Inhibition of cardiac delayed rectifier K+ current by overexpression of the long-QT syndrome HERG G628S mutation in transgenic mice.

Mutations in the HERG gene are linked to the LQT2 form of the inherited long-QT syndrome. Transgenic mice were generated expressing high myocardial levels of a particularly severe form of LQT2-associated HERG mutation (G628S). Hearts from G628S mice appeared normal except for a modest enlargement seen only in females. Ventricular myocytes isolated from adult wild-type hearts consistently exhibited an inwardly rectifying E-4031-sensitive K+ current resembling the rapidly activating cardiac delayed rectifier K+ current (Ikr) in its time and voltage dependence; this current was not found in cells isolated from G628S mice. Action potential duration was significantly prolonged in single myocytes from G628S ventricle (cycle length=1 second, 26 degrees C) but not in recordings from intact ventricular strips studied at more physiological rates and temperature (200 to 400 bpm, 37 degrees C). ECG intervals, including QT duration, were unchanged, although minor aberrancies were noted in 20% (16/80) of the G628S mice studied, primarily involving the QRS complex and, more rarely, T-wave morphology. The aberrations were more commonly observed in females than males but could not be correlated with sex-based differences in action potential duration. These results establish the presence of IKr in the adult mouse ventricle and demonstrate the ability of the G628S mutation to exert a dominant negative effect on endogenous IKr in vivo, leading to the expected LQT2 phenotype of prolonged repolarization at the single cell level but not QT prolongation in the intact animal. The model may be useful in dissecting repolarization currents in the mouse heart and as a means of examining the mechanism(s) by which the G628S mutation exerts its dominant negative effect on native cardiac cells in vivo.

Voltage-dependent inhibition of the ATP-sensitive K+ current by the class Ia agent disopyramide in cat ventricular myocytes.

The inhibition of the adenosine triphosphate-sensitive K+ (KATP) current by disopyramide, a class Ia antiarrhythmic drug, was studied using whole cell voltage clamp in cat ventricular myocytes at 37 degrees C and was compared to that seen with quinidine, a prototypical class Ia drug. The inhibition of the levcromakalim-induced KATP current was concentration dependent, with Ki, at -20 mV, of 4.9 +/- 0.6 and 1.5 +/- 0.1 microM for disopyramide and quinidine, respectively. Disopyramide also inhibited the KATP current elicited during the metabolic inhibition induced by the uncoupler CCCP. Disopyramide (9 microM) produced a 75% inhibition in comparison to the 82% inhibition of the levcromakalim-elicited current. The degree of inhibition increased with depolarization. This effect was quantified using the fractional electrical distance (delta) as an index of the voltage dependence, yielding equivalent voltage dependency values of 0.48 +/- 0.02 and 0.51 +/- 0.08 for disopyramide and quinidine, respectively. Depolarizing and repolarizing voltage steps in presence of tetrodotoxin (5-30 microM), nitrendipine (1 microM), and 4-aminopyridine (1.5 microM) failed to reveal clear kinetics of block and unblock, suggesting either a very fast block (< 3 ms) or a tonic (i.e., time-independent) inhibition. The preferential inhibition of KATP channels at depolarized potentials during metabolic inhibition may contribute to preserve the ability of class Ia agents to prolong action potential duration without compromising resting potential.

Block of the delayed rectifier current (IK) by the 5-HT3 antagonists ondansetron and granisetron in feline ventricular myocytes.

1. We investigated the effects of two 5-HT3 antagonists, ondansetron and granisetron, on the action potential duration (APD) and the delayed rectifier current (IK) of feline isolated ventricular myocytes. Whole-cell current and action potential recordings were performed at 37 degrees C with the patch clamp technique. 2. Ondansetron and granisetron blocked IK with a KD of 1.7 +/- 1.0 and 4.3 +/- 1.7 microM, respectively. At a higher concentration (30 microM), both drugs blocked the inward rectifier (IKl). 3. The block of IK was dependent on channel activation. Both drugs slowed the decay of IK tail currents and produced a crossover with the pre-drug current trace. These results are consistent with block and unblock from the open state of the channel. 4. Granisetron showed an intrinsic voltage-dependence as the block increased with depolarization. The equivalent voltage-dependency of block (delta) was 0.10 +/- 0.04, suggesting that granisetron blocks from the intracellular side at a binding site located 10% across the transmembrane electrical field. 5. Ondansetron (1 microM) and granisetron (3 microM) prolonged APD by about 30% at 0.5 Hz. The prolongation of APD by ondansetron was abolished at faster frequencies (3 Hz) showing reverse rate dependence. 6. In conclusion, the 5-HT3 antagonists, ondansetron and granisetron, are open state blockers of the ventricular delayed rectifier and show a clear class III action.

Inhibition of gramicidin channel activity by local anesthetics.

Ondrias et al. ((1986) Stud. Biophys. 115, 17-22) found that dibucaine, butacaine, and tetracaine reduce the conductance of membranes containing multiple (greater than 10(6)) gramicidin channels. Similar experiments with local anesthetics (LA's) added to the bath while gently stirring showed that the inhibition developed slowly over a time course of 5-10 min. We developed a many (10-20) channel membrane technique which demonstrated that when LA's were added to the bath and the membrane was repeatedly broken and reformed, the channel occurrence frequency declined promptly. In standard single-channel membrane experiments at lower gramicidin densities, the mean single channel conductance and lifetime distributions with LA's present in the bath did not differ from the controls. The predominant channel conductance amplitude was lower by 9.1% than those of controls, but channel amplitude distributions were also modified so that the net reduction in overall population channel conductance was only about 2.0%. Channel currents showed no evidence of flicker blocks. The lifetime histograms of control and LA-exposed channel populations were both satisfactorily fit by a single-exponential function with the same mean. Thus, inhibition is due primarily to a reduction in the frequency of occurrence of conducting channels, implying a reduced concentration of active monomers in the membrane.

Synthesis and channel properties of [Tau 16]gramicidin A.

Des(ethanolamine)-taurine16-gramicidin A ([Tau 16]gramicidin A) was synthesized by the solid phase method and its channel-forming behavior in planar lipid bilayers was examined. The purified monovalent anionic peptide formed channels when applied to the aqueous compartments on both sides of the bilayer, but not when applied to one side only. The single-channel conductance was measured for KCl concentrations between 0.1 and 1.0 M and was found to be higher than that of gramicidin A in each case. Single-channel lifetimes were similar to those of gramicidin A suggesting that the channels have the beta 6.3 helix structure.