Preferential Block of Late Sodium Current in the LQT3 DKPQ Mutant by the Class IC Antiarrhythmic Flecainide

Flecainide block of Na current (INa) was investigated in wildtype (WT) or the long QT syndrome 3 (LQT3) sodium channel a subunit mutation with three amino acids deleted (DKPQ) stably transfected into human embryonic kidney 293 cells using whole-cell, patch-clamp recordings. Flecainide (1–300 mM) caused tonic and use-dependent block (UDB) of INa in a concentration-dependent manner. Compared with WT, DKPQ INa was more sensitive to flecainide, and flecainide preferentially inhibited late INa (mean current between 20 and 23.5 ms after depolarization) compared with peak INa. The IC50 value of peak and late INa for WT was 127 6 6 and 44 6 2 mM (n 5 20) and for DKPQ was 80 6 9 and 19 6 2 mM (n 5 31) respectively. UDB of peak INa was greater and developed more slowly during pulse trains for DKPQ than for WT. The IC50 value for UDB of peak INa for WT was 29 6 4 mM (n 5 20) and for DKPQ was 11 6 1 mM (n 5 26). For DKPQ, UDB of late INa was greater than for peak INa. Recovery from block was slower for DKPQ than for WT. We conclude that DKPQ interacts differently with flecainide than with WT, leading to increased block and slowed recovery, especially for late INa. These data provide insights into mechanisms for flecainide block and provide a rationale at the cellular and molecular level that open channel block may be a useful pharmacological property for treatment of LQT3. The congenital long QT syndrome (LQT) is an inherited cardiac disorder that causes ventricular arrhythmias, resulting in syncope and sudden death. One form of the disease, LQT3, is caused by mutations in SCN5A, the gene that encodes the voltage-dependent cardiac Na channel a subunit in humans (hH1) (Jiang, 1994; George, 1995; Wang et al., 1995a,b). A KPQ deletion (DKPQ: lysine, proline, and glutamine at positions 1505–1507) in the linker between domains III and IV of hH1 is the most common mutation associated with LQT3. The DKPQ mutant channel exhibits late channel openings caused by a defect in inactivation (Bennett et al., 1995; An et al., 1996; Dumaine et al., 1996; Wang et al., 1996c), and the resulting late Na current (INa) would prolong the action potential and cause QT prolongation on the surface ECG. Gene-specific therapy for LQT3 (i.e., the use of drugs that target the Na channel, more specifically, late INa) is a logical approach. Most antiarrhythmic drugs block the Na channel in a use-dependent manner by preferential binding to either the inactivated state or the open state as described in the modulated receptor model (Hille, 1977; Hondeghem and Katzung, 1977). Inactivated state blockers of the Class Ib antiarrhythmic grouping (e.g., mexiletine) inhibit late INa at the cellular level (Wang et al., 1997), shorten action potential duration in a cellular model of LQT3 (Priori et al., 1996; Shimizu and Antzelevitch, 1997), and shorten the QT interval in LQT3 patients (Schwartz et al., 1995). We hypothesized that an open channel blocker would also be effective (perhaps more so) because of the prolonged dwell time of LQT3 channels in the open state. To test this hypothesis, we studied the Class Ic antiarrhythmic drug flecainide, a predominant open state blocker, comparing tonic block and usedependent block (UDB) by flecainide for peak and late INa for the wild-type (WT) human cardiac Na channel and the DKPQ mutant. Our findings may provide a molecular mechanism for the recently observed correction of the QTc interval in the electrocardiograms of DKPQ LQT3 patients by flecainide (Windle et al., 1999). Materials and Methods Clones and Construction of DKPQ Mutation. The human heart Na channel clone we used (hH1a) was kindly provided by Dr. H. Hartmann (Baylor College of Medicine, Houston, TX). The nucleThis work was supported by National Institutes of Health Grant HL56441 (JCM) and grants from the University of Wisconsin Cardiovascular Research Center, the Oscar Rennebohm Foundation, and by a travel grant from the Fukuda Memorial Foundation (TN). This work was published previously in abstract form: Nagatomo T, Fan Z, Ye B, January CT, Makielski JC (1996). Effects of flecainide on the long QT sodium channel syndrome. Circulation 96(Suppl):677. 1 Current affiliation: Second Department of Internal Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan. ABBREVIATIONS: LQT, long QT syndrome; DKPQ, LQT3 sodium channel a subunit mutation with three amino acids deleted; INa, Na 1 current; UDB, use-dependent block; WT, wild-type; MEM, minimal essential medium. 0026-895X/00/010101-07$3.00/0 Copyright © The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 57:101–107 (2000). 101 at A PE T Jornals on A uust 7, 2017 m oharm .aspeurnals.org D ow nladed from otide and amino acid numbering follow Hartmann et al. (1994). The DKPQ mutation was kindly provided by Drs. John W. Kyle and Gayle S. Tonkovich (University of Chicago, Chicago, IL). It was made by polymerase chain reaction techniques as described previously (Nagatomo et al., 1998). The entire polymerase chain reaction-generated region was completely sequenced to confirm the deletion and ensure that no unwanted changes were made in the channel. Cell Preparation and Transfection. Cells from transformed human embryonic kidney cell line 293 were used. Approximately 5 3 10 cells were seeded on a 60-mm diameter plate (Falcon 3001) with 3 ml of culture medium 1 day before the transfection. Culture medium was MEM complete medium containing: minimum essential medium (Eagle’s salts and L-glutamine), 10% fetal bovine serum, 2 mM L-glutamine, 0.1 mM MEM nonessential amino acids solution, 1 mM MEM pyruvate solution, 10,000 U of penicillin and 10,000 mg of streptomycin. Transfection was carried out using a cationic liposome method. Details have been described previously (Nagatomo et al.,