Global Proteomic Analysis Reveals Alterations in Differentially Expressed Proteins between Cardiopathic Lamin A/C Mutations.

Lamin A/C (LMNA) is an important component of nuclear lamina. Mutations cause arrhythmia, heart failure, and sudden cardiac death. While LMNA-associated cardiomyopathy typically has an aggressive course that responds poorly to conventional heart failure therapies, there is variability in severity and age of penetrance between and even within specific mutations, which is poorly understood at the cellular level. Further, this heterogeneity has not previously been captured to mimic the heterozygous state, nor have the hundreds of clinical LMNA mutations been represented. Herein, we have overexpressed cardiopathic LMNA variants in HEK cells and utilized state-of-the-art quantitative proteomics to compare the global proteomic profiles of (1) aggregating Q353 K alone, (2) Q353 K coexpressed with WT, (3) aggregating N195 K coexpressed with WT, and (4) nonaggregating E317 K coexpressed with WT to help capture some of the heterogeneity between mutations. We analyzed each data set to obtain the differentially expressed proteins (DEPs) and applied gene ontology (GO) and KEGG pathway analyses. We found a range of 162 to 324 DEPs from over 6000 total protein IDs with differences in GO terms, KEGG pathways, and DEPs important in cardiac function, further highlighting the complexity of cardiac laminopathies. Pathways disrupted by LMNA mutations were validated with redox, autophagy, and apoptosis functional assays in both HEK 293 cells and in induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) for LMNA N195 K. These proteomic profiles expand our repertoire for mutation-specific downstream cellular effects that may become useful as druggable targets for personalized medicine approach for cardiac laminopathies.

Mutation-Specific Differences in Kv7.1 (KCNQ1) and Kv11.1 (KCNH2) Channel Dysfunction and Long QT Syndrome Phenotypes.

The electrocardiogram (ECG) empowered clinician scientists to measure the electrical activity of the heart noninvasively to identify arrhythmias and heart disease. Shortly after the standardization of the 12-lead ECG for the diagnosis of heart disease, several families with autosomal recessive (Jervell and Lange-Nielsen Syndrome) and dominant (Romano-Ward Syndrome) forms of long QT syndrome (LQTS) were identified. An abnormally long heart rate-corrected QT-interval was established as a biomarker for the risk of sudden cardiac death. Since then, the International LQTS Registry was established; a phenotypic scoring system to identify LQTS patients was developed; the major genes that associate with typical forms of LQTS were identified; and guidelines for the successful management of patients advanced. In this review, we discuss the molecular and cellular mechanisms for LQTS associated with missense variants in KCNQ1 (LQT1) and KCNH2 (LQT2). We move beyond the "benign" to a "pathogenic" binary classification scheme for different KCNQ1 and KCNH2 missense variants and discuss gene- and mutation-specific differences in K+ channel dysfunction, which can predispose people to distinct clinical phenotypes (e.g., concealed, pleiotropic, severe, etc.). We conclude by discussing the emerging computational structural modeling strategies that will distinguish between dysfunctional subtypes of KCNQ1 and KCNH2 variants, with the goal of realizing a layered precision medicine approach focused on individuals.

A rapid solubility assay of protein domain misfolding for pathogenicity assessment of rare DNA sequence variants.

PURPOSE: DNA sequencing technology has unmasked a vast number of uncharacterized single-nucleotide variants in disease-associated genes, and efficient methods are needed to determine pathogenicity and enable clinical care. METHODS: We report an E. coli-based solubility assay for assessing the effects of variants on protein domain stability for three disease-associated proteins. RESULTS: First, we examined variants in the Kv11.1 channel PAS domain (PASD) associated with inherited long QT syndrome type 2 and found that protein solubility correlated well with reported in vitro protein stabilities. A comprehensive solubility analysis of 56 Kv11.1 PASD variants revealed that disruption of membrane trafficking, the dominant loss-of-function disease mechanism, is largely determined by domain stability. We further validated this assay by using it to identify second-site suppressor PASD variants that improve domain stability and Kv11.1 protein trafficking. Finally, we applied this assay to several cancer-linked P53 tumor suppressor DNA-binding domain and myopathy-linked Lamin A/C Ig-like domain variants, which also correlated well with reported protein stabilities and functional analyses. CONCLUSION: This simple solubility assay can aid in determining the likelihood of pathogenicity for sequence variants due to protein misfolding in structured domains of disease-associated genes as well as provide insights into the structural basis of disease.

Pharmacological correction of long QT-linked mutations in KCNH2 (hERG) increases the trafficking of Kv11.1 channels stored in the transitional endoplasmic reticulum.

KCNH2 encodes Kv11.1 and underlies the rapidly activating delayed rectifier K(+) current (IKr) in the heart. Loss-of-function KCNH2 mutations cause the type 2 long QT syndrome (LQT2), and most LQT2-linked missense mutations inhibit the trafficking of Kv11.1 channels. Drugs that bind to Kv11.1 and block IKr (e.g., E-4031) can act as pharmacological chaperones to increase the trafficking and functional expression for most LQT2 channels (pharmacological correction). We previously showed that LQT2 channels are selectively stored in a microtubule-dependent compartment within the endoplasmic reticulum (ER). We tested the hypothesis that pharmacological correction promotes the trafficking of LQT2 channels stored in this compartment. Confocal analyses of cells expressing the trafficking-deficient LQT2 channel G601S showed that the microtubule-dependent ER compartment is the transitional ER. Experiments with E-4031 and the protein synthesis inhibitor cycloheximide suggested that pharmacological correction promotes the trafficking of G601S stored in this compartment. Treating cells in E-4031 or ranolazine (a drug that blocks IKr and has a short half-life) for 30 min was sufficient to cause pharmacological correction. Moreover, the increased functional expression of G601S persisted 4-5 h after drug washout. Coexpression studies with a dominant-negative form of Rab11B, a small GTPase that regulates Kv11.1 trafficking, prevented the pharmacological correction of G601S trafficking from the transitional ER. These data suggest that pharmacological correction quickly increases the trafficking of LQT2 channels stored in the transitional ER via a Rab11B-dependent pathway, and we conclude that the pharmacological chaperone activity of drugs like ranolazine might have therapeutic potential.

How Functional Genomics Can Keep Pace With VUS Identification.

Over the last two decades, an exponentially expanding number of genetic variants have been identified associated with inherited cardiac conditions. These tremendous gains also present challenges in deciphering the clinical relevance of unclassified variants or variants of uncertain significance (VUS). This review provides an overview of the advancements (and challenges) in functional and computational approaches to characterize variants and help keep pace with VUS identification related to inherited heart diseases.

Most myopathic lamin variants aggregate: a functional genomics approach for assessing variants of uncertain significance.

Hundreds of LMNA variants have been associated with several distinct disease phenotypes. However, genotype-phenotype relationships remain largely undefined and the impact for most variants remains unknown. We performed a functional analysis for 178 variants across five structural domains using two different overexpression models. We found that lamin A aggregation is a major determinant for skeletal and cardiac laminopathies. An in vitro solubility assay shows that aggregation-prone variants in the immunoglobulin-like domain correlate with domain destabilization. Finally, we demonstrate that myopathic-associated LMNA variants show aggregation patterns in induced pluripotent stem cell derived-cardiomyocytes (iPSC-CMs) in contrast to non-myopathic LMNA variants. Our data-driven approach (1) reveals that striated muscle laminopathies are predominantly protein misfolding diseases, (2) demonstrates an iPSC-CM experimental platform for characterizing laminopathic variants in human cardiomyocytes, and (3) supports a functional assay to aid in assessing pathogenicity for myopathic variants of uncertain significance.

Long QT Syndrome KCNH2 Variant Induces hERG1a/1b Subunit Imbalance in Patient-Specific Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

[Figure: see text].

Rescue of mutated cardiac ion channels in inherited arrhythmia syndromes.

Inherited arrhythmia syndromes comprise an increasingly complex group of diseases involving mutations in multiple genes encoding ion channels, ion channel accessory subunits and channel interacting proteins, and various regulatory elements. These mutations serve to disrupt normal electrophysiology in the heart, leading to increased arrhythmogenic risk and death. These diseases have added impact as they often affect young people, sometimes without warning. Although originally thought to alter ion channel function, it is now increasingly recognized that mutations may alter ion channel protein and messenger RNA processing, to reduce the number of channels reaching the surface membrane. For many of these mutations, it is also known that several interventions may restore protein processing of mutant channels to increase their surface membrane expression toward normal. In this article, we reviewed inherited arrhythmia syndromes, focusing on long QT syndrome type 2, and discuss the complex biology of ion channel trafficking and pharmacological rescue of disease-causing mutant channels. Pharmacological rescue of misprocessed mutant channel proteins, or their transcripts providing appropriate small molecule drugs can be developed, has the potential for novel clinical therapies in some patients with inherited arrhythmia syndromes.

Long QT Syndrome Type 2: Emerging Strategies for Correcting Class 2 KCNH2 (hERG) Mutations and Identifying New Patients.

Significant advances in our understanding of the molecular mechanisms that cause congenital long QT syndrome (LQTS) have been made. A wide variety of experimental approaches, including heterologous expression of mutant ion channel proteins and the use of inducible pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from LQTS patients offer insights into etiology and new therapeutic strategies. This review briefly discusses the major molecular mechanisms underlying LQTS type 2 (LQT2), which is caused by loss-of-function (LOF) mutations in the KCNH2 gene (also known as the human ether-à-go-go-related gene or hERG). Almost half of suspected LQT2-causing mutations are missense mutations, and functional studies suggest that about 90% of these mutations disrupt the intracellular transport, or trafficking, of the KCNH2-encoded Kv11.1 channel protein to the cell surface membrane. In this review, we discuss emerging strategies that improve the trafficking and functional expression of trafficking-deficient LQT2 Kv11.1 channel proteins to the cell surface membrane and how new insights into the structure of the Kv11.1 channel protein will lead to computational approaches that identify which KCNH2 missense variants confer a high-risk for LQT2.

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.

Functional Invalidation of Putative Sudden Infant Death Syndrome-Associated Variants in the KCNH2-Encoded Kv11.1 Channel.

BACKGROUND: Heterologous functional validation studies of putative long-QT syndrome subtype 2-associated variants clarify their pathological potential and identify disease mechanism(s) for most variants studied. The purpose of this study is to clarify the pathological potential for rare nonsynonymous KCNH2 variants seemingly associated with sudden infant death syndrome. METHODS: Genetic testing of 292 sudden infant death syndrome cases identified 9 KCNH2 variants: E90K, R181Q, A190T, G294V, R791W, P967L, R1005W, R1047L, and Q1068R. Previous studies show R181Q-, P967L-, and R1047L-Kv11.1 channels function similar to wild-type Kv11.1 channels, whereas Q1068R-Kv11.1 channels accelerate inactivation gating. We studied the biochemical and biophysical properties for E90K-, G294V-, R791W-, and R1005W-Kv11.1 channels expressed in human embryonic kidney 293 cells; examined the electronic health records of patients who were genotype positive for the sudden infant death syndrome-linked KCNH2 variants; and simulated their functional impact using computational models of the human ventricular action potential. RESULTS: Western blot and voltage-clamping analyses of cells expressing E90K-, G294V-, R791W-, and R1005W-Kv11.1 channels demonstrated these variants express and generate peak Kv11.1 current levels similar to cells expressing wild-type-Kv11.1 channels, but R791W- and R1005W-Kv11.1 channels accelerated deactivation and activation gating, respectively. Electronic health records of patients with the sudden infant death syndrome-linked KCNH2 variants showed that the patients had median heart rate-corrected QT intervals <480 ms and none had been diagnosed with long-QT syndrome or experienced cardiac arrest. Simulating the impact of dysfunctional gating variants predicted that they have little impact on ventricular action potential duration. CONCLUSIONS: We conclude that these rare Kv11.1 missense variants are not long-QT syndrome subtype 2-causative variants and therefore do not represent the pathogenic substrate for sudden infant death syndrome in the variant-positive infants.

Cell-free complements in vivo expression of the E. coli membrane proteome.

Reconstituted cell-free (CF) protein expression systems hold the promise of overcoming the traditional barriers associated with in vivo systems. This is particularly true for membrane proteins, which are often cytotoxic and due to the nature of the membrane, difficult to work with. To evaluate the potential of cell-free expression, we cloned 120 membrane proteins from E. coli and compared their expression profiles in both an E. coli in vivo system and an E. coli-derived cell-free system. Our results indicate CF is a more robust system and we were able to express 63% of the targets in CF, compared to 44% in vivo. To benchmark the quality of CF produced protein, five target membrane proteins were purified and their homogeneity assayed by gel filtration chromatography. Finally, to demonstrate the ease of amino acid labeling with CF, a novel membrane protein was substituted with selenomethionine, purified, and shown to have 100% incorporation of the unnatural amino acid. We conclude that CF is a novel, robust expression system capable of expressing more proteins than an in vivo system and suitable for production of membrane proteins at the milligram level.

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.

Molecular pathogenesis of long QT syndrome type 2.

The molecular mechanisms underlying congenital long QT syndrome (LQTS) are now beginning to be understood. New insights into the etiology and therapeutic strategies are emerging from heterologous expression studies of LQTS-linked mutant proteins, as well as inducible pluripotent stem cell derived cardiomyocytes (iPSC-CMs) from LQTS patients. This review focuses on the major molecular mechanism that underlies LQTS type 2 (LQT2). LQT2 is caused by loss of function (LOF) mutations in KCNH2 (also known as the human Ether-à-go-go-Related Gene or hERG). Most LQT2-linked mutations are missense mutations and functional studies suggest that ~90% of them disrupt the intracellular transport (trafficking) of KCNH2-encoded Kv11.1 proteins to the cell membrane. Trafficking deficient LQT2 mutations disrupt Kv11.1 protein folding and misfolded Kv11.1 proteins are retained in the endoplasmic reticulum (ER) until they are degraded in the ER associated degradation pathway (ERAD). This review focuses on the quality control mechanisms in the ER that contribute to the folding and ERAD of Kv11.1 proteins; the mechanism for ER export of Kv11.1 proteins in the secretory pathway; different subclasses of trafficking deficient LQT2 mutations; and strategies being developed to mitigate or correct trafficking deficient LQT2-related phenotypes.

Pharmacological rescue of human K(+) channel long-QT2 mutations: human ether-a-go-go-related gene rescue without block.

BACKGROUND: Defective protein trafficking is a consequence of gene mutations. Human long-QT (LQT) syndrome results from mutations in several genes, including the human ether-a-go-go-related gene (HERG), which encodes a delayed rectifier K(+) current. Trafficking-defective mutant HERG protein is a mechanism for reduced delayed rectifier K(+) current in LQT2, and high-affinity HERG channel-blocking drugs can result in pharmacological rescue. Methods and Results- We postulated that drug molecules modified to remove high-affinity HERG block may still stabilize mutant proteins in a conformation required for rescue. We tested terfenadine carboxylate (fexofenadine) and terfenadine, structurally similar drugs with markedly different affinities for HERG block, for rescue of trafficking-defective LQT2 mutations. Terfenadine rescued the N470D mutation but blocked the channels. In contrast, fexofenadine rescued N470D with a half-maximal rescue concentration of 177 nmol/L, which is approximately 350-fold lower than the half-maximal channel block concentration. The G601S mutation was also rescued without channel block. CONCLUSIONS: Pharmacological rescue can occur without channel block. This could represent a new antiarrhythmic paradigm in the treatment of some trafficking-defective LQT2 mutations.

Large-scale mutational analysis of Kv11.1 reveals molecular insights into type 2 long QT syndrome.

It has been suggested that deficient protein trafficking to the cell membrane is the dominant mechanism associated with type 2 Long QT syndrome (LQT2) caused by Kv11.1 potassium channel missense mutations, and that for many mutations the trafficking defect can be corrected pharmacologically. However, this inference was based on expression of a small number of Kv11.1 mutations. We performed a comprehensive analysis of 167 LQT2-linked missense mutations in four Kv11.1 structural domains and found that deficient protein trafficking is the dominant mechanism for all domains except for the distal carboxy-terminus. Also, most pore mutations--in contrast to intracellular domain mutations--were found to have severe dominant-negative effects when co-expressed with wild-type subunits. Finally, pharmacological correction of the trafficking defect in homomeric mutant channels was possible for mutations within all structural domains. However, pharmacological correction is dramatically improved for pore mutants when co-expressed with wild-type subunits to form heteromeric channels.

Mouse ERG K(+) channel clones reveal differences in protein trafficking and function.

BACKGROUND: The mouse ether-a-go-go-related gene 1a (mERG1a, mKCNH2) encodes mERG K(+) channels in mouse cardiomyocytes. The mERG channels and their human analogue, hERG channels, conduct IKr. Mutations in hERG channels reduce IKr to cause congenital long-QT syndrome type 2, mostly by decreasing surface membrane expression of trafficking-deficient channels. Three cDNA sequences were originally reported for mERG channels that differ by 1 to 4 amino acid residues (mERG-London, mERG-Waterston, and mERG-Nie). We characterized these mERG channels to test the postulation that they would differ in their protein trafficking and biophysical function, based on previous findings in long-QT syndrome type 2. METHODS AND RESULTS: The 3 mERG and hERG channels were expressed in HEK293 cells and neonatal mouse cardiomyocytes and were studied using Western blot and whole-cell patch clamp. We then compared our findings with the recent sequencing results in the Welcome Trust Sanger Institute Mouse Genomes Project (WTSIMGP). CONCLUSIONS: First, the mERG-London channel with amino acid substitutions in regions of highly ordered structure is trafficking deficient and undergoes temperature-dependent and pharmacological correction of its trafficking deficiency. Second, the voltage dependence of channel gating would be different for the 3 mERG channels. Third, compared with the WTSIMGP data set, the mERG-Nie clone is likely to represent the wild-type mouse sequence and physiology. Fourth, the WTSIMGP analysis suggests that substrain-specific sequence differences in mERG are a common finding in mice. These findings with mERG channels support previous findings with hERG channel structure-function analyses in long-QT syndrome type 2, in which sequence changes in regions of highly ordered structure are likely to result in abnormal protein trafficking.

Specific serine proteases selectively damage KCNH2 (hERG1) potassium channels and I(Kr).

KCNH2 (hERG1) encodes the alpha-subunit proteins for the rapidly activating delayed rectifier K+ current (I(Kr)), a major K+ current for cardiac myocyte repolarization. In isolated myocytes I(Kr) frequently is small in amplitude or absent, yet KCNH2 channels and I(Kr) are targets for drug block or mutations to cause long QT syndrome. We hypothesized that KCNH2 channels and I(Kr) are uniquely sensitive to enzymatic damage. To test this hypothesis, we studied heterologously expressed K+, Na+, and L-type Ca2+ channels, and in ventricular myocytes I(Kr), slowly activating delayed rectifier K+ current (I(Ks)), and inward rectifier K+ current (I(K1)), by using electrophysiological and biochemical methods. 1) Specific exogenous serine proteases (protease XIV, XXIV, or proteinase K) selectively degraded KCNH2 current (I(KCNH2)) and its mature channel protein without damaging cell integrity and with minimal effects on the other channel currents; 2) immature KCNH2 channel protein remained intact; 3) smaller molecular mass KCNH2 degradation products appeared; 4) protease XXIV selectively abolished I(Kr); and 5) reculturing HEK-293 cells after protease exposure resulted in the gradual recovery of I(KCNH2) and its mature channel protein over several hours. Thus the channel protein for I(KCNH2) and I(Kr) is uniquely sensitive to proteolysis. Analysis of the degradation products suggests selective proteolysis within the S5-pore extracellular linker, which is structurally unique among Kv channels. These data provide 1) a new mechanism to account for low I(Kr) density in some isolated myocytes, 2) evidence that most complexly glycosylated KCNH2 channel protein is in the plasma membrane, and 3) new insight into the rate of biogenesis of KCNH2 channel protein within cells.

Intragenic suppression of trafficking-defective KCNH2 channels associated with long QT syndrome.

Mutations in the KCNH2 or human ether-a-go-go-related gene-encoded K(+) channel reduce functional KCNH2 current (I(KCNH2)) to cause long QT syndrome (LQT2) by multiple mechanisms, including defects in intracellular transport (trafficking). Trafficking-deficient, or class 2, LQT2 mutations reduce the Golgi processing and surface membrane expression of KCNH2 channel proteins. Drugs that associate with pore-S6 intracellular drug binding domain of KCNH2 channel proteins to cause high-affinity block of I(KCNH2) also can increase the processing of class 2 LQT2 channel proteins through the secretory pathway. We used a strategy of intragenic suppression to test the hypothesis that amino acid substitutions in the putative drug binding domain at residue Y652 could compensate for protein folding abnormalities caused by class 2 LQT2 mutations. We found that the Y652C substitution, and to lesser extent the Y652S substitution, resulted in intragenic suppression of the class 2 LQT2 G601S phenotype; these substitutions increased Golgi processing of G601S channel proteins. The Y652C substitution also caused intragenic suppression of the class 2 LQT2 V612L and F640V phenotypes but not the LQT2 N470D or F805C phenotypes. These are the first findings to demonstrate that a single amino acid substitution in the putative KCNH2 drug binding domain can cause intragenic suppression of several LQT2 mutations.

Role of glycosylation in cell surface expression and stability of HERG potassium channels.

The human ether-à-go-go-related gene (HERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel in the heart. We previously showed that HERG channel protein is modified by N-linked glycosylation. HERG protein sequence contains two extracellular consensus sites for N-linked glycosylation (N598, N629). In this study, we used the approaches of site-directed mutagenesis and biochemical modification to inhibit N-linked glycosylation and studied the role of glycosylation in the cell surface expression and turnover of HERG channels. Our results show that N598 is the only site for N-linked glycosylation and that glycosylation is not required for the cell surface expression of functional HERG channels. In contrast, N629 is not used for glycosylation, but mutation of this site (N629Q) causes a protein trafficking defect, which results in its intracellular retention. Pulse-chase experiments show that the turnover rate of nonglycosylated HERG channel is faster than that of the glycosylated form, suggesting that N-linked glycosylation plays an important role in HERG channel stability.