syndrome type 2 (LQT2). hERG encodes the pore-forming alpha-subunits that coassemble to form rapidly activating delayed rectifier K(+) current in the heart. LQT2-linked missense mutations have been extensively studied in noncardiac heterologous expression systems, where biogenic (protein trafficking) and biophysical (gating and permeation) abnormalities have been postulated to underlie the loss-of-function phenotype associated with LQT2 channels. Little is known about the properties of LQT2-linked hERG channel proteins in native cardiomyocyte systems. In this study, we expressed wild-type (WT) hERG and three LQT2-linked mutations in neonatal mouse cardiomyocytes and studied their electrophysiological and biochemical properties. Compared with WT hERG channels, the LQT2 missense mutations G601S and N470D hERG exhibited altered protein trafficking and underwent pharmacological correction, and N470D hERG channels gated at more negative voltages. The DeltaY475 hERG deletion mutation trafficked similar to WT hERG channels, gated at more negative voltages, and had rapid deactivation kinetics, and these properties were confirmed in both neonatal mouse cardiomyocyte and human embryonic kidney (HEK)-293 cell expression systems. Differences between the cardiomyocytes and HEK-293 cell expression systems were that hERG current densities were reduced 10-fold and deactivation kinetics were accelerated 1.5- to 2-fold in neonatal mouse cardiomyocytes. An important finding of this work is that pharmacological correction of trafficking-deficient LQT2 mutations, as a potential innovative approach to therapy, is possible in native cardiac tissue.

Fig. 1.

Cultured neonatal mouse cardiomyocytes. A: phase-contrast image of isolated, cultured cardiomyocytes. B: phase-contrast image of the same field showing green fluorescent protein (GFP) expression. Higher-power confocal images (C–F) show three isolated neonatal mouse cardiomyocytes expressing sacromeric actin (C), GFP (D), wild-type (WT) hERG (E), and a merged image (F). Calibration bars = 50 μm in A and B or 5 μm in C–F.

Fig. 2.

Currents in untranfected and transfected native cardiomyocytes. A: representative current traces from an untransfected cardiomyocyte. Scale = 25 pA by 0.5 s. The voltage-clamp protocol is shown above the currents. Inset a shows inward currents elicited with depolarization (scale = 200 pA by 0.05 s), and inset b shows block of tail current by E4031 (scale = 10 pA by 2 s). The activation relation for peak tail rapidly activating delayed rectifier K⁺ current is shown at the bottom. B and C: WT hERG-transfected cardiomyocytes. B: block of tail current by E4031. Scale = 50 pA by 2 s. C: a family of current traces and the activation relation for peak tail hERG current (I_hERG). Scale = 200 pA by 2 s. Dotted line = 0 pA.

Fig. 3.

Western blot analysis for hERG channels in neonatal mouse cardiomyocytes. Cardiomyocytes transfected with empty pcDNA3 vector (lane 1) have protein bands of 165 and 205 kDa, consistent with mouse ERG1a (mERG1a), and 95 and 114 kDa, consistent with mERG1b (small arrows). Cardiomyocytes transfected with WT hERG (hERG1a; lane 2) have additional protein bands at 135 and 155 kDa (large arrows). Lanes 3 and 4 show G601S hERG and lanes 5 and 6 show N470D hERG without (−) or with (+) E4031 incubation. E4031 incubation resulted in increased density of the 155-kDa bands. Lanes 7 and 8 show ΔY475 hERG without or with E4031 incubation and show both 135- and 155-kDa bands. Lane 9 shows that untransfected cardiomyocytes lack the 135- and 155-kDa bands. Quantitative Western blot density ratio analysis of hERG protein is shown at the bottom. See text for detail.

Fig. 4.

G601S, N470D, and ΔY475 hERG currents in cardiomyocytes. A: representative current traces for control conditions and after pharmacological correction with E4031. Scale = 50 pA by 2 s. Dotted line = 0 pA. B: mean peak tail I_hERG amplitudes for G601S, N470D, and ΔY475 hERG for control conditions and after pharmacological correction with E4031 (*P < 0.05). C: activation current-voltage relations for G601S, N470D, and ΔY475 hERG peak tail I_hERG after pharmacological correction with E4031.

Fig. 5.

ΔY475 hERG currents in cardiomyocytes and human embryonic kidney (HEK)-293 cells. A: current traces from ΔY475 hERG expressed in cardiomyocytes (scale = 50 pA by 2 s) and HEK-293 cells (scale = 200 pA by 2 s). Dotted line = 0 pA. B: mean peak tail I_hERG amplitudes for WT, control ΔY475, and ΔY475 hERG channels cultured in E4031 for 24 h followed by drug washout for 1 h and expressed in cardiomyocytes and HEK-293 cells (*P < 0.05, cardiomyocytes vs. HEK-293 cells). C: activation current-voltage relations for peak tail I_hERG for WT and control ΔY475 hERG channels expressed in cardiomyocytes and HEK-293 cells.