Long QT syndrome (LQT) is an electrophysiological disorder that can lead to sudden death from cardiac arrhythmias. One form of LQT has been attributed to mutations in the human ether‐a‐go‐go‐related gene (HERG) that encodes a voltage‐gated cardiac K+ channel. While a recent report indicates that LQT in some patients is associated with a mutation of HERG at a consensus extracellular N‐linked glycosylation site (N629), earlier studies failed to identify a role for N‐linked glycosylation in the functional expression of voltage‐gated K+ channels. In this study we used pharmacological agents and site‐directed mutagenesis to assess the contribution of N‐linked glycosylation to the surface localization of HERG channels. Tunicamycin, an inhibitor of N‐linked glycosylation, blocked normal surface membrane expression of a HERG‐green fluorescent protein (GFP) fusion protein (HERGGFP) transiently expressed in human embryonic kidney (HEK 293) cells imaged with confocal microscopy. Immunoblot analysis revealed that N‐glycosidase F shifted the molecular mass of HERGGFP, stably expressed in HEK 293 cells, indicating the presence of N‐linked carbohydrate moieties. Mutations at each of the two putative extracellular N‐linked glycosylation sites (N598Q and N629Q) led to a perinuclear subcellular localization of HERGGFP stably expressed in HEK 293 cells, with no surface membrane expression. Furthermore, patch clamp analysis revealed that there was a virtual absence of HERG current in the N‐glycosylation mutants. Taken together, these results strongly suggest that N‐linked glycosylation is required for surface membrane expression of HERG. These findings may provide insight into a mechanism responsible for LQT2 due to N‐linked glycosylation‐related mutations of HERG.

The role of the plasma membrane quality control machinery is demonstrated in the development of the long QT syndrome phenotype, caused by acquired and inherited conformational defects of the hERG potassium channel in multiple expression systems, including cardiac myocytes.

The Long QT Syndrome is a cardiac disorder associated with ventricular arrhythmias that can lead to syncope and sudden death. One prominent form of the Long QT syndrome has been linked to mutations in the HERG gene (KCNH2) that encodes the voltage-dependent delayed rectifier potassium channel (I Kr ). In order to search for HERG-interacting proteins important for HERG maturation and trafficking, we conducted a proteomics screen using myc-tagged HERG transfected into cardiac (HL-1) and non-cardiac (human embryonic kidney 293) cell lines. A partial list of putative HERG-interacting proteins includes several known components of the cytosolic chaperone system, including Hsc70 (70-kDa heat shock cognate protein), Hsp90 (90-kDa heat shock protein), Hdj-2, Hop (Hsp-organizing protein), and Bag-2 (BCL-associated athanogene 2). In addition, two membrane-integrated proteins were identified, calnexin and FKBP38 (38-kDa FK506-binding protein, FKBP8). We show that FKBP38 immunoprecipitates and co-localizes with HERG in our cellular system. Importantly, small interfering RNA knock down of FKBP38 causes a reduction of HERG trafficking, and overexpression of FKBP38 is able to partially rescue the LQT2 trafficking mutant F805C. We propose that FKBP38 is a co-chaperone of HERG and contributes via the Hsc70/Hsp90 chaperone system to the trafficking of wild type and mutant HERG potassium channels.

Loss of function mutations in the hERG (human ether-ago-go related gene or KCNH2) potassium channel underlie the proarrhythmic cardiac long QT syndrome type 2. Most often this is a consequence of defective trafficking of hERG mutants to the cell surface, with channel retention and degradation at the endoplasmic reticulum. Here, we identify the Hsp40 type 1 chaperones DJA1 (DNAJA1/Hdj2) and DJA2 (DNAJA2) as key modulators of hERG degradation. Overexpression of the DJAs reduces hERG trafficking efficiency, an effect eliminated by the proteasomal inhibitor lactacystin or with DJA mutants lacking their J domains essential for Hsc70/Hsp70 activation. Both DJA1 and DJA2 cause a decrease in the amount of hERG complexed with Hsc70, indicating a preferential degradation of the complex. Similar effects were observed with the E3 ubiquitin ligase CHIP. Both the DJAs and CHIP reduce hERG stability and act differentially on folding intermediates of hERG and the disease-related trafficking mutant G601S. We propose a novel role for the DJA proteins in regulating degradation and suggest that they act at a critical point in secretory pathway quality control.

The Na+/H+exchanger NHE1 isoform is an integral component of cardiac intracellular pH homeostasis that is critically important for myocardial contractility. To gain further insight into its physiological significance, we determined its cellular distribution in adult rat heart by using immunohistochemistry and confocal microscopy. NHE1 was localized predominantly at the intercalated disk regions in close proximity to the gap junction protein connexin 43 of atrial and ventricular muscle cells. Significant labeling of NHE1 was also observed along the transverse tubular systems, but not the lateral sarcolemmal membranes, of both cell types. In contrast, the Na+-K+-ATPase α1-subunit was readily labeled by a specific mouse monoclonal antibody (McK1) along the entire ventricular sarcolemma and intercalated disks and, to a lesser extent, in the transverse tubules. These results indicate that NHE1 has a distinct distribution in heart and may fulfill specialized roles by selectively regulating the pH microenvironment of pH-sensitive proteins at the intercalated disks (e.g., connexin 43) and near the cytosolic surface of sarcoplasmic reticulum cisternae (e.g., ryanodine receptor), thereby influencing impulse conduction and excitation-contraction coupling.

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.

Mutations in the potassium channel encoded by the human ether-a-go-go-related gene (HERG) have been linked to the congenital long QT syndrome (LQTS), a cardiac disease associated with an increased preponderance of ventricular arrhythmias and sudden death. The COOH terminus of HERG harbors a large number of LQTS mutations and its removal prevents functional expression for reasons that remain unknown. In this study, we show that the COOH terminus of HERG is required for normal trafficking of the ion channel. We have identified a region critical for trafficking between residues 860 and 899 that includes a novel missense mutation at amino acid 861 (HERG N861I ). Truncations or deletion of residues 860 -899, characterized in six different expression systems including a cardiac cell line, resulted in decreased expression levels and an absence of the mature glycosylated form of the HERG protein. Deletion of this region did not interfere with the formation of tetramers but caused retention of the assembled ion channels within the endoplasmic reticulum. Consequently, removal of residues 860 -899 resulted in the absence of the ion channels from the cell surface and a more rapid turnover rate than the wild type channels, which was evident very early in biogenesis. This study reveals a novel role of the COOH terminus in the normal biogenesis of HERG channels and suggests defective trafficking as a common mechanism for abnormal channel function resulting from mutations of critical COOH-terminal residues, including the LQTS mutant HERG N861I . The long QT syndrome (LQTS)1 is a congenital heart disorder characterized by delayed cardiac action potential repolarization and a prolongation of the QT interval. This leads to an increased susceptibility of the heart to potentially sustained ventricular tachyarrhythmias that cause syncope and sudden death. Molecular genetic studies have identified five genes linked to LQTS including the human ether-a-go-go-related gene (HERG) (1). HERG encodes the ␣-subunit of the rapidly activating delayed rectifier current I Kr and consists of six transmembrane domains as well as NH 2 -and COOH-terminal cytoplasmic tails (2, 3). Over 90 mutations distributed throughout HERG have been linked to the LQTS, most of which reside in the intracellular tail regions of the channel protein (4, 5). Earlier electrophysiological and structural analysis have emphasized abnormal HERG function as a common manifestation of mutations in the NH 2 terminus (6 -8). In contrast, studies of COOH-terminal mutations suggest that the pathology is because of the absence of HERG channels from the cell surface (9 -12). This phenotype may be caused by improper folding of newly synthesized HERG polypeptides, in a fashion similar to that described for the ⌬F508 allele of CFTR (CFTR⌬F508). CFTR⌬F508 is recognized by the endoplasmic reticulum (ER) quality control machinery and is rapidly degraded before being processed in the Golgi apparatus (13). As a consequence, these molecules are prevented from journeying through the secretory ...

The ability of ceruloplasmin, an important serum antioxidant, to reduce the vulnerability of the isolated rat heart to reperfusion arrhythmias has been investigated. Bovine plasma ceruloplasmin was purified by chromatography on aminoethyl-agarose. Isolated rat hearts were submitted to 15 min of regional ischemia and 10 min of reperfusion. The dose-effect relationship and the role of ceruloplasmin conformational integrity in cardioprotection were established by treatment of ischemic hearts with ceruloplasmin at various concentrations (0.25, 0.5, 1, and 2 microM) and at different degrees of conformational integrity (A610/A280 = 0.02, 0.04, and 0.06), 5 min before reperfusion. Deferoxamine (20-500 microM) was used as a positive control. As negative controls we used chemically inactivated ceruloplasmin (1 microM), heat-denatured ceruloplasmin (1 microM), and albumin (1-4 microM). In the control group during the first 5 min of reperfusion, the incidence of total ventricular fibrillation was 100% and of irreversible ventricular fibrillation was 83%. The incidence of reversible and irreversible ventricular fibrillation was significantly decreased in the ceruloplasmin-treated groups in both a dose and molecular integrity dependent manner. Ceruloplasmin had no effect on the incidence of ventricular tachycardia. Deferoxamine reduced the incidence of ventricular fibrillation to the same degree as ceruloplasmin but at concentrations much higher than those of ceruloplasmin. Chemically inactivated ceruloplasmin, heat-denatured ceruloplasmin, and albumin had no protective effects on reperfusion-induced arrhythmias.