Regulation of Kv11.1 Isoform Expression by Polyadenylate Binding Protein Nuclear 1.

The Kv11.1 voltage-gated potassium channel, encoded by the KCNH2 gene, conducts the rapidly activating delayed rectifier current in the heart. KCNH2 pre-mRNA undergoes alternative polyadenylation to generate two C-terminal Kv11.1 isoforms in the heart. Utilization of a poly(A) signal in exon 15 produces the full-length, functional Kv11.1a isoform, while intron 9 polyadenylation generates the C-terminally truncated, nonfunctional Kv11.1a-USO isoform. The relative expression of Kv11.1a and Kv11.1a-USO isoforms plays an important role in the regulation of Kv11.1 channel function. In this study, we tested the hypothesis that the RNA polyadenylate binding protein nuclear 1 (PABPN1) interacts with a unique 22 nt adenosine stretch adjacent to the intron 9 poly(A) signal and regulates KCNH2 pre-mRNA alternative polyadenylation and the relative expression of Kv11.1a C-terminal isoforms. We showed that PABPN1 inhibited intron 9 poly(A) activity using luciferase reporter assays, tandem poly(A) reporter assays, and RNA pulldown assays. We also showed that PABPN1 increased the relative expression level of the functional Kv11.1a isoform using RNase protection assays, immunoblot analyses, and patch clamp recordings. Our present findings suggest a novel role for the RNA-binding protein PABPN1 in the regulation of functional and nonfunctional Kv11.1 isoform expression.

Regulation of Kv11.1 potassium channel C-terminal isoform expression by the RNA-binding proteins HuR and HuD.

The potassium voltage-gated channel subfamily H member 2 (KCNH2) gene encodes the Kv11.1 potassium channel, which conducts the rapidly activating delayed rectifier current in the heart. KCNH2 pre-mRNA undergoes alternative polyadenylation and forms a functional, full-length Kv11.1a isoform if exon 15 is polyadenylated or a nonfunctional, C-terminally truncated Kv11.1a-USO isoform if intron 9 is polyadenylated. The molecular mechanisms that regulate Kv11.1 isoform expression are poorly understood. In this study, using HEK293 cells and reporter gene expression, pulldown assays, and RNase protection assays, we identified the RNA-binding proteins Hu antigen R (HuR) and Hu antigen D (HuD) as regulators of Kv11.1 isoform expression. We show that HuR and HuD inhibit activity at the intron 9 polyadenylation site. When co-expressed with the KCNH2 gene, HuR and HuD increased levels of the Kv11.1a isoform and decreased the Kv11.1a-USO isoform in the RNase protection assays and immunoblot analyses. In patch clamp experiments, HuR and HuD significantly increased the Kv11.1 current. siRNA-mediated knockdown of HuR protein decreased levels of the Kv11.1a isoform and increased those of the Kv11.1a-USO isoform. Our findings suggest that the relative expression levels of Kv11.1 C-terminal isoforms are regulated by the RNA-binding HuR and HuD proteins.

Regulation of Isoform Expression by Blocking Polyadenylation Signal Sequences with Morpholinos.

Alternative polyadenylation is increasingly being recognized as an important layer of gene regulation. Antisense-mediated modulation of alternative polyadenylation represents an attractive strategy for the regulation of gene expression as well as potential therapeutic applications. In this chapter, we describe methods to upregulate the functional Kv11.1 isoform expression by blocking intronic polyadenylation signal sequences with antisense morpholinos.

Pathogenesis of the Novel Autoimmune-Associated Long-QT Syndrome.

BACKGROUND: Emerging clinical evidence demonstrates high prevalence of QTc prolongation and complex ventricular arrhythmias in patients with anti-Ro antibody (anti-Ro Ab)-positive autoimmune diseases. We tested the hypothesis that anti-Ro Abs target the HERG (human ether-a-go-go-related gene) K(+) channel, which conducts the rapidly activating delayed K(+) current, IKr, thereby causing delayed repolarization seen as QT interval prolongation on the ECG. METHODS AND RESULTS: Anti-Ro Ab-positive sera, purified IgG, and affinity-purified anti-52kDa Ro Abs from patients with autoimmune diseases and QTc prolongation were tested on IKr using HEK293 cells expressing HERG channel and native cardiac myocytes. Electrophysiological and biochemical data demonstrate that anti-Ro Abs inhibit IKr to prolong action potential duration by directly binding to the HERG channel protein. The 52-kDa Ro antigen-immunized guinea pigs showed QTc prolongation on ECG after developing high titers of anti-Ro Abs, which inhibited native IKr and cross-reacted with guinea pig ERG channel. CONCLUSIONS: The data establish that anti-Ro Abs from patients with autoimmune diseases inhibit IKr by cross-reacting with the HERG channel likely at the pore region where homology between anti-52-kDa Ro antigen and HERG channel is present. The animal model of autoimmune-associated QTc prolongation is the first to provide strong evidence for a pathogenic role of anti-Ro Abs in the development of QTc prolongation. It is proposed that adult patients with anti-Ro Abs may benefit from routine ECG screening and that those with QTc prolongation should receive counseling about drugs that may increase the risk for life-threatening arrhythmias.

Upregulation of functional Kv11.1 isoform expression by inhibition of intronic polyadenylation with antisense morpholino oligonucleotides.

The KCNH2 gene encodes the Kv11.1 potassium channel that conducts the rapidly activating delayed rectifier current in the heart. KCNH2 pre-mRNA undergoes alternative processing; intron 9 splicing leads to the formation of a functional, full-length Kv11.1a isoform, while polyadenylation within intron 9 generates a non-functional, C-terminally truncated Kv11.1a-USO isoform. The relative expression of Kv11.1 isoforms plays an important role in the regulation of Kv11.1 channel function and the pathogenesis of long QT syndrome. In this study, we identified cis-acting elements that are required for KCNH2 intron 9 poly(A) signal activity. Mutation of these elements decreased Kv11.1a-USO expression and increased the expression of Kv11.1a mRNA, protein and channel current. More importantly, blocking these elements by antisense morpholino oligonucleotides shifted the alternative processing of KCNH2 intron 9 from the polyadenylation to the splicing pathway, leading to the predominant production of Kv11.1a and a significant increase in Kv11.1 current. Our findings indicate that the expression of the Kv11.1a isoform can be upregulated by an antisense approach. Antisense inhibition of KCNH2 intronic polyadenylation represents a novel approach to increase Kv11.1 channel function.

Identification of Kv11.1 isoform switch as a novel pathogenic mechanism of long-QT syndrome.

BACKGROUND: The KCNH2 gene encodes the Kv11.1 potassium channel that conducts the rapidly activating delayed rectifier current in the heart. The relative expression of the full-length Kv11.1a isoform and the C-terminally truncated Kv11.1a-USO isoform plays an important role in regulation of channel function. The formation of C-terminal isoforms is determined by competition between the splicing and alternative polyadenylation of KCNH2 intron 9. It is not known whether changes in the relative expression of Kv11.1a and Kv11.1a-USO can cause long-QT syndrome. METHODS AND RESULTS: We identified a novel KCNH2 splice site mutation in a large family. The mutation, IVS9-2delA, is a deletion of the A in the AG dinucleotide of the 3' acceptor site of intron 9. We designed an intron-containing full-length KCNH2 gene construct to study the effects of the mutation on the relative expression of Kv11.1a and Kv11.1a-USO at the mRNA, protein, and functional levels. We found that this mutation disrupted normal splicing and resulted in exclusive polyadenylation of intron 9, leading to a switch from the functional Kv11.1a to the nonfunctional Kv11.1a-USO isoform in HEK293 cells and HL-1 cardiomyocytes. We also showed that IVS9-2delA caused isoform switch in the mutant allele of mRNA isolated from patient lymphocytes. CONCLUSIONS: Our findings indicate that the IVS9-2delA mutation causes a switch in the expression of the functional Kv11.1a isoform to the nonfunctional Kv11.1a-USO isoform. Kv11.1 isoform switch represents a novel mechanism in the pathogenesis of long-QT syndrome.

Position of premature termination codons determines susceptibility of hERG mutations to nonsense-mediated mRNA decay in long QT syndrome.

The degradation of human ether-a-go-go-related gene (hERG, KCNH2) transcripts containing premature termination codon (PTC) mutations by nonsense-mediated mRNA decay (NMD) is an important mechanism of long QT syndrome type 2 (LQT2). The mechanisms governing the recognition of PTC-containing hERG transcripts as NMD substrates have not been established. We used a minigene system to study two frameshift mutations, R1032Gfs 25 and D1037Rfs 82. R1032Gfs 25 introduces a PTC in exon 14, whereas D1037Rfs 82 causes a PTC in the last exon (exon 15). We showed that R1032Gfs 25, but not D1037Rfs 82, reduced the level of mutant mRNA compared to the wild-type minigene in an NMD-dependent manner. The deletion of intron 14 prevented degradation of R1032Gfs 25 mRNA indicating that a downstream intron is required for NMD. The recognition and elimination of PTC-containing transcripts by NMD required that the mutation be positioned >54-60 nt upstream of the 3'-most exon-exon junction. Finally, we used a full-length hERG splicing-competent construct to show that inhibition of downstream intron splicing by antisense morpholino oligonucleotides inhibited NMD and rescued the functional expression of a third LQT2 mutation, Y1078. The present study defines the positional requirements for the susceptibility of LQT2 mutations to NMD and posits that the majority of reported LQT2 nonsense and frameshift mutations are potential targets of NMD.

LQT2 nonsense mutations generate trafficking defective NH2-terminally truncated channels by the reinitiation of translation.

The human ether-a-go-go-related gene (hERG) encodes a voltage-activated K(+) channel that contributes to the repolarization of the cardiac action potential. Long QT syndrome type 2 (LQT2) is an autosomal dominant disorder caused by mutations in hERG, and patients with LQT2 are susceptible to severe ventricular arrhythmias. We have previously shown that nonsense and frameshift LQT2 mutations caused a decrease in mutant mRNA by the nonsense-mediated mRNA decay (NMD) pathway. The Q81X nonsense mutation was recently found to be resistant to NMD. Translation of Q81X is reinitiated at Met(124), resulting in the generation of NH2-terminally truncated hERG channels with altered gating properties. In the present study, we identified two additional NMD-resistant LQT2 nonsense mutations, C39X and C44X, in which translation is reinitiated at Met(60). Deletion of the first 59 residues of the channel truncated nearly one-third of the highly structured Per-Arnt-Sim domain and resulted in the generation of trafficking-defective proteins and a complete loss of hERG current. Partial deletion of the Per-Arnt-Sim domain also resulted in the accelerated degradation of the mutant channel proteins. The coexpression of mutant and wild-type channels did not significantly disrupt the function and trafficking properties of wild-type hERG. Our present findings indicate that translation reinitiation may generate trafficking-defective as well as dysfunctional channels in patients with LQT2 premature termination codon mutations that occur early in the coding sequence.

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

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.

Isoform-specific dominant-negative effects associated with hERG1 G628S mutation in long QT syndrome.

BACKGROUND: Mutations in the human ether-a-go-go-related gene 1 (hERG1) cause type 2 long QT syndrome (LQT2). The hERG1 gene encodes a K(+) channel with properties similar to the rapidly activating delayed rectifying K(+) current in the heart. Several hERG1 isoforms with unique structural and functional properties have been identified. To date, the pathogenic mechanisms of LQT2 mutations have been predominantly described in the context of the hERG1a isoform. In the present study, we investigated the functional consequences of the LQT2 mutation G628S in the hERG1b and hERG1a(USO) isoforms. METHODS: A double-stable, mammalian expression system was developed to characterize isoform-specific dominant-negative effects of G628S-containing channels when co-expressed at equivalent levels with wild-type hERG1a. Western blot and co-immunoprecipitation studies were performed to study the trafficking and co-assembly of wild-type and mutant hERG1 isoforms. Patch-clamp electrophysiology was performed to characterize hERG1 channel function and the isoform-specific dominant-negative effects associated with the G628S mutation. CONCLUSIONS: The non-functional hERG1a-G628S and hERG1b-G628S channels co-assembled with wild-type hERG1a and dominantly suppressed hERG1 current. In contrast, G628S-induced dominant-negative effects were absent in the context of the hERG1a(USO) isoform. hERG1a(USO)-G628S channels did not appreciably associate with hERG1a and did not significantly suppress hERG1 current when co-expressed at equivalent ratios or at ratios that approximate those found in cardiac tissue. These results suggest that the dominant-negative effects of LQT2 mutations may primarily occur in the context of the hERG1a and hERG1b isoforms.

Nonsense-mediated mRNA decay caused by a frameshift mutation in a large kindred of type 2 long QT syndrome.

BACKGROUND: Nonsense and frameshift mutations are common in congenital long QT syndrome type 2 (LQT2). We previously demonstrated that hERG nonsense mutations cause degradation of mutant mRNA by nonsense-mediated mRNA decay (NMD) and are associated with mild clinical phenotypes. The impact of NMD on the expression of hERG frameshift mutations and their phenotypic severity is not clear. OBJECTIVE: The purpose of this study was to examine the role of NMD in the pathogenesis of a hERG frameshift mutation, P926AfsX14, identified in a large LQT2 kindred and characterize genotype-phenotype correlations. METHODS: Genetic screening was performed among family members. Phenotyping was performed by assessment of ECGs and LQTS-related cardiac events. The functional effect of P926AfsX14 was studied using hERG cDNA and minigene constructs expressed in HEK293 cells. RESULTS: Significant cardiac events occurred in carriers of the P926AfsX14 mutation. When expressed from cDNA, the P926AfsX14 mutant channel was only mildly defective. However, when expressed from a minigene, the P926AfsX14 mutation caused a significant reduction in mutant mRNA, protein, and hERG current. Inhibition of NMD by RNA interference knockdown of up-frameshift protein 1 partially restored expression of mutant mRNA and protein and led to a significant increase in hERG current in the mutant cells. These results suggest that NMD is involved in the pathogenic mechanism of the P926AfsX14 mutation. CONCLUSION: Our findings suggest that the hERG frameshift mutation P926AfsX14 primarily results in degradation of mutant mRNA by the NMD pathway rather than production of truncated proteins. When combined with environmental triggers and genetic modifiers, LQT2 frameshift mutations associated with NMD can manifest with a severe clinical phenotype.

Inhibition of nonsense-mediated mRNA decay by antisense morpholino oligonucleotides restores functional expression of hERG nonsense and frameshift mutations in long-QT syndrome.

Mutations in the human ether-a-go-go-related gene (hERG) cause long-QT syndrome type 2 (LQT2). We previously described a homozygous LQT2 nonsense mutation Q1070X in which the mutant mRNA is degraded by nonsense-mediated mRNA decay (NMD) leading to a severe clinical phenotype. The degradation of the Q1070X transcript precludes the expression of truncated but functional mutant channels. In the present study, we tested the hypothesis that inhibition of NMD can restore functional expression of LQT2 mutations that are targeted by NMD. We showed that inhibition of NMD by RNA interference-mediated knockdown of UPF1 increased Q1070X mutant channel protein expression and hERG current amplitude. More importantly, we found that specific inhibition of downstream intron splicing by antisense morpholino oligonucleotides prevented NMD of the Q1070X mutant mRNA and restored the expression of functional Q1070X mutant channels. The restoration of functional expression by antisense morpholino oligonucleotides was also observed in LQT2 frameshift mutations. Our findings suggest that inhibition of NMD by antisense morpholino oligonucleotides may be a potential therapeutic approach for some LQT2 patients carrying nonsense and frameshift mutations.

Multiple splicing defects caused by hERG splice site mutation 2592+1G>A associated with long QT syndrome.

Long QT syndrome type 2 (LQT2) is caused by mutations in the human ether-a-go-go-related gene (hERG). Cryptic splice site activation in hERG has recently been identified as a novel pathogenic mechanism of LQT2. In this report, we characterize a hERG splice site mutation, 2592+1G>A, which occurs at the 5' splice site of intron 10. Reverse transcription-PCR analyses using hERG minigenes transfected into human embryonic kidney-293 cells and HL-1 cardiomyocytes revealed that the 2592+1G>A mutation disrupted normal splicing and caused multiple splicing defects: the activation of cryptic splice sites within exon 10 and intron 10 and complete intron 10 retention. We performed functional and biochemical analyses of the major splice product, hERGΔ24, in which 24 amino acids within the cyclic nucleotide binding domain of the hERG channel COOH-terminus is deleted. Patch-clamp experiments revealed that the splice mutant did not generate hERG current. Western blot and immunostaining studies showed that mutant channels did not traffic to the cell surface. Coexpression of wild-type hERG and hERGΔ24 resulted in significant dominant-negative suppression of hERG current via the intracellular retention of the wild-type channels. Our results demonstrate that 2592+1G>A causes multiple splicing defects, consistent with the pathogenic mechanisms of long QT syndrome.

A major role for HERG in determining frequency of reentry in neonatal rat ventricular myocyte monolayer.

RATIONALE: the rapid delayed rectifier potassium current, I(Kr), which flows through the human ether-a-go-go-related (hERG) channel, is a major determinant of the shape and duration of the human cardiac action potential (APD). However, it is unknown whether the time dependency of I(Kr) enables it to control APD, conduction velocity (CV), and wavelength (WL) at the exceedingly high activation frequencies that are relevant to cardiac reentry and fibrillation. OBJECTIVE: to test the hypothesis that upregulation of hERG increases functional reentry frequency and contributes to its stability. METHODS AND RESULTS: using optical mapping, we investigated the effects of I(Kr) upregulation on reentry frequency, APD, CV, and WL in neonatal rat ventricular myocyte (NRVM) monolayers infected with GFP (control), hERG (I(Kr)), or dominant negative mutant hERG G628S. Reentry frequency was higher in the I(Kr)-infected monolayers (21.12 ± 0.8 Hz; n=43 versus 9.21 ± 0.58 Hz; n=16; P<0.001) but slightly reduced in G628S-infected monolayers. APD(80) in the I(Kr)-infected monolayers was shorter (>50%) than control during pacing at 1 to 5 Hz. CV was similar in both groups at low frequency pacing. In contrast, during high-frequency reentry, the CV measured at varying distances from the center of rotation was significantly faster in I(Kr)-infected monolayers than controls. Simulations using a modified NRVM model predicted that rotor acceleration was attributable, in part, to a transient hyperpolarization immediately following the AP. The transient hyperpolarization was confirmed experimentally. CONCLUSIONS: hERG overexpression dramatically accelerates reentry frequency in NRVM monolayers. Both APD and WL shortening, together with transient hyperpolarization, underlies the increased rotor frequency and stability.

Alternative splicing and polyadenylation contribute to the generation of hERG1 C-terminal isoforms.

The human ether-a-go-go-related gene 1 (hERG1) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel. Several hERG1 isoforms with different N- and C-terminal ends have been identified. The hERG1a, hERG1b, and hERG1-3.1 isoforms contain the full-length C terminus, whereas the hERG1(USO) isoforms, hERG1a(USO) and hERG1b(USO), lack most of the C-terminal domain and contain a unique C-terminal end. The mechanisms underlying the generation of hERG1(USO) isoforms are not understood. We show that hERG1 isoforms with different C-terminal ends are generated by alternative splicing and polyadenylation of hERG1 pre-mRNA. We identified an intrinsically weak, noncanonical poly(A) signal, AGUAAA, within intron 9 of hERG1 that modulates the expression of hERG1a and hERG1a(USO). Replacing AGUAAA with the strong, canonical poly(A) signal AAUAAA resulted in the predominant production of hERG1a(USO) and a marked decrease in hERG1 current. In contrast, eliminating the intron 9 poly(A) signal or increasing the strength of 5' splice site led to the predominant production of hERG1a and a significant increase in hERG1 current. We found significant variation in the relative abundance of hERG1 C-terminal isoforms in different human tissues. Taken together, these findings suggest that post-transcriptional regulation of hERG1 pre-mRNA may represent a novel mechanism to modulate the expression and function of hERG1 channels.

Recurrent intrauterine fetal loss due to near absence of HERG: clinical and functional characterization of a homozygous nonsense HERG Q1070X mutation.

BACKGROUND: Inherited arrhythmias may underlie intrauterine and neonatal arrhythmias. Resolving the molecular genetic nature of these rare cases provides significant insight into the role of the affected proteins in arrhythmogenesis and (extra-) cardiac development. OBJECTIVE: The purpose of this study was to perform clinical, molecular, and functional studies of a consanguineous Arabian family with repeated early miscarriages and two intrauterine fetal losses in the early part of the third trimester of pregnancy due to persistent arrhythmias. METHODS: In-depth clinical investigation was performed in two siblings, both of whom developed severe arrhythmia during the second trimester of pregnancy. Homozygosity mapping with microsatellite repeat polymorphic markers encompassing various cardiac ion channel genes linked to electrical instability of the heart was performed. Screening of the candidate gene in the homozygous locus was performed. Biochemical and electrophysiologic analysis was performed to elucidate the function of the mutated gene. RESULTS: Screening of the HERG gene in the homozygous locus detected a homozygous nonsense mutation Q1070X in the HERG C-terminus in affected children. Biochemical and functional analysis of the Q1070X mutant showed that although the mutant HERG had the ability to traffic to the plasma membrane and to form functional channels, it was destroyed by the nonsense-mediated decay (NMD) pathway before its translation. NMD leads to near absence of HERG in homozygous Q1070X mutation carriers, causing debilitating arrhythmias (prior to birth) in homozygous carriers but no apparent phenotype in heterozygous carriers. CONCLUSION: Homozygous HERG Q1070X is equivalent to near functional knockout of HERG. Clinical consequences appear early, originating during the early stages of embryonic life. The NMD pathway renders HERG Q1070X functionless before it can form a functional ion channel.

A splice site mutation in hERG leads to cryptic splicing in human long QT syndrome.

Mutations in the human ether-a-go-go-related gene (hERG) cause type 2 long QT syndrome. In this study, we investigated the pathogenic mechanism of the hERG splice site mutation 2398+1G>C and the genotype-phenotype relationship of mutation carriers in three unrelated kindreds with long QT syndrome. The effect of 2398+1G>C on mRNA splicing was studied by analysis of RNA isolated from lymphocytes of index patients and using minigenes expressed in HEK293 cells and neonatal rat ventricular myocytes. RT-PCR analysis revealed that the 2398+1G>C mutation disrupted the normal splicing and activated a cryptic splice donor site in intron 9, leading to the inclusion of 54 nt of the intron 9 sequence in hERG mRNA. The cryptic splicing resulted in an in-frame insertion of 18 amino acids in the middle of the cyclic nucleotide binding domain. In patch clamp experiments the splice mutant did not generate hERG current. Western blot and immunostaining studies showed that the mutant expressed an immature form of hERG protein that failed to reach the plasma membrane. Coexpression of the mutant and wild-type channels led to a dominant negative suppression of wild-type channel function by intracellular retention of heteromeric channels. Our results demonstrate that 2398+1G>C activates a cryptic site and generates a full-length hERG protein with an insertion of 18 amino acids, which leads to a trafficking defect of the mutant channel.

Nonsense mutations in hERG cause a decrease in mutant mRNA transcripts by nonsense-mediated mRNA decay in human long-QT syndrome.

BACKGROUND: Long-QT syndrome type 2 (LQT2) is caused by mutations in the human ether-a-go-go-related gene (hERG). More than 30% of the LQT2 mutations result in premature termination codons. Degradation of premature termination codon-containing mRNA transcripts by nonsense-mediated mRNA decay is increasingly recognized as a mechanism for reducing mRNA levels in a variety of human diseases. However, the role of nonsense-mediated mRNA decay in LQT2 mutations has not been explored. METHODS AND RESULTS: We examined the expression of hERG mRNA in lymphocytes from patients carrying the R1014X mutation using a technique of allele-specific transcript quantification. The R1014X mutation led to a reduced level of mutant mRNA compared with that of the wild-type allele. The decrease in mutant mRNA also was observed in the LQT2 nonsense mutations W1001X and R1014X using hERG minigenes expressed in HEK293 cells or neonatal rat ventricular myocytes. Treatment with the protein synthesis inhibitor cycloheximide or RNA interference-mediated knockdown of the Upf1 protein resulted in the restoration of mutant mRNA to levels comparable to that of the wild-type minigene, suggesting that hERG nonsense mutations are subject to nonsense-mediated mRNA decay. CONCLUSIONS: These results indicate that LQT2 nonsense mutations cause a decrease in mutant mRNA levels by nonsense-mediated mRNA decay rather than production of truncated proteins. Our findings suggest that the degradation of hERG mutant mRNA by nonsense-mediated mRNA decay is an important mechanism in LQT2 patients with nonsense or frameshift mutations.

[The mechanistic rote of KCNH2 gene L413P and L559H mutations in long QT syndrome].

OBJECTIVE: To investigate the molecular pathogenesis for two novel mutations L413P and L559H of KCNH2 found in Chinese patients with long QT syndrome. METHODS: L413P and L559H mutant constructs were generated by site-directed mutagenesis using human wild-type (WT) pcDNA3-HERG cDNA as a template. WT and mutant constructs were transiently transfected into human embryonic kidney 293 cells using lipofectamine method. After transfection, the recording of HERG current was performed using patch clamp technique. The expression and cellular localization of HERG protein were studied with Western blot and immunofluorescence methods. RESULTS: Electrophysiological recordings showed that L413P and L559H mutations did not express HERG current. Western blot analysis revealed that only 135 000 immature HERG protein was expressed in L413P and L559H-transfected cells, whereas both mature and immature forms of HERG protein were observed in WT-transfected cells. Immunofluorescence study showed that L413P and L559H mutant proteins were predominantly localized around the nucleus, suggesting that the mutant channels are retained in the endoplasmic reticulum. When L413P or L559H was co-transfected with equal amount of WT plasmids, both 135 000 and 155 000 forms of HERG protein were observed, and the HERG current was not significantly changed as compared with that of WT transfection alone. Low temperature and E-4031could not rescue these two mutant channels. CONCLUSIONS: The L413P and L559H mutations resulted in protein trafficking defects with failure of mutant proteins to reach the plasma membrane. However, both biochemical and electrophysiological results showed that the mutations did not have a dominant-negative effect on WT, indicating that the mechanism of the L413P and L559H mutations might be haploinsufficiency.

Most LQT2 mutations reduce Kv11.1 (hERG) current by a class 2 (trafficking-deficient) mechanism.

BACKGROUND: The KCNH2 or human ether-a-go-go related gene (hERG) encodes the Kv11.1 alpha-subunit of the rapidly activating delayed rectifier K+ current (IKr) in the heart. Type 2 congenital long-QT syndrome (LQT2) results from KCNH2 mutations that cause loss of Kv11.1 channel function. Several mechanisms have been identified, including disruption of Kv11.1 channel synthesis (class 1), protein trafficking (class 2), gating (class 3), or permeation (class 4). For a few class 2 LQT2-Kv11.1 channels, it is possible to increase surface membrane expression of Kv11.1 current (IKv11.1). We tested the hypotheses that (1) most LQT2 missense mutations generate trafficking-deficient Kv11.1 channels, and (2) their trafficking-deficient phenotype can be corrected. METHODS AND RESULTS: Wild-type (WT)-Kv11.1 channels and 34 missense LQT2-Kv11.1 channels were expressed in HEK293 cells. With Western blot analyses, 28 LQT2-Kv11.1 channels had a trafficking-deficient (class 2) phenotype. For the majority of these mutations, the class 2 phenotype could be corrected when cells were incubated for 24 hours at reduced temperature (27 degrees C) or in the drugs E4031 or thapsigargin. Four of the 6 LQT2-Kv11.1 channels that had a wild-type-like trafficking phenotype did not cause loss of Kv11.1 function, which suggests that these channels are uncommon sequence variants. CONCLUSIONS: This is the first study to identify a dominant mechanism, class 2, for the loss of Kv11.1 channel function in LQT2 and to report that the class 2 phenotype for many of these mutant channels can be corrected. This suggests that if therapeutic strategies to correct protein trafficking abnormalities can be developed, it may offer clinical benefits for LQT2 patients.