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Pharmacologic Approach to Defective Protein Trafficking in the E637K-hERG Mutant with PD-118057 and Thapsigargin.

Mao H, Lu X, Karush JM, Huang X, Yang X, Ba Y, Wang Y, Liu N, Zhou J, Lian J - PLoS ONE (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: LiHuiLi Hospital, Medical School of NingBo University, NingBo, China.

ABSTRACT

Background: 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.

Objective: 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.

Methods: 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.

Results: 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.

Conclusion: Our findings illustrate that neither PD-118057 nor thapsigargin play a role in correcting the dominant-negative effect of the E637K-hERG mutant.

No MeSH data available.


Related in: MedlinePlus

Effect of thapsigargin (1 µM) on voltage-dependent activation of hERG channel.Inset shows the voltage clamp protocol. a-f: Representative current traces in HEK293 cells transfected with WT-hERG, WT/E637K-hERG, and E637K-hERG in the presence or absence of thapsigargin. g, h: Current-voltage (I-V) relationships for peak and tail current amplitudes of WT-hERG and WT/E637K-hERG transfected cells in the presence or absence of thapsigargin. i: Amplitudes of tail currents of WT-hERG or WT/E637K-hERG channels in the presence or absence of thapsigargin are plotted as a function of the test potential and fitted to a Boltzmann function (n = 6).
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pone-0065481-g002: Effect of thapsigargin (1 µM) on voltage-dependent activation of hERG channel.Inset shows the voltage clamp protocol. a-f: Representative current traces in HEK293 cells transfected with WT-hERG, WT/E637K-hERG, and E637K-hERG in the presence or absence of thapsigargin. g, h: Current-voltage (I-V) relationships for peak and tail current amplitudes of WT-hERG and WT/E637K-hERG transfected cells in the presence or absence of thapsigargin. i: Amplitudes of tail currents of WT-hERG or WT/E637K-hERG channels in the presence or absence of thapsigargin are plotted as a function of the test potential and fitted to a Boltzmann function (n = 6).

Mentions: We first measured the peak and tail currents in untreated HEK293 cells expressing WT-hERG or WT/E637K-hERG channels. Currents were elicited by voltage clamp protocol as previously described [20], [21]. Cells were depolarized to test voltages between −60 mV and 60 mV in 10 mV increments for 2 s from a −80 mV holding potential, followed by −40 mV for 4 s to elicit tail currents. Figure 1 shows representative whole cell currents and the corresponding current–voltage (I–V) relationship of the maximal and tail current amplitudes. As expected, untreated WT/E637K-hERG cells demonstrated a significant reduction in maximal current amplitude compared to WT-hERG cells (198.66±33.39 pA versus 660.67±159.55 pA; n = 6, p<0.05, Fig. 1G and 2G). A similar reduction in tail current was observed in WT/E637K-hERG expressing cells compared to WT-hERG (333.12±1.48 pA versus 981.28±5.94 pA; n = 6, P<0.05, Fig. 1H and 2H), confirming the negative effect of WT/E637K-hERG expression.


Pharmacologic Approach to Defective Protein Trafficking in the E637K-hERG Mutant with PD-118057 and Thapsigargin.

Mao H, Lu X, Karush JM, Huang X, Yang X, Ba Y, Wang Y, Liu N, Zhou J, Lian J - PLoS ONE (2013)

Effect of thapsigargin (1 µM) on voltage-dependent activation of hERG channel.Inset shows the voltage clamp protocol. a-f: Representative current traces in HEK293 cells transfected with WT-hERG, WT/E637K-hERG, and E637K-hERG in the presence or absence of thapsigargin. g, h: Current-voltage (I-V) relationships for peak and tail current amplitudes of WT-hERG and WT/E637K-hERG transfected cells in the presence or absence of thapsigargin. i: Amplitudes of tail currents of WT-hERG or WT/E637K-hERG channels in the presence or absence of thapsigargin are plotted as a function of the test potential and fitted to a Boltzmann function (n = 6).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3686757&req=5

pone-0065481-g002: Effect of thapsigargin (1 µM) on voltage-dependent activation of hERG channel.Inset shows the voltage clamp protocol. a-f: Representative current traces in HEK293 cells transfected with WT-hERG, WT/E637K-hERG, and E637K-hERG in the presence or absence of thapsigargin. g, h: Current-voltage (I-V) relationships for peak and tail current amplitudes of WT-hERG and WT/E637K-hERG transfected cells in the presence or absence of thapsigargin. i: Amplitudes of tail currents of WT-hERG or WT/E637K-hERG channels in the presence or absence of thapsigargin are plotted as a function of the test potential and fitted to a Boltzmann function (n = 6).
Mentions: We first measured the peak and tail currents in untreated HEK293 cells expressing WT-hERG or WT/E637K-hERG channels. Currents were elicited by voltage clamp protocol as previously described [20], [21]. Cells were depolarized to test voltages between −60 mV and 60 mV in 10 mV increments for 2 s from a −80 mV holding potential, followed by −40 mV for 4 s to elicit tail currents. Figure 1 shows representative whole cell currents and the corresponding current–voltage (I–V) relationship of the maximal and tail current amplitudes. As expected, untreated WT/E637K-hERG cells demonstrated a significant reduction in maximal current amplitude compared to WT-hERG cells (198.66±33.39 pA versus 660.67±159.55 pA; n = 6, p<0.05, Fig. 1G and 2G). A similar reduction in tail current was observed in WT/E637K-hERG expressing cells compared to WT-hERG (333.12±1.48 pA versus 981.28±5.94 pA; n = 6, P<0.05, Fig. 1H and 2H), confirming the negative effect of WT/E637K-hERG expression.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: LiHuiLi Hospital, Medical School of NingBo University, NingBo, China.

ABSTRACT

Background: 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.

Objective: 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.

Methods: 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.

Results: 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.

Conclusion: Our findings illustrate that neither PD-118057 nor thapsigargin play a role in correcting the dominant-negative effect of the E637K-hERG mutant.

No MeSH data available.


Related in: MedlinePlus