Terence J. Campbell

HERG K+ channels: friend and foe

The K+ channel encoded by the human ether-à-go-go related gene (HERG) is one of many ion channels that are crucial for normal action potential repolarization in cardiac myocytes. HERG encodes the pore-forming subunit of the rapid component of the delayed rectifier K+ channel, I(K(Vr)). HERG K+ channels are of considerable pharmaceutical interest as possible therapeutic targets for anti-arrhythmic agents and as the molecular target responsible for the cardiac toxicity of a wide range of pharmaceutical agents. Recent studies of the molecular basis of the promiscuity of HERG K+ channel drug binding has not only started to shed light on this tricky pharmaceutical problem but has also provided further insights into the structure and function of HERG K+ channels.

The Intracellular Chloride Ion Channel Protein CLIC1 Undergoes a Redox-controlled Structural Transition*

Most proteins adopt a well defined three-dimensional structure; however, it is increasingly recognized that some proteins can exist with at least two stable conformations. Recently, a class of intracellular chloride ion channel proteins (CLICs) has been shown to exist in both soluble and integral membrane forms. The structure of the soluble form of CLIC1 is typical of a soluble glutathione S-transferase superfamily protein but contains a glutaredoxin-like active site. In this study we show that on oxidation CLIC1 undergoes a reversible transition from a monomeric to a non-covalent dimeric state due to the formation of an intramolecular disulfide bond (Cys-24–Cys-59). We have determined the crystal structure of this oxidized state and show that a major structural transition has occurred, exposing a large hydrophobic surface, which forms the dimer interface. The oxidized CLIC1 dimer maintains its ability to form chloride ion channels in artificial bilayers and vesicles, whereas a reducing environment prevents the formation of ion channels by CLIC1. Mutational studies show that both Cys-24 and Cys-59 are required for channel activity.

Inhibition of the human ether-a-go-go-related gene (HERG) potassium channel by cisapride : affinity for open and inactivated states

Cisapride is a prokinetic agent which has been associated with QT prolongation, torsades de pointes and cardiac arrest. The cellular mechanism for these observations is high affinity blockade of IKr (encoded by HERG). In a chronic transfection model using CHO‐K1 cells, cisapride inhibited HERG tail currents after a step to +25 mV with similar potency at room and physiological temperatures (IC50 16.4 nM at 20–22°C and 23.6 nM at 37°C). Channel inhibition exhibited time‐, voltage‐ and frequency‐dependence. In an envelope of tails test, channel blockade increased from 27±8% after a 120 ms depolarizing step to 50±4% after a 1.0 s step. These findings suggested affinity for open and/or inactivated channel states. Inactivation was significantly accelerated by cisapride in a concentration‐dependent manner and there was a small (−7 mV) shift in the voltage dependence of steady state inactivation. Channel blockade by cisapride was modulated by [K+]o, with a 26% reduction in the potency of channel blockade when [K+]o was increased from 1 to 10 mM. In conclusion, HERG channel inhibition by cisapride exhibits features consistent with open and inactivated state binding and is sensitive to external potassium concentration. These features may have significant clinical implications with regard to the mechanism and treatment of cisapride‐induced proarrhythmia.

Proarrhythmic effects of an oxygen-derived free radical generating system on action potentials recorded from guinea pig ventricular myocardium: a possible cause of reperfusion-induced arrhythmias.

Standard microelectrode techniques were used to study the effects of a free radical generating system on action potentials recorded from guinea pig ventricular myocardium. Free radicals were generated by mixing xanthine oxidase (0.02-0.04 mu/ml) with the superfusate-modified Lockes solution containing purine 2.3 mM. The system was validated by demonstrating that it could reduce cytochrome C at a rate of 15.9 +/- 1.5 mol/l/min. This rate was decreased to 3.0 +/- 0.3 (p less than 0.001) in the presence of superoxide dismutase (12 mg/100 ml), and the reaction was absent if xanthine oxidase and purine were premixed for 60 minutes prior to adding cytochrome C. Superfusion of guinea pig ventricular strips with the free radical generating system (20-30 minutes) resulted in a highly significant reduction in resting potential from -79.3 +/- 1.8 mV to -70.9 +/- 1.4 mV (p less than 0.0001, n = 6) and in action potential amplitude from 110.9 +/- 2.2 mV to 101.7 +/- 4.0 mV (p less than 0.0001). There was an accompanying fall in maximum rate of depolarization (Vmax) from 254.1 +/- 17.7 V/sec to 207.1 +/- 18.6 V/sec (p less than 0.01) and no significant change in action potential duration. These changes were accompanied by spontaneous activity in 3 of 6 preparations and reversed after 20-30 minutes washing in Lockes solution. They were largely abolished by adding superoxide dismutase (12 mg/100 ml) to the superfusate and completely absent if the xanthine oxidase and purine were premixed for 60 minutes before superfusing the myocardium. We conclude that the phenomena observed may contribute to the genesis of reperfusion arrhythmias.

The nuclear chloride ion channel NCC27 is involved in regulation of the cell cycle

1 NCC27 is a nuclear chloride ion channel, identified in the PMA‐activated U937 human monocyte cell line. NCC27 mRNA is expressed in virtually all cells and tissues and the gene encoding NCC27 is also highly conserved. Because of these factors, we have examined the hypothesis that NCC27 is involved in cell cycle regulation. 2 Electrophysiological studies in Chinese hamster ovary (CHO‐K1) cells indicated that NCC27 chloride conductance varied according to the stage of the cell cycle, being expressed only on the plasma membrane of cells in G2/M phase. 3 We also demonstrate that Cl− ion channel blockers known to block NCC27 led to arrest of CHO‐K1 cells in the G2/M stage of the cell cycle, the same stage at which this ion channel is selectively expressed on the plasma membrane. 4 These data strongly support the hypothesis that NCC27 is involved, in some as yet undetermined manner, in regulation of the cell cycle.

Resting and Rate-Dependent Depression of Maximum Rate of Depolarisation (Vmax) in Guinea Pig Ventricular Action Potentials by Mexiletine, Disopyramide, and Encainide

The influence of stimulation rate on the ability of mexiletine, disopyramide. and encainide to depress the maximum rate of depolarisation (Vmax) of intracellular action potentials has been studied in guinea pig ventricular myocardium, using standard microelectrode techniques. Mexiletine and disopyramide produced modest depression of Vmax even in the absence of stimulation (resting block), while encainide did not. All three drugs produced progressive depression of Vmax as stimulation rate was increased over a wide range of interstimulus intervals (rate-dependent block). This relationship between interstimulus interval (ISI) and depression of Vmax was steepest for mexiletine. least marked for disopyramide. and intermediate for encainide. Mexiletine also exhibited the fastest response to a sudden change of frequency. During a train of stimuli at ISI of 300ms after a rest period. Vmax fell rapidly, reaching 610 of its final value by the second beat. In response to a similar train of stimuli, disopyramide and encainide produced exponential falls of Vmax with rate constants of −0.113 AP′ (AP = action potential) and −0.025 AP′.respectively. Similar trends were seen in the recovery of cells from this rate-dependent block at the end of a train of stimuli. Time constants for this process for mexiletine. disopyramide. and encainide were 471.2 ms. 12.2 s, and 20.3 s. respectively. It is concluded that the rapid onset of and recovery from rate-dependent block seen with mexiletine may explain its lack of effect on conduction of sinus beats at concentrations that suppress extrasystoles and tachycardias.

Functional characterization of the NCC27 nuclear protein in stable transfected CHO-K1 cells

NCC27 belongs to a family of small, highly conserved, organellar ion channel proteins. It is constitutively expressed by native CHO‐K1 and dominantly localized to the nucleus and nuclear membrane. When CHO‐K1 cells are transfected with NCC27‐expressing constructs, synthesized proteins spill over into the cytoplasm and ion channel activity can then be detected on the plasma as well as nuclear membrane. This provided a unique opportunity to directly compare electrophysiological characteristics of the one cloned channel, both on the nuclear and cytoplasmic membranes. At the same time, as NCC27 is unusually small for an ion channel protein, we wished to directly determine whether it is a membrane‐resident channel in its own right. In CHO‐K1 cells transfected with epitope‐tagged NCC27 constructs, we have demonstrated that the NCC27 conductance is chloride dependent and that the electrophysiological characteristics of the channels are essentially identical whether expressed on plasma or nuclear membranes. In addition, we show that a monoclonal antibody directed at an epitope tag added to NCC27 rapidly inhibits the ability of the expressed protein to conduct chloride, but only when the antibody has access to the tag epitope. By selectively tagging either the amino or carboxyl terminus of NCC27 and varying the side of the membrane from which we record channel activity, we have demonstrated conclusively that NCC27 is a transmembrane protein that directly forms part of the ion channel and, further, that the amino terminus projects outward and the carboxyl terminus inward. We conclude that despite its relatively small size, NCC27 must form an integral part of an ion channel complex.—Tonini, R., Ferroni, A., Valenzuela, S. M., Warton, K., Campbell, T. J., Breit, S. N., Mazzanti, M. Functional characterization of the NCC27 nuclear protein in stable transfected CHO‐K1 cells. FASEB J. 14, 1171–1178 (2000)

Randomized controlled trial of intraaortic balloon counterpulsation in early myocardial infarction with acute heart failure

The value of intraaortic balloon counterpulsation in limiting infarct size and improving survival was studied in patients with early transmural myocardial infarction complicated by acute heart failure. Thirty such patients, previously well, were randomly assigned to counterpulsation (14 patients) or standard therapy (16 patients). Counterpulsation was begun 4.8 to 13.7 hours (mean 7.1) after the onset of pain and continued for less than 1 to 11 days (mean 4.5). Peak creatine kinase was 1,794 +/- 846 IU/liter (mean +/- standard deviation) in patients receiving counterpulsation compared with 1,688 +/- 908 for those receiving standard therapy; cumulative creatine kinase was 3,590 +/- 1,936 IU/liter for patients receiving counterpulsation and 2,945 +/- 1,803 for those receiving standard therapy. Hospital mortality was similar (counterpulsation, 7 of 14; standard therapy, 7 of 16 [p = 0.05 for 25 percent mortality reduction]) as was mortality at follow-up (counterpulsation, 8 of 14; standard therapy, 10 of 16 [p = 0.09 for 25 percent mortality reduction]). Functional class at follow-up examination 1 to 36 months (mean 15) after infarction was also similar in the two groups. Counterpulsation did not appear to modify infarct size or to alter morbidity or mortality when initiated as primary therapy 4.8 to 13.7 hours after the onset of symptoms of myocardial infarction.

Importance of physico-chemical properties in determining the kinetics of the effects of Class I antiarrhythmic drugs on maximum rate of depolarization in guinea-pig ventricle.

1 The effects of Class I antiarrhythmic drugs on the maximum rate of depolarization of guinea‐pig ventricular action potentials were studied by standard microelectrode techniques. 2 The ability of seven different drugs to depress in unstimulated tissue (‘resting block’) was found to correlate poorly with the lipophilicity (log P) of the compounds and only a little better with their molecular weights. 3 Depression of in stimulated tissue was studied for 11 drugs and found, in all cases, to increase with stimulation frequency (‘rate‐dependent block’). 4 The rapidity of onset of rate‐dependent block (at approximately equipotent concentrations) varied markedly between drugs. It correlated well with molecular weight (r = 0.83; P < 0.01). 5 The time constant of recovery from rate‐dependent block (ire) also correlated very well with molecular weight (r = 0.94; P < 0.001) for the seven drugs thus studied. 6 A simplified model for the interaction of Class I drugs with the fast sodium channel is proposed in which the drugs all act as ‘inactivation enhancers’; (as suggested by other workers) but in which their molecular weight plays a central role in determining the kinetics of this interaction.

Intravenous sotalol for the treatment of atrial fibrillation and flutter after cardiopulmonary bypass. Comparison with disopyramide and digoxin in a randomised trial.

The efficacy of sotalol in treating acute atrial fibrillation and flutter after open heart surgery was compared with that of a digoxin/disopyramide combination. Forty adult patients with postoperative atrial arrhythmias were randomised into either group 1 (sotalol 1 mg/kg bolus intravenously plus 0.2 mg/kg intravenously over 12 hours) or group 2 (digoxin 0.75 mg intravenously, then two hours later disopyramide 2 mg/kg intravenous bolus and 0.4 mg/kg/h intravenously for 10 hours). In each group, 17 out of 20 patients reverted to sinus or junctional rhythm within 12 hours. The time to reversion in group 1 was significantly shorter than in group 2. Systolic blood pressure fell by greater than or equal to 20 mm Hg or to less than or equal to 90 mm Hg during drug administration in 17 out of 20 patients in group 1 (sotalol withdrawn in two) and in none out of 20 in group 2. Two patients in group 1 developed transient bradycardia (sotalol withdrawn in one). None of 17 patients in group 1 and two of 17 in group 2 relapsed temporarily into atrial fibrillation during the 12 hours of intravenous treatment. On continued oral treatment, one late relapse occurred in group 1 and five in group 2, and five patients in group 2 had disopyramide withdrawn because of anticholinergic side effects (acute urinary retention in four). Sotalol was as effective as the digoxin/disopyramide combination and acted significantly faster. Sensitivity to beta blockade in these patients may be related to high plasma catecholamine concentrations known to occur after cardiopulmonary bypass.