Thapsigargin selectively rescues the trafficking defective LQT2 channels G601S and F805C.
ABSTRACT Several mutations in the human ether-a-go-go-related K+ channel gene (HERG or KCNH2) cause long QT syndrome (LQT2) by reducing the intracellular transport (trafficking) of the channel protein to the cell surface. Drugs that bind to and block HERG channels (i.e. E4031) rescue the surface expression of some trafficking defective LQT2 mutations. Because these drugs potently block HERG current, their ability to correct congenital LQT is confounded by their risk of causing acquired LQT. We tested the hypothesis that pharmacological rescue can occur without HERG channel block. Thapsigargin (1 microM), a sarcoplasmic/endoplasmic reticulum Ca2+-ATPase inhibitor, rescued the surface expression of G601S, and it did so without blocking current. Thapsigargin-induced rescue and E4031-induced rescue caused complex glycosylation that was evident within 3 h of drug exposure. Disruption of the Golgi apparatus with brefeldin A prevented thapsigargin- and E4031-induced rescue of IG01S. Confocal imaging showed that G601S protein is predominantly "trapped" intracellularly and that both thapsigargin and E4031 promote its relocation to the surface membrane. We also studied two other trafficking defective LQT2 mutations. Thapsigargin rescued the C terminus mutation F805C but not N470D, whereas E4031 rescued N470D but not F805C. Other sarcoplasmic/endoplasmic reticulum Ca2+-ATPase inhibitors did not rescue G601S or F805C. This study 1) supports the hypothesis that the LQT2 trafficking defective phenotype can be reversed without blocking the channel; 2) demonstrates pharmacological rescue of a C terminus LQT2 mutation; and 3) shows that thapsigargin can correct trafficking defective phenotypes in more than one channel type and disease (i.e. LQT2 and cystic fibrosis).
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ABSTRACT: Membrane trafficking in concert with the peripheral quality control machinery plays a critical role in preserving plasma membrane (PM) protein homeostasis. Adversely, the peripheral quality control may also dispose partially or transiently unfolded polypeptides and thereby contribute to the loss-of-expression phenotype of conformational diseases. Defective functional PM expression of the human ether-a-go-go related gene (hERG) K(+)-channel leads to the prolongation of the ventricular action potential that causes the long QT syndrome 2 (LQT2) with increased propensity for arrhythmia and sudden cardiac arrest. LQT2 syndrome is attributed to the channel biosynthetic processing defect due mutation, drug-induced misfolding, or direct channel blockade. Here we provide evidence that a peripheral quality control mechanism can contribute to development of the LQT2 syndrome. We show that PM hERG structural and metabolic stability are compromised by the reduction of extracellular or intracellular K(+) concentration. Cardiac glycoside-induced intracellular K(+)-depletion conformationally impairs the complex-glycosylated channel, which provokes chaperone- and CHIP-dependent poly-ubiquitination, accelerated internalization and ESCRT-dependent lysosomal degradation. A similar mechanism contributes to the down-regulation PM hERG harboring LQT2 missense mutations with incomplete secretion defect. These results suggest that the PM quality control plays a determinant role in the loss-of-expression phenotype of hERG in certain hereditary and acquired LTQ2 syndromes.Molecular biology of the cell 10/2013; · 5.98 Impact Factor
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ABSTRACT: The human ether-a-go-go - related gene (hERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel (IKr), which is important for cardiac repolarization. Reduction of hERG current due to genetic mutations or drug interferences causes long QT syndrome (LQTS), leading to cardiac arrhythmias and sudden death. To date, there is no effective therapeutic method to restore or enhance hERG channel function. Using cell biology and electrophysiological methods, we found that muscarinic receptor agonist carbachol increased the expression and function of hERG, but not EAG or Kv1.5 channels stably expressed in HEK cells. The carbachol-mediated increase in hERG expression was abolished by the selective M3 antagonist 4-DAMP but not by the M2 antagonist AF-DX 166. Treatment of cells with carbachol reduced the hERG-ubiquitin interaction and slowed the rate of hERG degradation. We previously showed that the E3 ubiquitin ligase Nedd4-2 mediates degradation of hERG channels. Here, we found that disrupting the Nedd4-2 binding domain in hERG completely eliminated the effect of carbachol on hERG channels. Carbachol treatment enhanced the phosphorylation level, but not the total level, of Nedd4-2. Blockade of the PKC pathway abolished the carbachol-induced enhancement of hERG channels. We conclude that muscarinic activation increases hERG channel expression by phosphorylating Nedd4-2 via the PKC pathway.Molecular pharmacology 03/2014; · 4.12 Impact Factor
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ABSTRACT: Abnormal functioning of cardiac ion channels can disrupt cardiac myocyte action potentials and thus cause potentially lethal cardiac arrhythmias. Ion channel dysfunction has been observed at all stages in channel ontogeny, from biogenesis to regulation, and arises from genetic or environmental factors, or both. Acquired arrhythmias - including those that are drug induced - are more common than solely inherited arrhythmias but, in some cases, also contain an identifiable genetic component. This interplay between the pharmacology and genetics - known as 'pharmacogenetics' - of cardiac ion channels and the systems that impact them presents both challenges and opportunities to academics, pharmaceutical companies and clinicians seeking to develop and utilize therapies for cardiac rhythm disorders. In this review, we discuss ion channel pharmacogenetics in the context of both causation and treatment of cardiac arrhythmias, focusing on the long QT syndromes.Expert Review of Clinical Pharmacology 01/2008; 1(1):93-104.