thapsigargin and Long-QT-Syndrome

Treatment of LQT2 is inadequate. Many drugs which can pharmacologically rescue defective protein trafficking in LQT2 also result in potent blockade of HERG current, negating their therapeutic benefit. It is reported that PD-118057 and thapsigargin can rescue LQT2 without hERG channel blockade, but the precise mechanism of action is unknown. Furthermore, the effect of PD-118057 and thapsigargin on the dominant negative E637K-hERG mutant has not been previously investigated.. IN THIS STUDY, WE INVESTIGATED: (a) the effect of PD-118057 and thapsigargin on the current amplitudes of WT-hERG and WT/E637K-hERG channels; (b) the effect of PD-118057 and thapsigargin on the biophysical properties of WT-hERG and WT/E637K-hERG channels; (c) whether drug treatment can rescue channel processing and trafficking defects of the WT/E637K-hERG mutant.. The whole-cell Patch-clamp technique was used to assess the effect of PD-118057 and thapsigargin on the electrophysiological characteristics of the rapidly activating delayed rectifier K(+) current (Ikr) of the hERG protein channel. Western blot was done to investigate pharmacological rescue on hERG protein channel function.. In our study, PD-118057 was shown to significantly enhance both the maximum current amplitude and tail current amplitude, but did not alter the gating and kinetic properties of the WT-hERG channel, with the exception of accelerating steady-state inactivation. Additionally, thapsigargin shows a similar result as PD-118057 for the WT-hERG channel, but with the exception of attenuating steady-state inactivation. However, for the WT/E637K-hERG channel, PD-118057 had no effect on either the current or on the gating and kinetic properties. Furthermore, thapsigargin treatment did not alter the current or the gating and kinetic properties of the WT/E637K-hERG channel, with the exception of opening at more positive voltages.. Our findings illustrate that neither PD-118057 nor thapsigargin play a role in correcting the dominant-negative effect of the E637K-hERG mutant.

Topics: Amino Acid Substitution; Chlorobenzenes; Endoplasmic Reticulum Stress; ERG1 Potassium Channel; Glutamic Acid; HEK293 Cells; Humans; Long QT Syndrome; Lysine; Mutant Proteins; Mutation, Missense; ortho-Aminobenzoates; Patch-Clamp Techniques; Protein Transport; Thapsigargin; Unfolded Protein Response

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.. 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.. 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.

Topics: Cell Line; Enzyme Inhibitors; ERG1 Potassium Channel; Ether-A-Go-Go Potassium Channels; Genes, Dominant; Humans; Kidney; Long QT Syndrome; Mutation, Missense; Patch-Clamp Techniques; Phenotype; Potassium Channels, Voltage-Gated; Protein Transport; Thapsigargin

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).

Topics: Amino Acid Substitution; Asparagine; Aspartic Acid; Cation Transport Proteins; Cell Line; Cell Membrane; Cell Nucleus; DNA-Binding Proteins; ERG1 Potassium Channel; Ether-A-Go-Go Potassium Channels; Glycine; Humans; Long QT Syndrome; Mutagenesis, Site-Directed; Patch-Clamp Techniques; Potassium Channel Blockers; Potassium Channels; Potassium Channels, Voltage-Gated; Protein Transport; Recombinant Proteins; Serine; Thapsigargin; Trans-Activators; Transcriptional Regulator ERG