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.