Thapsigargin Selectively Rescues the Trafficking Defective LQT2 Channels G601S and F805C

Department of Medicine (Cardiology), University of Wisconsin, Madison, Wisconsin 53706, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 10/2003; 278(37):35749-54. DOI: 10.1074/jbc.M305787200
Source: PubMed


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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    • "The incubation of rat SK1-transfected HEK293 cells with thapsigargin (1 lM) for 4-6 h prior to recording had no effect on the expressed current level (Fig. 1E). Other treatments attempted to rescue expression of functional rat SK1 channels, such as incubation of cells with chloroquine (Zhou et al., 1999), tacrine (Delisle et al., 2003) or dequalinium (Stocker & Pedarzani, 2000), also had no effect on the expressed current level (Fig. 1E). "
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    ABSTRACT: The activation of small conductance calcium-dependent (SK) channels regulates membrane excitability by causing membrane hyperpolarization. Three subtypes (SK1-3) have been cloned, with each subtype expressed within the nervous system. The locations of channel subunits overlap, with SK1 and SK2 subunits often expressed in the same brain region. We showed that expressed homomeric rat SK1 subunits did not form functional channels, because subunits accumulated in the Golgi. This raised the question of whether heteromeric channels could form with SK1 subunits. The co-expression of SK1 and SK2 subunits in HEK293 cells preferentially co-assembled to produce heteromeric channels with a fixed stoichiometry of alternating subunits. The expression in hippocampal CA1 neurons of mutant rat SK1 subunits [rat SK1(LV213/4YA)] that produced an apamin-sensitive current changed the amplitude and pharmacology of the medium afterhyperpolarization. The overexpression of rat SK1(LV213/4YA) subunits reduced the sensitivity of the medium afterhyperpolarization to apamin, substantiating the preferential co-assembly of SK1 and SK2 subunits to form heteromeric channels. Species-specific channel assembly occurred as the co-expression of human SK1 with rat SK2 did not form functional heteromeric channels. The replacement of two amino acids within the C-terminus of rat SK2 with those from human SK2 permitted the assembly of heteromeric channels when co-expressed with human SK1. These data showed that species-specific co-assembly was mediated by interaction between the C-termini of SK channel subunits. The finding that SK channels preferentially co-assembled to form heteromeric channels suggested that native heteromeric channels will predominate in cells expressing multiple SK channel subunits. © 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.
    Full-text · Article · Nov 2014 · European Journal of Neuroscience
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    • "Additionally, thapsigargin is a compound which inhibits endoplasmic reticulum Ca+-ATPase activity and has been shown to correct trafficking defects without hERG blockade [16]. We chose to assess thapsigargin because it promotes the relocation of intracellular proteins through its effect on Ca+ dependent molecular chaperone activity, not through binding to the hERG channel itself. "
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    ABSTRACT: 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.
    Full-text · Article · Jun 2013 · PLoS ONE
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    • "In general, these nonsense LQT2 mutants reside at the distal C-terminus, downstream of highly conserved stretches of amino acids including the pore region and domains required for tetramerization, maturation, stability and surface expression of Kv11.1channels [11]–[13]. Interestingly, numerous LQT2 trafficking-deficient mutants can be rescued following specific non-physiologic manipulations of the cell culture conditions [14]. For example, functional rescue has been achieved following 24 h incubation at reduced temperature (∼27°C) [15], incubation with high-affinity pore-blockers (E-4031, cisapride) [15]–[17], proteasomal inhibitors (lactacystin, MG132, ALLN) [18]–[20], lysosome inhibitors (leupeptin; bafilomycin) [18], [20], [21], or aminoglycoside antibiotics (G-418, gentamicin) [10]. "
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    ABSTRACT: The Kv11.1 (hERG) K+ channel plays a fundamental role in cardiac repolarization. Missense mutations in KCNH2, the gene encoding Kv11.1, cause long QT syndrome (LQTS) and frequently cause channel trafficking-deficiencies. This study characterized the properties of a novel KCNH2 mutation discovered in a LQT2 patient resuscitated from a ventricular fibrillation arrest. Proband genotyping was performed by SSCP and DNA sequencing. The electrophysiological and biochemical properties of the mutant channel were investigated after expression in HEK293 cells. The proband manifested a QTc of 554 ms prior to electrolyte normalization. Mutation analysis revealed an autosomal dominant frameshift mutation at proline 1086 (P1086fs+32X; 3256InsG). Co-immunoprecipitation demonstrated that wild-type Kv11.1 and mutant channels coassemble. Western blot showed that the mutation did not produce mature complex-glycosylated Kv11.1 channels and coexpression resulted in reduced channel maturation. Electrophysiological recordings revealed mutant channel peak currents to be similar to untransfected cells. Co-expression of channels in a 1∶1 ratio demonstrated dominant negative suppression of peak Kv11.1 currents. Immunocytochemistry confirmed that mutant channels were not present at the plasma membrane. Mutant channel trafficking rescue was attempted by incubation at reduced temperature or with the pharmacological agents E-4031. These treatments did not significantly increase peak mutant currents or induce the formation of mature complex-glycosylated channels. The proteasomal inhibitor lactacystin increased the protein levels of the mutant channels demonstrating proteasomal degradation, but failed to induce mutant Kv11.1 protein trafficking. Our study demonstrates a novel dominant-negative Kv11.1 mutation, which results in degraded non-functional channels leading to a LQT2 phenotype.
    Full-text · Article · Mar 2011 · PLoS ONE
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