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Cardiovascular Research 2005 67(3):459-466; doi:10.1016/j.cardiores.2005.01.017
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Copyright © 2005, European Society of Cardiology

Substitution of a conserved alanine in the domain IIIS4–S5 linker of the cardiac sodium channel causes long QT syndrome

Jeroen P.P. Smitsa, Marieke W. Veldkampa, Connie R. Bezzinaa, Zahir A. Bhuiyana,b, Horst Wedekindc,d, Eric Schulze-Bahrc,d and Arthur A.M. Wildea,*

aExperimental and Molecular Cardiology Group, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
bDepartment of Clinical Genetics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
cDepartment of Cardiology and Angiology, University of Münster, Münster, Germany
dDepartment of Molecular Cardiology, Institute for Arteriosclerosis Research of the University of Münster, Münster, Germany

* Corresponding author. Department of Clinical and Experimental Cardiology, Academic Medical Center, University of Amsterdam, Room M-0-107, Meibergdreef 9, 1105 AZ Amsterdam, PO Box 22700, 1100 DE Amsterdam, The Netherlands. Tel.: +31 20 5663265; fax: +31 20 6975458. Email address: a.a.wilde{at}amc.uva.nl

Received 16 March 2004; revised 29 December 2004; accepted 7 January 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Summary
 References
 
Objective: Congenital long QT syndrome type 3 (LQT3) is an inherited cardiac arrhythmia disorder due to mutations in the cardiac sodium channel gene, SCN5A. Although most LQT3 mutations cause a persistent sodium current, increasing diversity in the disease mechanism is shown. Here we present the electrophysiological properties of the A1330T sodium channel mutation (DIIIS4–S5 linker). Like the A1330P, LQT3 mutation, A1330T, causes LQT3 in the absence of a persistent current.

Methods: A1330T, A1330P and wild-type sodium channels were expressed in HEK-293 cells and characterized using the whole-cell configuration of the patch-clamp technique.

Results: The A1330T mutation shifts positively the voltage-dependence of inactivation and speeds recovery from inactivation. Measurements of sodium window (INa, window) currents revealed a positive shift of the INa, window voltage range for both 1330 mutants, with in addition an increase in INa, window magnitude for the A1330P mutant. Action potential (AP) clamp experiments revealed that these changes in INa, window properties cause an increased inward current during the initial part of phase 4 repolarization of the AP.

Conclusions: Our findings indicate that the alanine at position 1330 in the DIIIS4–S5 linker of the cardiac sodium channel has a role in channel fast inactivation. Substitution by a threonine shifts the voltage range of INa, window activity to more positive potentials. Here the counter-acting effect of outward K+ current is reduced and may delay AP repolarization, explaining the LQT3 phenotype.

KEYWORDS Long QT syndrome; Ion channel; Action potential


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 5. Summary
 References
 
The congenital long QT syndrome is a cardiac rhythm disorder that is caused by mutations in specific ion channel genes [1,2] or by a mutation encoding a cytoskeletal protein, ankyrin B [3]. This syndrome is clinically characterized by prolonged QT-intervals on the body surface ECG. Patients are at risk for syncope and sudden cardiac death due to the development of life-threatening ventricular arrhythmias such as torsades de pointes [2]. The prolongation of the QT-interval reflects an increased action potential (AP) duration, which results from an increase in net inward current. LQT variant type 3 (LQT3) arises from mutations in the SCN5A gene encoding the {alpha}-subunit of the cardiac sodium channel. The majority of these mutations produce a persistent inward sodium current (Ipst) by affecting the inactivation properties [1]. The presence of Ipst in the voltage range of the AP plateau underlies delayed repolarization and prolongation of AP duration. However, LQT3 sodium channel mutations are increasingly being identified that lack Ipst and produce QT-interval prolongation through alternative mechanisms. Presently, three LQT3 mutations have been identified that lack Ipst: E1295K [4], A1330P [5] and I1768V [6,7]. For the D1790G sodium channel mutation, the absence of Ipst is debated [8]. These findings demonstrate the existence of mutation-specific consequences on sodium channel properties and the consequent heterogeneity in the LQT3 disease mechanism.

In this study, we present a novel sodium channel mutation (A1330T), which we identified in a family with LQT3 phenotype. In this mutation, a highly conserved alanine in the intracellular DIIIS4–S5 linker of the human cardiac sodium channel is substituted by a threonine (Fig. 1). We have previously described the occurrence and biophysical characteristics of a de novo mutation at this same position (A1330P) [5]. Interestingly, like the A1330P mutation, A1330T lacks persistent inward sodium current. Using the patch-clamp technique, we characterized the electrophysiological properties of the A1330T mutation to get insight into the mechanism underlying QT-prolongation in carriers of this mutation.


Figure 1
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  		Fig. 1 Schematic drawing of the {alpha}-subunit of the cardiac sodium channel depicting the position of the A1330T amino acid substitution in the intracellular S4–S5 linker of domain III. Below, the amino acid composition of the DIIIS4–S5 linker from different voltage-gated sodium channel isoforms and species emphasizing the conserved nature of the linker, including the alanine.

 

   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 5. Summary
 References
 
2.1. Genetic analysis
Genomic DNA was isolated from peripheral lymphocytes following standard procedures. Genetic studies were performed in concordance with the recommendations of the Medical Ethics Committee of the AMC and with the consent of the parents and family members involved. Our study and all experiments conform to the Declaration of Helsinki. The coding region of the SCN5A gene was amplified by PCR using previously published primers [9] and screened for the presence of mutations by single-strand conformation polymorphism (SSCP) analysis.

2.2. Site-directed mutagenesis
Mutant sodium channel cDNA was prepared by mutagenesis on the pSP64T-hH1 plasmid [10]. Primers used for the site-directed mutagenesis were 5'-CAATGCCCTGGTGGGCACCATCCCGTCCATC-3' and 5'-CATGATGGACGGGATGGTGCCCACCAGGGCA-3'. An AccI–KpnI fragment was subcloned into wild-type pSP64T-hH1 and the mutant insert and ligation regions were completely analyzed by sequencing. The A1330T cDNA was then subcloned into the HindIII–XbaI sites of the expression vector pCGI (kindly provided by David Johns and Eduardo Marbán, Johns Hopkins University, Baltimore, MD) for bicistronic expression of the channel protein and GFP reporter in a Human Embryonic Kidney cell line (HEK-293). The pCGI A1330P construct was produced as described previously [11,12].

2.3. Heterologous expression of mutant and wild-type sodium channels
To express mutant A1330T and wild-type cardiac sodium channels (hH1), HEK-293 cells were cotransfected with 2 µg of sodium channel {alpha}-subunit cDNA (WT or mutant, respectively) and 2 µg hβ1-subunit cDNA using lipofectamine (Gibco BRL, Life Technologies, Scotland). For the experiments to determine sodium window current or persistent inward current, HEK-293 cells were co-transfected with 4 µg of sodium channel {alpha}-subunit cDNA (WT, A1330P or A1330T mutant, respectively) and 4 µg hβ1-subunit cDNA. Transfected cells were cultured in minimum essential medium (MEM) (Earles salts and L-glutamine) supplemented with non-essential amino acid solution, fetal bovine serum (10%), penicillin (100 IU/ml) and streptomycin (100 µg/ml) for 1 to 2 days in a 5% CO2 incubator at 37 °C. Only cells exhibiting green fluorescence were selected for electrophysiological experiments.

2.4. Electrophysiology
Sodium currents were measured in the whole-cell configuration of the patch-clamp technique using an Axopatch 200B amplifier (Axon Instruments) and the following solutions (mmol/l). Bath (external) solution: NaCl 140, KCl 4.7, CaCl2 1.8, MgCl2 2.0, NaHCO3 4.3, Na2HPO4 1.4, glucose 11.0, HEPES 16.8, pH adjusted to 7.4 (NaOH). Pipette (internal) solution: CsF 100, CsCl 40, EGTA 10, NaCl 10, MgCl2 1.2, HEPES 10, pH adjusted to 7.3 (NaOH 22.5). Electrophysiological experiments were carried out at a room temperature of 21 °C. Patch electrodes were pulled from borosilicate glass and had a tip resistance of 2–3 M{Omega} when filled with pipette solution. Series resistance was compensated to values of 80%. Whole-cell peak sodium currents were low-pass filtered at 5 kHz and digitized at 30 kHz. Sodium window currents were filtered at 1 kHz.

2.5. Voltage protocols and data analysis
Voltage protocols for voltage-dependence of activation/inactivation and recovery from inactivation are provided as insets in the relevant figures. The pulse protocol cycle time was 5 s for all three protocols. The steady-state activation and inactivation curves were fitted using the Boltzmann equation: I/Imax=A/{1.0+exp[(V1/2–V)/k]} to determine the membrane potential for the half maximal (in)activation (V1/2) and the slope factor k. The time course of inactivation was determined by fitting current decay with a two-exponential function: I/Imax=Afexp(–t/{tau}f)+Asexp(–t/{tau}s), where Af and As are fractions of fast and slow inactivating components and {tau}f and {tau}s are the time constants of the fast and slow inactivating components, respectively.

The time constant ({tau}) of recovery from inactivation was obtained by fitting the data with the function I/Imax=A[1–exp(–t/{tau})] where t is the recovery time interval.

The presence of a persistent inward sodium current was determined from the tetrodotoxin (TTX)-sensitive current (30 µM, Alomone Labs, Israel) by taking the average current amplitude during the last 50 ms of a 300 ms depolarization to –20 mV, relative to the holding current at –120 mV. Sodium window currents were measured as TTX-sensitive currents during a depolarizing ramp protocol from –120 mV to +50 mV at a rate of 0.1 mV/ms. The results are expressed as mean ± S.E.M. and statistical comparisons were made using an unpaired Student's t-test with P<0.05 indicating statistical significance.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 5. Summary
 References
 
3.1. Clinical characteristics and genetic analysis
The index patient (Fig. 2, subject III-2) was a 14-year-old girl who died suddenly at rest. Investigation of the ECG obtained during her stay in the intensive care unit revealed a prolonged QT-interval. Her previous medical history was unremarkable. Clinical examination of the parents, brother and two sisters (Fig. 2) by standard and 24-h holter ECG recording revealed a prolonged QT-interval in all the siblings. The clinical history of all family members was inconspicuous and they were all asymptomatic. Echocardiographic investigations revealed no structural cardiac abnormalities.


Figure 2
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  		Fig. 2 Pedigree of the A1330T family. Solid symbols represent affected individuals, ECG lead V5 of the index patient and her three siblings are shown. Note that the QTc-intervals in all siblings are prolonged.

 
With SSCP analysis of SCN5A on DNA from the proband, an aberrant conformer in exon 23 was identified. Sequence analysis revealed the presence of a g>a substitution (gcc>acc) leading to the replacement of the hydrophobic, non-polar residue alanine at position 1330 by the hydrophilic, polar residue threonine (A1330T). This nucleotide change could not be identified in 100 control individuals. The mother, the brother and the two sisters of the index patient were also heterozygous for this mutation (Fig. 2).

3.2. Functional properties of the A1330T sodium channel mutation
To determine the functional consequences of the A1330T mutation on activation and inactivation properties, wild-type (WT) and mutant sodium channels were expressed in HEK-293 cells. Because the majority of LQT3 sodium channel mutations prolong the action potential by inducing a persistent inward current, we first investigated whether A1330T mutant channels also exhibit this property. TTX-sensitive currents recorded during prolonged depolarizations (Fig. 3, inset) show that A1330T sodium channels lack such a Ipst. The amplitude of Ipst as a percentage of peak inward current during the last 50 ms of the 300 ms depolarization was 0.10 ± 0.14 % (n = 5) for A1330T mutant channels and 0.010 ± 0.02% (n = 4, ns) in wild-type controls.


Figure 3
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  		Fig. 3 WT (A) and A1330P (B) sodium currents recorded during 300 ms depolarizations to –20 mV from –120 mV in the absence and presence of 30 µM TTX. TTX-sensitive currents obtained by subtraction are shown in insets. No significant differences in TTX-sensitive current amplitude were found between WT and A1330T sodium channels. For values see text.

 
Comparison of wild-type and A1330T mutant peak sodium currents recorded during depolarizations (Fig. 4A), and the corresponding current–voltage (I–V) relationships (Fig. 4B), reveals no apparent difference in peak current amplitudes over the whole voltage-range tested. The average amplitude of the maximum peak sodium current was 3.5 ± 0.8 nA (n = 9) for WT and 4.5 ± 0.6 nA (n = 15, ns) for A1330T mutant channels (Fig. 4B). Quantification of the time course of inactivation by fitting current decay with a bi-exponential function confirmed that time constants of fast and slow inactivation were not significantly different for WT and mutant sodium channels (Fig. 4C).


Figure 4
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  		Fig. 4 (A) Whole cell sodium currents recorded from HEK-293 cells expressing WT (upper panel) and the A1330T (lower panel) mutant sodium channels. Sodium currents were recorded during depolarizing steps (5 mV increments) from a holding potential of –120 mV. (B) Average current–voltage (I–V) relationship for WT and A1330T channels. (C) Fast and slow time constants of current decay as a function of membrane potential. There was no significant differences between WT and the A1330T mutant channels.

 
The recovery from inactivation was investigated using a double pulse protocol. Fig. 5 shows the normalized peak currents as a function of the recovery time interval. Measurement of the time course of recovery from inactivation revealed that A1330T mutant channels recovered significantly faster from inactivation than WT channels. The time constant ({tau}) of recovery was 9.5 ± 0.84 ms (n = 14) for A1330T mutant channels compared to 13.4 ± 0.13 ms (n = 9, P<0.005), for WT channels.


Figure 5
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  		Fig. 5 Recovery from inactivation for WT and A1330P sodium channels. Relative current is plotted as a function of the recovery time interval. See text for values of time constants of inactivation.

 
Next we studied the effect of the A1330T mutation on the voltage-dependence of activation and steady-state inactivation. Fig. 6A shows that the A1330T mutation causes a positive shift of the inactivation curve (+6.9 mV), while the activation curve is unaffected. The half-maximal voltage (V1/2) of activation was –43.7 ± 3.0 mV (n = 9) and –41.3 ± 2.0 mV (n = 15, ns) for WT and A1330T mutant channels, respectively. The V1/2 of steady-state inactivation was –98.8 ± 2.1 mV (n = 13) and –91.9 ± 1.2 mV (n = 13, P<0.005) for WT and A1330T channels, respectively. The slope factors (k) of voltage-dependence of activation and inactivation were similar for wild-type and A1330T mutant channels (activation: WT, k = 6.7 ± 0.6 (n = 9), A1330T, k = 6.8 ± 0.5 (n = 15, ns); inactivation: WT, k = –5.0 ± 0.2 (n = 13), A1330T, k = –5.1 ± 0.2 (n = 13, ns)).


Figure 6
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  		Fig. 6 (A) Voltage-dependent properties of activation and steady-state inactivation for WT and A1330T mutant sodium channels. Voltage clamp protocols are shown as insets. See text for values of potential of half-maximal (in)activation (V1/2) and slope factor (k). (B) Enlarged reproduction of the lower part of Fig. 6A, depicting the overlap of activation and inactivation curves for WT and A1330P mutant sodium channels.

 
The positive shift of the inactivation curve observed in the A1330T mutant channels is expected to cause a positive shift of the voltage range of the INa window current, where activation and inactivation curves overlap. This can be appreciated from Fig. 6B, where the overlap of (in)activation curves is depicted on an enlarged scale. To test this hypothesis, sodium window currents were measured using a ramp protocol. In addition to the A1330T mutation, we also studied the window current of another LQT3 sodium channel mutation, A1330P, which we previously characterized [5]. Like the A1330T mutation, A1330P also lacks Ipst, shifts positively the voltage-dependence of steady-state inactivation and hastens recovery from inactivation. In addition, unlike the A1330T, it also shows a slowed time course of inactivation. Fig. 7 shows that a gradual depolarization from –120 mV to +20 mV induces a small inward sodium window current between –80 and –40 mV (maximum amplitude at about –65 mV) for WT sodium channels. For A1330T sodium channels, the voltage range of the sodium window current was shifted towards more positive potentials (maximum amplitude at about –50 mV), while its amplitude was unaffected. The largest alterations in sodium window current were observed for the A1330P mutation. The positive shift in voltage-dependence of inactivation alone gave rise to a broadening of the voltage range of sodium window current activity (maximum amplitude at about –58 mV) and resulted in a threefold increase in its amplitude.


Figure 7
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  		Fig. 7 Averaged WT (n = 4), A1330T (n = 4) and A1330P (n = 5) sodium window currents measured as TTX-sensitive currents during a slow ramp protocol as shown in inset.

 
To get insight into the consequences of these changes in sodium window current for the action potential, we performed action potential clamp experiments with the A1330P mutant sodium channel. A run of epicardial ventricular action potentials (0.5 Hz), obtained from the Priebe-Beuckelman human ventricular AP model [14], was applied as voltage clamp command potential to HEK-293 cells expressing either wild-type or A1330P sodium channels. This command potential (Fig. 8A) was applied from a holding potential of –120 mV. Fig. 8B shows typical recordings of wild-type and A1330P TTX-sensitive currents, expressed as the percentage of the maximum peak current. Initially, during the upstroke of the action potential, there is a large fast inward sodium peak current which rapidly declines (peak currents are off-scale). Subsequently, during phase 1 repolarization of the action potential, due to the combination of an increase in driving force for sodium ions and the fact that inactivation is not fully completed yet, a second small inward peak current is observed. This second small peak is larger for A1330P than for WT sodium channels, probably because of the slower inactivation time course of the former. Finally, during the initial phase of phase 4 repolarization, there is a slow transient increase in current for A1330P sodium channels which is absent for WT sodium channels. This A1330P slow transient inward current reaches a maximum amplitude around –20 to –30 mV, the voltage range where inward rectification of the inward rectifier potassium current is prominent and thus the counter-acting effect repolarizing outward current reduced.


Figure 8
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  		Fig. 8 Action potential clamp recordings of TTX-sensitive currents from HEK-293 cells expressing wild-type or A1330P mutant sodium channels. (A) Human ventricular action potential used as command potential in voltage clamp mode. (B) Averaged wild-type sodium current (n = 4, thin black line) and mutant sodium current (n = 4, gray thick line).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
5. Summary
 References
 
4.1. Mechanism of action potential prolongation of the A1330T and A1330P sodium channel mutations
In this study, we have investigated the functional consequences of a novel sodium channel mutation, A1330T, identified in an LQT family. The main findings are that the A1330T mutation induces a positive shift in the voltage-dependence of steady-state inactivation and accelerates recovery from inactivation. In contrast to the majority of LQT sodium channel mutations, A1330T did not display a persistent inward current. Similar biophysical properties were previously established for the A1330P mutation, which in addition displayed a slowed inactivation [5]. It was further demonstrated that the positive shift of the inactivation curve resulted in a shift of the voltage range of the sodium window current towards more positive potentials (for A1330T and A1330P) and also in an increase in the window current amplitude (for A1330P). The latter probably resulting from a slowed inactivation time course. During the action potential, this resulted in an increased sodium inward current during the initial part of phase 4 repolarization. Thus, the activity of the sodium window current will be shifted to the voltage range where the rectifying properties of the inward rectifier potassium currents are more pronounced and the counterbalancing effect of outward potassium current is reduced [13]. The resultant increase in net inward current in this phase of the action potential is thus likely to cause a delay in repolarization [4]. Thus far, three other LQT3 sodium channel mutations have been identified that cause QT-prolongation through mechanisms other than persistent inward current. The E1295K (DIIIS4) [4] and the I1768V (DIVS6) [6,7] SCN5A mutations have both been shown to shift the voltage range of the window current towards more positive potentials and to recover faster from inactivation. The I1768V has additionally been shown to have less slow inactivation [6]. Also the A1330P (DIIIS4-S5) ([5] and this this study) displays similar characteristics. These data suggest that action potential prolongation due to changes in sodium window current is at present not a rare mechanism in LQT3.

4.2. The alanine at position 1330 in DIIIS4–S5 and its role in sodium channel fast inactivation
The fact that both the A1330T and A1330P mutations affect the inactivation properties of the cardiac sodium channel in a similar way, i.e. positive shift of voltage-dependence of inactivation and faster recovery from inactivation, suggests that alanine at this position has an important role in sodium channel fast inactivation.

There is ample evidence that the amino-acid sequence of the DIIIS4–S5 linker, including the alanine, is highly conserved and forms part of the docking site for the inactivation particle (DIII–DIV linker) in voltage-gated sodium channels in several tissues and species [14]. For example, the A1156T mutation in the DIIIS4–S5 linker of the SCN4A gene, encoding the human skeletal muscle sodium channel, affects channel inactivation properties in a way similar to the A1330T mutation in the SCN5A gene [15]. That is, it shifts the V1/2 of inactivation positive, slows the inactivation process and makes channels recover faster from inactivation. The latter mutation was found to be causally involved in the muscle disease Paramyotonia Congenita, an autosomal dominant inherited disease characterized by muscle stiffness or myotonia due to sarcolemmal hyperexcitability [15]. Further evidence for the role of the DIIIS4–S5 linker in fast inactivation comes from site-directed mutagenesis studies involving several voltage-gated sodium channel isoforms. These experiments demonstrate that fast inactivation requires hydrophobic interactions between the proposed sodium channel inactivation particle, the DIII–DIV linker, and the proposed docking site, the DIIIS4–S5 linker [14–21]. These hydrophobic interactions require the presence of the three hydrophobic amino acids isoleucine, phenylalanine and methionine (IFM) in DIII–DIV, and an alanine in the DIIIS4–S5 linker [18]. Miyamoto et al. [21] showed that the alanine at position 1329 in DIIIS4–S5 of the human brain sodium channel, equivalent to position 1326 in human cardiac sodium channel, directly interacts with the phenylalanine (F1489) in the IFM motive of the proposed inactivation particle [21]. The presence of an amino acid with a small side chain like the alanine at position 1325, 4 amino acids before, and the alanine at position 1333 (equivalent to A1330 in the cardiac sodium channel), 4 amino acids after A1329, are thought to be essential to enable interaction between A1329 (A1326 in the cardiac sodium channel) and the bulky IFM motive [21]. When the DIII–S4 segment, one of four voltage sensors present in sodium channels, moves outward upon depolarization, interacting {alpha}-helixes are formed by the two DIIIS4–S5 linkers. This enables interaction between the IFM motive and the alanine at position 1329 [21]. Also, the alanine at position 1329 in the DIIIS4–S5 of the {alpha}-subunit of the rat brain sodium channel was found to have a similar role [18].

Our finding that the substitution of the alanine at position 1330 in hH1, by either the hydrophilic, polar residue threonine or even by the hydrophobic, non-polar residue proline affects the inactivation process of hH1 provides additional evidence for the important role of the DIIIS4–S5 linker in voltage-gated sodium channels isoforms [5,15,17,18,21].

Considering the proposed role of the DIIIS4–S5 linker in channel fast inactivation, it may be surprising that both the A1330T and the A1330P mutant channels were found to inactivate completely and to lack an Ipst. It may be speculated that neither substitution disturbs the hydrophobic interaction to such an extent that complete inactivation is hindered. An explanation could be that neither substitution disturbs the hydrophobic interaction of the DIIIS4–S5 linker with the DIII–DIV linker enough to prevent complete inactivation of the channels, but only slows it and alters its voltage-dependence. Proline is a large, neutral cyclic amino acid that replaces the smaller neutral amino acid alanine in the A1330P mutation. This replacement may hinder the hydrophobic interactions between the DIII–IV linker by the difference in size and probably the substitution will affect protein structure. Although this hindrance delays inactivation and destabilizes it, it does not prevent complete fast inactivation [5]. Threonine, with one extra carbon atom and a hydroxyl group, may affect interaction with LIII–IV because it is more hydrophilic than alanine, thereby affecting the normal hydrophobic interaction.

Theoretically, a structural change in the DIIIS4–S5 linker could affect the movement of the nearby voltage sensor, segment 4 of domain III (S4 DIII) [20]. This may explain the effects on the DIIIS4–S5 linker and the shift in voltage-dependence of inactivation in both mutations. Because alanine, proline and threonine are electrically neutral amino acids, an additional electrostatic interaction with the nearby voltage sensor is unlikely.


   5. Summary
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Summary
References
 
By characterization of the biophysical characteristics of the A1330T and A1330P sodium channel mutations, we have gained insight into the mechanisms underlying AP prolongation and arrhythmogenesis in carriers of these mutations. In addition, the obtained results have provided information on the structure–function relationship in hH1 and confirm the importance of the DIIIS4–S5 linker in channel fast inactivation in voltage-gated sodium channels. Although both missense mutations at position 1330 showed overlap in the effects on channel gating, differences were also present. This illustrates that even closely related amino acids (alanine and threonine) may differently affect channel gating. Therefore, one might speculate that future therapies should not only be gene-specific, but also mutation-specific. Finally, the fact that two different missense mutations were identified at the same position 1330 may point to a mutation hotspot.


   Acknowledgement
 
This work was supported by grants from the Netherlands Organisation for Scientific Research, NWO grant no. 902-16-193 (J.P.P.S., M.W.V. and A.A.M.W.), the Netherlands Heart Foundation, NHS grant 2000.059 (C.R.B., Z.A.B. and A.A.M.W.), Fondation Leducq, Paris, France DFG Schu1082/2-2 and 1982/3-1, Bonn, Germany (E.S.-B. and H.W.).


   Notes
 
Time for primary review 21 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Summary
 References
 

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