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Cardiovascular Research 2005 65(1):138-147; doi:10.1016/j.cardiores.2004.09.025
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Copyright © 2004, European Society of Cardiology

Intrinsic mechanism of the enhanced rate-dependent QT shortening in the R1623Q mutant of the LQT3 syndrome

Yasushi Oginosawaa, Toshihisa Nagatomoa,*, Haruhiko Abea, Naomasa Makitab, Jonathan C. Makielskic and Yasuhide Nakashimaa

aSecond Department of Internal Medicine, University of Occupational and Environmental Health Japan, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan
bDepartment of Cardiovascular Medicine, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan
cDepartment of Medicine, Section of Cardiovascular Medicine, University of Wisconsin, Madison, WI 53792, USA

* Corresponding author. Tel.: +81 93 691 7436; fax: +81 93 691 6913. Email address: toshi{at}med.uoeh-u.ac.jp

Received 14 April 2004; revised 23 September 2004; accepted 24 September 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: In the type 3 long QT syndrome (LQT3), arrhythmia events tend to occur at rest or during sleep. One of the mutations, R1623Q, is located in the voltage sensor of the cardiac sodium channel (hH1), and patients with R1623Q mutation have been also reported to show bradycardia-dependent cardiac events. Although the mutant channel has been characterized by inactivation gating defects, the intrinsic mechanism(s) that might explain why arrhythmia attack is most prevalent at slower heart rates has not been investigated.

Methods: cDNA encoding either the wild-type or the R1623Q mutant of hH1 was stably transfected into HEK293 cells. INa was recorded using a whole-cell patch-clamp technique at 23 °C.

Results: A train of 50 depolarizing pulses from holding potentials (–120 and –80 mV) to –20 mV or a train of 50 action potential waveforms was applied at different frequencies. When using a rectangular waveform voltage clamp protocol, rate-dependent reduction of INa was holding voltage-dependent but was not different between peak INa and late INa. However, using the action potential clamp, preferential rate-dependent reduction of the phase 3 INa was obvious as compared with peak INa. The discrepancy in the rate-dependent reduction between protocols was attributed to accelerated recovery from inactivation under non-equilibrium condition.

Conclusion: The rate dependency of phase 3 INa under non-equilibrium gating is a novel mechanism to explain the enhanced rate-dependent QT-shortening in LQT3 patients. Our findings are important for genotype–phenotype correlations in LQT3 mutants as well as for understanding the function of S4 segment of domain IV region in the cardiac Na+ channel.

KEYWORDS Arrhythmia (mechanisms); Long QT syndrome; Na channel; Membrane currents; Repolarization


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Congenital long QT syndrome (LQTS) is an inherited cardiac disorder that causes syncope and sudden death resulting from ventricular tachyarrhythmias. Type 3 of the long QT syndrome (LQT3) is caused by mutations in SCN5A, the gene that encodes the {alpha}-subunit of the human voltage-dependent cardiac Na+ channel (hH1, also called hNav1.5) [1–3]. Functional studies of SCN5A mutants revealed that most of the mutations induced gain of Na+ channel function. At least three mechanisms have been proposed so far to explain the gain of function. (1) Modal gating termed bursting; an alternative gating mode resulting from a common transient defect of inactivation increasing sustained Na+ channel inward current [4–6]. (2) Window current; steady-state Na+ channel reopening due to overlapping of activation and inactivation [7–9]. (3) Channel reopening under non-equilibrium (nonsteady-state) condition; resulting from faster recovery from inactivation at membrane potentials, that facilitates the activation transition [10]. These mechanisms of increased Na+ current (INa) in the SCN5A mutant result in prolongation of the cardiac action potential duration and predispose to the development of arrhythmogenic early afterdepolarization.

Genotype–phenotype relationships are important for the diagnosis and therapeutic strategy in congenital LQT syndrome. Different from LQT1 and LQT2 patients, ventricular tachyarrhythmias and sudden cardiac death in LQT3 patients tend to occur during sleep or at rest when the heart rate is slow [11]. Enhanced QT interval shortening at faster heart rate was observed in LQT3 patients compared with other types of LQT or normal individuals [12]. The biophysical properties related to this clinical finding have been reported in some of the LQT3 mutants [13–18]. Preferential rate-dependent reduction of the late INa caused by its slower recovery from inactivation compared with peak INa may be one of the mechanisms to explain the enhanced QT interval shortening in the {Delta}KPQ mutant (KPQ deletion in the III–IV linker) of LQT3 [16]. However, the cellular and molecular mechanisms for these clinically important features are not fully understood in LQT3 patients.

One missense mutation of SCN5A (R1623Q), in which a charged arginine residue is replaced with a neutral glutamine at an external position of S4 segment of domain IV (DIV-S4), was identified in a Japanese girl [19]. This patient has been also reported to develop cardiac events during sleep and at rest and cardiac pacing combined with sodium channel blocker, mexiletine, effectively prevented the cardiac events [19]. The R1623Q mutant is unique compared with other reported mutations because the site of the mutation is located in the external portion of the voltage sensor of the Na+ channel. Although the mutant channel has been characterized by inactivation gating defects that differ mechanistically from those caused by other LQT3 mutations [20–22], the intrinsic mechanism(s) that explains why arrhythmia attack is most prevalent at slower heart rates has not been investigated.

In the present study, we investigated the biophysical consequences of enhanced rate-dependent QT shortening in the R1623Q mutant by using whole-cell patch clamp techniques. R1623Q mutant channels were characterized by slowing of the time course of inactivation and expanded window current due to less voltage dependence of the inactivation curve. Notably, compared with peak INa, preferential rate-dependent reduction of the late INa during phase 3 of action potential was obvious in the action potential clamp protocol. The mechanism was attributed to the accelerated recovery from inactivation under non-equilibrium condition.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1. Clones and construction of R1623Q mutation
Amino acid substitution of glutamine for arginine-1623 (R1623Q) of human cardiac Na+ channel {alpha}-subunit (hH1) was performed by an overlap extension polymerase chain reaction (PCR) as reported in detail previously [20]. The entire PCR generated region was sequenced completely.

2.2. Cell preparation and transfection
Approximately 5 x 105 cells from a transformed human embryo kidney cell line (HEK293) were seeded on a 60-mm diameter plate with 3 ml of culture medium a day before the transfection. Culture medium was MEM complete medium containing minimum essential medium (Eagle's salts and L-glutamine), 10% of fetal bovine serum, 2 mM L-glutamine, 0.1 mM MEM non-essential amino acids solution, 1 mM MEM pyruvate solution, 10,000 units penicillin and 10,000 µg streptomycin. Transfection for WT-hH1 was carried out by a cationic liposome method, as described previously [23]. cDNA for R1623Q-hH1 was transfected into HEK293 cells using LipofectAMINETM2000 (Invitrogen) as directed by the manufacturer. To select stably transfected cells, geneticine (G418 Sulfate) at a concentration of 800 µg/ml was added for approximately 15 days, at which time surviving single colonies were isolated and cultured with 400 µg/ml geneticine for 1–3 weeks.

2.3. Electrophysiological recordings
INa was recorded using the whole-cell patch-clamp technique at room temperature (23 ± 1 °C). The bath solution contained (in mM): NaCl 140, KCl 4, CaCl2 1.8, MgCl2 0.75 and HEPES 5 (pH 7.4 set with NaOH). The pipette solution contained (in mM): CsF 120, CsCl 20, EGTA 5 and HEPES 5 (pH 7.4 set with CsOH). Methods to achieve and verify voltage control methods were as published previously [23].

2.4. Data analysis
Passive leak subtraction of peak and late currents was performed as previously described [23]. Data were fit to model equations using non-linear regression with pClamp ver. 8.1 or Sigma Plot ver. 7.0. Fitting procedures for the time course of macroscopic current decay, steady-state inactivation and activation data and recovery from inactivation were described in detail previously [23]. Mean data were expressed with their standard error (S.E.M.) with n representing the number of cells. All determinations of statistical significance were performed by using the Student's t-test for comparisons of two means or when appropriate one-way ANOVA for comparison of multiple means. A P value of <0.05 was considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1. Macroscopic current
Fig. 1A shows representative INa recorded from HEK 293 cells expressing WT or R1623Q mutant sodium channel during a 300 ms rectangular step depolarization to –20 mV from a holding potential (HP) of –120 mV. The current was normalized to peak amplitude and then averaged. Decreased rate of macroscopic current decay and a persistent component of late INa were observed in R1623Q mutant channels. Values of the time constant for fast ({tau}f) and slow ({tau}s) components of the current decay were, WT: {tau}f=0.7 ± 0.1 ms, {tau}s=3.7 ± 0.3 ms, n=6; R1623Q: {tau}f=3.5 ± 0.3 ms, {tau}s=13.0 ± 1.4 ms, n=7 (P<0.05).


Figure 1
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  		Fig. 1 (A) Normalized whole cell Na+ currents (INa) elicited by 300 ms step pulse to –20 mV from a holding potential of –120 mV in cells expressing wild-type (WT) and R1623Q mutant Na+ channels. (B) INa elicited by an action potential voltage clamp in WT and R1623Q mutant. Peak INa is off scale. The waveform was digitized from a published action potential recorded from an intact human heart ventricular cell [35]. Vertical dotted lines at 240 and 260 ms indicate repolarization phase of the action potential where Na+ current was measured for later analysis.

 
To evaluate the late INa during the repolarization phase of the action potential (phase 3 INa), we used an action potential waveform as a voltage clamp command signal. Fig. 1B shows representative INa for WT and R1623Q mutant during the action potential clamp. The INa was normalized to peak INa. In both the WT and R1623Q mutant, the INa persisted throughout the action potential plateau and increased further during the repolarization phase of an action potential. However, the amplitude of the phase 3 INa was larger in R1623Q mutant compared with WT.

3.2. Rate dependency of INa with rectangular pulse stimulation
Fig. 2 shows the time courses of peak and late INa for R1623Q mutant in response to 50 pulse trains at different frequencies. Currents were elicited by 48 ms rectangular step pulses to –20 mV from a holding potential of –120 mV (Fig. 2A) or –80 mV (Fig. 2B). Both the peak and the late INa showed similar time courses at each frequency. Fig. 3 shows summary data of the frequency dependence of INa at a holding potential of –120 mV (Fig. 3A) and –80 mV (Fig. 3B) for WT and R1623Q mutant. With the holding potential of –120 mV, the relative amplitudes were, R1623Q: 99 ± 4.0%, 98 ± 1.4%, 95 ± 1.0%, 88 ± 1.8% for peak INa and 105 ± 7.6%, 100 ± 4.9%, 89 ± 1.1%, 84 ± 2.7% for late INa; WT: 98 ± 1.7%, 98 ± 0.8%, 97 ± 0.3%, 93 ± 0.9% for peak INa and 99 ± 3.5%, 90 ± 5.2%, 87 ± 2.9%, 86 ± 6.1% for late INa, at 0.5, 1, 2, 5 Hz stimulation, respectively (n=7). The decrease in current amplitude with increasing rate was qualitatively similar for peak and late INa and there was no significant difference between the peak INa and late INa except for at 2 Hz stimulation in both WT and R1623Q mutant. With a holding potential of –80 mV, the relative amplitudes were, R1623Q: 87 ± 6.1%, 83 ± 2.5%, 71 ± 2.2%, 65 ± 3.6% for peak INa and 93 ± 6.3%, 83 ± 2.4%, 73 ± 5.7%, 63 ± 2.6% for late INa; WT: 87 ± 4.5%, 79 ± 3.2%, 71 ± 4.0%, 44 ± 3.4% for peak INa and 82 ± 9.1%, 80 ± 8.0%, 68 ± 4.8%, 61 ± 7.5% for late INa, at 0.5, 1, 2, 5 Hz stimulation, respectively (n=7). The decrease in current amplitude with increasing rate was quantitatively similar for peak and late INa and there was no significant difference between the peak INa and late INa at each frequency in both WT and R1623Q mutant. To further evaluate the late INa comparable to the repolarization phase of an action potential, longer rectangular step pulses (300 ms) were applied in R1623Q mutant (Fig. 3A and B, right side). The late INa in WT channels was not included in this study because of its small amplitude. The decrease in current amplitude with increasing rate for peak INa and late INa were larger than that with a 48-ms rectangular step pulse but there was no significant difference between the peak INa and late INa (HP=–120 mV: 99 ± 2.4%, 95 ± 2.4%, 88 ± 2.4% for peak INa and 99 ± 2.1%, 93 ± 5.0%, 87 ± 5.6% for late INa; HP=–80 mV: 83 ± 1.6%, 79 ± 2.1%, 67 ± 5.2% for peak INa and 89 ± 2.2%, 81 ± 6.5%, 71 ± 6.1% for late INa, at 0.5, 1, 2 Hz stimulation, respectively, n=7).


Figure 2
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  		Fig. 2 Time course of rate-dependent reduction of INa in R1623Q mutant. Currents were elicited by rectangular step pulse to –20 mV for 48 ms from holding potentials of –120 mV (A) and –80 mV (B). Peak (closed circles) and late (open circles) INa were normalized to the first pulse amplitude and plotted against pulse number. Late INa amplitude was evaluated by using the mean value measured between 39 and 41 ms. Data are mean ± S.E.M. (n=7). Solid lines represent exponential fits.

 

Figure 3
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  		Fig. 3 Summary data showing rate-dependent reduction of peak and late INa at holding potentials (HP) of –120 mV (A) and –80 mV (B). Data with longer rectangular step pulses, which evaluated current amplitude by using the mean value measured between 240 and 260 ms, were also shown (R1623Q250). Current amplitude for peak or late INa in response to the last five pulses of the train were averaged and normalized to the first pulse in the train. Data are mean ± S.E.M. (n=7). *Significant (P<0.05) difference between peak and late INa during repetitive depolarization. {dagger}, {ddagger}, # P<0.05, compared with 0.5, 1 and 2 Hz, respectively.

 
3.3. Rate dependency of INa with action potential stimulation
Fig. 4A shows representative current recordings of R1623Q mutant INa with action potential clamp in response to 50 pulse trains at different frequencies. In order to quantitatively determine the rate dependency of the phase 3 INa, the current amplitude was evaluated as mean value between 240 and 260 ms, represented by the longitudinal dotted line in Fig. 1B. Clamp voltage was changed continuously from –20 to –33 mV at this region (see Fig. 1B). The time courses of normalized INa were plotted against pulse number (Fig. 4B). In contrast to the time course with rectangular stimulation wave, the phase 3 INa cumulatively decreased during the pulse train. Fig. 5A shows summary for WT and R1623Q mutant (R1623Q: 97 ± 1.8%, 94 ± 2.0%, 84 ± 3.2% for peak INa and 95 ± 3.6%, 65 ± 9.4%, 48 ± 5.5% for phase 3 INa, n=7; WT: 97 ± 1.1%, 91 ± 1.0%, 82 ± 1.6% for peak INa and 92 ± 6.1%, 93 ± 2.8%, 76 ± 6.0% for phase 3 INa, n=7, at 0.5, 1, 2 Hz stimulation, respectively). The phase 3 INa in R1623Q mutant was significantly decreased at 1 and 2 Hz compared with peak INa. To further characterize the rate dependency of the phase 3 INa, we compared the ratio of phase 3 INa to peak INa (Fig. 5B). Fraction of the phase 3 INa was significantly decreased by higher pulse frequency in R1623Q mutant (P <0.01), but not in WT (P=0.07).


Figure 4
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  		Fig. 4 (A) Representative current recordings in response to pulse trains of action potential stimulation at frequencies of 0.5, 1 and 2 Hz in R1623Q mutant. The pulse protocol was the same as in Fig. 1. Currents from the 1st, 2nd, 10th, 25th and 50th pulses in the train are superimposed. The INa was normalized to the peak INa and peak components are off scale in order to show late components. (B) Time course of rate-dependent reduction of the phase 3 INa with action potential clamp. Data are mean ± S.E.M. (n=7). Solid lines represent exponential fits.

 

Figure 5
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  		Fig. 5 Summary data showing rate-dependent reduction of phase 3 INa in response to action potential stimulation. Current amplitude for peak or phase 3 INa in response to the last five pulses of the train were averaged and normalized to the first pulse in the train (A). *Significant (P<0.05) reduction of phase 3 INa compared to peak INa. (B) Ratio of phase 3 INa to peak INa. Data are mean ± S.E.M. (n=7). {dagger}, {ddagger} P<0.05, compared with 0.5 and 1 Hz, respectively.

 
3.4. Kinetics of R1623Q mutant channels
Clancy et al. [10] recently demonstrated that channel reopening resulting from faster recovery from inactivation during repolarization of the action potential (non-equilibrium condition) is a potential mechanism of arrhythmia. We hypothesized that the discrepancy of rate-dependent reduction of late INa between protocols (rectangular waveform and action potential) might be explained by changes in gating kinetics in the R1623Q mutant channels.

Comparison of steady-state inactivation and activation for WT and R1623Q mutant are presented in Fig. 6A. Half-maximum voltages (V1/2) of the steady-state inactivation for WT and R1623Q were not significantly different (WT: –79.9 ± 1.4 mV, n=6; R1623Q: –80.0 ± 2.4 mV, n=6, P=0.96). The slope factor ({kappa}) was significantly larger in R1623Q mutant than WT (WT: 5.5 ± 0.2, n=6; R1623Q: 9.4 ± 0.2, n=6, P<0.001), resulting in less voltage dependence in the R1623Q mutant. Activation curves for WT and R1623Q mutant were almost superimposable (WT: V1/2=–43.0 ± 1.4mV, {kappa}=5.9 ± 0.2, n=6; R1623Q: V1/2=–40.7 ± 4.4 mV, {kappa}=5.9 ± 0.3, n=6, P=0.24 and 0.98 for V1/2 and {kappa}, respectively). Consequently, the extent of the window current resulting from overlapping of activation and inactivation was expanded in R1623Q mutant compared with WT.


Figure 6
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  		Fig. 6 (A) Steady-state inactivation and activation relationships. Clamp protocols for activation and for inactivation are illustrated in insets. Data are mean ± S.E.M. Lines (WT: dotted lines, R1623Q: solid lines) represent fits to a Boltzmann function. See text for each value of parameters. (B) Time course of recovery from inactivation for peak INa in WT (closed symbols) and R1623Q (open symbols). Membrane potential was stepped to –20 mV from various holding potentials (–120, –100 and –80 mV) for 500 ms, and a test pulse to –20 mV was delivered followed by a variable recovery interval ({Delta}T) to holding potentials, as indicated in inset. Currents were normalized to the maximum current amplitude recorded in the absence of a conditioning pulse and plotted against the recovery time on a logarithmic axis. Solid (R1623Q) and dotted (WT) lines indicate the fitting with double-exponential function at each holding potential. Values of parameters are summarized in Table 1.

 
Fig. 6B shows the recovery from inactivation at various holding potentials (–120, –100 and –80 mV). The time course of recovery from inactivation was studied with a two-pulse protocol (see legend for Fig. 6B). The time constants and their fractions are summarized in Table 1. When the holding potential was –120 mV, the time courses of recovery from inactivation for WT and R1623Q mutant were nearly superimposable. The time constants were significantly increased at depolarized holding potentials in both WT and the R1623Q mutant. However, changes in the recovery time constant based on the depolarized holding potentials were significantly smaller in R1623Q mutant resulting in a faster recovery from inactivation in the R1623Q mutant compared with WT at depolarized holding potentials (P<0.05 at –100 mV and P<0.0001 at –80 mV, respectively).


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  		Table 1 Parameters for recovery from inactivation at different holding potentials

 
Since the late INa for the {Delta}KPQ mutant channels recovered more slowly compared with peak INa [23], we also studied the recovery from inactivation of the late component of INa in the R1623Qmutant (Fig. 7). The time constants were not significantly different between the peak and late INa at holding potentials of –120 mV and –80 mV (Table 1). However, fraction of the fast component of recovery was significantly decreased at a holding potential of –80 mV compared with –120 mV (P<0.001).


Figure 7
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  		Fig. 7 Time course of recovery from inactivation for late INa in the R1623Q mutant (open symbols). The same pulse protocol as for peak INa was used (see inset), and the INa measured at 50 ms was normalized and plotted for recovery time. Recovery for the peak INa (closed symbols) was duplicated from Fig. 6 for comparison. Lines indicate the fitting with double-exponential function with offset at each holding potential. Values of parameters are summarized in Table 1.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
The main finding of the present study was that R1623Q mutant channels showed preferential rate-dependent reduction of the phase 3 INa compared with peak INa in the action potential clamp protocol. Although the R1623Q mutant channels were characterized by slowing of the time course of inactivation (caused by inactivation gating defect) and expanded window current (due to less voltage dependence of the inactivation curve), the mechanism of the preferential rate-dependent reduction of phase 3 INa was explained by the accelerated recovery from inactivation under non-equilibrium condition.

We have reported that preferential rate-dependent reduction of the late INa, compared with peak INa, may underlie the enhanced shortening of the prolonged QT interval at faster heart rates in the {Delta}KPQ mutant [16]. The mechanism of the preferential rate-dependent reduction of late INa was attributed to the slower recovery from inactivation for the late INa compared with peak INa [16,23]. Although the preferential rate-dependent reduction of late INa was observed in both the rectangular waveform voltage clamp and the action potential clamp protocols in the {Delta}KPQ mutant [16], we could not observe this rate-dependent phenomenon in the R1623Q mutant when we used rectangular waveform voltage clamp and it was only observed in action potential clamp protocol. Moreover, the time constants of recovery from inactivation were not significantly different between peak INa and late INa (Fig. 7), which were in agreement with the result that the rate dependency in response to repetitive stimulation was similar in both the peak INa and late INa in the rectangular step pulse protocol (Fig. 3). These results led us to propose that different mechanisms might explain the enhanced QT shortening in the R1623Q mutant. What is the mechanism of the different reactions to the two protocols in R1623Q mutant? R1623Q mutant channels showed a predominant increase of the late INa during the repolarization phase of an action potential compared with WT channels (Fig. 1B). This increased late INa may be explained by inactivation gating defect caused by a decreased rate of transition from the open state to the inactivated state [20,21], and by expanded window current due to the less voltage dependence of the inactivation curve (Fig. 6). If these are the mechanisms for the rate dependency of late INa, similar phenomena should be observed in both protocols (rectangular and action potential voltage clamp). Moreover, since the window current reflects steady-state channel reopening, it cannot account for the gating under non-equilibrium condition like repetitive pulse stimulations. Recently, Clancy et al. [10] reported a novel gain-of-function mechanism in I1768V mutant (S6 segment of domain IV) of LQT3 by using negative voltage ramp protocol and computational analysis. In I1768V mutant channels, increase of phase 3 INa was obvious, albeit the late INa was not observed under steady-state rectangular waveform voltage clamp protocol. The mechanism has been explained by channel reopening at membrane potentials (nearby –20 mV) that facilitates the activation transition resulting from faster recovery from open-state inactivation. The same group also confirmed by computational analysis that this phase 3 INa under non-equilibrium condition caused prominent prolongation of the action potential duration and arrhythmic trigger. Therefore, we considered that mutation-induced changes in recovery time constants might underlie the rate dependency as well as augmentation of the phase 3 INa in an action potential clamp. Since the INa of ventricular myocytes recovers from inactivation more slowly at depolarized potentials and the holding potential greatly affects the time constant of recovery [24], we carefully analyzed the effect of holding potential on the time course of recovery in the mutant channels and compared the response to that of WT channels. Notably, R1623Q mutant channels showed smaller voltage dependence of the time course of recovery compared with WT, resulting in a faster recovery from inactivation at a depolarized holding potential (–80 mV), albeit the recovery time constants were similar in both WT and R1623Q mutant channels at a holding potential of –120 mV. These results suggest that the faster recovery from inactivation in the R1623Q mutant channels compared with WT may account for the increased phase 3 INa, because the resting membrane potential is usually from –80 to –100 mV. It is also conceivable that frequent stimulations make fewer fractions of sodium channel recover, resulting in a rate-dependent reduction of phase 3 INa.

The R1623Q mutation results in the replacement of neutral glutamine for positively charged arginine-1623Q located at the outermost of the S4 segment of domain IV (DIV-S4). Arginine-1623 in hH1 corresponds to the arginine-1448 (R1448) in the human skeletal muscle Na+ channel {alpha}-subunit (hSkM1) and two missense mutations (R1448H, R1448C) of this residue have been identified in patients with paramyotonia congenita [25]. This region is not only part of the voltage sensor of channel activation, but also important for activation–inactivation coupling [26,27]. Loss of positive charge of this site may change the voltage dependence of channel gating resulting in less voltage sensitivity of steady-state inactivation and alternative recovery from inactivation in the R1623Q mutant. In fact, R1448H/C mutant channels also showed that the recovery from inactivation was less voltage-dependent and accelerated compared with WT [28,29].

The present study also provides additional rationale for novel therapeutic strategies. Kambouris et al. [22] reported that R1623Q mutant channels have an intrinsically higher affinity than WT to Na+ channel blocker, lidocaine. In contrast to WT channels, lidocaine speeded the rate of R1623Q INa decay by enhancing closed-state inactivation and also delayed the recovery of R1623Q mutant channels [22]. Hence, administration of Na+ channel blocker may reduce the phase 3 INa under non-equilibrium condition by delaying recovery from inactivation as well as restoring rapid decay of INa.

Mechanisms other than those intrinsic to the Na+ channels have been proposed for the enhanced rate dependence of the QT interval in LQT3 patients. Priori et al. [30] suggested the importance of the slowly activating inward rectifying K+ current IKs, the repolarizing current defective in LQT1, in rate-related QT shortening because IKs is increased at shorter cycle lengths. However, action potential shortening in response to fast pacing was significantly greater for experimental models of LQT3 compared with LQT1 or LQT2 [31]. As IKs is maintained in these models, and presumably in LQT2 and LQT3 patients, the IKs does not account for enhanced rate-related QT shortening in LQT3. Recently, Schwartz et al. [11] suggested that Na+ accumulation in myocytes at higher heart rates might lower the Na+ gradient and thereby decrease both peak and late INa. Although this could play a role, the preferential inactivation of phase 3 INa over peak INa at higher rates is a more powerful mechanism for action potential shortening, decreasing phase 3 INa more than 50% while peak INa is relatively unaffected. Sympathetic-mediated modulations of Na+ channels in the heart have been also reported. Stimulation of protein kinase A increases the late INa in {Delta}KPQ and D1790G mutants but not in Y1795C mutant of LQT3 [32,33]. {alpha}-Adrenergic receptor-mediated stimulation of protein kinase C inhibits bursting in {Delta}KPQ and Y1795C mutants of LQT3 [34]. Complex interactions between the cytoplasmic loops of the mutated channels may be present and further investigation of this issue is necessary.

In conclusion, the intrinsic kinetic property of the INa in the LQT3 R1623Q mutant, preferential rate-dependent reduction of phase 3 INa under non-equilibrium condition, may account for the enhanced shortening of QT interval at faster heart rates. Our findings are important for genotype–phenotype correlations and therapeutic strategies in LQT3 mutants as well as for understanding the function of DIV-S4 region in the cardiac Na+ channel.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
This work was supported by a grant from Japan Heart Foundation, Pfizer Pharmaceuticals Grant, and by UOEH Research Grants for Promotion of Occupational Health (to T.N.).


   Notes
 
Time for primary review 27 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

  1. Jiang C., Atkinson D., Towbin J.A., Splawski I., Lehmann M.H., Li H., et al. Two long QT syndrome loci map to chromosomes 3 and 7 with evidence for further heterogeneity. Nat. Genet. (1994) 8:141–147.[CrossRef][ISI][Medline]
  2. George A.L. Jr., Varkony T.A., Drabkin H.A., Han J., Knops J.F., Finley W.H., et al. Assignment of the human heart tetrodotoxin-resistant voltage-gated Na+ channel {alpha}-subunit gene (SCN5A) to band 3p21. Cytogenet. Cell Genet. (1995) 68:67–70.[ISI][Medline]
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