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State-dependent blockade of human ether-a-go-go-related gene (hERG) K(+) channels by changrolin in stably transfected HEK293 cells.

ABSTRACT: To study the effect of changrolin on the K(+) channels encoded by the human ether-a-go-go-related gene (hERG).hERG channels were heterologously stably expressed in human embryonic kidney 293 cells, and the hERG K(+) currents were recorded using a standard whole-cell patch-clamp technique.Changrolin inhibited hERG channels in a concentration-dependent and reversible manner (IC(50)=18.23 mumol/L, 95% CI: 9.27-35.9 mumol/L; Hill coefficient=-0.9446). In addition, changrolin shifted the activation curve of hERG channels by 14.3+/-1.5 mV to more negative potentials (P<0.01, n=9) but did not significantly affect the steady-state inactivation of hERG (n=5, P>0.05). The relative block of hERG channels by changrolin was close to zero at the time point of channel opening by the depolarizing voltage step and quickly increased afterwards. The maximal block was achieved in the inactivated state, with no further development of the open channel block. In the "envelope of tails" experiments, the time constants of activation were found to be 287.8+/-46.2 ms and 174.2+/-18.4 ms, respectively, for the absence and presence of 30 mumol/L changrolin (P<0.05, n=7). The onset of inactivation was accelerated significantly by changrolin between -40 mV and +60 mV (P<0.05, n=7).The results demonstrate that changrolin is a potent hERG blocker that preferentially binds to hERG channels in the open and inactivated states.

SUBMITTER: Chen WH

PROVIDER: S-EPMC4007811 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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State-dependent blockade of human ether-a-go-go-related gene (hERG) K(+) channels by changrolin in stably transfected HEK293 cells.

Chen Wei-hai WH Wang Wen-yi WY Zhang Jie J Yang Ding D Wang Yi-ping YP

Acta pharmacologica Sinica 20100801 8

<h4>Aim</h4>To study the effect of changrolin on the K(+) channels encoded by the human ether-a-go-go-related gene (hERG).<h4>Methods</h4>hERG channels were heterologously stably expressed in human embryonic kidney 293 cells, and the hERG K(+) currents were recorded using a standard whole-cell patch-clamp technique.<h4>Results</h4>Changrolin inhibited hERG channels in a concentration-dependent and reversible manner (IC(50)=18.23 mumol/L, 95% CI: 9.27-35.9 mumol/L; Hill coefficient=-0.9446). In a ...[more]

PMID: 20686516

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Project description:Current regulatory guidelines for assessing the risk of QT prolongation include in vitro assays assessing drug effects on the human ether-à-go-go-related (hERG; also known as Kv11.1) channel expressed in cell lines. These assays are typically conducted at room temperature to promote the ease and stability of recording hERG currents. However, the new Comprehensive in vitro Proarrhythmia Assay (CiPA) paradigm proposes to use an in silico model of the human ventricular myocyte to assess risk, requiring as input hERG channel pharmacology data obtained at physiological temperatures. To accommodate current industry safety pharmacology practices for measuring hERG channel activity, an in silico model of hERG channel that allows for the extrapolation of hERG assay data across different temperatures is desired. Because temperature may have an effect on both channel gating and drug binding rate, such models may need to have two components: a base model dealing with temperature-dependent gating changes without drug, and a pharmacodynamic component simulating temperature-dependent drug binding kinetics. As a first step, a base mode that can capture temperature effects on hERG channel gating without drug is needed.To meet this need for a temperature-dependent base model, a Markov model of the hERG channel with state transition rates explicitly dependent on temperature was developed and calibrated using data from a variety of published experiments conducted over a range of temperatures. The model was able to reproduce observed temperature-dependent changes in key channel gating properties and also to predict the results obtained in independent sets of new experiments.This new temperature-sensitive model of hERG gating represents an attempt to improve the predictivity of safety pharmacology testing by enabling the translation of room temperature hERG assay data to more physiological conditions. With further development, this model can be incorporated into the CiPA paradigm and also be used as a tool for developing insights into the thermodynamics of hERG channel gating mechanisms and the temperature-dependence of hERG channel block by drugs.

Project description:The human ether-a-go-go-related gene (hERG) potassium channel conducts rapid delayed rectifier potassium currents (IKr) and contributes to phase III cardiac action potential repolarization. Drugs inhibit hERG channels by binding to aromatic residues in hERG helixes. Berberine (BBR) has multiple actions, and its hydrogenated derivative dihydroberberine (DHB) is a potential candidate for developing new drugs. Previous studies have demonstrated that BBR blocks hERG channels and prolongs action potential duration (APD). Our present study aimed to investigate the effects and mechanism of DHB on hERG channels. Protein expression and the hERG current were analyzed using western blotting and patch-clamp, respectively. DHB inhibited the hERG current concentration-dependently after instantaneous perfusion, accelerated channel inactivation by directly binding tyrosine (Tyr652) and phenylalanine (Phe656), and decreased mature (155-kDa) and simultaneously increased immature (135-kDa) hERG expression, respectively. This suggests disruption of forward trafficking of hERG channels. Besides, DHB remarkably reduced heat shock protein 90 (Hsp90) expression and its interaction with hERG, indicating that DHB disrupted hERG trafficking by impairing channel folding. Meanwhie, DHB enhanced the expression of cleaved activating transcription factor-6 (ATF-6), a biomarker of unfolded protein response (UPR). Expression of calnexin and calreticulin, chaperones activated by ATF-6 to facilitate channel folding, were also increased, which indicating UPR activation. Additionally, the degradation rate of mature 155-kDa hERG increased following DHB exposure. In conclusion, we demonstrated that DHB acutely blocked hERG channels by binding the aromatic Tyr652 and Phe656. DHB may decrease hERG plasma membrane expression through two pathways involving disruption of forward trafficking of immature hERG channels and enhanced degradation of mature hERG channels. Furthermore, forward trafficking was disrupted by impaired channel folding associated with altered interactions between hERG proteins and chaperones. Finally, trafficking inhibition activated UPR, and mature hERG channel degradation was increased by DHB.

Dataset Information

State-dependent blockade of human ether-a-go-go-related gene (hERG) K(+) channels by changrolin in stably transfected HEK293 cells.

Publications

State-dependent blockade of human ether-a-go-go-related gene (hERG) K(+) channels by changrolin in stably transfected HEK293 cells.

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