Direct block of hERG potassium channels by the protein kinase C inhibitor bisindolylmaleimide I (GF109203X).

Department of Cardiology, Medical University Hospital Heidelberg, Im Neuenheimer Feld 410, D-69120 Heidelberg, Germany.
Cardiovascular Research (Impact Factor: 5.94). 12/2004; 64(3):467-76. DOI: 10.1016/j.cardiores.2004.07.023
Source: PubMed

ABSTRACT The human ether-a-go-go-related gene (hERG) encodes the rapid component of the cardiac repolarizing delayed rectifier potassium current, I(Kr). The direct interaction of the commonly used protein kinase C (PKC) inhibitor bisindolylmaleimide I (BIM I) with hERG, KvLQT1/minK, and I(Kr) currents was investigated in this study.
hERG and KvLQT1/minK channels were heterologously expressed in Xenopus laevis oocytes, and currents were measured using the two-microelectrode voltage clamp technique. In addition, hERG currents in stably transfected human embryonic kidney (HEK 293) cells, native I(Kr) currents and action potentials in isolated guinea pig ventricular cardiomyocytes were recorded using whole-cell patch clamp electrophysiology.
Bisindolylmaleimide I blocked hERG currents in HEK 293 cells and Xenopus oocytes in a concentration-dependent manner with IC(50) values of 1.0 and 13.2 muM, respectively. hERG channels were primarily blocked in the open state in a frequency-independent manner. Analysis of the voltage-dependence of block revealed a reduction of inhibition at positive membrane potentials. BIM I caused a shift of -20.3 mV in the voltage-dependence of inactivation. The point mutations tyrosine 652 alanine (Y652A) and phenylalanine 656 alanine (F656A) attenuated hERG current blockade, indicating that BIM I binds to a common drug receptor within the pore region. KvLQT1/minK currents were not significantly altered by BIM I. Finally, 1 muM BIM I reduced native I(Kr) currents by 69.2% and lead to action potential prolongation.
In summary, PKC-independent effects have to be carefully considered when using BIM I as PKC inhibitor in experimental models involving hERG channels and I(Kr) currents.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Cardiac muscle contraction, triggered by the action potential, is mediated by the release of Ca(2+) from the sarcoplasmic reticulum through ryanodine receptor (RyR)2 channels. In situ, RyR2 gating is modulated by numerous physiological and pharmacological agents, and altered RyR2 function underlies the occurrence of arrhythmias in both inherited and acquired diseases. To understand fully the mechanisms underpinning the regulation of RyR2 in the normal heart and how these systems are altered in pathological conditions, we must first gain a detailed knowledge of the fundamental processes of RyR2 gating. In this investigation, we provide key novel mechanistic insights into the physical reality of RyR2 gating revealed by new experimental and analytical approaches. We have examined in detail the single-channel gating kinetics of the purified human RyR2 when activated by cytosolic Ca(2+) in a stringently regulated environment where the modulatory influence of factors external to the channel were minimized. The resulting gating schemes are based on an accurate description of single-channel kinetics using hidden Markov model analysis and reveal several novel aspects of RyR2 gating behavior: (a) constitutive gating is observed as unliganded opening events; (b) binding of Ca(2+) to the channel stabilizes it in different open states; (c) RyR2 exists in two preopening closed conformations in equilibrium, one of which binds Ca(2+) more readily than the other; (d) the gating of RyR2 when bound to Ca(2+) can be described by a kinetic scheme incorporating bursts; and (e) analysis of flicker closing events within bursts reveals gating activity that is not influenced by ligand binding. The gating schemes generated in this investigation provide a framework for future studies in which the mechanisms of action of key physiological regulatory factors, disease-linked mutations, and potential therapeutic compounds can be described precisely.
    The Journal of General Physiology 07/2012; 140(2):139-58. · 4.73 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The mixed ion channel blocker, vernakalant (RSD1235), is effective in rapid conversion of atrial fibrillation (AF) to sinus rhythm (SR). Suppression of cardiac two-pore-domain potassium (K2P) channels causes action potential prolongation and has recently been proposed as novel antiarrhythmic strategy. The objective of this study was to investigate acute effects of vernakalant on human K2P2.1 (TREK-1) and K2P3.1 (TASK-1) channels to provide a more complete picture of its antiarrhythmic mechanism of action. The class IC antiarrhythmic drug flecainide was studied as comparator agent. Two-electrode voltage clamp and whole-cell patch clamp electrophysiology was used to record K2P currents from Xenopus oocytes and Chinese hamster ovary (CHO) cells. Vernakalant inhibited cardiac K2P2.1 channels expressed in Xenopus oocytes and in CHO cells. The IC50 value obtained from mammalian cells (13.3µM) was close to the range of vernakalant levels reported in patients (2-8µM), indicating potential clinical significance of K2P2.1 blockade. Open rectification characteristics and current-voltage relationships of K2P2.1 currents were not affected by vernakalant. Vernakalant did not significantly reduce K2P3.1 currents. Finally, the class I antiarrhythmic drug flecainide had no effect on K2P2.1 or K2P3.1 channels. In conclusion, the recently developed antiarrhythmic drug vernakalant targets human K2P2.1 K(+) background channels. This previously unrecognized inhibitory property adds to the multichannel blocking profile of vernakalant and extends the mechanistic basis for its anti-fibrillatory effect.
    European journal of pharmacology 12/2013; · 2.59 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The inhibition of the cardiac rapid delayed rectifier potassium current (IKr ) and its cloned equivalent human ether-a-go-go-related gene (hERG) channel illustrate QT interval prolonging effects of a wide range of clinically used drugs. In this study, the direct interaction of the intravenous anaesthetic ketamine with wild-type (WT) and mutation hERG currents (IhERG ) was investigated. The hERG channel (WT, Y652A and F656A) was expressed in Xenopus oocytes and studied using standard two-microelectrode voltage-clamp techniques. WT hERG is blocked in a concentration-dependent manner with IC50 = 12.05 ± 1.38 μm by ketamine, and the steady-state inactivation curves are shifted to more negative potentials (about -27 mV). The mutation to Ala of Y652 and F656 located on the S6 domain attenuate IhERG blockade by ketamine, and produced approximately 9-fold and 2.5-fold increases in IC50 compared with that of WT hERG channel, respectively. Ketamine blocks WT IhERG expressed in Xenopus oocytes in a concentration-dependent manner and predominantly interacts with the open hERG channels. The interaction of ketamine with hERG channel may involve the aromatic residues Tyr652 and Phe656.
    The Journal of pharmacy and pharmacology. 09/2013; 65(9):1321-8.


Available from