HERG and KvLQT 1 / IsK , the Cardiac K 1 Channels Involved in Long QT Syndromes , Are Targets for Calcium Channel Blockers

We examined the effects of the calcium channel blockers nitrendipine, diltiazem, verapamil, bepridil, and mibefradil on the cloned HERG and KvLQT1/IsK K channels. These channels generate the rapid and slow components of the cardiac delayed rectifier K current, and mutations can affect them, which leads to long QT syndromes. When expressed in transfected COS cells, HERG is blocked in a concentration-dependent manner by bepridil (EC50 5 0.55 mM), verapamil (EC50 5 0.83 mM), and mibefradil (EC50 5 1.43 mM), whereas nitrendipine and diltiazem have negligible effects. Steady state activation and inactivation parameters are shifted to more negative values in the presence of the blockers. Similarly, KvLQT1/IsK is inhibited by bepridil (EC50 5 10.0 mM) and mibefradil (EC50 5 11.8 mM), while being insensitive to nitrendipine, diltiazem, or verapamil. These results demonstrate that both cloned K channels HERG and KvLQT1/IsK, which represent together the cardiac delayed rectifier K current, are sensitive targets to calcium channel blockers. This work may help in understanding the mechanisms of action of verapamil in certain ventricular tachycardia, as well as some of the deleterious adverse cardiac events associated with bepridil. CCBs constitute an heterogeneous class of compounds with different chemical structures and varying potencies for blocking voltage-dependent Ca channels (Hosey and Lazdunski, 1988). Their primary sites of action include cardiac muscle, systemic, and coronary arterial smooth muscle cells. Their largest domain of prescription is the management of hypertension, angina, and supraventricular arrhythmias. According to the type of voltage-dependent Ca channel they block, one can distinguish Tor L-type calcium antagonists. Mibefradil is the only T-type calcium antagonist approved by the Food and Drug Administration (Clozel et al., 1997). L-type calcium antagonists include the 1,4-dihydropyridines such as nitrendipine and isradipine, the phenylalkylamines such as verapamil, and the benzothiazepines as typified by diltiazem. Many other drugs have effects on L-type Ca channels, like the diarylaminopropylamine bepridil. Verapamil, diltiazem, bepridil, and mibefradil block Ca channels in cardiac cells at clinically relevant concentrations, resulting in a decrease in heart rate and atrioventricular nodal conduction velocity. This forms the basis of their antiarrhythmic efficacy in reentrant arrhythmias or in slowing the ventricular rate in the case of atrial flutter or fibrillation (at least for phenylalkylamines and benzothiazepines) (Roden, 1996). With few exceptions, calcium antagonists do shorten the cell action potential duration; this is why they do not prolong the refractory period and all except bepridil (Campbell et al., 1990) are considered ineffective as ventricular antiarrhythmic agents. Hence, verapamil, diltiazem, and mibefradil can increase the rate and duration of experimentally induced ventricular tachycardia (Billman and Hamlin, 1996), and inappropriate use of verapamil has been shown to jeopardize the outcome of Wolff-Parkinson-White syndrome by further shortening the refractory period of the accessory pathway and increasing the ventricular rate in the case of atrial fibrillation (Gulamhusein et al., 1983). Conversely, verapamil also has been shown to be of use in certain forms of ventricular tachycardia known as “verapamil sensitive” that seem to be triggered by delayed afterdepolarizations (Lauer et al., 1992; Gill et al., 1993; Lee et al., 1996). Bepridil, because of its ability to prolong the refractory period of ventricular myocytes and the QT interval on the electrocardiogram, has been successfully used in ventricular arrhythmias (Roden, 1996). This classic feature of class III antiarrhythmic drugs results from a blockade of IK, which is considered to be an This work was supported by the Centre National de la Recherche Scientifique. C.C. is a recipient of a Grant from the Association Française contre les Myopathies. ABBREVIATIONS IK, cardiac delayed rectifier K 1 current; IKr, rapidly activating component of cardiac delayed rectifier K 1 current; IKs, slowly activating component of cardiac delayed rectifier K current; EGTA, ethylene glycol bis(b-aminoethyl ether)-N,N,N9,N9-tetraacetic acid; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; CCB, calcium channel blocker. 0026-895X/98/040695-09$3.00/0 Copyright © by The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 54:695–703 (1998). 695 at A PE T Jornals on Jne 9, 2017 m oharm . aspeurnals.org D ow nladed from important modulator of the cardiac action potential repolarization. In most mammalian species, including humans, IK is recognized as being composed of at least two currents: IKr and IKs (Sanguinetti and Jurkiewicz, 1990; Li et al., 1996). IKr (rapid component) activates quickly and exhibits inward rectification. IKs (slow component) has much slower kinetics and shows no inactivation. Regarding class III antiarrhythmic drugs, with excessive prolongation of the QT interval, bepridil also can induce polymorphic ventricular tachycardias known as torsades de pointes, especially in the setting of hypokaliemia, bradycardia, or both (Manouvrier et al., 1986). The possibility for verapamil to share, with bepridil, some class III type of activity gives those calcium antagonists a role in modulating the action potential duration (i.e., shortening or lengthening the action potential duration according to the pathophysiological settings). This also raises concerns about their use in populations at risk, such as patients with long QT syndrome (Napolitano et al., 1994). Because previous studies have suggested that CCBs can interact with cardiac voltage-dependent K channels (Hume, 1985), we aimed to extensively explore the potency to block IK of the most prescribed CCBs available on the market. The two types of K channels involved in the generation of IKr (Sanguinetti et al., 1995) and IKs (Attali, 1996; Barhanin et al., 1996; Sanguinetti et al., 1996) have been cloned, and mutations in the corresponding genes have been associated with different manifestations of the long QT syndrome (Roden et al., 1996; Chouabe et al., 1997). The IKr and IKs currents are generated by, respectively, the human ethera-go go-related gene HERG product (Sanguinetti et al., 1995) and a K channel resulting from the assembly of two different proteins, KvLQT1 and IsK (Attali, 1996; Barhanin et al., 1996; Sanguinetti et al., 1996). To evaluate separately the effects of calcium antagonists on the two components of IK, we used an in vitro mammalian cell model (COS-7 cells) transfected with either HERG or KvLQT1 and IsK coding sequences. Materials and Methods Expression in COS cells. The pSI HERG expressing vector was kindly provided by D. J. Snyders (Snyders and Chaudhary, 1996). The human KvLQT1 and IsK coding sequences were amplified by polymerase chain reaction and respectively subcloned into the pCI and pCMV expression vector as described previously (Barhanin et