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. 2012 Nov;53(5):725-33.
doi: 10.1016/j.yjmcc.2012.08.021. Epub 2012 Sep 3.

Early LQT2 nonsense mutation generates N-terminally truncated hERG channels with altered gating properties by the reinitiation of translation

Matthew R Stump  1 , Qiuming Gong, Jonathan D Packer, Zhengfeng Zhou
Affiliations

Early LQT2 nonsense mutation generates N-terminally truncated hERG channels with altered gating properties by the reinitiation of translation

Matthew R Stump et al. J Mol Cell Cardiol. 2012 Nov.

Abstract

Mutations in the human ether-a-go-go-related gene (hERG) result in long QT syndrome type 2 (LQT2). The hERG gene encodes a K(+) channel that contributes to the repolarization of the cardiac action potential. We have previously shown that hERG mRNA transcripts that contain premature termination codon mutations are rapidly degraded by nonsense-mediated mRNA decay (NMD). In this study, we identified a LQT2 nonsense mutation, Q81X, which escapes degradation by the reinitiation of translation and generates N-terminally truncated channels. RNA analysis of hERG minigenes revealed equivalent levels of wild-type and Q81X mRNA while the mRNA expressed from minigenes containing the LQT2 frameshift mutation, P141fs+2X, was significantly reduced by NMD. Western blot analysis revealed that Q81X minigenes expressed truncated channels. Q81X channels exhibited decreased tail current levels and increased deactivation kinetics compared to wild-type channels. These results are consistent with the disruption of the N-terminus, which is known to regulate hERG deactivation. Site-specific mutagenesis studies showed that translation of the Q81X transcript is reinitiated at Met124 following premature termination. Q81X co-assembled with hERG to form heteromeric channels that exhibited increased deactivation rates compared to wild-type channels. Mutant channels also generated less outward current and transferred less charge at late phases of repolarization during ventricular action potential clamp. These results provide new mechanistic insight into the prolongation of the QT interval in LQT2 patients. Our findings indicate that the reinitiation of translation may be an important pathogenic mechanism in patients with nonsense and frameshift LQT2 mutations near the 5' end of the hERG gene.

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Figures

Fig. 1
Fig. 1
Analysis of the Q81X and P141fs+2X mutations using minigene constructs. (A) Structure of the full-length hERG minigene construct. The positions of the wild-type termination codon (TER) and premature termination codons are indicated. (B) Analysis of Q81X and P141fs+2X mRNA. HEK293 cells were transiently transfected with wild-type (WT), Q81X, or P141fs+2X minigenes and the expressed mRNA was analyzed by the RNase protection assay using a probe specific to 277 nt within exons 12 and 13 of hERG. Cells expressing wild-type and mutant minigenes were treated (+) with 100 μg/ml cycloheximide (CHX) for 3 h prior to RNA isolation or left untreated (−).The level of hygromycin resistance gene transcripts (Hygro) served as a loading control. (C) Normalized signals were quantified and plotted as the mean percentage of wild-type ± SEM (n = 4).
Fig. 2
Fig. 2
Western blot analysis of wild-type, Q81X, and P141fs+2X channels. (A) HEK293 cells were transiently transfected with wild-type, Q81X, and P141fs+2X hERG minigenes. Proteins from whole cell lysates were subjected to SDS-PAGE and probed with anti-hERG antibody. (B) HL-1 cardiomyocytes were transiently transfected with HA-tagged wild-type and Q81X constructs. Proteins were detected with anti-HA antibody. Mature, fully glycosylated wild-type channels are 155 kDa; immature, core-glycosylated wild-type channels are 135 kDa. Mature, fully-glycosylated Q81X channels are 140 kDa; immature, core-glycosylated Q81X channels are 120 kDa. Untransfected cells, Unt. The results shown are representative of three independent experiments.
Fig. 3
Fig. 3
Electrophysiological properties of wild-type and Q81X channels. (A) Representative currents recorded from HEK293 cells transiently transfected with wild-type and Q81X minigenes. hERG channels were activated with 4 s test potentials between −70 and +60 mV and tail current was recorded upon repolarization to −50 mV. (B) I–V plot of wild-type and Q81X current density measured at the end of the depolarizing pulse. (C) Activation curves determined using peak tail currents, normalized to maximum tail currents, and fit with a Boltzmann function (solid line). (D) I–V plot of peak tail current densities recorded at −50 mV, following depolarizing test potentials. Data were plotted as mean ± SEM, wild-type (n = 11) and Q81X (n = 13).
Fig. 4
Fig. 4
Deactivation kinetics of wild-type and Q81X channels. (A) Representative currents recorded from HEK293 cells transiently transfected with wild-type and Q81X minigenes. hERG channels were activated with a 1 s test potential to +60 mV and the deactivation tail currents were recorded upon repolarization to potentials ranging between −40 and −120 mV. The tail current was fit with a double exponential function and the fast and slow components of the deactivation time constants of wild-type and Q81X channels are shown in (B) and (C). The time constants for the fast and slow components of the deactivation rate were significantly faster for Q81X channels at all test potentials (P < 0.05, ANOVA). (D) Relative contribution of the fast component of the deactivation time constants at −40 and −120 mV for wild-type and Q81X channels. All values were plotted as mean ± SEM, wild-type (n = 6) and Q81X (n = 7). *, P < 0.05 compared to Q81X, ANOVA.
Fig. 5
Fig. 5
Voltage-dependent inactivation of wild-type and Q81X channels. (A) Representative currents from HEK293 cells transiently transfected with wild-type and Q81X minigenes. Current was recorded using a three-pulse protocol: channels were activated with a 0.5 s pulse to +40 mV followed by either a 20 ms or 5 ms hyperpolarizing pulse to potentials between −120 and +40 mV, the inactivation currents were recorded following a final pulse to +40 mV. (B) The corrected peak currents (described in the method), normalized to maximum currents, were plotted versus voltage and fit with a Boltzmann function. Values were plotted as mean ± SEM, wild-type (n = 7) and Q81X (n = 5).
Fig. 6
Fig. 6
Western blot analysis of translation reinitiation. (A) Schematic illustrating putative reinitiation sites downstream of the Q81X mutation. The partial cDNA sequence of hERG exon 3 reveals three downstream methionine codons (underlined) which are numbered according to the hERG cDNA sequence. (B) Western blot analysis of HEK293 cells transiently transfected with wild-type and the Q81X, Q81X+M124V, Q81X+M133V+M137V, and Q81X+M124V+M133V+M137V cDNA constructs. Proteins were detected with anti-hERG antibody. The results shown are representative of three independent experiments.
Fig. 7
Fig. 7
Dominant-negative effects of Q81X. (A) Representative current trace from HEK293 cells transiently co-transfected with equimolar amounts of wild-type and Q81X minigenes. hERG current was recorded using the protocols in the legend of Fig. 4. (B) Voltage-dependence of the slow (circles) and fast (diamonds) components of the deactivation time constants for the heteromeric wild-type + Q81X channels are indicated as the black symbols (n = 6). The fast and slow components of deactivation of wild-type channels from Fig. 4 are shown as white symbols for comparison. The time constants for the fast component of the deactivation rate were significantly faster for Q81X channels at test potentials positive to −100 mV and for the slow components of the deactivation rate at test potentials positive to −70 mV (P < 0.05, ANOVA).
Fig. 8
Fig. 8
Co-assembly of wild-type and Q81X channels. HA-tagged Q81X cDNA constructs were transiently transfected into HEK293 cells stably expressing Flag-tagged wild-type hERG channels. Cell lysates were subjected to immunoprecipitation using anti-Flag, followed by western blot with anti-HA and anti-Flag antibody. HEK293 cells stably expressing Flag-tagged wild-type or transiently expressing HA-tagged Q81X were used as controls. The lower two panels are inputs and are probed with anti-Flag and anti-HA antibody. The results shown are representative of three independent experiments.
Fig. 9
Fig. 9
hERG current during ventricular action potential clamp. (A) The ventricular action potential clamp waveform (upper trace) used to generate the representative current traces of wild-type hERG, Q81X and wild-type + Q81X channels (lower traces). (B) Plot of the averaged current density versus time during the ventricular action potential clamp. (C) Plot of the averaged current density as a function of action potential voltage. (D) Peak current density plotted as a function of peak potential. (E) Plot of the charge density transferred during the late phases of repolarization of the ventricular action potential clamp at potentials negative to −40 mV. Data were plotted as mean ± SEM, wild-type (n = 6), Q81X (n = 4), wild-type + Q81X (n = 5), *, P < 0.05 compared to wild-type hERG, ANOVA.
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