The pro-arrhythmic Long QT syndrome (LQT) is linked to 10 different genes (LQT1-10). Approximately 40% of genotype-positive LQT patients have LQT2, which is characterized by mutations in the human ether-a-go-go related gene (hERG). hERG encodes the voltage-gated K ؉ channel ␣-subunits that form the pore of the rapidly activating delayed rectifier K ؉ current in the heart. The purpose of this study was to elucidate the mechanisms that regulate the intracellular transport or trafficking of hERG, because trafficking is impaired for about 90% of LQT2 missense mutations. Protein trafficking is regulated by small GTPases. To identify the small GTPases that are critical for hERG trafficking, we coexpressed hERG and dominant negative (DN) GTPase mutations in HEK293 cells. The GTPases Sar1 and ARF1 regulate the endoplasmic reticulum (ER) export of proteins in COPII and COPI vesicles, respectively. Expression of DN Sar1 inhibited the Golgi processing of hERG, decreased hERG current (I hERG ) by 85% (n > 8 cells per group, *, p < 0.01),andreducedtheplasmalemmalstainingofhERG.Thecoexpression of DN ARF1 had relatively small effects on hERG trafficking. Surprisingly, the coexpression of DN Rab11B, which regulates the endosomal recycling, inhibited the Golgi processing of hERG, decreased I hERG by 79% (n > 8 cells per group; *, p < 0.01), and reduced the plasmalemmal staining of hERG. These data suggest that hERG undergoes ER export in COPII vesicles and endosomal recycling prior to being processed in the Golgi. We conclude that hERG trafficking involves a pathway between the ER and endosomal compartments that influences expression in the plasmalemma.The human KCNH2 or ether-a-go-go related gene (hERG) 3 encodes the voltage-gated K ϩ channel ␣-subunits that oligomerize to form the pore of the rapidly activating delayed rectifier K ϩ current (I Kr ) in cardiac myocytes (1-3). Hundreds of hERG mutations are linked to the congenital pro-arrhythmic Type 2 Long QT syndrome (LQT2) and functional studies suggest that these mutations result in a loss of normal hERG K ϩ channel (hERG) function (4, 5). In LQT2, missense mutations are the dominant abnormality and many LQT2 missense mutations reduce hERG K ϩ current (I hERG ) by decreasing the intracellular transport or trafficking of hERG to the Golgi apparatus (Golgi) and the cell surface membrane (plasmalemma) (6). Therefore, disruption of hERG K ϩ channel trafficking appears to be a principal mechanism for disease.Movement of proteins between membrane-bound intracellular compartments is mediated by small transport vesicles, which bud from a donor compartment to fuse with an appropriate acceptor compartment. The trafficking of many transmembrane and secretory proteins between the ER and Golgi compartments is dependent on the small GTPases ADP-ribosylation factor 1 (ARF1) and Sar1, which regulate the formation of coat-associated protein complex I (COPI) and II (COPII) vesicles, respectively (7-19). These small GTPases facilitate the polymerization of transport vesicle protein coats on t...
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Mutations in humanether-a-go-go-related gene 1 (hERG) are linked to long QT syndrome type 2 (LQT2). hERG encodes the pore-forming ␣-subunits that coassemble to form rapidly activating delayed rectifier K ϩ 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 ⌬Y475 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.
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