Kathryn Houseman

Electrophysiologic characterization of a novel hERG channel activator

Activators of the human ether-a-go-go-related gene (hERG) K(+) channel have been reported recently to enhance hERG current amplitude (five synthetic small molecules and one naturally occurring substance). Here, we characterize the effects of a novel compound A-935142 ({4-[4-(5-trifluoromethyl-1H-pyrazol-3-yl)-phenyl]-cyclohexyl}-acetic acid) on guinea-pig atrial and canine ventricular action potentials (microelectrode techniques) and hERG channels expressed in HEK-293 cells (whole-cell patch clamp techniques). A-935142 shortened cardiac action potentials and enhanced the amplitude of the hERG current in a concentration- and voltage-dependent manner. The fully activated current-voltage relationship revealed that this compound (60 microM) increased both outward and inward K(+) current as well as the slope conductance of the linear portion of the fully activated I-V relation. A-935142 significantly reduced the time constants (tau) of hERG channel activation at two example voltages (-10 mV: tau=100+/-17 ms vs. 164+/-24 ms, n=6, P<0.01; +30 mV: tau=16.7+/-1.8 ms vs. 18.9+/-1.8 ms, n=5, P<0.05) and shifted the voltage-dependence for hERG activation in the hyperpolarizing direction by 9 mV. The time course of hERG channel deactivation was slowed at multiple potentials (-120 to -70 mV). A-935142 also reduced the rate of inactivation and shifted the voltage-dependence of inactivation in the depolarizing direction by 15 mV. Recovery of hERG channel from inactivation was not affected by A-935142. In conclusion, A-935142 enhances hERG current in a complex manner by facilitation of activation, reduction of inactivation, and slowing of deactivation, and abbreviates atrial and ventricular repolarization.

Characterization of A-935142, a hERG enhancer, in the presence and absence of standard hERG blockers.

AIMS In a previous study we found that A-935142 enhanced hERG current in a concentration-dependent manner by facilitating activation, reducing inactivation, and slowing deactivation (Su et al., 2009). A-935142 also shortened action potential duration (APD90) in canine Purkinje fibers and guinea pig atrial tissue. This study focused on the combined effects of the prototypical hERG enhancer, A-935142 and two hERG current blockers (sotalol and terfenadine). MAIN METHODS The whole-cell voltage clamp method with HEK 293 cells heterologously expressing the hERG channel (Kv 11.1) was used. KEY FINDINGS A-935142 did not compete with 3H-dofetilide binding, suggesting that A-935142 does not overlap the binding site of typical hERG blockers. In whole-cell voltage clamp studies we found: 1) 60 μM A-935142 enhanced hERG current in the presence of 150 μM sotalol (57.5±5.8%) to a similar extent as seen with A-935142 alone (55.6±5.1%); 2) 150 μM sotalol blocked hERG current in the presence of 60 μM A-935142 (43.5±1.5%) to a similar extent as that seen with sotalol alone (42.0±3.2%) and 3) during co-application, hERG current enhancement was followed by current blockade. Similar results were obtained with 60 nM terfenadine combined with A-935142. SIGNIFICANCE These results suggest that the hERG enhancer, A-935142 does not compete with these two known hERG blockers at their binding site within the hERG channel. This selective hERG current enhancement may be useful as a treatment for inherited or acquired LQTS (Casis et al., 2006).

Drug-Induced hERG Current Enhancement is Modulated by Extracellular Proton and Potassium

Acid-base disturbances and hypokalemia or hyperkalemia are frequently encountered in a variety of clinical scenarios. Extracellular proton and potassium concentrations are important modulators of hERG channel functions and biophysical properties such as deactivation and inactivation. Slowing deactivation and inactivation kinetics of hERG channel are two important components for drug-induced hERG current enhancement. This study examined if drug-induced hERG current enhancement is affected by acid-base and electrolyte aberrations at physiological temperature (37° C). Whole-cell voltage clamp technique was used to measure hERG current (step-ramp protocol: initial 1-sec depolarization step to 0 mV from −80 mV (VH) followed by a 2-sec repolarization ramp back to VH) and a typical hERG activator A-935142 was used as a tool drug. Drug-induced hERG current enhancement was potentiated by extracellular acidification (% increase for step current: 132 ± 5 vs. 58.4 ± 4.2, pH 6.8 vs. 7.4, P < 0.01; % increase for tail current: 115 ± 3 vs. 43.2 ± 3.2, P < 0.01). In contrast, alkalosis (pH 8.4) weakened the drug-induced hERG current enhancement (% increase for step: 30.3 ± 3.1 vs. 58.4 ± 4.2, pH 8.4 vs 7.4, P < 0.01; % increase for tail: 21.9 ± 2.3 vs. 43.2 ± 3.2, P < 0.01). The measured bath drug concentrations were the same at normal pH, acidosis, and alkalosis. Hypokalemia (1 mM K+) did not affect drug-induced hERG current enhancement but hyperkalemia (10 mM K+) attenuated the drug-induced hERG current enhancement (increase for step: 41.1 ± 1.6% vs. 58.4 ± 4.2%, 10 mM vs. 5 mM K+, P < 0.01; increase for tail: 31.5 ± 1.3 % vs. 43.2 ± 3.2 %, P < 0.05). These results demonstrate that hERG current enhancement by A-935142 is modulated by extracellular proton concentrations and hyperkalemia.

Electrophysiologic Characterization of a Complex hERG Channel Activator

Novel and specific activators of the human ether-a-go-go-related gene (hERG) K+ channel have been reported recently to enhance hERG current amplitude (4 synthetic small molecules and one naturally occurring substance). Here, we characterize the effects of a novel compound (Abbott-x) on atrial and ventricular action potentials (using microelectrode techniques) and cloned hERG channels stably expressed in HEK-293 cells (using whole cell patch clamp techniques). Abbott-x shortened cardiac action potentials and enhanced the amplitude of the hERG current in a concentration- and voltage-dependent manner. The fully activated current-voltage relationship revealed that this compound (60 uM) increased both outward and inward K+ current. The slope conductance of the linear portion of the fully activated I-V relation was increased in the presence of the compound. Abbott-x significantly reduced the time constants (τ) of hERG channel activation at two example voltages tested (-10 mV: τ =100 ± 17 vs 164 ± 24 ms, n = 6, P < 0.01; +30 mV: τ = 16.7 ± 1.8 vs 18.9 ± 1.8, n = 5, P < 0.05) and shifted the voltage-dependence for hERG activation in the hyperpolarizing direction by 9 mV (n = 7, P < 0.01). The time course of hERG channel deactivation was significantly slowed at multiple potentials tested (−120 to −70 mV). Abbott-x also significantly reduced the rate of inactivation and shifted the voltage dependence of inactivation in the depolarizing direction by 15 mV (n = 5, P < 0.05). Recovery of hERG channel from inactivation was not significantly affected by Abbott-x. In conclusion, Abbott-x enhances hERG current in a complex manner by facilitation of activation, reduction of inactivation, and slowing of deactivation, and abbreviates atrial and ventricular repolarization.