Pharmacological targeting of long QT mutant sodium channels. D W Wang, … , A L George Jr, P B Bennett J Clin Invest. 1997;99(7):1714-1720. https://doi.org/10.1172/JCI119335. Research Article The congenital long QT syndrome (LQTS) is an inherited disorder characterized by a delay in cardiac cellular repolarization leading to cardiac arrhythmias and sudden death often in young people. One form of the disease (LQT3) involves mutations in the voltage-gated cardiac sodium channel. The potential for targeted suppression of the LQT defect was explored by heterologous expression of mutant channels in cultured human cells. Kinetic and steady state analysis revealed an enhanced apparent affinity for the predominantly charged, primary amine compound, mexiletine. The affinity of the mutant channels in the inactivated state was similar to the wild type (WT) channels (IC50 approximately 15-20 microM), but the late-opening channels were inhibited at significantly lower concentrations (IC50 = 2-3 microM) causing a preferential suppression of the late openings. The targeting of the defective behavior of the mutant channels has important implications for therapeutic intervention in this disease. The results provide insights for the selective suppression of the mutant phenotype by very low concentrations of drug and indicate that mexiletine equally suppresses the defect in all three known LQT3 mutants. Find the latest version: https://jci.me/119335/pdf Pharmacological Targeting of Long QT Mutant Sodium Channels D.W. Wang, K. Yazawa, N. Makita, A.L. George, Jr., and P.B. Bennett Department of Pharmacology and Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6602 Abstract cently been described (7–9). The mutations cause the normally transiently opening sodium channels to continue to reopen at The congenital long QT syndrome (LQTS) is an inherited depolarized membrane potentials giving rise to a sustained in- disorder characterized by a delay in cardiac cellular repo- ward current which prolongs the cardiac action potential. larization leading to cardiac arrhythmias and sudden death An important goal for rational treatment of this disorder is often in young people. One form of the disease (LQT3) in- to define the molecular basis and conditions for suppression of volves mutations in the voltage-gated cardiac sodium chan- this defect (10). One strategy is to target and selectively sup- nel. The potential for targeted suppression of the LQT defect press the phenotype produced by the mutation. A recent study was explored by heterologous expression of mutant chan- suggests that this may be possible. An et al. (11) demonstrated nels in cultured human cells. Kinetic and steady state analy- suppression of late-opening sodium channels by high concen- sis revealed an enhanced apparent affinity for the predomi- trations of lidocaine in the ⌬KPQ mutant channels. However, nantly charged, primary amine compound, mexiletine. The lidocaine may not be the optimal drug for this purpose because affinity of the mutant channels in the inactivated state was its mechanism of action is to block inactivated rather than ف similar to the wild type (WT) channels (IC50 15–20 ␮M), open sodium channels (12, 13) and it must be given intrave- but the late-opening channels were inhibited at significantly nously to patients. Dumaine et al. (8) have recently reported lower concentrations (IC50 ϭ 2–3 ␮M) causing a preferen- that there are multiple distinct mechanisms for the three LQT3 tial suppression of the late openings. The targeting of the mutant channels and that high concentrations of mexiletine defective behavior of the mutant channels has important could inhibit these mutant channels when expressed in Xeno- implications for therapeutic intervention in this disease. The pus laevis oocytes. However, the range of therapeutic plasma results provide insights for the selective suppression of the concentrations of mexiletine for treatment of arrhythmias is mutant phenotype by very low concentrations of drug and much lower than they tested (4–11 ␮M), and X. laevis oocytes indicate that mexiletine equally suppresses the defect in all can be problematic for studies of drug effects possibly due to three known LQT3 mutants. (J. Clin. Invest. 1997. 99:1714– their large lipophilic yolk. Thus, it is unknown whether thera- 1720.) Key words: SCN5A • human heart • hH1 • LQT3 • peutic levels of an antiarrhythmic agent can affect these chan- Naϩ channel • antiarrhythmic agent • mexiletine nels, how the inhibition compares to wild type (WT) channels, or whether such a drug will affect each of the mutant channels Introduction differently. Mexiletine is an orally available antiarrhythmic agent which The congenital long QT syndrome (LQTS)1 is a myocardial like lidocaine inhibits sodium channels (4, 12–27). The charged disease caused by mutations at distinct genetic loci (1, 2). One form interacts with the open state and the uncharged form form of the disease (LQT3) is caused by mutations in the hu- interacts with the inactivated state of the channel (14–17). The man voltage-gated cardiac sodium channel gene (SCN5A) (3–6). primary mechanism of inhibition of WT Naϩ channels by mex- Three different mutations in SCN5A were identified in subsets iletine involves the uncharged drug interacting with the in- of long QT syndrome patients (5, 6). These include an in- activated state of the channel (25, 26, 28). The open state is frame deletion of three amino acids (lys 1,505, proline 1,506, normally too brief to permit significant interactions with mex- and glutamine 1,507, ⌬KPQ), and two point mutations: iletine (13, 22, 29–32). Nevertheless, the pKa of mexiletine is N1325S (asparagine 1,325 converted to serine) and R1644H 9.3, therefore, 99% of the drug is charged at physiological pH. (arginine 1,644 converted to histidine). The functional defects Thus, under normal conditions, only 1% of mexiletine is in the caused by these sodium channel mutations on gating have re- form that blocks inactivated channels. We reasoned that the major charged species of mexiletine may opportunistically sup- press the abnormal repetitive reopenings of sodium channels in LQT3. To test this hypothesis, LQT3 sodium channel mutations Address correspondence to Paul B. Bennett, Ph.D., Department of were engineered in the recombinant human heart sodium Pharmacology, 558 MRB II, Vanderbilt University School of Medi- channel (hH1) by site-directed mutagenesis (9). WT and mu- cine, Nashville, TN 37232-6602. Phone: 615-936-1683; FAX: 615-322- tant channels were expressed heterologously in human embry- 4707; E-mail: [email protected] Received for publication 3 September 1996 and accepted in revised onic kidney cells. Channel behavior and inhibition by mexile- form 21 January 1997. tine were investigated by whole-cell patch clamp methods. Voltage clamp protocols were used to favor occupancy of dis- 1. Abbreviations used in this paper: LQT, long QT; WT, wild type. tinct conformational states of the antiarrhythmic drug receptor (i.e., primarily rested or primarily inactivated). Remarkably, J. Clin. Invest. the results show that LQT3 mutant channels are much more © The American Society for Clinical Investigation, Inc. sensitive to inhibition by mexiletine than are WT sodium chan- 0021-9738/97/04/1714/07 $2.00 nels. The defective late openings are selectively suppressed Volume 99, Number 7, April 1997, 1714–1720 more than the peak sodium current and these late openings 1714 Wang et al. can be suppressed by concentrations at the lower end of the where Imax is the unblocked current in the absence of mexiletine (M) therapeutic range. Furthermore, the defect is suppressed in and I is the magnitude of the current in the presence of a given con- each of the three mutant channels to approximately the same centration ([M]) of the drug. Alternatively, Eq. 2 can be fitted to con- extent indicating that mexiletine may be useful for treatment centration-block data to estimate the apparent KD (IC50). Assuming a of this disorder regardless of the specific sodium channel muta- first-order binding interaction between the drug and the channel, the time constant for block development should depend on the mexile- tion. tine concentration ([M]) according to the following relationship where k and l represent the association and dissociation rate con- stants, respectively: Methods τ ([] )Ϫ1 Block = M k+1 . (4) Voltage clamp methods. The methods used have been described pre- viously (9, 27, 32, 33). Sodium currents were recorded using the whole-cell patch clamp technique (34). Electrodes resistances ranged from 0.8–2 M⍀. Voltage clamp command pulses were generated us- ing pCLAMP software (v6.03; Axon Instruments, Inc., Foster City, CA). Currents were filtered at 5 kHz (Ϫ3 dB, 4-pole Bessel filter). An Axopatch 200 patch clamp amplifier was used with series resis- tance compensation (Axon Instruments, Inc.). The holding potential for all pulse protocols was Ϫ120 mV. Experiments were done at room temperature (20–22ЊC). The bath solution contained: 145 mM NaCl; 4 mM KCl; 1.8 nM CaCl2; 1.0 mM MgCl2; 10 mM Hepes; and 10 mM glucose, pH 7.35. The pipette solution contained (intracellular solu- tion): 10 mM NaF; 110 nM CsF; 20 nM CsCl; 10 nM EGTA ; and 10 nM Hepes, pH 7.35. Data are presented as meanϮSEM. Site-directed mutagenesis of human heart sodium channel (3) was performed as described (7, 9). Mutant and WT cDNAs were sub- cloned into pRc/CMV (Invitrogen Corp., San Diego, CA) for expres- sion in mammalian cells (9, 27, 33). Multiple independent recombinants were sequenced thoroughly in the mutated region and and tested to confirm expression and identical behavior. At least three indepen- dent clones were tested for expression studies. The calcium phosphate method was used to express mutant and WT human heart sodium channel in a transformed human kidney cell line (HEK 293t, also called tsA-201) stably expressing the SV40 T-antigen.