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Enhanced fast-inactivated state stability of cardiac sodium channels by a novel voltage sensor SCN5A mutation, R1632C, as a cause of atypical Brugada syndrome
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Enhanced fast-inactivated state stability of cardiac sodium channels by a novel voltage sensor SCN5A mutation, R1632C, as a cause of atypical Brugada syndrome Tadashi Nakajima, MD, PhD, Yoshiaki Kaneko, MD, PhD, Akihiro Saito, MD, PhD, Masaki Ota, MD, Takafumi Iijima, MD, PhD, Masahiko Kurabayashi, MD, PhD From the Department of Medicine and Biological Science, Gunma University Graduate School of Medicine, Maebashi, Japan. BACKGROUND Mutations in SCN5A, which encodes the cardiac voltage-gated sodium channels, can be associated with multiple electrophysiological phenotypes. A novel SCN5A R1632C mutation, located in the domain IV-segment 4 voltage sensor, was identified in a young male patient who had a syncopal episode during exercise and presented with atrial tachycardia, sinus node dysfunction, and Brugada syndrome. OBJECTIVE We sought to elucidate the functional consequences of the R1632C mutation. METHODS The wild-type (WT) or R1632C SCN5A mutation was coexpressed with β1 subunit in tsA201 cells, and whole-cell sodium currents (INa) were recorded using patch-clamp methods. RESULTS INa density, measured at 20 mV from a holding potential of 120 mV, for R1632C was significantly lower than that for WT (R1632C: 433 ⫾ 52 pA/pF, n ¼ 14; WT: 672 ⫾ 90 pA/pF, n ¼ 15; P o .05); however, no significant changes were observed in the steady-state activation rate and fast inactivation rate. The steadystate inactivation curve for R1632C was remarkably shifted to hyperpolarizing potentials compared with that for WT (R1632C: V1/2 ¼ 110.7 ⫾ 0.8 mV, n ¼ 16; WT: V1/2 ¼ 85.9 ⫾ 2.5 mV, n ¼ 17; P o .01). The steady-state fast inactivation curve for R1632C was also
Introduction Brugada syndrome (BrS), characterized by ST-segment elevations in the right precordial electrocardiographic (ECG) leads and a high incidence of syncope or sudden death due to ventricular tachyarrhythmias, is an inherited arrhythmia syndrome that predominantly affects adult male patients.1,2 Approximately 30% of BrS cases are attributed to defects in genes encoding the cardiac ion channels or their modifiers.1–5 In contrast, supraventricular arrhythmias, such This work was supported, in part, by JSPS KAKENHI (grant no. 26461056). Address reprint requests and correspondence: Dr Tadashi Nakajima, Department of Medicine and Biological Science, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan. E-mail address: [email protected].
1547-5271/$-see front matter B 2015 Heart Rhythm Society. All rights reserved.
shifted to the same degree. Recovery from fast inactivation after a 20ms depolarizing pulse for R1632C was remarkably delayed compared with that for WT (R1632C: τ ¼ 246.7 ⫾ 14.3 ms, n ¼ 8; WT: τ ¼ 3.7 ⫾ 0.3 ms, n ¼ 8; P o .01). Repetitive depolarizing pulses at various cycle lengths greatly attenuated INa for R1632C than that for WT. CONCLUSION R1632C showed a loss of function of INa by an enhanced fast-inactivated state stability because of a pronounced impairment of recovery from fast inactivation, which may explain the phenotypic manifestation observed in our patient. KEYWORDS SCN5A; Mutation; Voltage sensor; Sodium currents; Atrial tachycardia; Sinus node dysfunction; Brugada syndrome ABBREVIATIONS AF ¼ atrial fibrillation; AT ¼ atrial tachycardia; BrS ¼ Brugada syndrome; cDNA ¼ complementary DNA; DIV-S4 ¼ fourth segment of domain IV; ECG ¼ electrocardiogram/ electrocardiographic; INa ¼ sodium current; IRES ¼ internal ribosome entry site; LQTS ¼ long QT syndrome; PCR ¼ polymerase chain reaction; SND ¼ sinus node dysfunction; WT ¼ wild type (Heart Rhythm 2015;0:-1–9) rights reserved.
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2015 Heart Rhythm Society. All
as sinus node dysfunction (SND), atrial fibrillation (AF), and atrial tachycardia (AT), are common electrophysiological disturbances becoming prevalent as one grows older. However, these supraventricular arrhythmias, especially recognized in younger populations, could also be associated with defects of cardiac ion channel or its modifying genes.6–9 Mutations in SCN5A, which encodes the α subunit of the cardiac voltage-gated sodium channels, are associated with several phenotypically distinct electrophysiological disturbances in both the ventricle and the atrium, including long QT syndrome (LQTS), BrS, SND, and AF.3,6,10 Loss of function of cardiac sodium currents (INa) during the early phase of action potential can lead to BrS, SND, and AF.3,6,10 In contrast, gain of function of INa during the late phase of action potential can cause LQTS and AF.7,11 Intriguingly, a http://dx.doi.org/10.1016/j.hrthm.2015.05.032
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compromised loss and gain of function of INa is reported to be associated with AF8 or with mixed phenotypes of BrS, LQTS, and SND.12,13 We recently encountered a young male patient who had a syncopal episode during exercise and presented with multiple electrophysiological disturbances including AT during exercise, SND, and BrS, in whom we identified a novel SCN5A mutation, R1632C. R1632C in SCN5A is located in the fourth segment (S4) of domain IV (DIV) (DIV-S4), where it is postulated to function as a voltage sensor.14–16 Since SCN5A is composed of 4 homologous but nonidentical domains, DI–DIV, there are distinct functional roles of each S4 segment.16 Thus, SCN5A mutations in each S4 segment and even those in the same S4 segment may cause diverse functional abnormalities and phenotypic manifestations.6,8,17–23 It has been reported that recovery from fast inactivation of INa may be associated with immobilized gating charge caused by the slow movement of the S4 segments in domains III and IV, but not in domains I and II,15 which suggested that recovery from fast inactivation of INa would be impaired by R1632C mutation. Therefore, we examined functional abnormalities of the SCN5A R1632C mutation using whole-cell patch-clamp methods. The obtained kinetic changes in R1632C INa were thought to be associated with multiple electrophysiological phenotypes observed in our patient.
Methods Clinical data Appropriate approval from the institutional review board and written informed consent for the genetic analysis from the patient and his mother were obtained. The pilsicainide provocation test was performed as described previously.24
Mutation analysis Genomic DNA was extracted from peripheral blood lymphocytes using the QIAamp DNA Blood Midi Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. All coding exons of KCNQ1 (NM_000218.2), KCNH2 (NM_000238.2), and SCN5A (NM_198056.2) and their splice sites were amplified by polymerase chain reaction (PCR) using primers flanking the intronic sequences as reported previously.25,26 The PCR products were purified using the QIAquick PCR Purification Kit (QIAGEN) and directly sequenced using an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA). The mutation was analyzed twice by independent PCR amplification and sequencing reactions.
Mutagenesis and heterologous expression Wild-type (WT) human heart sodium channel complementary DNA (cDNA) (NM_000335) subcloned into pcDNA3.1 vector (hH1-pcDNA3.1) and a plasmid (pGFP-IRES-hβ1) encoding both GFP and the human β1 subunit (hβ1) under the control of a single CMV promoter with the 2 coding regions separated by a viral internal ribosome entry
Heart Rhythm, Vol 0, No 0, Month 2015 site (IRES) were kindly provided by Prof Naomasa Makita (Nagasaki University).12 Site-directed mutagenesis (R1632C-pcDNA3.1) was performed by the QuikChangeII Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA) according to the manufacturer’s instructions. hH1-pcDNA3.1 (0.5 μg) or R1632C-pcDNA3.1 (0.5 μg) in combination with pGFP-IRES-hβ1 (0.5 μg) was transiently transfected into tsA201 cells growing on 6-cm plates using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and maintained in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in a 5% CO2 incubator at 371C for 24–36 hours before current recordings. Cells exhibiting green fluorescence were chosen for the current recordings.
Electrophysiology Membrane INa densities were recorded using whole-cell patch-clamp methods at room temperature (231C–251C) as described previously.27 The bath solution for recording membrane currents contained 145 mM NaCl, 4 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 5 mM glucose (pH 7.35 with NaOH), and the pipette solution contained 10 mM NaF, 110 mM CsF, 20 mM CsCl, 10 mM EGTA, 10 mM HEPES (pH 7.35 with CsOH). The electrode resistance ranged from 1.1 to 2.0 MΩ. Data acquisition was done using an Axopatch 200B amplifier and pCLAMP software version 10.3 (Molecular Devices, Sunnyvale, CA). Currents were acquired at 20–50 kHz and low-pass filtered at 5 kHz using an analog-to-digital interface (Digidata 1440A acquisition system, Molecular Devices). To avoid the possible time-dependent shift of activation and steady-state inactivation curves,28 all pulse protocols were applied more than 5 minutes after membrane rupture. Therefore, the time-dependent shift became negligible. Recordings from the cells exhibiting peak current amplitudes less than 0.8 nA were excluded from the analysis to avoid potential endogenous current contamination. Cells exhibiting large currents (peak current 4 10 nA) were also excluded if the voltage control was compromised. All pulse protocols are described in figures or figure legends.
Data analysis Pulse protocols were applied from a holding potential of 120 mV, unless otherwise indicated. Current densities at each test potential were obtained by dividing the INa by cell capacitance. The steady-state activation and steady-state inactivation curves were fitted with Boltzmann functions of the following forms: y ¼ 1 1/{1 þ exp[(Vm V1/2)/K]} or y ¼ 1/{1 þ exp[(Vm V1/2)/K]}, respectively, where y is the relative current, Vm is the membrane potential, V1/2 is the voltage at which half of the channels are available to open, and K is the slope factor. The time course of fast inactivation was fitted with a single exponential function of the following form: I(t)/Imax ¼ A0 þ A1 exp(t/τ), where A is the amplitude, τ is the time constant, I is the current, and t is the time. Recovery from inactivation was fitted with a single
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Nakajima et al 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 P 229 R 230 I 231 N 232 T 233 & W 234 E 235 B 236 4 237 C 238 / 239 F Q16 240 P O 241
Stabilized Fast Inactivation of INa by SCN5A R1632C
or double exponential function of the following form: I(t)/ Imax ¼ A0 þ A1 exp(t/τ) or I(t)/Imax ¼ A0 þ A1 exp(t/τf) þ A2 exp(t/τs), where τf and τs are the fast and slow components of τ, respectively.
Statistical analysis All data are expressed as mean ⫾ standard error, and statistical comparisons were made using the unpaired Student t test, with P o .05 considered to be statistically significant. In some figures, the standard error bars are smaller than the data symbols.
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Results Phenotypic characterization A 17-year-old male patient, who had a history of syncope after having palpitations during exercise 1 year ago, was transferred to us because he had again lost consciousness after having palpitations for several minutes during a longdistance race. His 12-lead ECG showed AT with 2-to-1 conduction (heart rate 110 beats/min) (Figure 1A), which changed to 1-to-1 conduction (heart rate 220 beats/min) (Figure 1B). At that time, he had palpitations accompanied by faintness, suggesting that AT with 1-to-1 conduction may be a cause of syncope. The next day, AT spontaneously
Figure 1 Multiple electrophysiological phenotypes in the patient. A: A 12-lead ECG recorded just after arrival showed atrial tachycardia (AT) with 2-to-1 conduction. Red arrows indicate P waves. B: The 12-lead ECG changed to AT with 1-to-1 conduction. C: The ECG monitor recording showing sinus arrest of 5.7 seconds. D and E: The 12-lead ECG before (panel D) and precordial ECG leads after (panel E) the pilsicainide provocation test. ECG ¼ electrocardiogram; ics ¼ intercostal space.
242 243 244 245 246 247 248 Q8249 QF1 250 251 Q9252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298
4 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 F2 329 330Q10 331 332 333 334 335 336 337 338 339 340 341 342 P 343 R 344 I 345 N 346 T 347 & W 348 E 349 B 350 4 351 C 352 / 353 F 354 P O 355
returned to sinus rhythm. An ECG monitor recording revealed that he had SND (sinus arrest of 5.7 seconds) (Figure 1C). Lead V2 during sinus rhythm (heart rate 68 beats/min; PQ interval 200 ms; QRS duration 118 ms) showed a saddleback-type ST-segment elevation (Figure 1D), and a coved-type ST-segment elevation in the right precordial ECG leads appeared after provocation with pilsicainide (50 mg) (Figure 1E). The echocardiogram and conventional left heart catheterization, including acetylcholine provocation to the coronary arteries, revealed no structural heart disease. A cardiac electrophysiological study was also performed. Although the AH interval (95 ms) was not prolonged, the HV interval (73 ms) was slightly prolonged. Ventricular tachyarrhythmias were not induced by up to 3 ventricular extrastimuli at both the right ventricular apex and the right ventricular outflow tract. AT was induced by high-frequency atrial stimuli (230 beats/min) during isoproterenol infusion (50 μg/h), and it was thought to originate from the left atrium. Considering the patient’s young age, we did not perform catheter ablation. Under exercise restrictions, he has currently not experienced palpitations or syncope for more than 1 year. His mother also had a history of syncope during a longdistance race when she was a high-school student. Her ECG exhibited a Brugada pattern (coved-type) after provocation with pilsicainide (Online Supplemental Figure S1).
Identification of a novel SCN5A mutation The genetic analysis of the patient identified a novel SCN5A mutation, R1632C (Figure 2A). The SCN5A R1632C mutation was absent in our 200 control alleles, and the R1632 position in SCN5A was found to be highly conserved among different species (Figure 2B), suggesting that it is a diseasecausing mutation. The R1632 position is located in DIV-S4
Heart Rhythm, Vol 0, No 0, Month 2015 (Figure 2C), where it is postulated to function as a voltage sensor.14–16,29,30 The patient’s mother also carries the same mutation.
Biophysical characterization of the SCN5A R1632C mutation To determine the functional consequences of the SCN5A R1632C mutation, we engineered the mutation, then expressed WT or the mutant (R1632C) in tsA201 cells in combination with hβ1 subunit, and finally recorded wholecell INa. R1632C exhibited typical INa resembling that of WT (Figures 3A and 3B). The peak INa density, measured at 20 mV from a holding potential of 120 mV, for R1632C was significantly lower than that for WT (Table 1 and Figure 3C), while the INa density for R1632C from a holding potential of 150 mV was not different from that for WT (Online Supplemental Table S1 and Online Supplemental Figure S2). Of note, R1632C INa from a holding potential of 90 mV was low. No significant changes were observed in steadystate activation (Table 1 and Figure 3D) or fast inactivation rate (Figure 3E). In contrast, the steady-state inactivation curve, assessed using prepulses of 500 ms, for R1632C was remarkably shifted to hyperpolarizing potentials ( 25 mV) as compared with that for WT (Table 1) (Figure 4A). A hyperpolarizing shift in the steady-state inactivation curve for R1632C could be attributable to enhanced fast and/or slow/intermediate inactivation.31 In order to assess voltage dependence of fast inactivation, brief prepulses of 10 ms were applied to avoid slow inactivation (Figure 4B). Similar to the results obtained after applying prepulses of 500 ms, the steady-state fast inactivation curve for R1632C was remarkably shifted to hyperpolarizing potentials compared with that for WT (Table 1 and Figure 4B), thus suggesting that R1632C may have an enhanced fast-inactivated state
Figure 2 Identification of an SCN5A R1632C mutation located in the DIV-S4 voltage sensor. A: A sequence electropherogram showing the detected R1632C mutation in SCN5A. Nucleotide and amino acid substitutions are indicated below the sequence. B: A comparison of the amino acid sequences of DIV-S4 in SCN5A among different species. The arrow indicates the position of R1632 in SCN5A. C: Location of R1632C mutation (red filled circle) in the predicted topology of SCN5A. DIV-S4 ¼ fourth segment of domain IV.
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Nakajima et al 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 T2 456 457 458 459 460Q17 461 462 463 464 465 466 467 468 469
Stabilized Fast Inactivation of INa by SCN5A R1632C
5
Figure 3 Reduced sodium current density for SCN5A R1632C without affecting the steady-state activation and fast inactivation rates. A and B: Representative current tracings for WT (panel A) and R1632C (panel B). C: The current-voltage relationship for WT (filled squares, n ¼ 15) and R1632C (open circles, n ¼ 14). Peak currents obtained by the pulse protocol were normalized to cell capacitances. D: The voltage dependence of activation for WT (filled squares, n ¼ 15) and R1632C (open circles, n ¼ 14). E: The time constant of the voltage dependence of fast inactivation rate for WT (filled squares, n ¼ 15) and R1632C (open circles, n ¼ 14). WT ¼ wild type.
mV for 20 ms (P1) was applied. Recovery from fast inactivation, fitted with a single exponential function, for R1632C was also significantly delayed compared with that for WT (Table 2) (Figure 4D), which also suggested that R1632C had an enhanced fast-inactivated state stability. We also examined if recovery from fast inactivation for R1632C was accelerated by using a holding potential of 150 mV. Recovery from fast inactivation for both R1632C and WT was accelerated to the same degree, but recovery from fast inactivation for R1632C did not reach the same level as did recovery for WT (Table 2 and Figure 4). Development into the inactivated state was assessed using a double pulse protocol (Online Supplemental Figure S3A). A 10-ms repolarization pulse of 120 mV was interposed between varying intervals of prepulse (P1) and test pulse (P2) to allow recovery from fast inactivation. R1632C
stability. We could not assess voltage dependence of slow inactivation because of a delayed recovery from fast inactivation for R1632C (refer to below). Recovery from inactivation was assessed using a conventional double-pulse protocol with applying a depolarizing prepulse of 20 mV for 500 ms (P1) and varying hyperpolarizing interpulse before test pulse (P2) (Figure 4C). Recovery from inactivation for WT was well-fitted with a double exponential function, but that for R1632C was wellfitted with a single, rather than a double, exponential function. As shown in Figure 4C, recovery from inactivation for R1632C was apparently delayed compared with that for WT (Table 2). The assessment of recovery from inactivation using this protocol may have contained recoveries from both fast and slow/intermediate inactivation. To assess recovery from fast inactivation, a brief depolarizing prepulse of 20 Table 1
Parameters of activation and steady-state inactivation for WT and R1632C Activation INa density (pA/pF) at 20 mV
V1/2 (mV)
Steady-state inactivationv K (mV)
V1/2 (mV)
K (mV)
WT 672 ⫾ 90 (n ¼ 15) 38.7 ⫾ 0.8 6.4 ⫾ 0.3 85.9 ⫾ 0.6 (n ¼ 17) 4.8 ⫾ 0.1 R1632C 433 ⫾ 52* (n ¼ 14) 36.5 ⫾ 1.0 7.2 ⫾ 0.3 110.7 ⫾ 0.8† (n ¼ 16) 6.0 ⫾ 0.1† WT ¼ wild type. * P o .05 vs WT. † P o .01 vs WT.
Steady-state fast inactivation V1/2 (mV)
K (mV)
58.2 ⫾ 0.9 (n ¼ 8) 6.5 ⫾ 0.5 80.2 ⫾ 1.6† (n ¼ 8) 12.6 ⫾ 0.3†
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Heart Rhythm, Vol 0, No 0, Month 2015
Figure 4 A hyperpolarizing shift in the steady-state availability and a delayed recovery from inactivation for SCN5A R1632C. A: The voltage dependence of steady-state inactivation for WT (filled squares, n ¼ 17) and R1632C (open circles, n ¼ 16). B: The voltage dependence of steady-state fast inactivation for WT (filled squares, n ¼ 8) and R1632C (open circles, n ¼ 8). C: The time course of recovery from inactivation for WT (filled squares, n ¼ 15) and R1632C (open circles, n ¼ 16). D: Time course of recovery from fast inactivation for WT using a holding potential of 120 mV (filled squares, n ¼ 8) and 150 mV (filled reverse triangles, n ¼ 11) and that for R1632C using a holding potential of 120 mV (open circles, n ¼ 8) and 150 mV (open reverse triangles, n ¼ 11). WT ¼ wild type.
showed a pronounced reduction in the P2/P1 ratio (an enhanced development into the inactivated state) (Online Supplemental Figure S3A), which may be primarily due to an impaired recovery from inactivation because less than 10% of R1632C channels recover during a 10-ms repolarizing pulse (Figures 4C and 4D) and the fast inactivation rate for R1632C was not different from that for WT (Figure 3E). To assess activity-dependent loss of INa availability, depolarizing potentials of 20 mV for 500 ms at cycle lengths of 0.52, 1, or 2 seconds were applied (Online Supplemental Figure S3B). At a long cycle length of 2 seconds, both WT and R1632C INa retained the current amplitudes during the train of stimuli. At a cycle length of 1 Table 2
second, while WT INa retained the current amplitudes during the train of stimuli, R1632C INa decreased to 85% of the initial current amplitude at the second pulse and remained almost constant throughout the following train of stimuli. At a short cycle length of 0.52 seconds, while WT INa gradually decreased during successive stimuli (96% of the initial current amplitude at the second pulse and 78% at the twentieth pulse), R1632C INa decreased to 0.08% of the initial current amplitude at the second pulse and remained almost constant throughout the following train of stimuli. Although R1632C INa decreased current amplitudes at the second pulse at shorter cycle lengths, it remained stable after the second pulse, suggesting that fast inactivation, rather than
Parameters of recovery from inactivation for WT and R1632C Recovery from inactivation HP ¼ 120 mV
WT R1632C
Recovery from fast inactivation HP ¼ 120 mV
HP ¼ 150 mV
τ fast (ms)
τ slow (ms)
τ (ms)
Τ (ms)
3.1 ⫾ 0.2 (n ¼ 15) 254.5 ⫾ 7.4* (n ¼ 16)
36.2 ⫾ 3.4 (n ¼ 15) —
3.7 ⫾ 0.3 (n ¼ 8) 246.7 ⫾ 14.3* (n ¼ 8)
1.4 ⫾ 0.1 (n ¼ 11) 71.1 ⫾ 3.2* (n ¼ 11)
HP ¼ holding potential; WT ¼ wild type. * P o .01 vs WT.
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Stabilized Fast Inactivation of INa by SCN5A R1632C
slow/intermediate inactivation, affected its channel availability. Therefore, to assess activity-dependent loss of INa availability implying fast inactivation, brief depolarizing potentials of 20 mV for 20 ms at cycle lengths of 0.1, 0.5, or 2 seconds were applied (Online Supplemental Figure S3C). WT INa remained constant during successive stimuli at all cycle lengths. However, at cycle lengths of 0.5 and 0.1 seconds, R1632C INa at the second pulses decreased to 85% and 0.08% of the initial current amplitude, respectively, but remained almost constant throughout the following train of stimuli. These findings suggest that activity-dependent loss of availability for R1632C may be due to an enhanced fastinactivated state stability. Since some SCN5A mutations in DIV-S4 show persistent late INa during prolonged depolarization, tetrodotoxinsensitive currents were compared between R1632C and WT. The amplitude of tetrodotoxin-sensitive currents for R1632C was not significantly different from that for WT (Online Supplemental Figure S4).
Discussion Functional roles of S4 segments in hSkM1/SCN4A, the α subunit of skeletal muscle voltage-gated sodium channels, have been examined intensely. S4 segments in each of the 4 domains are postulated to function as a voltage sensor.14–16 Since the α subunits are composed of homologous but nonidentical domains, DI-DIV, there are distinct functional roles for the specific domains.15,16 The recovery from fast inactivation of INa was reported to be associated with a slow component in the time course of gating charge during repolarization, resulting from the slow movement of S4 segments in domains III and IV, but not in domains I and II.15 We observed that R1632C had an enhanced fast-inactivated state stability due to an impaired recovery from fast inactivation, resulting in a remarkable hyperpolarizing shift in the steady-state inactivation curve and a severe activity–dependent loss of INa availability, without affecting the fast inactivation rate. These findings are in agreement with the previous report demonstrating that hSkM1/SCN4A R1457C, a mutation at the same position as R1632C in SCN5A, showed a pronounced hyperpolarizing shift in the steady-state inactivation curve without affecting the inactivation rate.32 Therefore, our data imply that impaired recovery from fast inactivation for R1632C may be associated with the gating charge immobilization of DIV-S4 in SCN5A during depolarization and subsequent repolarization as was the gating charge immobilization of DIV-S4 in hSkM1/SCN4A.15,32 SCN5A mutations can cause loss of function of INa by multiple mechanisms, including a production of nonfunctional channels, a trafficking defect of and kinetic changes in INa such as a negative shift in steady-state inactivation, a positive shift in steady-state activation, an enhanced fast inactivation, and an enhanced intermediate/slow inactivation.4,6,31,33 In addition to the kinetic changes in R1632C INa, a trafficking defect of R1632C might cause loss of function of INa. In our experiments, INa density for R1632C, measured
7
at 20 mV test potential from a holding potential of 120 mV, was significantly low compared with that for WT. However, INa density measured at 20 mV from a holding potential of 150 mV was comparable between R1632C and WT (Online Supplemental Table S1 and Online Supplemental Figure S2), indicating that reduced INa density for R1632C was not caused by a trafficking defect but rather by an enhanced fast-inactivated state stability. Loss of function of INa underlies the cause of both BrS and supraventricular arrhythmias. SCN5A mutations that cause loss of function of INa in the ventricle are associated with approximately 20% of BrS cases.2,4 Moreover, loss of function of INa in the atrium may lead to a failure of impulse conduction from the sinus node into the adjacent myocardium (exit block), which may be a plausible mechanism of SND caused by SCN5A mutations6 and may also form the substrate for the reentry of supraventricular arrhythmias, such as AT. R1632C INa availability in both the atrium and the ventricle, whose resting membrane potentials are approximately 90 mV, is likely to be substantially reduced (Online Supplemental Figure S2), although the current reduction caused by the hyperpolarizing shift in voltage dependence of inactivation might be enhanced in our experimental condition.28 A pronounced reduction of INa in both the atrium and the ventricle may be associated with multiple electrophysiological phenotypes (BrS, AT, and SND) observed in our patient. An increased HV interval and QRS duration could also be attributable to the R1632C mutation. Of note, repetitive depolarizing pulses rapidly attenuated R1632C INa compared with WT INa (Online Supplemental Figures S3B and S3C), and β-adrenergic stimulation augments the frequency-dependent reduction of INa.28 These findings may explain the clinical phenotypes of our patient such that AT preferentially occurred during exercise or was induced by high-frequency atrial stimuli during isoproterenol infusion. Several SCN5A mutations in DIV-S4 have previously been identified in patients with BrS or LQTS alone or BrS with SSS or LQTS, and some of these mutations have been functionally characterized. The functional consequences of R1623Q and R1644H, identified in patients with LQTS, were gain of function of INa due to a delayed fast inactivation rate and/or increased late INa.20–22 In contrast, R1629Q, identified in patients with BrS, resulted in loss of function of INa with a hyperpolarizing shift in the steady-state inactivation curve, an increased fast inactivation rate, and a delayed recovery from inactivation.23 However, although Zeng et al23 insisted that functional abnormality of R1629Q was an enhanced intermediate inactivation, whether fast inactivation is also enhanced should be examined. Intriguingly, R1626H, identified in a patient with AF, exhibited a positive shift in steady-state activation and a negative shift in steadystate inactivation, together with a decreased fast inactivation rate.8 The functional consequence of R1632H, identified in patients with SND or first-degree heart block, resembles that of R1632C identified in our patient.6 R1632H exhibited a large hyperpolarizing shift in the steady-state inactivation curve, a delayed fast inactivation rate, and a delayed recovery from fast inactivation (enhanced fast inactivation),
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suggesting that R1632H had a mixture of loss and gain of function with a predominance of functional loss.6 R1632H carriers did not have spontaneous BrS phenotype. A provocative test should be performed to confirm whether R1632H carriers manifest BrS phenotype.
Conclusion We identified a novel SCN5A mutation, R1632C, located in the DIV-S4 voltage sensor, in a young male patient with AT, SND, and BrS. The functional consequence of SCN5A R1632C was an enhanced fast-inactivated state stability of INa because of a pronounced impairment of recovery from fast inactivation, resulting in a severe activity–dependent loss of INa availability, which may explain the phenotypic manifestation observed in our patient. These findings provide valuable insight into the pathophysiological role of the mutation.
Acknowledgments We thank Naomasa Makita, XX (Nagasaki University), for kindly providing us with hH1 and hβ1 plasmids, and Kenshi Hayashi, XX (Kanazawa University), for valuable advices on performing patch-clamp methods. We also thank Yukiyo Tosaka, XX, and Takako Kobayashi, XX, for their helpful technical assistances.
Appendix Supplementary data Supplementary material cited in this article is available online at http://dx.doi.org/10.1016/j.hrthm.2015.05.032.
References 1. Brugada P, Brugada J. Right bundle branch block, persistent ST segment elevation and sudden cardiac death: a distinct clinical and electrocardiographic syndrome. A multicenter report. J Am Coll Cardiol 1992;20:1391–1396. 2. Shimizu W. Update of diagnosis and management of inherited cardiac arrhythmias. Circ J 2013;77:2867–2872. 3. Chen Q, Kirsch GE, Zhang D, et al. Genetic basis and molecular mechanism for idiopathic ventricular fibrillation. Nature 1998;392:293–296. 4. Zimmer T, Surber R. SCN5A channelopathies—an update on mutations and mechanisms. Prog Biophys Mol Biol 2008;98:120–136. 5. Berne P, Brugada J. Brugada syndrome 2012. Circ J 2012;76:1563–1571. 6. Benson DW, Wang DW, Dyment M, Knilans TK, Fish FA, Strieper MJ, Rhodes TH, George AL Jr. Congenital sick sinus syndrome caused by recessive mutations in the cardiac sodium channel gene (SCN5A). J Clin Invest 2003;112: 1019–1028. 7. Makiyama T, Akao M, Shizuta S, et al. A novel SCN5A gain-of-function mutation M1875T associated with familial atrial fibrillation. J Am Coll Cardiol 2008;52:1326–1334. 8. Olesen MS, Yuan L, Liang B, et al. High prevalence of long QT syndromeassociated SCN5A variants in patients with early-onset lone atrial fibrillation. Circ Cardiovasc Genet 2012;5:450–459. 9. Watanabe H, Darbar D, Kaiser DW, Jiramongkolchai K, Chopra S, Donahue BS, Kannankeril PJ, Roden DM. Mutations in sodium channel β1- and β2-subunits associated with atrial fibrillation. Circ Arrhythm Electrophysiol 2009;2:268–275. 10. Ellinor PT, Nam EG, Shea MA, Milan DJ, Ruskin JN, MacRae CA. Cardiac sodium channel mutation in atrial fibrillation. Heart Rhythm 2008;5:99–105.
Heart Rhythm, Vol 0, No 0, Month 2015 11. Bennett PB, Yazawa K, Makita N, George AL Jr. Molecular mechanism for an inherited cardiac arrhythmia. Nature 1995;376:683–685. 12. Makita N, Behr E, Shimizu W, et al. The E1784K mutation in SCN5A is associated with mixed clinical phenotype of type 3 long QT syndrome. J Clin Invest 2008;118:2219–2229. 13. Veldkamp MW, Viswanathan PC, Bezzina C, Baartscheer A, Wilde AA, Balser JR. Two distinct congenital arrhythmias evoked by a multidysfunctional Naþ channel. Circ Res 2000;86:E91–E97. 14. Yang N, Horn R. Evidence for voltage-dependent S4 movement in sodium channels. Neuron 1995;15:213–218. 15. Cha A, Ruben PC, George AL Jr, Fujimoto E, Bezanilla F. Voltage sensors in domains III and IV, but not I and II, are immobilized by Naþ channel fast inactivation. Neuron 1999;22:73–87. 16. Bezanilla F. The voltage sensor in voltage-dependent ion channels. Physiol Rev 2000;80:555–592. 17. McNair WP, Sinagra G, Taylor MR, Di Lenarda A, Ferguson DA, Salcedo EE, Slavov D, Zhu X, Caldwell JH, Mestroni L; Familial Cardiomyopathy Registry Research Group. SCN5A mutations associate with arrhythmic dilated cardiomyopathy and commonly localize to the voltage-sensing mechanism. J Am Coll Cardiol 2011;57:2160–2168. 18. Nair K, Pekhletski R, Harris L, Care M, Morel C, Farid T, Backx PH, Szabo E, Nanthakumar K. Escape capture bigeminy: phenotypic marker of cardiac sodium channel voltage sensor mutation R222Q. Heart Rhythm 2012;9: 1681–1688. e1681. 19. Beckermann TM, McLeod K, Murday V, Potet F, George AL Jr. Novel SCN5A mutation in amiodarone-responsive multifocal ventricular ectopy-associated cardiomyopathy. Heart Rhythm 2014;11:1446–1453. 20. Makita N, Shirai N, Nagashima M, Matsuoka R, Yamada Y, Tohse N, Kitabatake A. A de novo missense mutation of human cardiac Naþ channel exhibiting novel molecular mechanisms of long QT syndrome. FEBS Lett 1998;423:5–9. 21. 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. 22. Dumaine R, Wang Q, Keating MT, Hartmann HA, Schwartz PJ, Brown AM, Kirsch GE. Multiple mechanisms of Naþ channel–linked long-QT syndrome. Circ Res 1996;78:916–924. 23. Zeng Z, Zhou J, Hou Y, Liang X, Zhang Z, Xu X, Xie Q, Li W, Huang Z. Electrophysiological characteristics of a SCN5A voltage sensors mutation R1629Q associated with Brugada syndrome. PLoS One 2013;8:e78382. 24. Nakajima T, Kaneko Y, Saito A, Irie T, Tange S, Iso T, Kurabayashi M. Identification of six novel SCN5A mutations in Japanese patients with Brugada syndrome. Int Heart J 2011;52:27–31. 25. Nakajima T, Wu J, Kaneko Y, Ashihara T, Ohno S, Irie T, Ding WG, Matsuura H, Kurabayashi M, Horie M. KCNE3 T4A as the genetic basis of Brugada-pattern electrocardiogram. Circ J 2012;76:2763–2772. 26. Imai M, Nakajima T, Kaneko Y, Niwamae N, Irie T, Ota M, Iijima T, Tange S, Kurabayashi M. A novel KCNQ1 splicing mutation in patients with forme fruste LQT1 aggravated by hypokalemia. J Cardiol 2014;64:121–126. 27. Nakajima T, Davies SS, Matafonova E, Potet F, Amarnath V, Tallman KA, Serwa RA, Porter NA, Balser JR, Kupershmidt S, Roberts LJ III. Selective γketoaldehyde scavengers protect NaV1.5 from oxidant-induced inactivation. J Mol Cell Cardiol 2010;48:352–359. 28. Tsurugi T, Nagatomo T, Abe H, Oginosawa Y, Takemasa H, Kohno R, Makita N, Makielski JC, Otsuji Y. Differential modulation of late sodium current by protein kinase A in R1623Q mutant of LQT3. Life Sci 2009;84:380–387. 29. Sheets MF, Hanck DA. Charge immobilization of the voltage sensor in domain IV is independent of sodium current inactivation. J Physiol 2005;563:83–93. 30. Goldschen-Ohm MP, Capes DL, Oelstrom KM, Chanda B. Multiple pore conformations driven by asynchronous movements of voltage sensors in a eukaryotic sodium channel. Nat Commun 2013;4:1350. 31. Wang DW, Makita N, Kitabatake A, Balser JR, George AL Jr. Enhanced Naþ channel intermediate inactivation in Brugada syndrome. Circ Res 2000;87: E37–E43. 32. Yang N, George AL Jr, Horn R. Molecular basis of charge movement in voltagegated sodium channels. Neuron 1996;16:113–122. 33. Makiyama T, Akao M, Tsuji K, et al. High risk for bradyarrhythmic complications in patients with Brugada syndrome caused by SCN5A gene mutations. J Am Coll Cardiol 2005;46:2100–2106.
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Stabilized Fast Inactivation of INa by SCN5A R1632C
CLINICAL PERSPECTIVES
Q14
SCN5A, which encodes the α subunit of cardiac voltage-gated sodium channels, consists of 4 homologous but nonidentical domains, DI–DIV. The fourth segment (S4) in each domain is postulated to function as a voltage sensor, but each S4 segment is thought to have distinct functional roles. SCN5A mutations in each S4 segment and even those in the same S4 segment may cause diverse functional abnormalities and phenotypic manifestations. We identified a novel SCN5A mutation, R1632C, located in the DIV-S4 voltage sensor, in a young male patient who had a syncopal episode during exercise and presented with multiple electrophysiological phenotypes including atrial tachycardia during exercise, sinus node dysfunction, and Brugada syndrome. Functional analysis of the SCN5A R1632C mutation expressed in tsA201 cells using a patch-clamp method revealed that the R1632C mutation caused a loss of function of sodium currents by an enhanced fast-inactivated state stability due to a pronounced impairment of recovery from fast inactivation, resulting in a severe activity–dependent loss of sodium current availability. The obtained kinetic changes in the R1632C mutation can be the cause of multiple electrophysiological phenotypes observed in our patient. These findings provided us with valuable insight into the structure-function relationship of the DIV-S4 voltage sensor in SCN5A and shed light on the pathophysiological role of the R1632C mutation. Further studies are necessary to fully clarify the structure-function relationship of the voltage sensor in SCN5A.
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Enhanced fast-inactivated state stability of cardiac sodium channels by a novel voltage sensor SCN5A mutation, R1632C, as a cause of atypical Brugada syndrome Tadashi Nakajima, MD, PhD, Yoshiaki Kaneko, MD, PhD, Akihiro Saito, MD, PhD, Masaki Ota, MD, Takafumi Iijima, MD, PhD, Masahiko Kurabayashi, MD, PhD From the Department of Medicine and Biological Science, Gunma University Graduate School of Medicine, Maebashi, Japan. BACKGROUND Mutations in SCN5A, which encodes the cardiac voltage-gated sodium channels, can be associated with multiple electrophysiological phenotypes. A novel SCN5A R1632C mutation, located in the domain IV-segment 4 voltage sensor, was identified in a young male patient who had a syncopal episode during exercise and presented with atrial tachycardia, sinus node dysfunction, and Brugada syndrome. OBJECTIVE We sought to elucidate the functional consequences of the R1632C mutation. METHODS The wild-type (WT) or R1632C SCN5A mutation was coexpressed with β1 subunit in tsA201 cells, and whole-cell sodium currents (INa) were recorded using patch-clamp methods. RESULTS INa density, measured at 20 mV from a holding potential of 120 mV, for R1632C was significantly lower than that for WT (R1632C: 433 ⫾ 52 pA/pF, n ¼ 14; WT: 672 ⫾ 90 pA/pF, n ¼ 15; P o .05); however, no significant changes were observed in the steady-state activation rate and fast inactivation rate. The steadystate inactivation curve for R1632C was remarkably shifted to hyperpolarizing potentials compared with that for WT (R1632C: V1/2 ¼ 110.7 ⫾ 0.8 mV, n ¼ 16; WT: V1/2 ¼ 85.9 ⫾ 2.5 mV, n ¼ 17; P o .01). The steady-state fast inactivation curve for R1632C was also
Introduction Brugada syndrome (BrS), characterized by ST-segment elevations in the right precordial electrocardiographic (ECG) leads and a high incidence of syncope or sudden death due to ventricular tachyarrhythmias, is an inherited arrhythmia syndrome that predominantly affects adult male patients.1,2 Approximately 30% of BrS cases are attributed to defects in genes encoding the cardiac ion channels or their modifiers.1–5 In contrast, supraventricular arrhythmias, such This work was supported, in part, by JSPS KAKENHI (grant no. 26461056). Address reprint requests and correspondence: Dr Tadashi Nakajima, Department of Medicine and Biological Science, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan. E-mail address: [email protected].
1547-5271/$-see front matter B 2015 Heart Rhythm Society. All rights reserved.
shifted to the same degree. Recovery from fast inactivation after a 20ms depolarizing pulse for R1632C was remarkably delayed compared with that for WT (R1632C: τ ¼ 246.7 ⫾ 14.3 ms, n ¼ 8; WT: τ ¼ 3.7 ⫾ 0.3 ms, n ¼ 8; P o .01). Repetitive depolarizing pulses at various cycle lengths greatly attenuated INa for R1632C than that for WT. CONCLUSION R1632C showed a loss of function of INa by an enhanced fast-inactivated state stability because of a pronounced impairment of recovery from fast inactivation, which may explain the phenotypic manifestation observed in our patient. KEYWORDS SCN5A; Mutation; Voltage sensor; Sodium currents; Atrial tachycardia; Sinus node dysfunction; Brugada syndrome ABBREVIATIONS AF ¼ atrial fibrillation; AT ¼ atrial tachycardia; BrS ¼ Brugada syndrome; cDNA ¼ complementary DNA; DIV-S4 ¼ fourth segment of domain IV; ECG ¼ electrocardiogram/ electrocardiographic; INa ¼ sodium current; IRES ¼ internal ribosome entry site; LQTS ¼ long QT syndrome; PCR ¼ polymerase chain reaction; SND ¼ sinus node dysfunction; WT ¼ wild type (Heart Rhythm 2015;0:-1–9) rights reserved.
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2015 Heart Rhythm Society. All
as sinus node dysfunction (SND), atrial fibrillation (AF), and atrial tachycardia (AT), are common electrophysiological disturbances becoming prevalent as one grows older. However, these supraventricular arrhythmias, especially recognized in younger populations, could also be associated with defects of cardiac ion channel or its modifying genes.6–9 Mutations in SCN5A, which encodes the α subunit of the cardiac voltage-gated sodium channels, are associated with several phenotypically distinct electrophysiological disturbances in both the ventricle and the atrium, including long QT syndrome (LQTS), BrS, SND, and AF.3,6,10 Loss of function of cardiac sodium currents (INa) during the early phase of action potential can lead to BrS, SND, and AF.3,6,10 In contrast, gain of function of INa during the late phase of action potential can cause LQTS and AF.7,11 Intriguingly, a http://dx.doi.org/10.1016/j.hrthm.2015.05.032
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2 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94Q4 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125Q5 126Q6 127
compromised loss and gain of function of INa is reported to be associated with AF8 or with mixed phenotypes of BrS, LQTS, and SND.12,13 We recently encountered a young male patient who had a syncopal episode during exercise and presented with multiple electrophysiological disturbances including AT during exercise, SND, and BrS, in whom we identified a novel SCN5A mutation, R1632C. R1632C in SCN5A is located in the fourth segment (S4) of domain IV (DIV) (DIV-S4), where it is postulated to function as a voltage sensor.14–16 Since SCN5A is composed of 4 homologous but nonidentical domains, DI–DIV, there are distinct functional roles of each S4 segment.16 Thus, SCN5A mutations in each S4 segment and even those in the same S4 segment may cause diverse functional abnormalities and phenotypic manifestations.6,8,17–23 It has been reported that recovery from fast inactivation of INa may be associated with immobilized gating charge caused by the slow movement of the S4 segments in domains III and IV, but not in domains I and II,15 which suggested that recovery from fast inactivation of INa would be impaired by R1632C mutation. Therefore, we examined functional abnormalities of the SCN5A R1632C mutation using whole-cell patch-clamp methods. The obtained kinetic changes in R1632C INa were thought to be associated with multiple electrophysiological phenotypes observed in our patient.
Methods Clinical data Appropriate approval from the institutional review board and written informed consent for the genetic analysis from the patient and his mother were obtained. The pilsicainide provocation test was performed as described previously.24
Mutation analysis Genomic DNA was extracted from peripheral blood lymphocytes using the QIAamp DNA Blood Midi Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. All coding exons of KCNQ1 (NM_000218.2), KCNH2 (NM_000238.2), and SCN5A (NM_198056.2) and their splice sites were amplified by polymerase chain reaction (PCR) using primers flanking the intronic sequences as reported previously.25,26 The PCR products were purified using the QIAquick PCR Purification Kit (QIAGEN) and directly sequenced using an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA). The mutation was analyzed twice by independent PCR amplification and sequencing reactions.
Mutagenesis and heterologous expression Wild-type (WT) human heart sodium channel complementary DNA (cDNA) (NM_000335) subcloned into pcDNA3.1 vector (hH1-pcDNA3.1) and a plasmid (pGFP-IRES-hβ1) encoding both GFP and the human β1 subunit (hβ1) under the control of a single CMV promoter with the 2 coding regions separated by a viral internal ribosome entry
Heart Rhythm, Vol 0, No 0, Month 2015 site (IRES) were kindly provided by Prof Naomasa Makita (Nagasaki University).12 Site-directed mutagenesis (R1632C-pcDNA3.1) was performed by the QuikChangeII Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA) according to the manufacturer’s instructions. hH1-pcDNA3.1 (0.5 μg) or R1632C-pcDNA3.1 (0.5 μg) in combination with pGFP-IRES-hβ1 (0.5 μg) was transiently transfected into tsA201 cells growing on 6-cm plates using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and maintained in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in a 5% CO2 incubator at 371C for 24–36 hours before current recordings. Cells exhibiting green fluorescence were chosen for the current recordings.
Electrophysiology Membrane INa densities were recorded using whole-cell patch-clamp methods at room temperature (231C–251C) as described previously.27 The bath solution for recording membrane currents contained 145 mM NaCl, 4 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 5 mM glucose (pH 7.35 with NaOH), and the pipette solution contained 10 mM NaF, 110 mM CsF, 20 mM CsCl, 10 mM EGTA, 10 mM HEPES (pH 7.35 with CsOH). The electrode resistance ranged from 1.1 to 2.0 MΩ. Data acquisition was done using an Axopatch 200B amplifier and pCLAMP software version 10.3 (Molecular Devices, Sunnyvale, CA). Currents were acquired at 20–50 kHz and low-pass filtered at 5 kHz using an analog-to-digital interface (Digidata 1440A acquisition system, Molecular Devices). To avoid the possible time-dependent shift of activation and steady-state inactivation curves,28 all pulse protocols were applied more than 5 minutes after membrane rupture. Therefore, the time-dependent shift became negligible. Recordings from the cells exhibiting peak current amplitudes less than 0.8 nA were excluded from the analysis to avoid potential endogenous current contamination. Cells exhibiting large currents (peak current 4 10 nA) were also excluded if the voltage control was compromised. All pulse protocols are described in figures or figure legends.
Data analysis Pulse protocols were applied from a holding potential of 120 mV, unless otherwise indicated. Current densities at each test potential were obtained by dividing the INa by cell capacitance. The steady-state activation and steady-state inactivation curves were fitted with Boltzmann functions of the following forms: y ¼ 1 1/{1 þ exp[(Vm V1/2)/K]} or y ¼ 1/{1 þ exp[(Vm V1/2)/K]}, respectively, where y is the relative current, Vm is the membrane potential, V1/2 is the voltage at which half of the channels are available to open, and K is the slope factor. The time course of fast inactivation was fitted with a single exponential function of the following form: I(t)/Imax ¼ A0 þ A1 exp(t/τ), where A is the amplitude, τ is the time constant, I is the current, and t is the time. Recovery from inactivation was fitted with a single
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Nakajima et al 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 P 229 R 230 I 231 N 232 T 233 & W 234 E 235 B 236 4 237 C 238 / 239 F Q16 240 P O 241
Stabilized Fast Inactivation of INa by SCN5A R1632C
or double exponential function of the following form: I(t)/ Imax ¼ A0 þ A1 exp(t/τ) or I(t)/Imax ¼ A0 þ A1 exp(t/τf) þ A2 exp(t/τs), where τf and τs are the fast and slow components of τ, respectively.
Statistical analysis All data are expressed as mean ⫾ standard error, and statistical comparisons were made using the unpaired Student t test, with P o .05 considered to be statistically significant. In some figures, the standard error bars are smaller than the data symbols.
3
Results Phenotypic characterization A 17-year-old male patient, who had a history of syncope after having palpitations during exercise 1 year ago, was transferred to us because he had again lost consciousness after having palpitations for several minutes during a longdistance race. His 12-lead ECG showed AT with 2-to-1 conduction (heart rate 110 beats/min) (Figure 1A), which changed to 1-to-1 conduction (heart rate 220 beats/min) (Figure 1B). At that time, he had palpitations accompanied by faintness, suggesting that AT with 1-to-1 conduction may be a cause of syncope. The next day, AT spontaneously
Figure 1 Multiple electrophysiological phenotypes in the patient. A: A 12-lead ECG recorded just after arrival showed atrial tachycardia (AT) with 2-to-1 conduction. Red arrows indicate P waves. B: The 12-lead ECG changed to AT with 1-to-1 conduction. C: The ECG monitor recording showing sinus arrest of 5.7 seconds. D and E: The 12-lead ECG before (panel D) and precordial ECG leads after (panel E) the pilsicainide provocation test. ECG ¼ electrocardiogram; ics ¼ intercostal space.
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4 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 F2 329 330Q10 331 332 333 334 335 336 337 338 339 340 341 342 P 343 R 344 I 345 N 346 T 347 & W 348 E 349 B 350 4 351 C 352 / 353 F 354 P O 355
returned to sinus rhythm. An ECG monitor recording revealed that he had SND (sinus arrest of 5.7 seconds) (Figure 1C). Lead V2 during sinus rhythm (heart rate 68 beats/min; PQ interval 200 ms; QRS duration 118 ms) showed a saddleback-type ST-segment elevation (Figure 1D), and a coved-type ST-segment elevation in the right precordial ECG leads appeared after provocation with pilsicainide (50 mg) (Figure 1E). The echocardiogram and conventional left heart catheterization, including acetylcholine provocation to the coronary arteries, revealed no structural heart disease. A cardiac electrophysiological study was also performed. Although the AH interval (95 ms) was not prolonged, the HV interval (73 ms) was slightly prolonged. Ventricular tachyarrhythmias were not induced by up to 3 ventricular extrastimuli at both the right ventricular apex and the right ventricular outflow tract. AT was induced by high-frequency atrial stimuli (230 beats/min) during isoproterenol infusion (50 μg/h), and it was thought to originate from the left atrium. Considering the patient’s young age, we did not perform catheter ablation. Under exercise restrictions, he has currently not experienced palpitations or syncope for more than 1 year. His mother also had a history of syncope during a longdistance race when she was a high-school student. Her ECG exhibited a Brugada pattern (coved-type) after provocation with pilsicainide (Online Supplemental Figure S1).
Identification of a novel SCN5A mutation The genetic analysis of the patient identified a novel SCN5A mutation, R1632C (Figure 2A). The SCN5A R1632C mutation was absent in our 200 control alleles, and the R1632 position in SCN5A was found to be highly conserved among different species (Figure 2B), suggesting that it is a diseasecausing mutation. The R1632 position is located in DIV-S4
Heart Rhythm, Vol 0, No 0, Month 2015 (Figure 2C), where it is postulated to function as a voltage sensor.14–16,29,30 The patient’s mother also carries the same mutation.
Biophysical characterization of the SCN5A R1632C mutation To determine the functional consequences of the SCN5A R1632C mutation, we engineered the mutation, then expressed WT or the mutant (R1632C) in tsA201 cells in combination with hβ1 subunit, and finally recorded wholecell INa. R1632C exhibited typical INa resembling that of WT (Figures 3A and 3B). The peak INa density, measured at 20 mV from a holding potential of 120 mV, for R1632C was significantly lower than that for WT (Table 1 and Figure 3C), while the INa density for R1632C from a holding potential of 150 mV was not different from that for WT (Online Supplemental Table S1 and Online Supplemental Figure S2). Of note, R1632C INa from a holding potential of 90 mV was low. No significant changes were observed in steadystate activation (Table 1 and Figure 3D) or fast inactivation rate (Figure 3E). In contrast, the steady-state inactivation curve, assessed using prepulses of 500 ms, for R1632C was remarkably shifted to hyperpolarizing potentials ( 25 mV) as compared with that for WT (Table 1) (Figure 4A). A hyperpolarizing shift in the steady-state inactivation curve for R1632C could be attributable to enhanced fast and/or slow/intermediate inactivation.31 In order to assess voltage dependence of fast inactivation, brief prepulses of 10 ms were applied to avoid slow inactivation (Figure 4B). Similar to the results obtained after applying prepulses of 500 ms, the steady-state fast inactivation curve for R1632C was remarkably shifted to hyperpolarizing potentials compared with that for WT (Table 1 and Figure 4B), thus suggesting that R1632C may have an enhanced fast-inactivated state
Figure 2 Identification of an SCN5A R1632C mutation located in the DIV-S4 voltage sensor. A: A sequence electropherogram showing the detected R1632C mutation in SCN5A. Nucleotide and amino acid substitutions are indicated below the sequence. B: A comparison of the amino acid sequences of DIV-S4 in SCN5A among different species. The arrow indicates the position of R1632 in SCN5A. C: Location of R1632C mutation (red filled circle) in the predicted topology of SCN5A. DIV-S4 ¼ fourth segment of domain IV.
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Stabilized Fast Inactivation of INa by SCN5A R1632C
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Figure 3 Reduced sodium current density for SCN5A R1632C without affecting the steady-state activation and fast inactivation rates. A and B: Representative current tracings for WT (panel A) and R1632C (panel B). C: The current-voltage relationship for WT (filled squares, n ¼ 15) and R1632C (open circles, n ¼ 14). Peak currents obtained by the pulse protocol were normalized to cell capacitances. D: The voltage dependence of activation for WT (filled squares, n ¼ 15) and R1632C (open circles, n ¼ 14). E: The time constant of the voltage dependence of fast inactivation rate for WT (filled squares, n ¼ 15) and R1632C (open circles, n ¼ 14). WT ¼ wild type.
mV for 20 ms (P1) was applied. Recovery from fast inactivation, fitted with a single exponential function, for R1632C was also significantly delayed compared with that for WT (Table 2) (Figure 4D), which also suggested that R1632C had an enhanced fast-inactivated state stability. We also examined if recovery from fast inactivation for R1632C was accelerated by using a holding potential of 150 mV. Recovery from fast inactivation for both R1632C and WT was accelerated to the same degree, but recovery from fast inactivation for R1632C did not reach the same level as did recovery for WT (Table 2 and Figure 4). Development into the inactivated state was assessed using a double pulse protocol (Online Supplemental Figure S3A). A 10-ms repolarization pulse of 120 mV was interposed between varying intervals of prepulse (P1) and test pulse (P2) to allow recovery from fast inactivation. R1632C
stability. We could not assess voltage dependence of slow inactivation because of a delayed recovery from fast inactivation for R1632C (refer to below). Recovery from inactivation was assessed using a conventional double-pulse protocol with applying a depolarizing prepulse of 20 mV for 500 ms (P1) and varying hyperpolarizing interpulse before test pulse (P2) (Figure 4C). Recovery from inactivation for WT was well-fitted with a double exponential function, but that for R1632C was wellfitted with a single, rather than a double, exponential function. As shown in Figure 4C, recovery from inactivation for R1632C was apparently delayed compared with that for WT (Table 2). The assessment of recovery from inactivation using this protocol may have contained recoveries from both fast and slow/intermediate inactivation. To assess recovery from fast inactivation, a brief depolarizing prepulse of 20 Table 1
Parameters of activation and steady-state inactivation for WT and R1632C Activation INa density (pA/pF) at 20 mV
V1/2 (mV)
Steady-state inactivationv K (mV)
V1/2 (mV)
K (mV)
WT 672 ⫾ 90 (n ¼ 15) 38.7 ⫾ 0.8 6.4 ⫾ 0.3 85.9 ⫾ 0.6 (n ¼ 17) 4.8 ⫾ 0.1 R1632C 433 ⫾ 52* (n ¼ 14) 36.5 ⫾ 1.0 7.2 ⫾ 0.3 110.7 ⫾ 0.8† (n ¼ 16) 6.0 ⫾ 0.1† WT ¼ wild type. * P o .05 vs WT. † P o .01 vs WT.
Steady-state fast inactivation V1/2 (mV)
K (mV)
58.2 ⫾ 0.9 (n ¼ 8) 6.5 ⫾ 0.5 80.2 ⫾ 1.6† (n ¼ 8) 12.6 ⫾ 0.3†
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Heart Rhythm, Vol 0, No 0, Month 2015
Figure 4 A hyperpolarizing shift in the steady-state availability and a delayed recovery from inactivation for SCN5A R1632C. A: The voltage dependence of steady-state inactivation for WT (filled squares, n ¼ 17) and R1632C (open circles, n ¼ 16). B: The voltage dependence of steady-state fast inactivation for WT (filled squares, n ¼ 8) and R1632C (open circles, n ¼ 8). C: The time course of recovery from inactivation for WT (filled squares, n ¼ 15) and R1632C (open circles, n ¼ 16). D: Time course of recovery from fast inactivation for WT using a holding potential of 120 mV (filled squares, n ¼ 8) and 150 mV (filled reverse triangles, n ¼ 11) and that for R1632C using a holding potential of 120 mV (open circles, n ¼ 8) and 150 mV (open reverse triangles, n ¼ 11). WT ¼ wild type.
showed a pronounced reduction in the P2/P1 ratio (an enhanced development into the inactivated state) (Online Supplemental Figure S3A), which may be primarily due to an impaired recovery from inactivation because less than 10% of R1632C channels recover during a 10-ms repolarizing pulse (Figures 4C and 4D) and the fast inactivation rate for R1632C was not different from that for WT (Figure 3E). To assess activity-dependent loss of INa availability, depolarizing potentials of 20 mV for 500 ms at cycle lengths of 0.52, 1, or 2 seconds were applied (Online Supplemental Figure S3B). At a long cycle length of 2 seconds, both WT and R1632C INa retained the current amplitudes during the train of stimuli. At a cycle length of 1 Table 2
second, while WT INa retained the current amplitudes during the train of stimuli, R1632C INa decreased to 85% of the initial current amplitude at the second pulse and remained almost constant throughout the following train of stimuli. At a short cycle length of 0.52 seconds, while WT INa gradually decreased during successive stimuli (96% of the initial current amplitude at the second pulse and 78% at the twentieth pulse), R1632C INa decreased to 0.08% of the initial current amplitude at the second pulse and remained almost constant throughout the following train of stimuli. Although R1632C INa decreased current amplitudes at the second pulse at shorter cycle lengths, it remained stable after the second pulse, suggesting that fast inactivation, rather than
Parameters of recovery from inactivation for WT and R1632C Recovery from inactivation HP ¼ 120 mV
WT R1632C
Recovery from fast inactivation HP ¼ 120 mV
HP ¼ 150 mV
τ fast (ms)
τ slow (ms)
τ (ms)
Τ (ms)
3.1 ⫾ 0.2 (n ¼ 15) 254.5 ⫾ 7.4* (n ¼ 16)
36.2 ⫾ 3.4 (n ¼ 15) —
3.7 ⫾ 0.3 (n ¼ 8) 246.7 ⫾ 14.3* (n ¼ 8)
1.4 ⫾ 0.1 (n ¼ 11) 71.1 ⫾ 3.2* (n ¼ 11)
HP ¼ holding potential; WT ¼ wild type. * P o .01 vs WT.
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Stabilized Fast Inactivation of INa by SCN5A R1632C
slow/intermediate inactivation, affected its channel availability. Therefore, to assess activity-dependent loss of INa availability implying fast inactivation, brief depolarizing potentials of 20 mV for 20 ms at cycle lengths of 0.1, 0.5, or 2 seconds were applied (Online Supplemental Figure S3C). WT INa remained constant during successive stimuli at all cycle lengths. However, at cycle lengths of 0.5 and 0.1 seconds, R1632C INa at the second pulses decreased to 85% and 0.08% of the initial current amplitude, respectively, but remained almost constant throughout the following train of stimuli. These findings suggest that activity-dependent loss of availability for R1632C may be due to an enhanced fastinactivated state stability. Since some SCN5A mutations in DIV-S4 show persistent late INa during prolonged depolarization, tetrodotoxinsensitive currents were compared between R1632C and WT. The amplitude of tetrodotoxin-sensitive currents for R1632C was not significantly different from that for WT (Online Supplemental Figure S4).
Discussion Functional roles of S4 segments in hSkM1/SCN4A, the α subunit of skeletal muscle voltage-gated sodium channels, have been examined intensely. S4 segments in each of the 4 domains are postulated to function as a voltage sensor.14–16 Since the α subunits are composed of homologous but nonidentical domains, DI-DIV, there are distinct functional roles for the specific domains.15,16 The recovery from fast inactivation of INa was reported to be associated with a slow component in the time course of gating charge during repolarization, resulting from the slow movement of S4 segments in domains III and IV, but not in domains I and II.15 We observed that R1632C had an enhanced fast-inactivated state stability due to an impaired recovery from fast inactivation, resulting in a remarkable hyperpolarizing shift in the steady-state inactivation curve and a severe activity–dependent loss of INa availability, without affecting the fast inactivation rate. These findings are in agreement with the previous report demonstrating that hSkM1/SCN4A R1457C, a mutation at the same position as R1632C in SCN5A, showed a pronounced hyperpolarizing shift in the steady-state inactivation curve without affecting the inactivation rate.32 Therefore, our data imply that impaired recovery from fast inactivation for R1632C may be associated with the gating charge immobilization of DIV-S4 in SCN5A during depolarization and subsequent repolarization as was the gating charge immobilization of DIV-S4 in hSkM1/SCN4A.15,32 SCN5A mutations can cause loss of function of INa by multiple mechanisms, including a production of nonfunctional channels, a trafficking defect of and kinetic changes in INa such as a negative shift in steady-state inactivation, a positive shift in steady-state activation, an enhanced fast inactivation, and an enhanced intermediate/slow inactivation.4,6,31,33 In addition to the kinetic changes in R1632C INa, a trafficking defect of R1632C might cause loss of function of INa. In our experiments, INa density for R1632C, measured
7
at 20 mV test potential from a holding potential of 120 mV, was significantly low compared with that for WT. However, INa density measured at 20 mV from a holding potential of 150 mV was comparable between R1632C and WT (Online Supplemental Table S1 and Online Supplemental Figure S2), indicating that reduced INa density for R1632C was not caused by a trafficking defect but rather by an enhanced fast-inactivated state stability. Loss of function of INa underlies the cause of both BrS and supraventricular arrhythmias. SCN5A mutations that cause loss of function of INa in the ventricle are associated with approximately 20% of BrS cases.2,4 Moreover, loss of function of INa in the atrium may lead to a failure of impulse conduction from the sinus node into the adjacent myocardium (exit block), which may be a plausible mechanism of SND caused by SCN5A mutations6 and may also form the substrate for the reentry of supraventricular arrhythmias, such as AT. R1632C INa availability in both the atrium and the ventricle, whose resting membrane potentials are approximately 90 mV, is likely to be substantially reduced (Online Supplemental Figure S2), although the current reduction caused by the hyperpolarizing shift in voltage dependence of inactivation might be enhanced in our experimental condition.28 A pronounced reduction of INa in both the atrium and the ventricle may be associated with multiple electrophysiological phenotypes (BrS, AT, and SND) observed in our patient. An increased HV interval and QRS duration could also be attributable to the R1632C mutation. Of note, repetitive depolarizing pulses rapidly attenuated R1632C INa compared with WT INa (Online Supplemental Figures S3B and S3C), and β-adrenergic stimulation augments the frequency-dependent reduction of INa.28 These findings may explain the clinical phenotypes of our patient such that AT preferentially occurred during exercise or was induced by high-frequency atrial stimuli during isoproterenol infusion. Several SCN5A mutations in DIV-S4 have previously been identified in patients with BrS or LQTS alone or BrS with SSS or LQTS, and some of these mutations have been functionally characterized. The functional consequences of R1623Q and R1644H, identified in patients with LQTS, were gain of function of INa due to a delayed fast inactivation rate and/or increased late INa.20–22 In contrast, R1629Q, identified in patients with BrS, resulted in loss of function of INa with a hyperpolarizing shift in the steady-state inactivation curve, an increased fast inactivation rate, and a delayed recovery from inactivation.23 However, although Zeng et al23 insisted that functional abnormality of R1629Q was an enhanced intermediate inactivation, whether fast inactivation is also enhanced should be examined. Intriguingly, R1626H, identified in a patient with AF, exhibited a positive shift in steady-state activation and a negative shift in steadystate inactivation, together with a decreased fast inactivation rate.8 The functional consequence of R1632H, identified in patients with SND or first-degree heart block, resembles that of R1632C identified in our patient.6 R1632H exhibited a large hyperpolarizing shift in the steady-state inactivation curve, a delayed fast inactivation rate, and a delayed recovery from fast inactivation (enhanced fast inactivation),
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suggesting that R1632H had a mixture of loss and gain of function with a predominance of functional loss.6 R1632H carriers did not have spontaneous BrS phenotype. A provocative test should be performed to confirm whether R1632H carriers manifest BrS phenotype.
Conclusion We identified a novel SCN5A mutation, R1632C, located in the DIV-S4 voltage sensor, in a young male patient with AT, SND, and BrS. The functional consequence of SCN5A R1632C was an enhanced fast-inactivated state stability of INa because of a pronounced impairment of recovery from fast inactivation, resulting in a severe activity–dependent loss of INa availability, which may explain the phenotypic manifestation observed in our patient. These findings provide valuable insight into the pathophysiological role of the mutation.
Acknowledgments We thank Naomasa Makita, XX (Nagasaki University), for kindly providing us with hH1 and hβ1 plasmids, and Kenshi Hayashi, XX (Kanazawa University), for valuable advices on performing patch-clamp methods. We also thank Yukiyo Tosaka, XX, and Takako Kobayashi, XX, for their helpful technical assistances.
Appendix Supplementary data Supplementary material cited in this article is available online at http://dx.doi.org/10.1016/j.hrthm.2015.05.032.
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Heart Rhythm, Vol 0, No 0, Month 2015 11. Bennett PB, Yazawa K, Makita N, George AL Jr. Molecular mechanism for an inherited cardiac arrhythmia. Nature 1995;376:683–685. 12. Makita N, Behr E, Shimizu W, et al. The E1784K mutation in SCN5A is associated with mixed clinical phenotype of type 3 long QT syndrome. J Clin Invest 2008;118:2219–2229. 13. Veldkamp MW, Viswanathan PC, Bezzina C, Baartscheer A, Wilde AA, Balser JR. Two distinct congenital arrhythmias evoked by a multidysfunctional Naþ channel. Circ Res 2000;86:E91–E97. 14. Yang N, Horn R. Evidence for voltage-dependent S4 movement in sodium channels. Neuron 1995;15:213–218. 15. Cha A, Ruben PC, George AL Jr, Fujimoto E, Bezanilla F. Voltage sensors in domains III and IV, but not I and II, are immobilized by Naþ channel fast inactivation. Neuron 1999;22:73–87. 16. Bezanilla F. The voltage sensor in voltage-dependent ion channels. Physiol Rev 2000;80:555–592. 17. McNair WP, Sinagra G, Taylor MR, Di Lenarda A, Ferguson DA, Salcedo EE, Slavov D, Zhu X, Caldwell JH, Mestroni L; Familial Cardiomyopathy Registry Research Group. SCN5A mutations associate with arrhythmic dilated cardiomyopathy and commonly localize to the voltage-sensing mechanism. J Am Coll Cardiol 2011;57:2160–2168. 18. Nair K, Pekhletski R, Harris L, Care M, Morel C, Farid T, Backx PH, Szabo E, Nanthakumar K. Escape capture bigeminy: phenotypic marker of cardiac sodium channel voltage sensor mutation R222Q. Heart Rhythm 2012;9: 1681–1688. e1681. 19. Beckermann TM, McLeod K, Murday V, Potet F, George AL Jr. Novel SCN5A mutation in amiodarone-responsive multifocal ventricular ectopy-associated cardiomyopathy. Heart Rhythm 2014;11:1446–1453. 20. Makita N, Shirai N, Nagashima M, Matsuoka R, Yamada Y, Tohse N, Kitabatake A. A de novo missense mutation of human cardiac Naþ channel exhibiting novel molecular mechanisms of long QT syndrome. FEBS Lett 1998;423:5–9. 21. 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. 22. Dumaine R, Wang Q, Keating MT, Hartmann HA, Schwartz PJ, Brown AM, Kirsch GE. Multiple mechanisms of Naþ channel–linked long-QT syndrome. Circ Res 1996;78:916–924. 23. Zeng Z, Zhou J, Hou Y, Liang X, Zhang Z, Xu X, Xie Q, Li W, Huang Z. Electrophysiological characteristics of a SCN5A voltage sensors mutation R1629Q associated with Brugada syndrome. PLoS One 2013;8:e78382. 24. Nakajima T, Kaneko Y, Saito A, Irie T, Tange S, Iso T, Kurabayashi M. Identification of six novel SCN5A mutations in Japanese patients with Brugada syndrome. Int Heart J 2011;52:27–31. 25. Nakajima T, Wu J, Kaneko Y, Ashihara T, Ohno S, Irie T, Ding WG, Matsuura H, Kurabayashi M, Horie M. KCNE3 T4A as the genetic basis of Brugada-pattern electrocardiogram. Circ J 2012;76:2763–2772. 26. Imai M, Nakajima T, Kaneko Y, Niwamae N, Irie T, Ota M, Iijima T, Tange S, Kurabayashi M. A novel KCNQ1 splicing mutation in patients with forme fruste LQT1 aggravated by hypokalemia. J Cardiol 2014;64:121–126. 27. Nakajima T, Davies SS, Matafonova E, Potet F, Amarnath V, Tallman KA, Serwa RA, Porter NA, Balser JR, Kupershmidt S, Roberts LJ III. Selective γketoaldehyde scavengers protect NaV1.5 from oxidant-induced inactivation. J Mol Cell Cardiol 2010;48:352–359. 28. Tsurugi T, Nagatomo T, Abe H, Oginosawa Y, Takemasa H, Kohno R, Makita N, Makielski JC, Otsuji Y. Differential modulation of late sodium current by protein kinase A in R1623Q mutant of LQT3. Life Sci 2009;84:380–387. 29. Sheets MF, Hanck DA. Charge immobilization of the voltage sensor in domain IV is independent of sodium current inactivation. J Physiol 2005;563:83–93. 30. Goldschen-Ohm MP, Capes DL, Oelstrom KM, Chanda B. Multiple pore conformations driven by asynchronous movements of voltage sensors in a eukaryotic sodium channel. Nat Commun 2013;4:1350. 31. Wang DW, Makita N, Kitabatake A, Balser JR, George AL Jr. Enhanced Naþ channel intermediate inactivation in Brugada syndrome. Circ Res 2000;87: E37–E43. 32. Yang N, George AL Jr, Horn R. Molecular basis of charge movement in voltagegated sodium channels. Neuron 1996;16:113–122. 33. Makiyama T, Akao M, Tsuji K, et al. High risk for bradyarrhythmic complications in patients with Brugada syndrome caused by SCN5A gene mutations. J Am Coll Cardiol 2005;46:2100–2106.
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Stabilized Fast Inactivation of INa by SCN5A R1632C
CLINICAL PERSPECTIVES
Q14
SCN5A, which encodes the α subunit of cardiac voltage-gated sodium channels, consists of 4 homologous but nonidentical domains, DI–DIV. The fourth segment (S4) in each domain is postulated to function as a voltage sensor, but each S4 segment is thought to have distinct functional roles. SCN5A mutations in each S4 segment and even those in the same S4 segment may cause diverse functional abnormalities and phenotypic manifestations. We identified a novel SCN5A mutation, R1632C, located in the DIV-S4 voltage sensor, in a young male patient who had a syncopal episode during exercise and presented with multiple electrophysiological phenotypes including atrial tachycardia during exercise, sinus node dysfunction, and Brugada syndrome. Functional analysis of the SCN5A R1632C mutation expressed in tsA201 cells using a patch-clamp method revealed that the R1632C mutation caused a loss of function of sodium currents by an enhanced fast-inactivated state stability due to a pronounced impairment of recovery from fast inactivation, resulting in a severe activity–dependent loss of sodium current availability. The obtained kinetic changes in the R1632C mutation can be the cause of multiple electrophysiological phenotypes observed in our patient. These findings provided us with valuable insight into the structure-function relationship of the DIV-S4 voltage sensor in SCN5A and shed light on the pathophysiological role of the R1632C mutation. Further studies are necessary to fully clarify the structure-function relationship of the voltage sensor in SCN5A.
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