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

<div><p>Background</p><p>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.</p><p>Objective</p><p>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.</p><p>Methods</p><p>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 (I_kr) of the hERG protein channel. Western blot was done to investigate pharmacological rescue on hERG protein channel function.</p><p>Results</p><p>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.</p><p>Conclusion</p><p>Our findings illustrate that neither PD-118057 nor thapsigargin play a role in correcting the dominant-negative effect of the E637K-hERG mutant.</p></div

Effect of PD-118057 (3 µM) on voltage-dependent activation of hERG channel.

<p>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 PD-118057. 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 and absence of PD-118057. i: Amplitudes of tail currents of WT-hERG and WT/E637K-hERG channels in the presence or absence of PD-119057 are plotted as a function of the test potential and fitted to a Boltzmann function (n = 6).</p

Effect of PD-118057 (3 µM) and thapsigargin (1 µM) on steady-state inactivation of hERG channel.

<p>Inset shows the voltage clamp protocol. a-d: Representative current traces in HEK293 cells transfected with WT-hERG or WT/E637K-hERG plasmids in the presence or absence of PD-118057 and thapsigargin. e: Normalized steady-state inactivation curves in cells transfected with WT-hERG or WT/E637K-hERG plasmids in the presence or absence of drug (n = 6).</p

Effect of PD-118057 (3 µM) and thapsigargin (1 µM) on deactivation of hERG channel.

<p>Insert shows the voltage clamp protocol. a-d: Representative deactivation traces in HEK293 cells transfected with WT-hERG or WT/E637K-hERG in the presence or absence of PD-118057 and thapsigargin (arrow marks the deactivation phase). e: Fast and slow components of deactivation time constants (tau, τ) are plotted as a function of test potentials for WT-hERG or WT/E637K-hERG plasmids in the presence or absence of drug (n = 6).</p

Analysis of hERG protein expression in HEK293 cells.

<p>a. Representative protein expression of untreated WT-hERG, WT/E637K-hERG, and E637K-hERG channels. b–d. Representative protein expression of WT-hERG, WT/E637K-hERG and E637K-hERG channels treated with 1 uM thapsigargin for 24 h to 48 h, respectively. Thapsigargin has no effect on the protein expression profile of either WT/E637K-hERG or E637K-hERG. e–f. Representative protein expression of WT-hERG, WT/E637K-hERG and E637K-hERG channels treated with 3 µM PD-118057 for 24 h to 48 h, respectively. As demonstrated, PD-118057 also has no effect on the protein expression profile of either WT/E637K-hERG or E637K-hERG channels.</p

Effect of PD-118057 (3 µM) and thapsigargin (1 µM) on recovery from inactivation of hERG channel.

<p>Insert shows the voltage clamp protocol. a-d: Representative recovery from inactivation traces in HEK293 cells transfected with WT-hERG or WT/E637K-hERG plasmids in the presence or absence of PD-118057 and thapsigargin. e: Time constants (tau, τ) for hERG channel recovery from inactivation are plotted as a function of test voltages for WT-hERG or WT/E637K-hERG plasmids in the presence or absence of drug (n = 6).</p

Effect of PD-118057 (3 µM) and thapsigargin (1 µM) on time courses of inactivation of hERG channel.

<p>Inset shows the voltage clamp protocol. a-d: Representative current traces of time courses and inactivation time constants (tau, τ) in HEK293 cells transfected with WT-hERG or WT-hERG/E637K-hERG plasmids in the presence or absence of PD-118057 and thapsigargin. e: τ was measured by fitting inactivation currents of WT-hERG or WT-hERG/E637K-hERG channel, in the presence or absence of PD-118057 and thapsigargin during test pulse at each potential, with a single exponential function (n = 6).</p