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Cardiovascular Research Advance Access originally published online on December 7, 2007
Cardiovascular Research 2008 77(4):740-748; doi:10.1093/cvr/cvm096
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2007. For permissions please email: journals.permissions@oxfordjournals.org

Combination of cardiac conduction disease and long QT syndrome caused by mutation T1620K in the cardiac sodium channel

Ralf Surber1,{dagger}, Sabine Hensellek2,{dagger}, Dirk Prochnau1, Gerald S. Werner1, Klaus Benndorf2, Hans R. Figulla1 and Thomas Zimmer2,*

1 Department of Internal Medicine I, Friedrich Schiller University Jena, 07740 Jena, Germany
2 Institute of Physiology II, Friedrich Schiller University Jena, Kollegiengasse 9, 07743 Jena, Germany

* Corresponding author. Tel: +49 3641 934372; fax: +49 3641 933202. E-mail address: thomas.zimmer{at}mti.uni-jena.de

Received 16 May 2007; revised 26 November 2007; accepted 30 November 2007

Time for primary review: 21 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
Aims: The aim of the present study was to elucidate the molecular mechanism underlying the concomitant occurrence of cardiac conduction disease and long QT syndrome (LQT3), two SCN5A channelopathies that are explained by loss-of-function and gain-of-function, respectively, in the cardiac Na+ channel.

Methods and results: A Caucasian family with prolonged QT interval, intermittent bundle-branch block, sudden cardiac death, and syncope was investigated. Lidocaine (1 mg/kg i.v.) normalized the prolonged QT interval and rescued bundle-branch block. An SCN5A mutation analysis was performed that revealed a C-to-A mutation at position 4859 (exon 28), predicted to change a highly conserved threonine for a lysine at position 1620. Mutant channels were characterized both in Xenopus oocytes and HEK293 cells. The T1620K mutation remarkably altered the properties of Nav1.5 channels. In particular, the voltage-dependence of the current decay time constants was largely lost. As a consequence, mutant channels inactivated faster than wild-type channels at potentials negative to –30 mV, resulting in less Na+ inward current (loss-of-function), but significantly slower at potentials positive to –30 mV, resulting in an increased Na+ inward current (gain-of-function). Moreover, we found a hyperpolarized shift of steady-state activation and an accelerated recovery from inactivation (gain-of-function). At the same time, channel availability was significantly reduced at the resting membrane potential (loss-of-function).

Conclusion: We conclude that lysine at position 1620 leads to both loss-of-function and gain-of-function properties in hNav1.5 channels, which may consequently cause in the same individuals impaired impulse propagation in the conduction system and prolonged QTc intervals, respectively.

KEYWORDS Conduction block; Ion channels; Long QT syndrome; Na+ channels


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
The human cardiac sodium channel plays a key role for excitability in cardiac myocytes and for impulse propagation through the specific conduction system. Mutations in the SCN5A gene, encoding the major cardiac sodium channel {alpha} subunit (hNav1.5), cause several inherited arrhythmogenic syndromes including long QT syndrome subtype 3 (LQT3), Brugada syndrome, and several cardiac conduction defects. Most of the mutations resulting in LQT3 cause the respective prolongation of the ventricular action potential (AP) by generating an increased persistent Na+ current fraction (gain-of-function).1,2 In Brugada syndrome, which is characterized by an overt or transient ST elevation in the right precordial leads, ventricular fibrillation occurs in the absence of a demonstrable structural heart disease.3,4 All SCN5A mutations in Brugada syndrome result in a loss-of-function of the cardiac sodium channel by several mechanisms.5,6 However, SCN5A gene mutations are found in only 20–30% of patients with Brugada syndrome, suggesting the involvement of other mechanisms.6 Cardiac conduction disease (CCD), the third disease entity associated with mutations in the SCN5A gene, is characterized by sinus pauses, bundle-branch block, or atrioventricular block in the absence of a structural heart disease.7–12 The SCN5A mutations in CCD lead to a reduced sodium current (loss-of-function) by different mechanisms, e.g. by reduction in the current density13 or even complete abolition of the current through mutant channels.9

Some studies suggested a significant overlap in the phenotype of individuals with SCN5A mutations. LQT3 may be associated either with atrioventricular block14–17 or Brugada syndrome.18–20 Both CCD and Brugada syndrome were reported in case of a single missense mutation (E161K),13 whereas another amino acid exchange (G1406R) resulted in either CCD or Brugada syndrome.9 Interestingly, even all three SCN5A-caused disorders were linked to a single amino acid deletion ({Delta}K1500) in a large French family, although this phenotype was not detectable in all family members.8

In the present study, we describe the identification and electrophysiological characterization of a novel SCN5A missense mutation (T1620K) that causes another combination of SCN5A-related cardiac disorders: We observed both prolonged QTc intervals (LQT3) and a pronounced conduction deficiency (bundle-branch block) in the same individuals. Electrophysiological measurements indicated both gain-of-function mechanisms that may underlie prolonged QTc intervals as well as loss-of-function mechanisms that may cause defective impulse propagation in the conduction system.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
2.1 Clinical investigation
All members of the three-generation Caucasian family were investigated, and informed, written consent was obtained from each family member in accordance with local institutional guidelines. Investigations included at least two 12-lead ECG recordings at different times. The QT interval corrected for heart rate (QTc) was measured in limb lead II (or lead I or III if it could not be measured exactly in lead II) with the use of Bazet's formula. QTc intervals were averaged from five consecutive beats of at least two 12-lead ECG recordings at different time points. An echocardiographic study and two 24 h Holter-ECG were performed in each affected individual. Furthermore, lidocaine (1 mg per kg body weight, i.v.) was administered during continuous ECG monitoring. The HV interval was investigated during an electrophysiological study in all genetically affected individuals. Ajmaline (1 mg per kg body weight, i.v.) was administered in two of the three affected individuals to provoke ECG signs of Brugada syndrome.21 Peripheral blood samples for genotype analysis were taken twice at intervals of 6 months for repeated examination.

2.2 SCN5A mutation analysis
Genomic DNA was prepared from peripheral blood lymphocytes using a standard DNA extraction kit (QIAamp, QIAGEN GmbH, Hilden, Germany). All of the 27 SCN5A exons encoding the hNav1.5 channel of patient II-2 were amplified by polymerase chain reaction (PCR) using intronic primers, as suggested by Wang et al.,22 with the exception of exon 15 (forward primer: 5'-CACAGCAAGAGTCAAGAGGCAGGT-3'; reverse primer: 5'-CCCCACCATCCCCCATGCAGT-3'), and exon 25 (forward primer: 5'-GCCTGTCTGATCTCCCTGTGTGA-3'; reverse primer: 5'-CCCTCAATCCCCTGGCACCC-3'). PCR products were subject to DNA sequencing (Sequence Laboratories Göttingen GmbH, Germany). Upon detection of H558R and T1620K in patient II-2, we analysed respective exons 12 and 28 of all other family members.

2.3 Recombinant DNA procedures
Plasmid pSP64T-hH1 coding for human Nav1.5 (hNav1.5 or hH1, accession no. M77235 [GenBank] ) was kindly provided by Dr A.L. George (Vanderbilt University). Mutations at amino acid positions 558 and 1620 were introduced into the background of the hNav1.5 (hH1) clone using the recombinant PCR technique and the following internal primer pairs: 5'-AGAGCCACCGCACATCACTGCTGGTGCCCTGG-3' and 5'-ACCAGCAGTGATGTGCGGTGGCTCTCGCTCTCCCCC-3' (exchange A1673G to obtain H558R), and 5'-TTCTTCTCCCCGAAGCTCTTCCGAGTCATCCGCCT-3' and 5'-CGGATGACTCGGAAGAGCTTCGGGGAGAAGAAGTAC-3' (exchange C4859A to obtain T1620K). The recombinant PCR products were inserted as SdaI/BseJI (H558R) and BstEII/SfiI (T1620K) fragments into the corresponding sites of the hNav1.5 clone. To obtain the hNav1.5 variant containing both exchanges (H558R/T1620K) the SdaI/BseJI fragment of H558R was inserted into the T1620K clone. Constructs coding for T1620R and T1620H were obtained as described earlier, except that triplets AGG (for R) and CAC (for H) were incorporated into the PCR primer sequences. All DNA constructs were checked by restriction analysis and nucleotide exchanges were confirmed by DNA sequencing.

2.4 Expression in Xenopus oocytes
Preparation of Xenopus laevis oocytes, in vitro transcription, and cRNA injection was done as previously described.23 Fluorescence intensities of the cRNA bands were measured with the gel documentation system from Herolab (Wiesloch, Germany) to adjust the cRNA concentration for each variant to ~0.02 µg/ µL. After 3 days incubation at 18°C in Barth medium, the peak current amplitude of the whole-cell Na+ current was between 0.5 to 5.0 µA. Measurements were performed in at least three different batches of oocytes. For the measurements of persistent Na+ currents, we injected undiluted cRNA preparations (~1–2 µg/ µL) in order to increase this small current fraction, which resulted in transient Na+ currents >15 µA in 96 mM external Na+.

2.5 Electrophysiology
Whole-cell Na+ currents were recorded with the two-microelectrode voltage clamp technique as previously described,23 using the following bath solution (in mmol/L): 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 10 Hepes/KOH, pH 7.4. To reduce peak current amplitudes in oocytes that were injected with undiluted cRNA and thus to insure adequate voltage control, the following bath solution was used (in mmol/L): 20 NaCl, 78 KCl, 1.8 CaCl2, 1 MgCl2, 10 Hepes/KOH, pH 7.4. The currents were elicited by 200 ms test potentials from –80 to 0 mV in 5 mV increments, and from 10 to 80 mV in 10 mV increments (holding potential –120 mV, pulsing frequency 1.0 Hz).

The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996) and with the principles outlined in the Declaration of Helsinki.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Supplementary material
 Funding
 References
 
3.1 Clinical characteristics
The family came to our attention after the resuscitation of patient III-1 (Figures 1 and 3A), a 14-year-old boy who experienced ventricular fibrillation during shopping. He had had no syncopal attacks before. The initially depressed left ventricular function after prolonged cardiopulmonal resuscitation recovered completely after 1 week. Abnormalities in the coronary angiogram could not be detected. In contrast, the 12-lead ECG (Figures 1, left) demonstrated intermittent atrial rhythm, alternating incomplete/complete right bundle-branch block and a prolonged QTc interval (535 ms). The HV interval was normal (40 ms) with no evidence of His bundle conduction disturbances during decremental atrial pacing. Lidocaine markedly shortened the QT interval, restored sinus rhythm and eliminated intermittent complete right bundle-branch block (Figure 1, middle). Interestingly, an ajmaline challenge showed a paradoxical shortening of the QTc interval but no ECG signs of Brugada syndrome (Figure 1, right). The patient was treated with an implantable cardioverter defibrillator (ICD) and beta-blockers (bisoprolol, 5 mg per day).


Figure 1
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  		Figure 1 ECG tracings from patient III-1, resuscitated from ventricular fibrillation. The basal ECG (left panel) showing atrial rhythm, intermittent complete right bundle-branch block, and a prolonged QT interval. Acute administration of lidocaine (1 mg per kg body weight, i.v.) (middle panel) restored sinus rhythm, abolished complete right bundle-branch block, and shortened QT interval. An ajmaline challenge (1 mg per kg body weight, i.v.) also significantly shortened the QT interval (right panel), but failed to demonstrate signs of a Brugada syndrome.

 
The father of patient III-1, patient II-2 (Figures 2 and 3A), had recurrent syncope despite medical treatment with the beta-blocker metoprolol (95 mg per day). The last syncopal attack occurred at the third day of antibiotic treatment with moxifloxacin (400 mg per day). The 12-lead ECG showed ‘fatigue’ of the left anterior fascicle, intermittent right bundle-branch block and a slightly prolonged QTc interval. The HV interval was normal. A lidocaine challenge completely reversed conduction abnormalities and normalized also the QTc interval (Figure 2, right). An ICD was implanted because of syncope despite beta-blocker therapy.


Figure 2
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  		Figure 2 ECG tracings from patient II-2, who experienced recurrent syncope. The basal ECG (left and middle panel) showing variable degrees of left anterior fascicular block and intermittent right bundle-branch block, but also a normal conducted beat. A lidocaine challenge reversed conduction abnormalities and normalized the QT interval (right panel).

 


Figure 3
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  		Figure 3 Family pedigree and sequence analysis of exons 12 and 28 of SCN5A. (A) Pedigree of the family and averaged QTc intervals. Open symbols indicate clinically unaffected family members, gray symbols indicate the presence of polymorphism H558R, and black symbols are for patients with LQT syndrome and cardiac conduction disease carrying the mutation T1620K in addition to H558R. Circles indicate female, and squares, male. (B and C). The heterozygous A1673G exchange leading to H558R (exon 12), and mutation C4859A leading to T1620K (exon 28) are indicated by asterisks. (D) Putative membrane topology of the Nav1.5 channel. Position 1620 is located at the beginning of S4 in domain IV. The first positively charged amino acid of this S4 occurs at position 1623.

 
The 18-year-old sister of patient III-1, patient III-2 (Figure 3A), had the ECG signs of LQT syndrome (QTc 485 ms), but was completely asymptomatic. During 24 h Holter-ECG, frequent bundle-branch block was detected. Again, His bundle conduction abnormalities were not found, and Brugada syndrome was excluded by ajmaline administration. Similarly as observed in the case of her father and her brother, lidocaine significantly shortened the QTc interval. A therapy with the beta-blocker propranolol (75 mg per day) was initiated.

The 10-year-old cousin (patient III-3; Figure 3A) had a borderline QTc interval of 465 ms. She was asymptomatic, and acute lidocaine challenge had no significant effect on the QTc interval. Similarly, all other family members were asymptomatic to date. Averaged QTc intervals of all family members are reported in Figure 3A.

In none of the patients, signs of sinus node dysfunction were observed during a 24-h Holter-ECG and in the electrophysiological study. Moreover, acute lidocaine challenge had no obvious effect on heart rate. We noticed only slight alterations of the heart rate in the three patients without any correlation to the T1620K genotype (+4% in patient III-1, ±0% in patient III-2, and –16% in patient II-2).

3.2 Mutation analysis of SCN5A
The prolonged QTc intervals together with the effect of lidocaine on the ECG suggested a SCN5A mutation in, respectively, affected family members. PCR amplification and sequencing of all hNav1.5 encoding exons including exon–intron boundaries of patient II-2 revealed a novel SCN5A mutation in one allele of exon 28 (C4859A), leading to missense mutation T1620K (Figure 3). Additionally, we detected the polymorphism H558R (A1673G in exon 12),24 which occurred frequently in this family and which could not be linked to an abnormal clinical phenotype (gray and black symbols in Figure 3A). However, mutation T1620K occurred specifically in all three patients with prolonged QTc intervals and conduction abnormalities (black symbols in Figure 3A). The pedigree of the family shows that T1620K occurred as a de novo mutation in patient II-2 in the chromosome carrying the H558R exchange (Figure 3A).

As demonstrated in Figure 3D, position 1620 is located at the extracellular side. This is interesting insofar that most other known LQT3 missense mutations affecting Na+ channel function occurred at positions coding either for intracellularly exposed amino acids or for positions within transmembrane segments. In particular, the intracellular Na+ channel linkers are responsible for Na+ channel inactivation and mutations within these regions often cause severe inactivation defects resulting in persistent or late currents, and consequently, in LQT3 syndrome. In the following, we addressed the questions whether there is a similar inactivation deficiency despite the fact that an extracellular amino acid is mutated, and whether the H558R exchange affects the properties of T1620K channels. We introduced the respective mutations in the hNav1.5 sequence (hH1 clone), expressed the wild-type and mutant channels in Xenopus oocytes, and recorded whole-cell Na+ currents with the two-microelectrode voltage clamp technique.

3.3 Electrophysiological characterization of the mutant hNav1.5 channels
Mutant T1620K and H558R/T1620K channels produced a slightly increased persistent current compared with wild-type hNav1.5 only at test potentials between –40 and –20 mV, whereas H558R channels themselves did not generate a larger non-inactivating current component (Figure 4A and B). The significant increase of the sustained Na+ current component in T1620K and H558R/T1620K channels could be due to a shift of steady-state activation to more negative potentials, a voltage-independent increase of channel activity, or a mixture of both. In the case of a relevant contribution of shift of steady-state activation, the ratio of persistent and transient Na+ current would be unchanged, whereas in the case of a voltage-independent increase of channel activity, the persistent current would become relatively larger. To clarify this point, we normalized the persistent current with respect to the transient current for each oocyte. First, we measured the inward current at the end of a 200 ms test pulse that could be blocked by 10 µM TTX in 96 mM external Na+ (Ipersistent). Then, we reduced the extracellular Na+ concentration to 20 mM in order to insure adequate voltage control also for the first few milliseconds of the test pulse and determined the peak current amplitude in the same oocyte (Itransient). As shown in Figure 4C, mutant H558R/T1620K channels were not characterized by a larger ratio of persistent to transient current, compared with wild-type hNav1.5. In control experiments, we also investigated {Delta}KPQ channels, the first known LQT3 mutant,1 and found several-fold larger persistent Na+ currents compared with hNav1.5, T1620K, or H558R/T1620K. We conclude that the threonine/lysine exchange at position 1620 does not result in an increase of the persistent current, and consequently, that alternative mechanisms underlie the LQT phenotype in the patients.


Figure 4
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  		Figure 4 Mutant H558R/T1620K channels did not generate an increased persistent Na+ current fraction. (A) Representative Na+ currents recorded at –20 mV. (B) The total persistent current component was slightly larger in T1620K (not shown) and H558R/T1620K channels, compared with wild-type hNav1.5 at test potentials between –40 and –20 mV (asterisk indicates P < 0.05 for hNav1.5 vs. H558R/T1620K; n = 10 for hNav1.5, n = 13 for H558R/T1620K, and n = 5 for {Delta}KPQ). All currents were determined at the end of 200 ms test pulses in the absence and presence of 10 µM TTX. (C) The ratio of persistent to transient current was not affected by the mutation at position 1620, in contrast to {Delta}KPQ channels that generated several-fold larger persistent currents (n = 14 for hNav1.5, n = 13 for H558R/T1620K, and n = 9 for {Delta}KPQ). The ratio was calculated from the respective current fraction blocked by 10 µM TTX.

 
When investigating the current decay in T1620K and H558R/T1620K channels at the different test potentials, we noticed that the voltage dependence of the inactivation process was severely affected compared with wild-type hNav1.5 (Table 1, Figure 5A–C). Inactivation time constants were not significantly voltage-dependent over the wide voltage range between –35 and –5 mV. As a consequence, T1620K and H558R/T1620K channels inactivated significantly faster than the wild-type channels at potentials negative to –30 mV, resulting in less Na+ inward current (Figure 5A and C), and significantly slower at potentials positive to –30 mV, resulting in more Na+ current (Figure 5B and C). Thus, the mutation at position 1620 resulted in a loss-of-function at less depolarized membrane potentials, but in a gain-of-function at more depolarized potentials.


Figure 5
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  		Figure 5 Electrophysiological properties of wild-type and mutant Nav1.5 channels. (A and B). Na+ current fraction as function of the test pulse duration at –35 and –15 mV, respectively. Representative whole-cell currents are shown in the insets. Currents were normalized with respect to the peak current. (C) Time constants of inactivation as function of voltage. The Na+ current decay was fitted with a monoexponential function. Statistically, the H558R exchange had no effect on both hNav1.5 and T1620K channels (Table 1). (D and E) Steady-state activation and inactivation as function of voltage. (F) Recovery from inactivation. The voltage protocol is shown in the inset. Bars indicate SEM. For individual values and statistical data evaluation see Tables 1 and 2. Peak current densities were similar for all hNav1.5 variants.

 


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  		Table 1 Inactivation properties of wild-type and mutant hNav1.5 channels in Xenopus oocytes

 
When investigating steady-state activation, we observed a significant shift by –5.8 ± 0.96 mV towards hyperpolarized potentials in T1620K vs. hNav1.5 channels (Figure 5D; Tables 2 and 3). A similar shift of the mid-activation potential Vm was observed in H558R/T1620K (–36.5 ± 1.4 vs. –31.5 ± 1.4 mV in hNav1.5; P < 0.05). At the same time, channel availability at potentials more positive to –95 mV was significantly reduced (Figure 5E). This reduction was produced both by a hyperpolarizing shift of the mid-inactivation potential Vh by –4.14 ± 0.4 mV in T1620K vs. hNav1.5 and by an altered slope of the steady-state inactivation curve (Table 2, Figure 5E). When considering recovery from inactivation, we observed an acceleration in both of the mutant channels (Figure 5F): The time constants for both the fast and the slow recovery component ({tau}f and {tau}s) were significantly smaller compared with the respective hNav1.5 data (Table 2).


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  		Table 2 Electrophysiological properties of wild-type and mutant hNav1.5 channels in Xenopus oocytes and HEK293 cells

 


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  		Table 3 Differential effects of the three positively charged amino acids K, R, and H at position 1620 on hNav1.5 kinetics

 
In contrast to the channels mutated at position 1620, inactivation time constants, steady-state activation, steady-state inactivation, and recovery from inactivation were not altered in H558R compared with hNav1.5 channels (Tables 1 and 2), suggesting that the respective polymorphism is not related to the pathophysiological phenotype and that the critical amino acid exchange in the patients Na+ channel occurred at position 1620.

Finally, we expressed wild-type hNav1.5 and mutant H558R/T1620K channels also in HEK293 cells and performed co-expression studies with the accessory β1 subunit in oocytes (see Supplementary Material). These electrophysiological recordings confirmed the partial loss of the voltage dependence of inactivation, the significant hyperpolarizing shifts of steady-state inactivation/activation, and the enhanced recovery from inactivation in mutant H558R/T1620K channels (Table 2 and Supplementary Material).

3.4 The additional charge in T1620K critically affects Na+ channel kinetics
In order to prove whether or not the additional positive charge at position 1620 is indeed responsible for the alteration of hNav1.5 kinetics in T1620K, we also analysed T1620R and T1620H channels expressed in oocytes. Histidine contains an imidazole group that is only partially charged at physiological pH. Consequently, it should cause only a mild effect. In contrast, arginine and lysine provide a strong positive charge under physiological conditions, and both amino acids should therefore cause a similar dramatic alteration of hNv1.5 function.

As expected, we observed distinct effects with the different amino acids (Figure 6). Arginine at position 1620 led to a significant negative shift of steady-state inactivation and activation (–13.48 ± 0.39 and –6.56 ± 0.69 mV, respectively, vs. hNav1.5), and to a loss of the voltage dependence of open-state inactivation (Figure 6; Table 3). In contrast, histidine at position 1620 did not shift steady-state inactivation and activation (Figure 6A and B; Table 3). This amino acid caused only a reduced voltage dependence of channel inactivation (Figure 6C).


Figure 6
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  		Figure 6 Differential effects of lysine, arginine, and histidine at position 1620 on hNav1.5 channel gating. (A) Steady-state activation as function of voltage. (B) Steady-state inactivation as function of voltage. (C) Time constants of inactivation as function of voltage. Bars indicate SEM. For changes relative to hNav1.5 (dotted line) see Table 3.

 
We conclude that the introduction of a strong positive charge at position 1620 rather than unspecific steric effects underlie the altered electrophysiological properties of T1620K channels.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Supplementary material
 Funding
 References
 
The concomitant occurrence of disordered cardiac conduction and prolonged QT intervals is unusual insofar that the two SCN5A-related channelopathies are explained by opposite mechanisms: LQT3 syndrome is generally expected to result from a gain-of-function mutation, whereas loss-of-function is supposed to cause CCD (or Brugada syndrome).25 Following this concept, the Thr/Lys exchange at position 1620 should produce channels characterized by both features. Although a precise understanding of the clinical picture is not yet possible, our electrophysiological data indeed suggest both gain-of-function and loss-of-function properties of mutant T1620K channels that could be of significant importance for the simultaneous presence of LQT3 syndrome and CCD, respectively.

We suggest two gain-of-function phenomena that may contribute in vivo to the LQT3 syndrome. First, we hypothesize that the slowed onset of inactivation at potentials more positive than –30 mV generates an additional Na+ inward current during the early phase of the AP plateau, similarly as reported for other LQT3 mutations.26–28 Such an increase of the net ionic current may modify the shape of the AP and thus the activity of other depolarizing (Ca++) or repolarizing (K+) currents, which could finally prolong AP repolarization. In case of another LQT3 mutant (D1790G) lacking a detectable persistent Na+ current, a computational analysis suggested a Ca++-dependent mechanism as the primary cause for an AP prolongation.29 Second, we suggest that the hyperpolarizing shift of steady-state activation in combination with the increased slope of the steady-state inactivation curve results in an increase in the amplitude and voltage range of the window current. As previously discussed by other authors,26,30,31 such a maintained inward current, caused by an overlap in the activation and inactivation relations, may prolong AP repolarization.

Most of the mutant channels reported to cause CCD are characterized by the absence or by a clear reduction of the peak Na+ current in the heterologous expression system (for an overview see http://www.fsm.it/cardmoc/). In case of T1620K channels, we did not find a respective reduction of the transient current (also see Supplementary Material). Nevertheless, our electrophysiological measurements suggest two alternative loss-of-function mechanisms that would not reverse a long QT phenotype, but that may lead to the conduction disturbances in the patients. First, we observed reduced channel availability at the holding potential which should result in a reduction of the AP upstroke velocity, a primary determinant of conduction velocity. However, the lower availability would be antagonized by facilitated excitability, as suggested from the hyperpolarizing shift of steady-state activation. Therefore, a second and more important loss-of-function mechanism should exist in the conduction system. We speculate that the unusual inactivation properties at less depolarized membrane potentials (below –30 mV) significantly contribute to delayed conduction: An accelerated onset of inactivation at these potentials may significantly reduce the net Na+ inward current leading to a delayed upstroke of an AP in the conduction system. Interestingly, two other LQT3 mutations in the S3/S4 linker (delF1617) and in the S4 of domain IV (R1623Q) also resulted in delayed inactivation of macroscopic currents and in reduced channel availability.27,28,32 However, the reduced voltage dependence of inactivation was much less pronounced compared with T1620K channels, which could explain the absence of CCD in delF1617 and R1623Q carriers.

Threonine at position 1620 is highly conserved among the mammalian voltage-gated Na+ channels, implicating an important role of this amino acid for normal channel gating. It is therefore not surprising that another SCN5A mutation at the same site, T1620M, has been already reported.5 Interestingly, methionine at this position neither leads to LQT3 nor to CCD, but to Brugada syndrome. Thus, amino acid position 1620 is of high criticality for driving hNav1.5 channels into different pathophysiological directions. In the present study, we show that a weak positive charge (histidine) at this position already disturbs the voltage dependency of open-state inactivation. Strongly charged amino acids (arginine, lysine) even lead to a nearly voltage independent open-state inactivation and, moreover, to a shift of steady-state activation and inactivation.

How could a positive charge at position 1620 attenuate the voltage-dependence of the inactivation process? It has been established that the S4 of domain IV (S4-DIV) moves after the other three S4 helices, when the pore already permits Na+ permeation.33,34 This delayed movement is coupled to open-state inactivation, i.e. it precedes the action of the inactivation gate located in the DIII/DIV linker. Thus, S4-DIV plays a unique role in coupling activation to fast inactivation. Assuming that the S3/S4 linker of domain IV including position 1620 is located at the extracellular side and thus outside the electrical field,35 the additional positive charge would capture the S4-DIV in a more outward position at the resting membrane potential. At less depolarized potentials (negative to –30 mV), a less pronounced S4 movement would be sufficient to initiate open-state inactivation in T1620K, compared with wild-type Nav1.5. In contrast to this, S4 flexibility (and thus the resulting gating charge) should be larger in wild-type vs. T1620K channels at stronger membrane depolarization, resulting in facilitated open-state inactivation in wild-type Nav1.5. This hypothesis is strongly supported by studies using neurotoxin receptor site 3 toxins like {alpha}-scorpion and sea-anemone toxins.35 These compounds bind efficiently to the S3/S4 loop of domain IV, thereby reducing S4 flexibility and inhibiting the outward movement of the respective gating charge.35 Thus, site-3 toxins disturb normal activation–inactivation coupling and inhibit conformational changes required for fast inactivation.36 Interestingly, sea anemone toxin ATXII caused a loss of the voltage-dependence of the current decay time constants ({tau}h), a faster recovery from inactivation, and even a larger slope factor for the steady-state inactivation curve in hNav1.5 channels.35,37 All these phenomena appeared also in T1620K channels.

Concerning the lidocaine and ajmaline effect on the ECG of the patients, we have to notice that our present results do not allow to establish a respective genotype–phenotype link. A normalization of QT intervals might be explained by the action of the drugs as blockers of both wild-type and mutant Nav1.5 channels in ventricular cardiomyocytes. At the same time, however, both drugs must exert a specific gain-of-function effect in the conduction system. Future research including drug application to transfected HEK293 cells and computer modelling may provide more insight into this novel effect of the antiarrhythmic drugs lidocaine and ajmaline.


   Supplementary material
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
Funding
 References
 
Supplementary material is available at Cardiovascular Research online.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
References
 
This work was supported by the Friedrich Schiller University of Jena (budget account no. 923409).


   Acknowledgements
 
The authors would like to thank all family members for their participation in this study. We are also grateful to Karin Schoknecht for her excellent contribution to the electrophysiological measurements and to Annett Schmidt, Birgit Tietsch, Sandra Bernhardt, and Andrea Kolchmeier for excellent technical assistance.

Conflict of interest: none declared.


   Notes
 
{dagger} These authors contributed equally to this work. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 

  1. Wang Q, Shen J, Splawski I, Atkinson D, Li Z, Robinson JL, et al. SCN5A mutations associated with an inherited cardiac arrhythmia, long QT syndrome. Cell (1995) 80:805–811.[CrossRef][Web of Science][Medline]
  2. Bennett PB, Yazawa K, Makita N, George AL Jr. Molecular mechanism for an inherited cardiac arrhythmia. Nature (1995) 376:683–685.[CrossRef][Medline]
  3. Brugada J, Brugada P. Further characterization of the syndrome of right bundle branch block, ST segment elevation, and sudden cardiac death. J Cardiovasc Electrophysiol (1997) 8:325–331.[Web of Science][Medline]
  4. Brugada J, Brugada R, Brugada P. Right bundle-branch block and ST-segment elevation in leads V1 through V3: a marker for sudden death in patients without demonstrable structural heart disease. Circulation (1998) 97:457–460.[Abstract/Free Full Text]
  5. Chen Q, Kirsch GE, Zhang D, Brugada R, Brugada J, Brugada P, et al. Genetic basis and molecular mechanism for idiopathic ventricular fibrillation. Nature (1998) 392:293–296.[CrossRef][Medline]
  6. Shimizu W. The Brugada syndrome—an update. Intern Med (2005) 44:1224–1231.[CrossRef][Web of Science][Medline]
  7. Schott JJ, Alshinawi C, Kyndt F, Probst V, Hoorntje TM, Hulsbeek M, et al. Cardiac conduction defects associate with mutations in SCN5A. Nat Genet (1999) 23:20–21.[Web of Science][Medline]
  8. Grant AO, Carboni MP, Neplioueva V, Starmer CF, Memmi M, Napolitano C, et al. Long QT syndrome, Brugada syndrome, and conduction system disease are linked to a single sodium channel mutation. J Clin Invest (2002) 110:1201–1209.[CrossRef][Web of Science][Medline]
  9. Kyndt F, Probst V, Potet F, Demolombe S, Chevallier JC, Baro I, et al. Novel SCN5A mutation leading either to isolated cardiac conduction defect or Brugada syndrome in a large French family. Circulation (2001) 104:3081–3086.[Abstract/Free Full Text]
  10. McNair WP, Ku L, Taylor MR, Fain PR, Dao D, Wolfel E, et al. SCN5A mutation associated with dilated cardiomyopathy, conduction disorder, and arrhythmia. Circulation (2004) 110:2163–2167.[Abstract/Free Full Text]
  11. Tan HL, Bink-Boelkens MT, Bezzina CR, Viswanathan PC, Beaufort-Krol GC, van Tintelen PJ, et al. A sodium-channel mutation causes isolated cardiac conduction disease. Nature (2001) 409:1043–1047.[CrossRef][Medline]
  12. Wang DW, Viswanathan PC, Balser JR, George AL, Benson DW. Clinical, genetic, and biophysical characterization of SCN5A mutations associated with atrioventricular conduction block. Circulation (2002) 105:341–346.[Abstract/Free Full Text]
  13. Smits JP, Koopmann TT, Wilders R, Veldkamp MW, Opthof T, Bhuiyan ZA, et al. A mutation in the human cardiac sodium channel (E161K) contributes to sick sinus syndrome, conduction disease and Brugada syndrome in two families. J Mol Cell Cardiol (2005) 38:969–981.[CrossRef][Web of Science][Medline]
  14. Kehl HG, Haverkamp W, Rellensmann G, Yelbuz TM, Krasemann T, Vogt J, et al. Life-threatening neonatal arrhythmia: successful treatment and confirmation of clinically suspected extreme long QT-syndrome-3. Circulation (2004) 109:e205–e206.[Free Full Text]
  15. Lupoglazoff JM, Cheav T, Baroudi G, Berthet M, Denjoy I, Cauchemez B, et al. Homozygous SCN5A mutation in long-QT syndrome with functional two-to-one atrioventricular block. Circ Res (2001) 89:e16–e21.[CrossRef][Web of Science][Medline]
  16. Valdivia CR, Ackerman MJ, Tester DJ, Wada T, McCormack J, Ye B, et al. A novel SCN5A arrhythmia mutation, M1766L, with expression defect rescued by mexiletine. Cardiovasc Res (2002) 55:279–289.[Abstract/Free Full Text]
  17. Chang CC, Acharfi S, Wu MH, Chiang FT, Wang JK, Sung TC, et al. A novel SCN5A mutation manifests as a malignant form of long QT syndrome with perinatal onset of tachycardia/bradycardia. Cardiovasc Res (2004) 64:268–278.[Abstract/Free Full Text]
  18. Bezzina C, Veldkamp MW, van Den Berg MP, Postma AV, Rook MB, Viersma JW, et al. A single Na+ channel mutation causing both long-QT and Brugada syndromes. Circ Res (1999) 85:1206–1213.[Abstract/Free Full Text]
  19. Veldkamp MW, Viswanathan PC, Bezzina C, Baartscheer A, Wilde AA, Balser JR. Two distinct congenital arrhythmias evoked by a multidysfunctional Na+ channel. Circ Res (2000) 86:e91–e97.[Web of Science][Medline]
  20. Rivolta I, Abriel H, Tateyama M, Liu H, Memmi M, Vardas P, et al. Inherited Brugada and long QT-3 syndrome mutations of a single residue of the cardiac sodium channel confer distinct channel and clinical phenotypes. J Biol Chem (2001) 276:30623–30630.[Abstract/Free Full Text]
  21. Rolf S, Bruns HJ, Wichter T, Kirchhof P, Ribbing M, Wasmer K, et al. The ajmaline challenge in Brugada syndrome: diagnostic impact, safety, and recommended protocol. Eur Heart J (2003) 24:1104–1112.[Abstract/Free Full Text]
  22. Wang Q, Li Z, Shen J, Keating MT. Genomic organization of the human SCN5A gene encoding the cardiac sodium channel. Genomics (1996) 34:9–16.[CrossRef][Web of Science][Medline]
  23. Zimmer T, Biskup C, Bollensdorff C, Benndorf K. The β1 subunit but not the β2 subunit colocalizes with the human heart Na+ channel (hH1) already within the endoplasmic reticulum. J Membr Biol (2002) 186:13–21.[CrossRef][Web of Science][Medline]
  24. Ackerman MJ, Splawski I, Makielski JC, Tester DJ, Will ML, Timothy KW, et al. Spectrum and prevalence of cardiac sodium channel variants among black, white, Asian, and Hispanic individuals: implications for arrhythmogenic susceptibility and Brugada/long QT syndrome genetic testing. Heart Rhythm (2004) 1:600–607.[CrossRef][Web of Science][Medline]
  25. Grant AO. Molecular biology of sodium channels and their role in cardiac arrhythmias. Am J Med (2001) 110:296–305.[CrossRef][Web of Science][Medline]
  26. Wedekind H, Smits JP, Schulze-Bahr E, Arnold R, Veldkamp MW, Bajanowski T, et al. De novo mutation in the SCN5A gene associated with early onset of sudden infant death. Circulation (2001) 104:1158–1164.[Abstract/Free Full Text]
  27. Kambouris NG, Nuss HB, Johns DC, Tomaselli GF, Marban E, Balser JR. Phenotypic characterization of a novel long-QT syndrome mutation (R1623Q) in the cardiac sodium channel. Circulation (1998) 97:640–644.[Abstract/Free Full Text]
  28. Makita N, Shirai N, Nagashima M, Matsuoka R, Yamada Y, Tohse N, et al. A de novo missense mutation of human cardiac Na+ channel exhibiting novel molecular mechanisms of long QT syndrome. FEBS Lett (1998) 423:5–9.[CrossRef][Web of Science][Medline]
  29. Wehrens XH, Abriel H, Cabo C, Benhorin J, Kass RS. Arrhythmogenic mechanism of an LQT-3 mutation of the human heart Na+ channel alpha-subunit: A computational analysis. Circulation (2000) 102:584–590.[Abstract/Free Full Text]
  30. Abriel H, Cabo C, Wehrens XH, Rivolta I, Motoike HK, Memmi M, et al. Novel arrhythmogenic mechanism revealed by a long-QT syndrome mutation in the cardiac Na+ channel. Circ Res (2001) 88:740–745.[Abstract/Free Full Text]
  31. Rivolta I, Clancy CE, Tateyama M, Liu H, Priori SG, Kass RS. A novel SCN5A mutation associated with long QT-3: altered inactivation kinetics and channel dysfunction. Physiol Genomics (2002) 10:191–197.[Abstract/Free Full Text]
  32. Chen T, Inoue M, Sheets MF. Reduced voltage dependence of inactivation in the SCN5A sodium channel mutation delF1617. Am J Physiol Heart Circ Physiol (2005) 288:H2666–H2676.[Abstract/Free Full Text]
  33. Sheets MF, Hanck DA. Voltage-dependent open-state inactivation of cardiac sodium channels: gating current studies with Anthopleurin-A toxin. J Gen Physiol (1995) 106:617–640.[Abstract/Free Full Text]
  34. Chanda B, Bezanilla F. Tracking voltage-dependent conformational changes in skeletal muscle sodium channel during activation. J Gen Physiol (2002) 120:629–645.[Abstract/Free Full Text]
  35. Hanck DA, Sheets MF. Site-3 toxins and cardiac sodium channels. Toxicon (2007) 49:181–193.[Medline]
  36. Rogers JC, Qu Y, Tanada TN, Scheuer T, Catterall WA. Molecular determinants of high affinity binding of alpha-scorpion toxin and sea anemone toxin in the S3-S4 extracellular loop in domain IV of the Na+ channel alpha subunit. J Biol Chem (1996) 271:15950–15962.[Abstract/Free Full Text]
  37. Chahine M, Plante E, Kallen RG. Sea anemone toxin (ATX II) modulation of heart and skeletal muscle sodium channel alpha-subunits expressed in tsA201 cells. J Membr Biol (1996) 152:39–48.[CrossRef][Web of Science][Medline]

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