Long QT Syndrome Type 2 Mutations Research Articles

Mutation-specific peripheral and ER quality control of hERG channel cell-surface expression

Impaired functional plasma membrane (PM) expression of the hERG K+-channel is associated with Long-QT syndrome type-2 (LQT2) and increased risk of cardiac arrhythmia. Reduced PM-expression is primarily attributed to retention and degradation of misfolded channels by endoplasmic reticulum (ER) protein quality control (QC) systems. However, as the molecular pathogenesis of LQT2 was defined using severely-misfolded hERG variants with limited PM-expression, the potential contribution of post-ER (peripheral) QC pathways to the disease phenotype remains poorly established. Here, we investigate the cellular processing of mildly-misfolded Per-Arnt-Sim (PAS)-domain mutant hERGs, which display incomplete ER-retention and PM-expression defects at physiological temperature. We show that the attenuated PM-expression of hERG is dictated by mutation-specific contributions from both the ER and peripheral QC systems. At the ER, PAS-mutants experience inefficient conformational maturation coupled with rapid ubiquitin-dependent proteasomal degradation. In post-ER compartments, they are rapidly endocytosed from the PM via a ubiquitin-independent mechanism and rapidly targeted for lysosomal degradation. Conformational destabilization underlies aberrant cellular processing at both ER- and post-ER compartments, since conformational correction by a hERG-specific pharmacochaperone or low-temperatures can restore WT-like trafficking. Our results demonstrate that the post-ER QC alone or jointly with the ER QC determines the loss-of-PM-expression phenotype of a subset of LQT2 mutations.

Visualizing Mutation-Specific Differences in the Trafficking-Deficient Phenotype of Kv11.1 Proteins Linked to Long QT Syndrome Type 2.

KCNH2 encodes the Kv11.1 α-subunit that underlies the rapidly activating delayed-rectifier K+ current in the heart. Loss-of-function KCNH2 mutations cause long QT syndrome type 2 (LQT2), and most LQT2-linked missense mutations inhibit the trafficking of Kv11.1 channel protein to the cell surface membrane. Several trafficking-deficient LQT2 mutations (e.g., G601S) generate Kv11.1 proteins that are sequestered in a microtubule-dependent quality control (QC) compartment in the transitional endoplasmic reticulum (ER). We tested the hypothesis that the QC mechanisms that regulate LQT2-linked Kv11.1 protein trafficking are mutation-specific. Confocal imaging analyses of HEK293 cells stably expressing the trafficking-deficient LQT2 mutation F805C showed that, unlike G601S-Kv11.1 protein, F805C-Kv11.1 protein was concentrated in several transitional ER subcompartments. The microtubule depolymerizing drug nocodazole differentially affected G601S- and F805C-Kv11.1 protein immunostaining. Nocodazole caused G601S-Kv11.1 protein to distribute into peripheral reticular structures, and it increased the diffuse immunostaining of F805C-Kv11.1 protein around the transitional ER subcompartments. Proteasome inhibition also affected the immunostaining of G601S- and F805C-Kv11.1 protein differently. Incubating cells in MG132 minimally impacted G601S-Kv11.1 immunostaining, but it dramatically increased the diffuse immunostaining of F805C-Kv11.1 protein in the transitional ER. Similar results were seen after incubating cells in the proteasome inhibitor lactacystin. Differences in the cellular distribution of G601S-Kv11.1 and F805C-Kv11.1 protein persisted in transfected human inducible pluripotent stem cell derived cardiomyocytes. These are the first data to visually demonstrate mutation-specific differences in the trafficking-deficient LQT2 phenotype, and this study has identified a novel way to categorize trafficking-deficient LQT2 mutations based on differences in intracellular retention.

Rescue of protein expression defects may not be enough to abolish the pro-arrhythmic phenotype of long QT type 2 mutations.

Most missense long QT syndrome type 2 (LQTS2) mutations result in Kv11.1 channels that show reduced levels of membrane expression. Pharmacological chaperones that rescue mutant channel expression could have therapeutic potential to reduce the risk of LQTS2-associated arrhythmias and sudden cardiac death, but only if the mutant Kv11.1 channels function normally (i.e. like WT channels) after membrane expression is restored. Fewer than half of mutant channels exhibit relatively normal function after rescue by low temperature. The remaining rescued missense mutant Kv11.1 channels have perturbed gating and/or ion selectivity characteristics. Co-expression of WT subunits with gating defective missense mutations ameliorates but does not eliminate the functional abnormalities observed for most mutant channels. For patients with mutations that affect gating in addition to expression, it may be necessary to use a combination therapy to restore both normal function and normal expression of the channel protein. In the heart, Kv11.1 channels pass the rapid delayed rectifier current (IKr ) which plays critical roles in repolarization of the cardiac action potential and in the suppression of arrhythmias caused by premature stimuli. Over 500 inherited mutations in Kv11.1 are known to cause long QT syndrome type 2 (LQTS2), a cardiac electrical disorder associated with an increased risk of life threatening arrhythmias. Most missense mutations in Kv11.1 reduce the amount of channel protein expressed at the membrane and, as a consequence, there has been considerable interest in developing pharmacological agents to rescue the expression of these channels. However, pharmacological chaperones will only have clinical utility if the mutant Kv11.1 channels function normally after membrane expression is restored. The aim of this study was to characterize the gating phenotype for a subset of LQTS2 mutations to assess what proportion of mutations may be suitable for rescue. As an initial screen we used reduced temperature to rescue expression defects of mutant channels expressed in Xenopus laevis oocytes. Over half (∼56%) of Kv11.1 mutants exhibited functional gating defects that either dramatically reduced the amount of current contributing to cardiac action potential repolarization and/or reduced the amount of protective current elicited in response to premature depolarizations. Our data demonstrate that if pharmacological rescue of protein expression defects is going to have clinical utility in the treatment of LQTS2 then it will be important to assess the gating phenotype of LQTS2 mutations before attempting rescue.

Role of the Cytoplasmic N-terminal Cap and Per-Arnt-Sim (PAS) Domain in Trafficking and Stabilization of Kv11.1 Channels

The N-terminal cytoplasmic region of the Kv11.1a potassium channel contains a Per-Arnt-Sim (PAS) domain that is essential for the unique slow deactivation gating kinetics of the channel. The PAS domain has also been implicated in the assembly and stabilization of the assembled tetrameric channel, with many clinical mutants in the PAS domain resulting in reduced stability of the domain and reduced trafficking. Here, we use quantitative Western blotting to show that the PAS domain is not required for normal channel trafficking nor for subunit-subunit interactions, and it is not necessary for stabilizing assembled channels. However, when the PAS domain is present, the N-Cap amphipathic helix must also be present for channels to traffic to the cell membrane. Serine scan mutagenesis of the N-Cap amphipathic helix identified Leu-15, Ile-18, and Ile-19 as residues critical for the stabilization of full-length proteins when the PAS domain is present. Furthermore, mutant cycle analysis experiments support recent crystallography studies, indicating that the hydrophobic face of the N-Cap amphipathic helix interacts with a surface-exposed hydrophobic patch on the core of the PAS domain to stabilize the structure of this critical gating domain. Our data demonstrate that the N-Cap amphipathic helix is critical for channel stability and trafficking.

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 >54–60nt 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.

The structural mechanism of KCNH-channel regulation by the eag domain

The KCNH voltage-dependent potassium channels (ether-á-go-go, EAG; EAG-related gene, ERG; EAG-like channels, ELK) are important regulators of cellular excitability1-3 and have key roles in diseases such as cardiac long QT syndrome type 2 (LQT2)4, epilepsy5, schizophrenia6 and cancer7. The intracellular domains of KCNH channels are structurally distinct from other voltage-gated channels. The amino-terminal region contains an eag domain, which is comprised of a Per-Arnt-Sim (PAS) domain and a PAS-cap domain8, while the carboxy-terminal region contains a cyclic nucleotide-binding homology domain (CNBHD) which is connected to the pore through a C-linker domain. Many disease-causing mutations localize to these specialized intracellular domains, which underlie the unique gating and regulation of KCNH channels9. It has been suggested that the eag domain may regulate the channel by interacting with either the S4-S5 linker or the CNBHD8,10. Here we present a 2-Å resolution crystal structure of the eag domain-CNBHD complex of the mouse EAG1 (mEAG1) channel. It displays extensive interactions between the eag domain and the CNBHD, indicating that the regulatory mechanism of the eag domain involves primarily the CNBHD. Surprisingly, the structure reveals that a number of LQT2 mutations at homologous positions in hERG, and cancer-associated mutations in EAG channels, localize to the eag domain-CNBHD interface. Furthermore, mutations at the interface produced dramatic effects on channel gating demonstrating the important physiological role of the eag domain-CNBHD interaction. Our structure of the eag domain-CNBHD complex of mEAG1 provides unique insights into the physiological and pathophysiological mechanisms of KCNH channels.

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.

LQT2 Nonsense Mutation Escapes NMD by the Reinitiation of Translation to Generate N-Terminally Truncated hERG Channels

Mutations in the human ether-a-go-go-related gene (hERG) result in long QT syndrome type 2 (LQT2). hERG mRNA transcripts that contain premature termination codon (PTC) mutations are known to be rapidly degraded by nonsense-mediated mRNA decay (NMD). We have identified a LQT2 nonsense mutation Q81X which escapes NMD-mediated degradation through the reinitiation of translation to generate an N-terminally truncated channel with altered gating properties. RNA analysis of full-length wild-type or Q81X hERG minigenes revealed equivalent mRNA levels, in contrast with transcripts containing the P141fs+2X frameshift mutation which undergo NMD. Western blot analysis revealed that Q81X channels are expressed at the cell surface but have a lower molecular weight than wild-type channels. Functional analysis of the Q81X channels revealed reduced current levels and accelerated deactivation kinetics compared with wild-type channels. These results are consistent with the disruption of the hERG N-terminus, including the Per, Arnt, and Sim domain, which is known to play an important role in regulating hERG channel deactivation. Site-specific mutagenesis showed that translation of the Q81X transcript is reinitiated at Met124 following premature termination. Furthermore, we found that Q81X co-assembles with wild-type hERG channels resulting in the dominant-negative acceleration of deactivation in heteromeric channels. Our findings indicate that the reinitiation of translation may be an important pathogenic mechanism in patients with early nonsense and frameshift LQT2 mutations.

Abstract 18298: Trafficking-Deficient hERG K+ Channels Linked to Long QT Syndrome are Regulated by a Quality Control Compartment in the ER

The human Ether-a-go-go Related Gene (hERG) encodes the pore-forming voltage-gated K+ channel (Kv) α-subunit that underlies the rapidly-activating delayed-rectifier current in cardiomyocytes. hERG is synthesized in the Endoplasmic Reticulum (ER) as “immature” 135 kDa asparagine-linked (N-linked) glycoprotein & is terminally glycosylated in the Golgi apparatus (Golgi) to form a “mature” 155 kDa glycoprotein. Most hERG missense mutations linked to Long QT syndrome Type 2 (LQT2) reduce terminal glycosylation & functional expression. E-4031 is a class III anti-arrhythmic that binds to hERG & blocks current (IhERG), & can increase terminal glycosylation & functional expression for many “trafficking-deficient” LQT2 mutations (pharmacological correction). Treating HEK293 cells expressing the trafficking-deficient LQT2 mutation G601S-hERG in 10 uM E-4031 (E4) for 6 or 12 hours showed a time-dependent increase in maximal IhERG after drug washout, Western blot analyses showed similar increase in the fractional density (FD) of terminally glycosylated G601S-hERG. Treating cells in 0.2mg/ml cycloheximide (cyclo), that inhibits protein translation, did not alter the E4 induced increase in terminally glycosylated G601S-hERG, suggesting pharmacological correction occurs post-translationally. Immunocytochemistry & colocalization analyses showed that in control conditions G601S-hERG primarily colocalized w/ BAP31, an itinerant ER protein that moves between the peripheral ER and perinuclear ER. G601S-hERG weakly colocalized w/ other ER proteins, like lecithin chaperone calnexin, ERAD complex protein Derlin, or transitional ER marker Sec31. Treating cells in E4 decreased G601S-hERG and BAP31 colocalization after 6 hrs & 12 hr. Treatment with microtubular depolymerizing agent nocodazole (10 uM) for 2 hrs increased G601S-hERG colocalization with BAP31 & caused them to accumulate into peripheral ER compartments. We conclude that in control conditions, G601S-hERG is an itinerant ER protein that moves between the peripheral and perinuclear ER via a microtubule-dependent mechanism, & pharmacological correction with E-4031 prevents this by allowing transport to the Golgi & cell surface membrane.

Trafficking-deficient hERG K+ channels linked to long QT syndrome are regulated by a microtubule-dependent quality control compartment in the ER

The human ether-a-go-go related gene (hERG) encodes the voltage-gated K(+) channel that underlies the rapidly activating delayed-rectifier current in cardiac myocytes. hERG is synthesized in the endoplasmic reticulum (ER) as an "immature" N-linked glycoprotein and is terminally glycosylated in the Golgi apparatus. Most hERG missense mutations linked to long QT syndrome type 2 (LQT2) reduce the terminal glycosylation and functional expression. We tested the hypothesis that a distinct pre-Golgi compartment negatively regulates the trafficking of some LQT2 mutations to the Golgi apparatus. We found that treating cells in nocodazole, a microtubule depolymerizing agent, altered the subcellular localization, functional expression, and glycosylation of the LQT2 mutation G601S-hERG differently from wild-type hERG (WT-hERG). G601S-hERG quickly redistributed to peripheral compartments that partially colocalized with KDEL (Lys-Asp-Glu-Leu) chaperones but not calnexin, Sec31, or the ER golgi intermediate compartment (ERGIC). Treating cells in E-4031, a drug that increases the functional expression of G601S-hERG, prevented the accumulation of G601S-hERG to the peripheral compartments and increased G601S-hERG colocalization with the ERGIC. Coexpressing the temperature-sensitive mutant G protein from vesicular stomatitis virus, a mutant N-linked glycoprotein that is retained in the ER, showed it was not restricted to the same peripheral compartments as G601S-hERG at nonpermissive temperatures. We conclude that the trafficking of G601S-hERG is negatively regulated by a microtubule-dependent compartment within the ER. Identifying mechanisms that prevent the sorting or promote the release of LQT2 channels from this compartment may represent a novel therapeutic strategy for LQT2.

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.

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

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.

Mutations at the Intron 9 Donor Splice Site in hERG Lead to Cryptic Splicing in LQT2

Long QT syndrome type 2 (LQT2) is caused by mutations in the human ether-a-go-go-related gene (hERG). More than 30% of LQT2 mutations are nonsense, frameshift, or splice site mutations that may affect mRNA stability and splicing. To date, relatively few studies have focused on the pathogenesis of hERG splice site mutations. We characterized three LQT2 mutations in the 5′ donor splice site of intron 9: 2398G>T, 2398+3A>T, and 2398+5G>T. G2398 is the last nucleotide of exon 9 and 2398G>T has been previously classified as a missense mutation (G800W). The functional consequences of these mutations were studied by RT-PCR analysis of RNA collected from HEK293 cells transfected with minigenes containing the wild-type or mutant genomic sequence spanning exon 8 to exon 11 of hERG. All three splice site mutants disrupt normal splicing and produce an aberrantly spliced transcript. Sequence analysis showed that this transcript results from the use of a cryptic 5′ donor splice site in intron 9 located 54 nt downstream of the normal site. Translation of this transcript would result in an in-frame insertion of 18 amino acids in the cyclic nucleotide binding domain. A full length hERG cDNA construct including the 2398G>T mutation and the additional 54 nt from intron 9 was expressed in HEK293 cells. Patch clamp studies revealed that the splice mutant channels did not produce hERG current. Western blot analysis showed that the mutant expressed the immature form of the hERG protein indicating defective channel trafficking. These studies underscore the importance of RNA analysis in describing the pathogenesis of LQT2. The intron 9 donor splice site appears to be a localized hot-spot for LQT2 mutations.

Nonsense Mutations in hERG Cause a Decrease in Mutant mRNA Transcripts by Nonsense-Mediated mRNA Decay in Human Long-QT Syndrome

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. 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. 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.

Mechanisms of Pharmacological Rescue of Trafficking-defective hERG Mutant Channels in Human Long QT Syndrome

Long QT syndrome type 2 is caused by mutations in the human ether-a-go-go-related gene (hERG). We previously reported that the N470D mutation is retained in the endoplasmic reticulum (ER) but can be rescued to the plasma membrane by hERG channel blocker E-4031. The mechanisms of ER retention and how E-4031 rescues the N470D mutant are poorly understood. In this study, we investigated the interaction of hERG channels with the ER chaperone protein calnexin. Using coimmunoprecipitation, we showed that the immature forms of both wild type hERG and N470D associated with calnexin. The association required N-linked glycosylation of hERG channels. Pulse-chase analysis revealed that N470D had a prolonged association with calnexin compared with wild type hERG and E-4031 shortened the time course of calnexin association with N470D. To test whether the prolonged association of N470D with calnexin is due to defective folding of mutant channels, we studied hERG channel folding using the trypsin digestion method. We found that N470D and the immature form of wild type hERG were more sensitive to trypsin digestion than the mature form of wild type hERG. In the presence of E-4031, N470D became more resistant to trypsin even when its ER-to-Golgi transport was blocked by brefeldin A. These results suggest that defective folding of N470D contributes to its prolonged association with calnexin and ER retention and that E-4031 may restore proper folding of the N470D channel leading to its cell surface expression.

Identification of the cyclic-nucleotide-binding domain as a conserved determinant of ion-channel cell-surface localization

Mutations of a putative cyclic-nucleotide-binding domain (CNBD) can disrupt the function of the hyperpolarization-activated cyclic-nucleotide-gated channel (HCN2) and the human ether-a-go-go-related gene potassium channel (HERG). Loss of function caused by C-terminal truncation, which includes all or part of the CNBD in HCN and HERG, has been related to abnormal channel trafficking. Similar defects have been reported for several of the missense mutations of HERG associated with long QT syndrome type 2 (LQT2). Thus, we postulate that normal processing of these channels depends upon the presence of the CNBD. Here, we show that removal of the entire CNBD prevents Golgi transit, surface localization and function of HERG channel tetramers. This is also true when any of the structural motifs of the CNBD is deleted, suggesting that deletion of any highly conserved region along the entire length of the CNBD can disrupt channel trafficking. Furthermore, we demonstrate that defective trafficking is a consequence of all LQT2 mutations in the CNBD, including two mutations not previously assessed and two others for which there are conflicting results in the literature. The trafficking sensitivity of the CNBD might be of general significance for other ion channels because complete deletion of the CNBD or mutations at highly conserved residues within the CNBD of the related ERG3 channel and HCN2 also prevent Golgi transit. These results broadly implicate the CNBD in ion-channel trafficking that accounts for the commonly observed loss of function associated with CNBD mutants and provides a rationale for distinct genetic disorders.

Defective assembly and trafficking of mutant HERG channels with C-terminal truncations in long QT syndrome

Mutations in the human ether-a-go-go-related gene ( HERG) cause long QT syndrome type 2 (LQT2). HERG encodes a voltage-gated potassium channel consisting of four subunits. Tetrameric assembly is required for the formation of functional HERG channels. In the present work, we studied the role of assembly in HERG channel dysfunction of LQT2 mutations Q725X and R1014X, both of which cause truncations of the C-terminus of HERG channels. When expressed in HEK293 cells, Q725X did not generate HERG current, while R1014X generated HERG current with markedly reduced amplitude. Western blot analysis showed that both mutations caused defective trafficking of HERG channel proteins. Using sucrose gradient centrifugation we showed that wild type HERG and R1014X formed a tetrameric structure, whereas Q725X was expressed as a monomer. When coexpressed with wild type HERG, R1014X, but not Q725X, caused dominant negative suppression of wild type HERG current. Coimmunoprecipitation experiments showed that the lack of dominant negative effect by Q725X was due to failure of mutant subunits to coassemble with wild type subunits. These results suggest that the Q725X mutation causes HERG channel dysfunction by disruption of tetrameric assembly of HERG channels. In contrast, the R1014X mutation is capable of forming tetrameric structure, and it causes HERG channel dysfunction by defective trafficking of the mutant protein.

The Binding Site for Channel Blockers That Rescue Misprocessed Human Long QT Syndrome Type 2 ether-a-gogo-related Gene (HERG) Mutations

Mutations in the human ether-a-gogo-related gene (HERG) K(+) channel gene cause chromosome 7-linked long QT syndrome type 2 (LQT2), which is characterized by a prolonged QT interval in the electrocardiogram and an increased susceptibility to life-threatening cardiac arrhythmias. LQT2 mutations produce loss-of-function phenotypes and reduce I(Kr) currents either by the heteromeric assembly of non- or malfunctioning channel subunits with wild type subunits at the cell surface or by retention of misprocessed mutant HERG channels in the endoplasmic reticulum. Misprocessed mutations often encode for channel proteins that are functional upon incorporation into the plasma membrane. As a result the pharmacological correction of folding defects and restoration of protein function are of considerable interest. Here we report that the trafficking-deficient pore mutation HERG G601S was rescued by a series of HERG channel blockers that increased cell surface expression. Rescue by these pharmacological chaperones varied directly with their blocking potency. We used structure-activity relationships and site-directed mutagenesis to define the binding site of the pharmacological chaperones. We found that binding occurred in the inner cavity and correlated with hydrophobicity and cationic charge. Rescue was domain-restricted because the trafficking of two misprocessed mutations in the C terminus, HERG F805C and HERG R823W, was not restored by channel blockers. Our findings represent a first step toward the design of pharmacological chaperones that will rescue HERG K(+) channels without block.