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Cardiovascular Research 2002 55(2):229-232; doi:10.1016/S0008-6363(02)00486-8
© 2002 by European Society of Cardiology
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Copyright © 2002, European Society of Cardiology

Pharmacological rescue of mutant ion channels

Connie R Bezzinaa,* and Hanno L Tana,b

aExperimental and Molecular Cardiology Group, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
bDepartment of Cardiology, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands

c.r.bezzina{at}amc.uva.nl

* Corresponding author. Tel.: +31-20-566-3265; fax: +31-20-697-5458

Received 13 May 2002; accepted 13 May 2002

See article by Valdivia et al. [1] (pages 279–289) in this issue.

The congenital Long QT (LQT) syndrome [1] is a disorder that probably affects 1 per 5000 to 10 000 individuals. The disorder presents clinically with syncope, seizures and a torsade de pointes ventricular tachycardia. It is a repolarization abnormality caused by a decrease in net outward current during the repolarization phase of the cardiac action potential, thereby prolonging action potential duration. Six genes have thus far been linked to the disorder. Mutations in the potassium channel subunits KCNQ1 (LQT1) and KCNE1 (LQT5) affect the slowly activating component of the delayed rectifier current (IKs), while mutations in the potassium channel subunits KCNH2 (LQT2) and KCNE2 (LQT6) affect the rapidly activating component of this current (IKr). Mutations in SCN5A (the gene that encodes the cardiac sodium [Na+] channel) are associated with one of the least common forms of LQT syndrome, type 3, affecting 5–10% of Long QT patients. LQT-associated SCN5A mutations typically lead to the delayed repolarization phenotype by a ‘gain-of-function’ mechanism, through a sustained non-inactivating (depolarizing) Na+ current during the plateau phase of the action potential. A variant of the LQT syndrome is the Jervell–Lange–Nielsen syndrome, wherein homozygous mutations in KCNQ1 or KCNE1 not only result in QT-prolongation but also in sensorineural deafness. Another variant, with mutations in the KCNJ2 gene that encodes the inward rectifier channel Kir2.1, presents with periodic paralysis and dysmorphic features [2].

Besides the LQT syndrome, mutations in SCN5A can lead to other clinical presentations [3]. One is the Brugada syndrome [4], a disorder with a prevalence presumed to be similar to that of LQT syndrome. In disparity to LQT syndrome, however, SCN5A mutations leading to Brugada syndrome are associated with a ‘loss-of-function’ mechanism, either through altered channel gating [5], or through inability of mutant channels to traffic efficiently to the sarcolemma secondary to presumed misfolding of the mutant channel [6,7]. This ‘loss-of-function’ is thought to hasten epicardial repolarization, thereby augmenting the transmural heterogeneity in action potential duration leading to ST-segment elevation in ECG leads V1–V3 and increasing the propensity for reentrant arrhythmias [8]. The third clinical expression of a cardiac sodium channelopathy is conduction disease [9]. SCN5A mutations associated with this phenotype also lead to ‘loss-of-function’, either through altered channel gating [10], or through formation of gene products that presumably fail to form functional membrane channels [9]. Yet-unknown factors are presumed to contribute to the difference in phenotypic expression (Brugada syndrome versus conduction disease) of variants that in vitro display a similar defect.

In this issue of Cardiovascular Research, Valdivia et al. [11], present an interesting study on a child who presented shortly after birth with torsade de pointes and marked QT interval prolongation. In this study, genetic investigation demonstrated that a mutation (M1766L) in SCN5A arose spontaneously in the boy. The electrophysiological analysis of the M1766L mutant Na+ channel in the human embryonic kidney cell line HEK293 uncovered several features that are of note.


   1. Gain-of-function and loss-of-function defect for the M1766L mutant channel
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 1. Gain-of-function and loss-of...
2. Augmentation of M1766L...
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The abnormal non-inactivating INa observed in this mutant has been found in virtually all LQT-associated mutant Na+ channels and ties up with a phenotype of QT prolongation. This gating change is considered a ‘gain of function’. While the additional depolarizing current that results from this gating defect is small (10% of the severely reduced peak INa, see below), its effect to prolong the cardiac action potential and possibly also to provide the trigger for the early afterdepolarizations that initiate torsade de pointes, stems from its auspicious timing. It occurs at a phase of the cardiac cycle (action potential plateau) when the balance of depolarizing and repolarizing forces is delicate and even small disruptions of this balance may have profound effects [12]. Due to the consistent presence of non-inactivating INa in the reported LQT3-associated mutants, it is quite acceptable that this gating defect may explain the clinical findings in the present mutant, i.e., QT prolongation and 2:1 AV block. However, in addition to non-inactivating INa, this mutant also exhibits a severely reduced density of INa (‘loss of function’). This is frankly a puzzling finding, because reduced INa cannot be reconciled with the clinical phenotype. One may even wonder whether such a dramatic reduction in INa would not overwhelm the small increase of depolarizing current due to non-inactivating INa and render it insignificant to the development of QT prolongation. INa reduction is expected to cause conduction disease or Brugada syndrome, but the present patient exhibits no clear indications of either. The presence or absence of conduction disease cannot be easily assessed, because all but the first ECG were recorded in the presence of antiarrhythmic drugs that cause conduction slowing. Still, the underlying sinus rate, PR interval, and QRS width in the first ECG appear normal (the 2:1 AV block presumably results from the fact that the ventricular refractory period exceeds the intervals of the sinus beats, and not from intrinsic slowing of the spread of excitation). The absence of Brugada syndrome appears more compelling. Not only does the first ECG carry no signs of Brugada syndrome, but neither do all successive ECGs, recorded during lidocaine and/or mexiletine treatment, although these drugs may be expected to exacerbate or uncover the ECG abnormalities of Brugada syndrome as other class I antiarrhythmic drugs (INa blockers) do [13]. However, lidocaine is less potent than these other drugs (ajmaline, flecainide, procainamide) [4], so the lack of Brugada syndrome-like ECG changes upon lidocaine administration does not rule out the presence of this disorder. Indeed, the authors speculate that INa reduction due to reduced current density of this mutant may have contributed to the maintenance of torsade de pointes by reentry, similar to the proposed mechanism of ventricular tachyarrhythmias in Brugada syndrome [8].

While this speculation implies that M1766L causes a mixed phenotype (LQT3 plus Brugada syndrome and/or conduction disease), clear evidence for this contention is lacking, as discussed. Still, the notion of a mixed phenotype arising from a single Na+ channel mutation with different, partly opposing, gating defects linked to distinct channel domains does merit consideration. The phenomenon of overlapping clinical manifestations of cardiac sodium channelopathy between LQT3, Brugada syndrome, and conduction disease has emerged consistently in the last 3 years. Some conduction abnormalities can be observed in patients with Brugada syndrome [14]. A kindred was described in whom conduction disease or Brugada syndrome segregated with the SCN5A mutant S1710L in different family branches [15]. Overlap is not restricted to the combination of Brugada syndrome with conduction disease. We have described features of LQT3 and Brugada syndrome in a family with the 1795insD mutation [16–18]. Moreover, it was found that flecainide may induce Brugada syndrome associated ECG changes (ST segment elevation) in LQT3 patients [19]. The similarity of the clinical response to flecainide in these apparently opposite syndromes may be explained by flecainide's action on particular gating changes shared by mutants in both syndromes [20].

Alternatively, if one surmises that M1766L lacks features of conduction disease or Brugada syndrome, one mechanism to explain the apparent discrepancy between the clinical phenotype of this patient and the biophysical changes of his mutant Na+ channel is the presence of a second, modifying, mutation. This mechanism has been proposed to explain variable expression of gene defects in general, but requires validation, certainly in the present study. It seems that the investigators did not analyse all coding exons of SCN5A. Thus, the occurrence of a second SCN5A mutation with a low penetrance (both parents were asymptomatic and had normal ECGs) in this boy cannot be excluded. The same applies to the possible occurrence of such a mutation in other (LQT syndrome associated) genes. This is particularly relevant since of the three genotyped cases of infants presenting with QT prolongation and 2:1 AV block, two were homozygous for mutations in KCNH2 [21,22], and the third was homozygous for mutations in SCN5A [23]. An additional mutation that resulted in QT prolongation (of sufficient magnitude to overcome the severe ‘loss of function’ due to reduced Na+ current density) could contribute to building a straighter bridge between the clinical manifestations of this mutant and its biophysical characterization using voltage-clamp studies.


   2. Augmentation of M1766L current by mexiletine
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 1. Gain-of-function and loss-of...
 2. Augmentation of M1766L...
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The most exciting finding in this paper is the (partial) rescue of INa of the mutant M1766L channel by incubation at lower temperature or with mexiletine. Why?

It is becoming increasingly evident that failure of proper trafficking of the Na+ channel protein to the plasma membrane contributes at least in part to the pathomechanism in Brugada syndrome [6,7] and maybe also conduction disease [9]. Baroudi and co-workers [6,7] have demonstrated accumulation of the Brugada syndrome mutant R1432G and the double mutant R1232W/T1620M within the endoplasmic reticulum (ER). An error in protein folding and failure of the protein product to reach its proper locale within the cell has been increasingly recognised as a mechanism for several genetically inherited diseases [24,25], including channelopathies such as cystic fibrosis, episodic ataxia, nephrogenic kidney disease, as well as types 2 [26] and 5 [27] of the LQT syndrome.

The ER is the first membrane compartment for the synthesis and processing of membrane (and secretory) proteins. When errors in folding of polypeptides occur, quality control mechanisms within the ER ensure that the misfolded polypeptides get recognised and degraded. Oftentimes the mutation is relatively minor and those nascent molecules that escape the ER quality control mechanisms and reach their proper cellular localisation retain some function [11,28,29]. Consequently, the identification of strategies to correct diseases of protein folding is presently at the forefront of clinical research [24,25]. Trafficking-deficient mutants have been rescued by lowering the incubation temperature or by using ‘chemical chaperones’ such as glycerol and dimethylsulfoxide. These strategies are believed to function by stabilizing conformations that escape degradation by the ER. The former is only applicable in vitro while the latter is not clinically applicable since concentrations that would be needed to attain the desired effect are too high to be achieved in vivo and are unsafe. More recently, it has been demonstrated that substrates and blockers that bind to the target protein can achieve similar stabilizing effects to rescue such mutant proteins [30–33]. The selectivity that can be achieved using such ‘pharmacological chaperones’ (ligand-mediated) makes it more likely that such a strategy could ultimately become clinically relevant.

Zhou et al. [30] have demonstrated that the HERG channel (protein product of KCNH2) blockers E-4031, astemizole and cisapride can function as ‘pharmacological chaperones’ to improve membrane expression of the trafficking-deficient HERG mutant N470D. More recently, Ficker et al. [31] demonstrated that defective trafficking of another HERG mutant, G601S, was also rescued by a series of HERG channel blockers. Can defective membrane trafficking of SCN5A mutants in Brugada syndrome or conduction disease be rescued by such a strategy? The findings by Valdivia et al. [11] of increased INa following incubation with mexiletine provide circumstantial evidence that this could be possible. However, before immunohistochemical experiments demonstrate that the M1766L protein is trafficking-deficient and that incubation with mexiletine actually increases membrane expression, one can only speculate that this is the case. Nevertheless, should rescue be possible, such a strategy is still far from clinical applicability, as is also the case for rescue by channel blockers in the case of trafficking-deficient HERG mutants. Firstly, pharmacological chaperones that achieve channel rescue without block need to be designed. Secondly, should this problem be overcome, one needs to consider the fact that at least in some cases, rescued mutant channels might display abnormal gating behaviour which could still be arrhythmogenic. Thirdly, rescue could be domain-specific or even mutation-specific, which would necessitate design of mutant-specific pharmacological chaperones. This would be an important factor to be reckoned with when one considers that mutations causing cardiac ion channelopathies are often private mutations, i.e., different mutations are responsible for the disorder in different families.

If the rescue of mutant Na+ channels as reported by Valdivia et al. [11] is confirmed in other mutants, this study may prove to be a seminal contribution towards new therapeutic modalities for that subset of sodium channelopathies associated with trafficking defects.


   References
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 2. Augmentation of M1766L...
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