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Physiol. Genomics 10: 191-197, 2002. First published July 16, 2002; doi:10.1152/physiolgenomics.00039.2002
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Received 9 April 2002; accepted in final form 12 July 2002.
Physiological Genomics 10:191-197 (2002)
1094-8341/02 $5.00 © 2002 American Physiological Society

A novel SCN5A mutation associated with long QT-3: altered inactivation kinetics and channel dysfunction

Ilaria Rivolta1, Colleen E. Clancy1, Michihiro Tateyama1, Huajun Liu1, Silvia G. Priori2 and Robert S. Kass1

1 Department of Pharmacology, College of Physicians and Surgeons of Columbia University, New York, New York 10032
2 University of Pavia and Molecular Cardiology Laboratory, Fondazione Salvatore Maugeri, Instituto di Ricovero e Cura a Carattere Scientifico, Pavia 27100, Italy


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mutations in the gene (SCN5A) encoding the {alpha}-subunit of the cardiac Na+ channel cause congenital long QT syndrome (LQT-3). Here we describe a novel LQT-3 mutation I1768V (I1768V) located in the sixth transmembrane spanning segment of domain IV. This mutation is unusual in that it is located within a transmembrane spanning domain and does not promote the typically observed sustained inward current corresponding to a gain of channel function (bursting). Rather, I1768V increases the rate of recovery from inactivation and increases the channel availability, observed as a positive shift of the steady-state inactivation curve (+7.6 mV). Using a Markovian model of the cardiac Na+ channel, we simulated these changes in gating behavior and demonstrated that a small increase in the rate of recovery from inactivation is sufficient to explain all of the experimentally observed current changes. The effect of these alterations in channel gating results in an increase in window current that may act to disrupt cardiac repolarization.

long QT syndrome; sodium channel; electrophysiology; genetics; arrhythmias


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE CARDIAC ISOFORM of the sodium channel {alpha}-subunit, encoded by the gene SCN5A (chromosome 3p21–23), may contain genetic defects that alter channel properties and underlie disease syndromes including long QT, Brugada, and conduction abnormalities (4, 6, 13, 24, 26, 30). Cardiac sodium current (INa) is primarily responsible the generation and propagation of the cardiac action potential, crucial for contractile synchronization (20). The elucidation of a role of Na+ channel defects in seemingly idiopathic syndromes is relatively recent (15). Electrophysiological characterization of such defects promotes understanding of the relationship between genotype and phenotype and provides insight into the discrete structures that are involved in channel gating.

In this paper we report the functional consequences of a new mutation identified in a patient with congenital long QT syndrome (LQT-3). The mutation is at position 1768, located within the sixth transmembrane spanning region in domain IV of the cardiac sodium channel {alpha}-subunit. The mutation results in the substitution of a valine in place of a highly conserved isoleucine (I1768V). We have previously reported a mutation in a different region (E1295K) (1) that has properties similar to those reported here. Namely, both mutations act to increase channel availability and increase the rate of channel recovery from inactivation, which results in a "window" of voltages over which noninactivating channel activity is observed and is distinct from bursting activity (3, 12, 23, 27). Similar changes were also observed in an SCN5A mutation recently reported (29) associated with sudden infant death syndrome (SIDS). Here we show that the I1768V mutation-induced changes in the kinetics of recovery from inactivation can account for all of the biophysical changes in the channel and consequently are likely to be key to mutation-induced rhythm dysfunction.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Description of a new LQT-3 mutation in a patient with QT prolongation.
The proband was a 15-yr-old male who experienced a syncopal episode at rest. He was then admitted to the hospital where a self-terminating polymorphic ventricular tachycardia with Torsade de Pointes morphology was observed (the K+ plasma level at admission was 4.3 meq/l). After sinus rhythm was restored spontaneously, QT interval prolongation was observed (QT = 455 ms, RR = 800 ms, QTc = 510 ms, lead II). All the possible causes of acquired QT interval prolongation (QT prolonging drugs, electrolyte imbalance, etc.) were excluded and therefore a congenital form of LQT syndrome (LQTS) was diagnosed. Genetic analysis of the proband performed by single-strand conformational polymorphisms (SSCP) (17) demonstrated an abnormal conformer identified in the exon 28 of the SCN5A gene. Subsequent DNA sequence analysis showed a single nucleotide transition (A->G at position 5302) leading to a nonconservative amino acid change with a valine replacing an isoleucine at codon 1768. The mutation occurs in the transmembrane segment S6 of domain IV of the channel. The entire coding region of all known LQTS-related genes (KCNE1, KCNE2, KCNH2, KCNQ1) were screened, and no mutations were identified. The family history was unremarkable, with no evidence of premature unexplained sudden death and/or history of syncope. Both parents were asymptomatic with normal QT intervals and QTc values. No other family member consented to genetic analysis.

Genomic DNA was extracted from peripheral blood lymphocytes by standard techniques. The entire coding region of the known LQTS genes was screened by SSCP and/or denaturing HPLC after PCR amplification of the 150- to 300-bp fragments using published primers pairs. The abnormal conformers were directly sequenced using an ABI model 310 genetic analyzer or cloned (TopoTA cloning, Invitrogen) and sequenced using plasmid-specific oligonucleotides. A panel of 350 (700 alleles) healthy individuals was used as control group.

Expression of recombinant Na+ channels.
Na+ channels were expressed in HEK 293 cells as previously described. Transient transfection was carried out with equal amounts of Na+ channel {alpha}-subunit cDNA [wild type (WT) or I1768V, respectively], with human cardiac Na+ channel ß 1-subunit (hß1) cDNA subcloned into the pcDNA3.1 (+)vector (Invitrogen) along with CD8, a commercially available reporter gene (EBo-pCD vector, American Type Culture Collection). Total cDNA was 2.5 µg, and we used a previously described lipofection procedure (2) to transfect cells. CD8-positive cells, identified using Dynabeads (Dynal, M-450) were patch clamped 48 h after transfection.

Electrophysiology.
Membrane currents were measured using whole cell patch-clamp procedures, with Axopatch 200B amplifiers (Axon Instruments, Foster City, CA). Capacity current and series resistance compensation were carried out using analog techniques according to the amplifier manufacture (Axon Instruments, Foster City, CA). All measurements were obtained at room temperature (22°C). Macroscopic whole cell Na+ current was recorded using the following solutions. The internal solution contained (in mmol/l) 50 aspartic acid, 60 CsCl, 5 disodium ATP, 11 EGTA, 10 HEPES, 1 CaCl2, and 1 MgCl2, with pH 7.4 adjusted with CsOH. The external solution contained (in mmol/l) 130 NaCl, 2 CaCl2, 5 CsCl, 1.2 MgCl2, 10 HEPES, and 5 glucose, with pH 7.4 adjusted with CsOH. The voltage dependence of inactivation was determined after application of conditioning pulses (500 ms) applied once every 2 s to a series of voltages. In experiments designed to measure the voltage dependence of activation, external Na+ was reduce to 10 mM using N-methyl-glucamine as a Na+ substitute. In experiments designed to test for sustained currents, tetrodotoxin (TTX) was applied at high concentrations (30 µM) to block expressed Na+ channel currents and reveal background currents, which were then subtracted digitally. This subtraction procedure was used both for voltage ramp experiments and experiments assaying for sustained currents in response to prolonged depolarizing voltage pulses. Holding potentials were -100 mV. Recovery from inactivation was measured in paired pulse experiments with a test pulse applied at variable times after a 500-ms conditioning pulse to -10 mV. PClamp8 (Axon Instruments), Excel (Microsoft, Seattle, WA), and Origin 6.0 (Microcal Software, Northampton, MA) were used for data acquisition and analysis. Data are represented as mean values ± SE. Two-tailed Student’s t-test was used to compare means; P < 0.05 was considered statistically significant.

Single channels.
Single channel experiments were carried out using the cell-attached configuration (seal resistance >10 G{Omega}). Test pulses (-30 mV, 100 ms) were applied every 0.5 s from a -120-mV holding potential. Single channel currents were low-pass filtered (5 kHz cutoff frequency) and digitized at a sampling frequency of 20 kHz. Capacitative and leak currents were eliminated by digital subtraction of averaged null sweeps. Idealization of single channel currents and the measurement of open time were carried out with the program SKM [QUB suite (18, 19)]. Further analysis was carried out using Excel (Microsoft) and Origin 7.0 (Microcal Software). Open probability (Po) for one or two channel patches was calculated by the following equation

where b, t, and n represent the number of blank sweeps, total sweeps, and channels, respectively (5). We used the same formulation to determine the frequency of bursting. Burst frequency was estimated as above by substituting b (in this case, the number of sweeps without bursting) and t, the total number of sweeps studied. Here the number of channels per patch (n) was estimated by counting overlapping unitary current from 1,000–2,000 total sweeps (n < 11). Data are represented as means ± SE. Statistical significance was determined using an unpaired Student’s t-test.

Computational methods.
We have used a previously developed Markovian model of the cardiac sodium channel(9). Simulations were conducted in isolated myocardial cells using voltage protocols as described for experiments. Computer code was written in C/C++ using Apple Developer Tools (Apple Computer). Simulations were run on a dual 800 mHz G4 Power PC processor Macintosh (Apple Computer).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Biophysical characterization of the I1768V mutation.
HEK 293 cells transiently cotransfected with hß1 plus either WT or I1768V cDNA expressed sodium currents, which had no obvious biophysical differences (Fig. 1A). Comparison of the properties of WT and I1768V channels revealed no significant difference between the peak current, voltage dependence of channel activation, or current density as reflected in the current voltage relationships shown in Fig. 1B. Unlike the majority of previously characterized LQT-3-linked mutations, I1768V did not result in an increase in sustained current due to channel bursting compared with WT as shown in Fig. 2 [0.24 pA/pF = 0.06 ± 0.02%, n = 8, for WT; 0.24 pA/pF = 0.06 ± 0.02% of peak, n = 5, for I1768V (not significant)]. We confirmed this result by measuring the frequency of bursting in single channels and found that the WT channels burst with the same frequency as I1768V channels (Fig. 2C; also, see Supplementary Fig. 2, 1 published online at the Physiological Genomics web site). At -20 mV, WT bursting frequency = 0.0029 ± 0.0006% (n = 3) and I1768V bursting frequency = 0.0027 ± 0.0010% (n = 4) (not significant). We also characterized the onset of inactivation kinetics (computed as {tau}1/2, the time to decay to half of peak current) (Fig. 3) of the I1768V mutant and WT and observed no significant difference. However, we did find that the mutation affected the kinetics of the recovery from inactivation, which we measured by repeated application of a two-pulse protocol. Channels were inactivated by conditioning pulses (500 ms, -10 mV) and allowed to recover from inactivation at the holding potential (-100 mV) for variable times before INa was measured in response to test pulses (-10 mV). Normalized test pulse current was plotted vs. recovery intervals (Fig. 4A). We observed a significant mutation-induced speeding of this process. Time to half recovery was 3.0 ± 0.1 ms (n = 8) for WT and 1.3 ± 0.1 ms for I1768V channels (n = 8) (P < 0.05). We reasoned that a twofold increase the rate of recovery from inactivation might increase channel availability and confirmed that this was indeed the case in measurements of the voltage dependence of availability for both WT and I1768V channels. The I1768V channel availability curve was shifted +7.6 mV compared with WT (Fig. 4B) [V1/2 = -62.8 ± 0.9; k = 5.4 ± 0.2 (WT, n = 12); V1/2 = -55.2 ± 0.8; k = 5.2 ± 0.1 (I1768V, n = 8); P < 0.01], consistent with predictions of the experimental data. We also measured the increased rate of recovery from inactivation in WT and I1768V channels at 22°C and 30°C, since previous studies have shown that some mutations may affect the temperature dependence of kinetic transitions. We found that increasing the temperature resulted in uniform increases of recovery kinetics [the time to 50% recovery ({tau}1/2) for WT at 22°C = 4.21 ± 0.06 ms (n = 6), 30°C = 2.31 ± 0.46 ms (n = 6); and I1768V at 22°C = 1.81 ± 0.11 (n = 5), and 30°C = 0.86 ± 0.19 (n = 4)], corresponding to a Q10 {approx} 3, for both WT and I1768V channels. The differences between WT and I1768V at both 22°C and 32°C were significant [P < 0.01 and P < 0.05, respectively (see Supplementary Fig. 1)].



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  		Fig. 1. The I1768V mutation does not alter the voltage dependence of activation. A: averaged currents recorded in response to a series of voltage pulses from -80 mV to +30 mV (5-mV increments) for wild-type (WT, n = 5) and I1768V (n = 8) channels. B: current-voltage relationship of peak inward current in HEK 293 cells expressing and I1768V channels (measured in 10 mM Na+, see METHODS) (for WT n = 5, and for I1768V n = 8).

 


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  		Fig. 2. The I1768V mutation does not increase sustained macroscopic Na+ channel activity or single channel bursting. A: background currents were recorded in the presence of tetrodotoxin (TTX, 30 µM) as described in METHODS. TTX-sensitive and averaged current traces recorded WT (n = 8) and 1768V (n = 5) channels during sustained (150 ms) depolarization (-10 mV). B: average TTX-sensitive current measured at 150 ms; WT 0.06 ± 0.02% of the peak current (n = 8); I1768V 0.06 ± 0.02% (n = 5) (P < 0.05). C: bursting probability determined as described in METHODS at -20 mV for WT and I1768V channels.

 


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  		Fig. 3. The I1768V mutation does not alter the time course of the onset of inactivation. A: families of current traces recorded from cells expressing WT or I1768V channels in response to a series of brief (25 ms) test pulses (-60, -25, -10, 0, and 10 mV). B: time to half inactivation ({tau}1/2, measured as time to 50% decay of transient inward current) during pulse plotted vs. test pulse voltage (WT, n = 5; I1768V, n = 8). Mean values are not significantly different comparing WT vs. I1768V data points.

 


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  		Fig. 4. The I1768V mutation speeds the recovery from inactivation. A: the recovery from inactivation induced by conditioning pulse (500 ms, -10 mV) was measured using a paired pulse protocol (see text and METHODS). Plots show amplitude of peak inward current, normalized to fully recovered current, as a function of time after imposition of the conditioning pulse for WT (open symbols) and I1768V (solid symbols) channels. There is a significant mutation-induced speeding of the process: the time to half recovery for WT is 3.01 ± 0.06 ms (n = 8) and for I1768V is 1.34 ± 0.14 ms (n = 8), P < 0.05. B: the I1768V mutation causes a positive shift in the voltage dependence of channel availability. Mean availability data were determined using 500-ms conditioning pulses to the voltages indicated along the abscissa for WT (open symbols) and I1768V (solid symbols) channels. Fits to experimental data (smooth curves) yielded the resulting Boltzmann parameters: V1/2 = -62.8 ± 0.9 mV; k = 5.4 ± 0.2 (WT, n = 12); V1/ 2 = -55.2 ± 0.8 mV; k = 5.2 ± 0.1 (I1768V, n = 8). Differences in inactivation voltage V1/2 are statistically significant with P < 0.01.

 
To determine whether this increase in the rate of channel recovery from inactivation might underlie the 7.6-mV shift in inactivation gating, we applied the same rate increase to recovery transitions in a computer simulated Markov model of the cardiac Na+ channel (9). Our simulation demonstrated that a doubling of the rate of recovery from inactivation results in a leftward shift in the time to half recovery from inactivation (Fig. 5B), a +7-mV shift in the voltage dependence of channel availability (Fig. 5C), and no effect on current density or activation (Fig. 5A). These results, which are remarkably similar to the experimental data of Fig. 1, demonstrate that the I1768V mutation acts to make recovery from inactivation more energetically favorable, and this singular alteration in kinetics is sufficient to account for all of the experimentally observed gating changes.



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  		Fig. 5. Computational simulation of gating: I1768V mutation-induced increase in rate of recovery from inactivation underlies positive shift of channel availability. A Markovian model of Na+ channel gating (METHODS and text) was used to simulate Na+ channel currents. Simulation of the I1768V mutation was carried out by increasing the rate of recovery from inactivation as determined experimentally (Fig. 4), and the effects of this change were examined on the peak inward current/voltage relationship (WT and I1768V curves are superimposed) (A); the time course of the recovery from inactivation (B); and the voltage dependence of steady-state channel availability (C). The change in rate is reflected in a speeding of the recovery from inactivation (B) and also a shift of +7 mV in the midpoint of the channel availability relationship (C).

 
We have demonstrated previously that mutation-induced shifts in the voltage dependence of activation and/or inactivation will affect the window of overlap between the activation and availability curves (1), an effect subsequently reported in a de novo SCN5A mutation linked to SIDS (29). The resulting current can be measured directly by using a slow positive ramp pulse protocol. We used this positive ramp protocol (25) (slow voltage ramp from -100 mV to +50 mV in 2 s) to investigate the effect of the I1768V mutation on the window current (Fig. 3). We measured a small, but significant, mutation-induced change in the voltage dependence of the window current [-44.6 ± 1.1 mV (n = 9), WT; -39.6 ± 0.6 mV (n = 15), I1768V P < 0.01] (Fig. 6). Our theoretical analysis indicates that this change in window current is a consequence of the I1768V mutation-induced change in the kinetics of the recovery from inactivation.



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  		Fig. 6. The I1768V mutation alters the voltage dependence of Na+ channel overlap ("window") current. "Window" noninactivating current for WT and I1768V channels was measured using a slow ramp protocol (-100 mV to +50 mV in 2 s). Averaged TTX-sensitive traces (METHODS) were measured in response to this protocol for WT (n = 5) and I1768V (n = 7) channels. The averaged voltage at which the window current peaks is slightly and significantly shifted as a result of the mutation (-39.6 ± 0.6 mV, I1768V, n = 15; -44.6 ± 1.1 mV, WT, n = 9; P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The I1768V mutation in the {alpha}-subunit of the cardiac Na+ channel results in an amino acidic substitution (I1768V) in the transmembrane segment S6 of domain IV proximal to the start of the COOH terminus. Interestingly, although isoleucine and valine have similar structural properties [both are aliphatic and have nonreactive side chain groups ( CH3)], the substitution mutation results in abnormalities in channel gating. Notably, isoleucine in position 1768 is very conserved between channel isoforms and species, suggesting an important structural role. The I1768V mutation results in two primary effects on channel biophysics: an increase in steady-state channel availability and an increase in the rate of recovery from inactivation. Importantly, although residue 1768 is within the S6 segment of domain IV of the channel, it is very close to the proximal end of the carboxy tail of the channel, a structure that has recently gained more attention as key modulator of Na channel inactivation in both brain and heart (10, 16). It is quite possible that the effects of the mutation on channel gating are a consequence of allosteric changes in structure that alter COOH-terminal interactions with the channel.

The I1768V mutation is unusual in that very few LQT-3-related mutations have been reported to reside in one of the transmembrane spanning segments. The three other mutations that have been reported in transmembrane regions are located in S4DIII (T1304M) (28) or in S4DI (R1623Q) (11, 14). The overwhelming majority of LQT-3-linked mutations are in cytoplasmic linkers (21). Interestingly, other mutations have been identified in S6 of the skeletal isoform of the Na+ channel and are associated with hypokalemic periodic paralysis (HypoPP) (7, 22).

The I1768V mutation is also unusual, but clearly not unique, in that it is an LQT-3-linked mutation that it does not result in an obvious gain of function from failure of channel inactivation (bursting). Instead, one of the functional consequences of this mutation is alteration in "overlap" or " window" that is critical to maintenance of the plateau phase of the cardiac action potential, similar to one other previously described LQT-3 (1) and SIDS (29) mutation. Thus this mechanism of channel dysfunction may be more common than previously considered (1).

Here we demonstrate that altered channel availability, which underlies this overlap current, may be caused by mutation-induced changes in the kinetics of recovery from inactivation. We show that I1768V channel expressed in HEK 293 cells produces an increase in the rate of channel recovery from inactivation and increase in channel availability. We also use a computational model to demonstrate that the observed increase in the rate of channel recovery from inactivation is fully sufficient to account for the shift in inactivation gating. The I1768V mutation acts to make recovery from inactivation more energetically favorable, and this causes a change in recovery kinetics. This alteration in kinetics is sufficient to account for all of the experimentally observed gating changes. The increase in channel availability, stemming from faster channel recovery, acts to increase the window of overlap between the inactivation and activation curve. This more extensive overlap occurs at voltages corresponding to the delicate plateau phase of the cardiac action potential.

It has been demonstrated and is now well-accepted that LQT-3 mutations that act to diminish transitions into a nonconducting inactivated state during the plateau phase of the cardiac action potential promote sustained whole cell current activity and underlie the primary disease phenotype: delayed repolarization of the ventricle (8). Not all SCN5A mutations linked to repolarization dysfunction disrupt inactivation in this manner and promote sustained current during the plateau (1, 2, 29). Here we show that mutation-induced changes in the energetics of transitions out of the inactivated state can also play an important role in key channel properties that underlie control of repolarization in the ventricle. As we learn more about the mechanisms underlying disorders of repolarization, we also gain insight into possible novel therapeutic strategies to treat them. A key implication of the analysis presented in this study is that compounds that act to slow transitions from inactivated to rested channels may prove uniquely useful in the treatment of repolarization dysfunction due to the I1768V and related SCN5A mutations.


   ACKNOWLEDGMENTS
 
This work was supported by National Heart, Lung, and Blood Institute Grants R01-HL-56810-05 and P01-HL-67849-01 (to R. S. Kass).


   FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: R. S. Kass, Dept. of Pharmacology, College of Physicians & Surgeons of Columbia Univ., 630 W. 168th St., PH 7W 318, New York, NY 10032 (E-mail: rsk20{at}columbia.edu).

10.1152/physiolgenomics.00039.2002.

1 Supplementary materials (APPENDIX and Supplementary Figs. 1 and 2) to this article are available online at http://physiolgenomics.physiology.org/cgi/content/full/10/3/191/DC1. Back


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 ABSTRACT
 INTRODUCTION
 METHODS
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 DISCUSSION
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