Inherited Brugada and Long QT-3 Syndrome Mutations of a Single Residue of the Cardiac Sodium Channel Confer Distinct Channel and Clinical Phenotypes
2001; Elsevier BV; Volume: 276; Issue: 33 Linguagem: Inglês
10.1074/jbc.m104471200
ISSN1083-351X
AutoresIlaria Rivolta, Hugues Abriel, Michihiro Tateyama, Huajun Liu, Mirella Memmi, Panos Vardas, Carlo Napolitano, Silvia G. Priori, Robert S. Kass,
Tópico(s)Neuroscience and Neural Engineering
ResumoDefects of the SCN5A gene encoding the cardiac sodium channel α-subunit are associated with both the long QT-3 (LQT-3) subtype of long-QT syndrome and Brugada syndrome (BrS). One previously described SCN5A mutation (1795insD) in the C terminus results in a clinical phenotype combining QT prolongation and ST segment elevation, indicating a close interrelationship between the two disorders. Here we provide additional evidence that these two disorders are closely related. We report the analysis of two novel mutations on the same codon, Y1795C (LQT-3) and Y1795H (BrS), expressed in HEK 293 cells and characterized using whole-cell patch clamp procedures. We find marked and opposing effects on channel gating consistent with activity associated with the cellular basis of each clinical disorder. Y1795H speeds and Y1795C slows the onset of inactivation. The Y1795H, but not the Y1795C, mutation causes a marked negative shift in the voltage dependence of inactivation, and neither mutation affects the kinetics of the recovery from inactivation. Interestingly, both mutations increase the expression of sustained Na+ channel activity compared with wild type (WT) channels, although this effect is most pronounced for the Y1795C mutation, and both mutations promote entrance into an intermediate or a slowly developing inactivated state. These data confirm the key role of the C-terminal tail of the cardiac Na+ channel in the control of channel gating, illustrate how subtle changes in channel biophysics can have significant and distinct effects in human disease, and, additionally, provide further evidence of the close interrelationship between BrS and LQT-3 at the molecular level. Defects of the SCN5A gene encoding the cardiac sodium channel α-subunit are associated with both the long QT-3 (LQT-3) subtype of long-QT syndrome and Brugada syndrome (BrS). One previously described SCN5A mutation (1795insD) in the C terminus results in a clinical phenotype combining QT prolongation and ST segment elevation, indicating a close interrelationship between the two disorders. Here we provide additional evidence that these two disorders are closely related. We report the analysis of two novel mutations on the same codon, Y1795C (LQT-3) and Y1795H (BrS), expressed in HEK 293 cells and characterized using whole-cell patch clamp procedures. We find marked and opposing effects on channel gating consistent with activity associated with the cellular basis of each clinical disorder. Y1795H speeds and Y1795C slows the onset of inactivation. The Y1795H, but not the Y1795C, mutation causes a marked negative shift in the voltage dependence of inactivation, and neither mutation affects the kinetics of the recovery from inactivation. Interestingly, both mutations increase the expression of sustained Na+ channel activity compared with wild type (WT) channels, although this effect is most pronounced for the Y1795C mutation, and both mutations promote entrance into an intermediate or a slowly developing inactivated state. These data confirm the key role of the C-terminal tail of the cardiac Na+ channel in the control of channel gating, illustrate how subtle changes in channel biophysics can have significant and distinct effects in human disease, and, additionally, provide further evidence of the close interrelationship between BrS and LQT-3 at the molecular level. long QT-3 Brugada syndrome electrocardiogram wild type intermediate tetrodotoxin action potential duration Mutations of SCN5A, the gene coding for the α-subunit of the cardiac sodium channel, have been linked to the following four human syndromes: congenital long QT syndrome type 3 (LQT-3),1 Brugada syndrome (BrS), different types of conduction block (2Schott J.J. Alshinawi C. Kyndt F. Probst V. Hoorntje T.M. Hulsbeek M. Wilde A.A. Escande D. Mannens M.M. Le Marec H. Nat. Genet. 1999; 23: 20-21Crossref PubMed Scopus (70) Google Scholar), and sudden infant death syndrome (3Tan H.L. Bink-Boelkens M.T. Bezzina C.R. Viswanathan P.C. Beaufort-Krol G.C. van Tintelen P.J. van den Berg M.P. Wilde A.A. Balser J.R. Nature. 2001; 409: 1043-1047Crossref PubMed Scopus (346) Google Scholar, 4Wang Q. Shen J. Splawski I. Atkinson D. Li Z. Robinson J.L. Moss A.J. Towbin J.A. Keating M.T. Cell. 1995; 80: 805-811Abstract Full Text PDF PubMed Scopus (1452) Google Scholar, 5Chen Q. Kirsch G.E. Zhang D. Brugada R. Brugada J. Brugada P. Potenza D. Moya A. Borggrefe M. Breithardt G. Ortiz-Lopez R. Wang Z. Antzelevitch C. O'Brien R.E. Schulze-Bahr E. Keating M.T. Towbin J.A. Wang Q. Nature. 1998; 392: 293-296Crossref PubMed Scopus (1566) Google Scholar, 6Schwartz P.J. Priori S.G. Dumaine R. Napolitano C. Antzelevitch C. Stramba-Badiale M. Richard T.A. Berti M.R. Bloise R. N. Engl. J. Med. 2000; 343: 262-267Crossref PubMed Scopus (319) Google Scholar, 7Priori S.G. Napolitano C. Giordano U. Collisani G. Memmi M. Lancet. 2000; 355: 808-809Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Despite this classification based on clinical characteristics such as ECG findings, age of first presentation, triggers of events, and others, significant clinical and pathophysiological overlap between these different disorders have been noted recently. This observation has led to the proposition that these syndromes are in fact only different appearances of a "unique"SCN5A disease. The most significant overlap exists between LQT-3 and the BrS (8Priori S.G. Napolitano C. Schwartz P.J. Bloise R. Crotti L. Ronchetti E. Circulation. 2000; 102: 945-947Crossref PubMed Scopus (243) Google Scholar). The most frequent presentation in both disorders is a history of syncope or cardiac arrests with a very high lethality, occurring mainly during rest or sleep and with a poor response to anti-adrenergic treatment such as β receptor blockade (9Priori S.G. J. Cardiovasc. Electrophysiol. 2000; 11: 1174-1178Crossref PubMed Scopus (23) Google Scholar, 10Moss A.J. Zareba W. Hall W.J. Schwartz P.J. Crampton R.S. Benhorin J. Vincent G.M. Locati E.H. Priori S.G. Napolitano C. Medina A. Zhang L. Robinson J.L. Timothy K. Towbin J.A. Andrews M.L. Circulation. 2000; 101: 616-623Crossref PubMed Scopus (730) Google Scholar, 11Priori S.G. Napolitano C. Gasparini M. Pappone C. Della B.P. Brignole M. Giordano U. Giovannini T. Menozzi C. Bloise R. Crotti L. Terreni L. Schwartz P.J. Circulation. 2000; 102: 2509-2515Crossref PubMed Scopus (450) Google Scholar, 12Wilde A.A. Priori S.G. Eur. Heart J. 2000; 21: 1483-1484Crossref PubMed Scopus (14) Google Scholar). However, the typical electrocardiographic manifestations of long-QT syndrome (QT interval prolongation) and BrS (ST segment elevation in leads V1 through V3) may coexist in the same patients, which raises questions about the actual differences between LQT3 and BrS (8Priori S.G. Napolitano C. Schwartz P.J. Bloise R. Crotti L. Ronchetti E. Circulation. 2000; 102: 945-947Crossref PubMed Scopus (243) Google Scholar). Moreover, in some LQT-3 patients, exposure to the sodium channel blocker flecainide generates an ECG pattern that is very similar to the BrS (8Priori S.G. Napolitano C. Schwartz P.J. Bloise R. Crotti L. Ronchetti E. Circulation. 2000; 102: 945-947Crossref PubMed Scopus (243) Google Scholar). By definition LQT-3 is caused by mutations found in SCN5A, while, to date, all mutations linked with BrS are also in this gene. Multiple SCN5A mutations linked to both disorders have now been reported and characterized (13Bezzina C.R. Rook M.B. Wilde A.A. Cardiovasc. Res. 2001; 49: 257-271Crossref PubMed Scopus (105) Google Scholar). 2Data base of SCN5A mutations and functional consequences. The most common alteration in channel function caused by LQT-3 mutations is the induction of sustained sodium channel activity over the time course and voltage range of the ventricular action potential plateau phase first described by Bennett et al. (15Bennett Jr., P.B. Makita N. George Jr., A.L. FEBS Lett. 1993; 326: 21-24Crossref PubMed Scopus (75) Google Scholar) for the ΔKPQ LQT-3 mutation (16Clancy C.E. Rudy Y. Nature. 1999; 400: 566-569Crossref PubMed Scopus (361) Google Scholar). It is generally thought that mutation-induced loss of channel activity underlies the cellular and clinical phenotype of BrS (5Chen Q. Kirsch G.E. Zhang D. Brugada R. Brugada J. Brugada P. Potenza D. Moya A. Borggrefe M. Breithardt G. Ortiz-Lopez R. Wang Z. Antzelevitch C. O'Brien R.E. Schulze-Bahr E. Keating M.T. Towbin J.A. Wang Q. Nature. 1998; 392: 293-296Crossref PubMed Scopus (1566) Google Scholar, 17Antzelevitch C. Curr. Opin. Cardiol. 1999; 14: 274-279Crossref PubMed Scopus (72) Google Scholar, 18Dumaine R. Towbin J.A. Brugada P. Vatta M. Nesterenko D.V. Nesterenko V.V. Brugada J. Brugada R. Antzelevitch C. Circ. Res. 1999; 85: 803-809Crossref PubMed Scopus (501) Google Scholar, 19Gussak I. Antzelevitch C. Bjerregaard P. Towbin J.A. Chaitman B.R. J. Am. Coll. Cardiol. 1999; 33: 5-15Crossref PubMed Scopus (447) Google Scholar, 20Yan G.X. Antzelevitch C. Circulation. 1999; 100: 1660-1666Crossref PubMed Scopus (958) Google Scholar). Nevertheless, these functional changes, although sufficient, may not always be necessary to cause clinically either LQT-3 or BrS (21An R.H. Wang X.L. Kerem B. Benhorin J. Medina A. Goldmit M. Kass R.S. Circ. Res. 1998; 83: 141-146Crossref PubMed Scopus (171) Google Scholar), and it is possible that other, as yet undiscovered, genetic factors are likely to contribute to the expression of the specific disease phenotypes in these patients. Here we report and characterize two novel point mutations of SCN5A in the same codon (Y-1795) that were identified in patients clinically classified as LQT-3 (Y1795C) and BrS (Y1795H) cases. Functional analysis of these cardiac sodium channel mutations expressed in HEK 293 cells indicates marked and opposing effects on the kinetics and voltage dependence of the inactivated state of the expressed channels consistent with the disease phenotypes. Interestingly, both mutations also confer some biophysical properties upon the expressed channels that are not generally associated with the observed clinical phenotypes. Genomic DNA was extracted from peripheral blood lymphocytes by standard techniques. The coding region of the SCN5A gene encoding the cardiac Na+channel α-subunit was screened using single strand conformation polymorphism on polymerase chain reaction-amplified genomic DNA samples. The abnormal conformers were directly sequenced on both strands using an ABI310 genetic analyzer or were cloned (TopoTA cloning, Invitrogen) and sequenced using plasmid-specific oligonucleotides. Single strand conformation polymorphism shifts were also checked against a panel of genomic DNA from 300 (600 chromosomes) healthy reference individuals and were not found in this group. The Y1795C and Y1795H mutations of SCN5A were engineered into WT cDNA cloned in pcDNA3.1 (Invitrogen) by overlap extension using mutation-specific primers and Quick Change Site-directed Mutagenesis kit (Stratagene). The presence of the mutation was confirmed by sequence analysis. WT and mutant Na+ channels were expressed in HEK 293 cells as described previously. Transient transfections were carried out with equal amounts of Na+ channel α-subunit and with hβ1, subcloned individually into the pcDNA3.1 (Invitrogen) vector (total cDNA 2.5 mg) using a previously described lipofection procedure. Expression of channels was studied using patch clamp procedures 48 h after transfection. Membrane currents were measured using whole-cell patch clamp procedures, with Axopatch 200B amplifiers (Axon Instruments, Foster City, CA). Recordings were made at room temperature (22 °C) or 32 °C where indicated. Internal pipette solution contained (in mmol/liter) aspartic acid 50, CsCl 60, Na2-ATP 5, EGTA 11, HEPES 10, CaCl2 1, and MgCl2 1, with pH 7.4 adjusted with CsOH (22Abriel H. Cabo C. Wehrens X.H. Rivolta I. Motoike H.K. Memmi M. Napolitano C. Priori S.G. Kass R.S. Circ. Res. 2001; 88: 740-745Crossref PubMed Scopus (108) Google Scholar). External solutions (full Na+) consisted of (mmol/liter) NaCl 130, CaCl2 2, CsCl 5, MgCl2 1.2, HEPES 10, and glucose 5, with pH 7.4 adjusted with CsOH. In experiments designed to measure the voltage dependence of activation, external Na+was reduced to 30 mm using n-methylglucamine as a Na+ substitute. Holding potentials were −100 mV or −120 mV as indicated in figure legends. Pclamp8 (Axon Instruments, Foster City, CA), Excel (Microsoft, Seattle, WA), and Origin6 (Microcal Software, Northampton, MA) were used for data acquisition and analysis. Data for the voltage dependence of activation were fitted with Boltzmann relationships, and data for the time course of recovery from inactivation were fitted with functions of two exponentials using Origin as described previously by us (23An R.H. Bangalore R. Rosero S.Z. Kass R.S. Circ. Res. 1996; 79: 103-108Crossref PubMed Scopus (99) Google Scholar). Data are presented as mean values ± S.E. Two-tailed Student's t test was used to compare means; p < 0.05 was considered statistically significant (23An R.H. Bangalore R. Rosero S.Z. Kass R.S. Circ. Res. 1996; 79: 103-108Crossref PubMed Scopus (99) Google Scholar). We report two novel mutations identified at codon Tyr-1795 of the SCN5Agene. The mutation Y1795C (substitution of a tyrosine by a cysteine, Y1795C) was found to be linked with an LQT syndrome clinical phenotype, and the mutation Y1795H (tyrosine replace by an histidine residue, Y1795H) was linked to a BrS phenotype (see below). Interestingly, these two mutations are found in the same codon of the (Tyr-1795) C terminus tail of the cardiac sodium channel (Fig.1) where an insertion mutation (1795insD) has been shown to be linked to both BrS and LQT-3, depending on physiological conditions of the mutation carrier such as heart rate (1Bezzina C. Veldkamp M.W. Van Den Berg M.P. Postma A.V. Rook M.B. Viersma J.W. Van Langen I.M. Tan-Sindhunata G. Bink-Boelkens M.T. Der Hout A.H. Mannens M.M. Wilde A.A. Circ. Res. 1999; 85: 1206-1213Crossref PubMed Scopus (557) Google Scholar). Additionally, other LQT-3 (i.e. E1748K,D1790G) (1Bezzina C. Veldkamp M.W. Van Den Berg M.P. Postma A.V. Rook M.B. Viersma J.W. Van Langen I.M. Tan-Sindhunata G. Bink-Boelkens M.T. Der Hout A.H. Mannens M.M. Wilde A.A. Circ. Res. 1999; 85: 1206-1213Crossref PubMed Scopus (557) Google Scholar,24Wei J. Wang D.W. Alings M. Fish F. Wathen M. Roden D.M. George Jr., A.L. Circulation. 1999; 99: 3165-3171Crossref PubMed Scopus (124) Google Scholar, 25Deschenes I. Baroudi G. Berthet M. Barde I. Chalvidan T. Denjoy I. Guicheney P. Chahine M. Cardiovasc. Res. 2000; 46: 55-65Crossref PubMed Scopus (138) Google Scholar, 26Benhorin J. Goldmit M. MacCluer J. Blangero J. Goffen R. Leibovitch A. Rahat A. Wang Q. Medina A. Towbin J. Kerem B. Hum. Mutat. 1998; 12: 72Crossref PubMed Google Scholar) and BrS (A1924T) (27Rook M.B. Alshinawi C.B. Groenewegen W.A. van Gelder I.C. van Ginneken A.C. Jongsma H.J. Mannens M.M. Wilde A.A. Cardiovasc. Res. 1999; 44: 507-517Crossref PubMed Scopus (173) Google Scholar) mutations have been reported in the C-terminal tail of the channel indicating its importance in the control of channel gating. The proband is a 42-year-old female asymptomatic for arrhythmias but presenting a long QT interval on the ECG (Fig.2 A). Two children of the proband (daughters) died suddenly in their sleep at 17 and 10 years of age, respectively. No ECG is available for the victims. Two other children of the proband (9 and 5 years old) are asymptomatic for syncope or cardiac arrest but have a prolonged QT interval (QTc 512 and 508 ms, respectively). The morphology of the QT-T interval in the proband and in the two girls shows long ST segment and small T wave as reported for LQT-3 patients (28Zhang L. Timothy K.W. Vincent G.M. Lehmann M.H. Fox J. Giuli L.C. Shen J. Splawski I. Priori S.G. Compton S.J. Yanowitz F. Benhorin J. Moss A.J. Schwartz P.J. Robinson J.L. Wang Q. Zareba W. Keating M.T. Towbin J.A. Napolitano C. Medina A. Circulation. 2000; 102: 2849-2855Crossref PubMed Scopus (404) Google Scholar). No other family members are available for evaluation. Genetic analysis demonstrated the presence of a mutation in the cardiac sodium channel gene SCN5A. The proband was tested with flecainide (intravenously) and demonstrated a significant reduction of QT interval (QTc shortened from 530 to 470); no ST segment elevation was observed. The proband is a 45-year-old white male asymptomatic for palpitations, syncope, or cardiac arrest with a negative family history for juvenile (<40 years) cardiac arrest, syncope, or known ventricular arrhythmias. The ECG recording (Fig. 2 B) was performed as routine screening and presented the typical pattern of ST segment elevation with a coved type morphology in V1 and saddle-back type in V2 and V3 (29Brugada P. Heart. 2000; 84: 1-2Crossref PubMed Scopus (20) Google Scholar, 30Brugada P. Brugada R. Brugada J. Curr. Cardiol. Rep. 2000; 2: 507-514Crossref PubMed Scopus (40) Google Scholar). ST segment elevation was intermittently present, and in some of the ECG recordings the ST morphology in V1–V3 was unremarkable, and incomplete right bundle branch block was present. PQ and QT intervals were within normal values (QTc 434 ms). A flecainide test significantly enhanced ST segment elevation with a conversion of the morphology of ST segment from saddle-back type to coved type elevation in V2 (maximum elevation 4 mm); no arrhythmias developed. Programmed electrical stimulation was performed at two sites (right ventricular apex and right ventricular outflow tract). Non-sustained ventricular tachycardia was induced with two premature beats (coupling interval 220–210 ms) delivered at the outflow tract on a drive of 500 ms. An implantable cardioverter defibrillator was implanted; at a follow up of 2 years no arrhythmic events occurred. Genetic analysis identified a mutation in the cardiac sodium channel gene (SCN5A) in the proband and in his son (13 years old, asymptomatic with normal ECG), sister (56 years old, asymptomatic with normal ECG), and nephew (33 years old, asymptomatic with normal ECG). The daughter of the proband refused genetic analysis and clinical evaluation. A flecainide test unmasked ST segment elevation in the son of the proband but not in the two female gene carriers, thus demonstrating variable expression of the disease in the family. We expressed Y1795H and Y1795C mutant channels in HEK 293 cells and investigated their functional properties using the whole-cell configuration of the patch clamp procedure. Fig.3 illustrates families of current traces for the two mutant as well as WT channels. The figure shows the effects of the mutations on inactivation kinetics and the current/voltage relationship. It is apparent from the current traces that the Y1795H mutation speeds the time course of the onset of inactivation, whereas the Y1795C mutation slows it. This can be seen more clearly in Fig.3 B which plots the half-time of decay of peak current as a function of test voltage over a broad voltage range. Interestingly, the effects of the Y1795H mutation on the time course of inactivation are most pronounced at voltages near threshold for channel activation, in contrast with the effects of the Y1795C mutation, which are marked at voltages more positive than −20 mV. Normalized current/voltage (I/V) relationships for WT, Y1795H, and Y1795C channels shown in Fig. 3 C superimpose suggesting that neither mutation affects the voltage dependence of activation. We also tested the effects of the mutations on current densities and found that the mutations had opposite effects on the magnitude of expressed current. YC mutant channels were expressed at a slightly higher density (−448 ± 43 pA/pF, n = 17) than WT channel (−370 ± 32 pA/pF, n = 29), but YH channels were expressed at significantly (p < 0.05) lower density (−244 ± 15 pA/pF, n = 30). These mutation-induced changes in current density are consistent with expected properties underlying each clinical disorder. Fig.4 A confirms little effect on the voltage dependence of activation caused by either the Y1795H or Y1795C mutation but, in contrast, shows that the Y1795H, but not the Y1795C, mutation causes a hyperpolarizing shift in the voltage dependence of inactivation. Inactivation curves shown in the figure were obtained from a −100 mV holding potential using 500-ms conditioning pulses. We obtained similar effects of the mutations on inactivation curves measured from −120 mV holding potentials and after 50- or 10-ms conditioning pulses (not shown) indicating that the effects of the YH mutation are primarily due to changes in the voltage dependence of fast inactivation. We tested for, but did not find, mutation-induced changes in the recovery from inactivation using a paired pulse protocol (Fig. 4 B).Figure 4A, averaged inactivation and activation curves are shown for WT, Y1795C, and Y1795H Na+ channels. The voltage dependence for the inactivation was measured by applying a series of conditioning pulses (500 ms) from −110 to + 20 mV in 10-mV increments before assaying current availability at a test voltage of −10 mV. Test voltage currents were normalized to current measured after the −120-mV conditioning pulse and plotted versusconditioning pulse voltage. The voltage dependence of activation was measured by normalizing currents measured during pulses (25 ms) from −80 to +50 mV (10-mV increments) to driving force. Experimental data were fitted with Boltzmann relationships ("Materials and Methods") to obtain the parameters that follow. For inactivationV 12 (mV) = −62.8 ± 0.8(WT); −65.6 ± 0.4(Y1795C); and −73.3 ± 0.8 (Y1795H p < 0.001). The slope factor, V K is 5.9 ± 0.2 (WT); 5.5 ± 0.1 (Y1795C); 5.4 ± 0.2 (Y1795H). For activation V 12 (mV) is −23.3 ± 1 (WT); −24.3 ± 1.4 (Y1795C); and −22.2 ± 1.3 (Y1795H). The slope factor, V K is 7.1 ± 0.7 (WT); 7.3 ± 0.8 (Y1795C); and 7.6 ± 0.9 (Y1795H). B, neither the Y1795H nor the Y1795C mutation affects the time course of recovery from inactivation using paired pulse analysis (see "Materials and Methods").View Large Image Figure ViewerDownload (PPT) We next tested for mutation-induced sustained Na+ activity, and we summarize these results in Fig. 5. In these experiments, we focused on currents measured at −10 mV which is near the peak voltage of the I/V relationship to optimize resolution of small currents and measured tetrodotoxin (TTX)-sensitive current to ensure subtraction of time-independent leakage currents. Similar to other LQT-3 mutant channels such as the ΔKPQ channel (31Bennett P.B. Yazawa K. Makita N. George A.L. Nature. 1995; 376: 683-685Crossref PubMed Scopus (794) Google Scholar), sustained TTX-sensitive current was detected for Y1795C (LQT-3) channels at a magnitude ∼4–5 times greater than for WT channels. This large effect on maintained current is consistent with a cellular phenotype of action potential prolongation necessary to cause LQT syndrome (16Clancy C.E. Rudy Y. Nature. 1999; 400: 566-569Crossref PubMed Scopus (361) Google Scholar). Surprisingly, we also found that the Y1795H (BrS) mutation causes a small but significant increase in sustained Na+ activity relative to WT channels, although this effect is only about half that caused by the Y1795C mutation. Both the 1795insD and T1620M BrS mutations of SCN5A have been shown to enhance the entry of expressed channels into an "intermediate" inactivated state, "Im," that develops slowly during membrane depolarization over times consistent with the plateau phase of the ventricular action potential (∼500 ms) (32Wang D.W. Makita N. Kitabatake A. Balser J.R. George A.L. Circ. Res. 2000; 87: E37-E43Crossref PubMed Google Scholar, 33Veldkamp M.W. Viswanathan P.C. Bezzina C. Baartscheer A. Wilde A.A. Balser J.R. Circ. Res. 2000; 86: E91-E97Crossref PubMed Google Scholar). Under conditions of rapid heart rate (tachycardia), an intermediate inactivated state might contribute to a decrease in Na+channel current, consistent with a cellular phenotype associated with the Brugada syndrome (17Antzelevitch C. Curr. Opin. Cardiol. 1999; 14: 274-279Crossref PubMed Scopus (72) Google Scholar). We thus tested for an effect of the Y1795H mutation on the slow development of inactivation using protocols described previously (33Veldkamp M.W. Viswanathan P.C. Bezzina C. Baartscheer A. Wilde A.A. Balser J.R. Circ. Res. 2000; 86: E91-E97Crossref PubMed Google Scholar), and we summarize the results in Fig.6. We used a protocol to detect changes in the onset of Im inactivation by applying conditioning pulses of a common amplitude but variable duration before assaying available current with a test pulse. A 20 ms return to the holding potential is used to remove most fast inactivation that develops during the prepulses. As was the case for previous studies, we found little Im inactivation developing for either WT or mutant channels at room temperature for conditioning pulses up to 3 s in duration (data not shown). However, at more physiological temperatures, we were able to detect significantly greater slowly developing Im inactivation for Y1795H versus WT channels (Fig. 6 A) similar to the previously investigated BrS mutant channels (32Wang D.W. Makita N. Kitabatake A. Balser J.R. George A.L. Circ. Res. 2000; 87: E37-E43Crossref PubMed Google Scholar). At sufficiently prolonged conditioning times, there was a greater tendency for YH channels to inactivate the YC channels. The observation of mutation-induced changes in intermediate inactivation raises the possibility that clinically relevant functional properties of the mutant channels may be influenced by stimulation rate in voltage-clamp experiments and heart rate in patients carrying the specific mutations. Consequently, we tested for the effect of pulse frequency on the amplitude (late current) and kinetics of mutant and wild type channels and summarize these data in Fig. 6 B and Fig. 7. Traces in the upper row are normalized to emphasize changes in kinetics; those in lower row are absolute currents to emphasize possible change in peak current. As expected due to slow inactivation, at this pulse rate there is reduction in peak current during the pulse train, but perhaps more interesting, the kinetics of YC, but not WT or YH, channels changes during the pulse train. Fig. 7 presents the effect of repetitive activity at two frequencies on expressed channels. These experiments were carried out by holding cells at −100 mV for 30 s under pulse-free conditions after which test pulses were applied at the indicated frequencies. Fig. 7 A shows superimposition of normalized currents measured in response to the first and last pulse of each train and at each pulse frequency. Only the wave forms of YC currents are markedly affected by pulse frequency; at 1.9 Hz, there is a reduction in the time course of inactivation that develops with repetitive pulsing. This is more apparent in Fig. 7 B which plots the time to 80% decay of initial current amplitude (t 80) versus pulse number during this protocol. As indicated in the traces (A), but seen clearly in the plot, there is no change in the time course of YC channels at the slower pulse rate (0.67 Hz) but a pulse-dependent speeding at 1.9 Hz. Indicated in the figure for comparison are t 80 values for WT channels recorded under the same conditions (open squares, circles). In the steady state at the 1.9-Hz pulse frequency, the onset kinetics of YC channel approach those of WT channels, but both following a pause in stimulation (pulse 1) or in the steady state at the slower frequency (0.67 Hz) YC channels decay almost twice as slowly as WT channels. A similar trend is seen in the analysis of maintained current which is illustrated in Fig. 7 C. Maintained current through YC channels is most prominent at the slower pulse frequency, is most problematic following a pause in stimulation, and approaches the magnitude of WT maintained current at higher stimulation frequencies. These experiments clearly show that, particularly for the case of the YC mutation, the stimulation rate of cellular experiments and heart rates of mutation carriers will be critical to expression of cellular and systems phenotypes. In this study we have presented further evidence of the close interrelationship in the molecular determinants of LQT-3 and BrS. We describe the functional consequences of two novel mutations of the same residue of the C-terminal portion of SCN5A linked to BrS (Y1795H) and LQT-3 (Y1795C). The most pronounced effects of the mutations are changes in channel gating that are consistent with the models that link channel to cellular to systems phenotypes for both LQT-3 (16Clancy C.E. Rudy Y. Nature. 1999; 400: 566-569Crossref PubMed Scopus (361) Google Scholar) and BrS (20Yan G.X. Antzelevitch C. Circulation. 1999; 100: 1660-1666Crossref PubMed Scopus (958) Google Scholar). Hence Y1795C mutant channels inactivate more slowly than WT channels and are characterized by a prominent fraction of current that is maintained during prolonged depolarization. Both of these changes in channel properties are expected to contribute more inward current during both the early and late phases of the action potential and hence are consistent with prolongation of the cellular action potential (16Clancy C.E. Rudy Y. Nature. 1999; 400: 566-569Crossref PubMed Scopus (361) Google Scholar, 34Clayton R.H. Bailey A. Biktashev V.N. Holden A.V. J. Theor. Biol. 2001; 208: 215-225Crossref PubMed Scopus (15) Google Scholar, 35Grant A.O. Am. J. Med. 2001; 110: 296-305Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Similarly, the most pronounced effects of the Y1795H mutation are a negative shift in channel availability, a speeding of the onset of inactivation, particularly at voltages near threshold for channel activation, and a reduction in peak current density. These effects are consistent with a reduction in inward current in cells expressing mutant channels as required in models proposed by Yan and Antzelevitch (20Yan G.X. Antzelevitch C. Circulation. 1999; 100: 1660-1666Crossref PubMed Scopus (958) Google Scholar) to explain ST segment elevation in BrS. We also found a small but consistent and statistically significant increase in maintained Na+ channel activity caused by the Y1795H mutation. By itself, this effect on channel gating would be expected to prolong the cellular action potential and accompanying QT interval in carriers of this gene defect. Despite this the dominant clinical phenotype leading to the discovery of this mutation was ST-segment elevation and not QT prolongation. Furthermore, when we investigated the effects of both mutations on the development of slow or intermediate inactivation, we found that both mutations promote this state of the channel to a degree that is significantly greater than WT channels. Because promotion of inactivation of the channel leads to eventual loss of channel activity, such a biophysical change would not be anticipated for an LQT-3 mutation, which would generally be considered linked to gain of function activity. However, our data indicate that these effects would only be anticipated under conditions of rapid stimulation (high heart rates), and the LQT-3 proband did not present clinical characteristics linked to BrS such as ST elevation and or other evidence of right bundle branch block even during challenges with flecainide indicating that this reduction in current is not likely to be important under physiologically relevant conditions. On the other hand, our data for the YC mutation show that biophysical properties that contribute to action potential duration (APD), prolongation, slowing of the onset of inactivation, and the promotion of maintained Na+ channel current are more pronounced at slower heart rates (Fig. 7) indicate that carriers of this mutation will be at greatest risk for prolonged APD-induced arrhythmias at slow heart rates. Thus the YC mutation, like other previously described LQT-3 mutations, is most problematic during bradycardia or following pauses in excitation of the ventricle (16Clancy C.E. Rudy Y. Nature. 1999; 400: 566-569Crossref PubMed Scopus (361) Google Scholar, 36Schwartz P.J. Priori S.G. Locati E.H. Napolitano C. Cantu F. Towbin J.A. Keating M.T. Hammoude H. Brown A.M. Chen L.S. Circulation. 1995; 92: 3381-3386Crossref PubMed Scopus (731) Google Scholar, 37Swan H. Saarinen K. Kontula K. Toivonen L. Viitasalo M. J. Am. Coll. Cardiol. 1998; 32: 486-491Crossref PubMed Scopus (56) Google Scholar, 38Wehrens X.H. Abriel H. Cabo C. Benhorin J. Kass R.S. Circulation. 2000; 102: 584-590Crossref PubMed Scopus (77) Google Scholar). The biophysical duality of the Y1795H and Y1795C mutations in some ways resembles the duality demonstrated for the 1795insD mutation described by Veldkamp et al. (33Veldkamp M.W. Viswanathan P.C. Bezzina C. Baartscheer A. Wilde A.A. Balser J.R. Circ. Res. 2000; 86: E91-E97Crossref PubMed Google Scholar). In the case of 1795insD, mutation-induced changes in an intermediate inactivated state of the channel reduce currents in a steeply heart rate-dependent manner. Thus at low heart rates a maintained current is revealed and potentiated relative to WT channels, and at faster heart rates, overall channel activity is reduced relative to WT (33Veldkamp M.W. Viswanathan P.C. Bezzina C. Baartscheer A. Wilde A.A. Balser J.R. Circ. Res. 2000; 86: E91-E97Crossref PubMed Google Scholar). We detected an increase in maintained Na+ channel activity for Y1795H mutant compared with WT channels which, even though much smaller in amplitude than similar activity induced by the Y1795C mutation, would contribute to delay in action potential repolarization. However, the overall reduction in the expression of YH channel activity (Fig. 5) apparently dominates the physiological impact of the mutation. The most pronounced biophysical consequence of the Y1795C mutation is the slowing of the onset of inactivation, which is accompanied by a marked increase in maintained channel activity. These two properties together are consistent with QT prolongation that characterizes the ECG of the proband, which as described above is also markedly dependent on heart rate. At sufficiently fast rates, the YC mutant channels will conduct less maintained current (Fig. 7), inactivate faster (Fig. 7), and, via enhanced Im inactivation (Fig. 6), express less peak current than WT channels. Taken together, these effects, although predicted for very fast stimulation rates, would predict BrS and not LQT-3 functional consequences. Together with the data of Veldkamp et al. (33Veldkamp M.W. Viswanathan P.C. Bezzina C. Baartscheer A. Wilde A.A. Balser J.R. Circ. Res. 2000; 86: E91-E97Crossref PubMed Google Scholar), the present results 1) demonstrate the critical importance of residue Tyr-1795 in controlling inactivation of the cardiac Na+ channel and 2) provide further evidence of the close interrelationship between Na+ channel properties that underlie BrS and LQT-3 in carriers of Na+ channel mutations. These mutations present an opportunity to dissect physiological conditions and pharmacological properties of the mutant channels that underlie expression of the distinct disease phenotypes both in the absence and presence of drug challenges. Further evidence for the crossover in mutation-induced channel properties and causal linkage to BrS and LQT-3 comes from a comparison of the properties of D1790G, another previously described LQT-3 mutation of the C terminus of the Na+ channel α-subunit, with those of the Y1795H BrS mutant channel. Both mutations speed the onset of inactivation, and both mutations cause a negative shift in the voltage dependence of steady state channel availability. Furthermore, under the same recording conditions, the Y1795H, but not the D1790G, mutation increases sustained channel activity, but clinically the two mutations have been linked distinctly to BrS (Y1795H) and LQT-3 (D1790G). A recent analysis of the D1790G channel using distinctly different intracellular recording conditions and a different cell line has revealed that, under these conditions, this mutation can promote sustained channel activity (14Baroudi G. Chahine M. FEBS Lett. 2000; 487: 224-228Crossref PubMed Scopus (59) Google Scholar). Computational analysis has suggested that the D1790G mutation prolongs APD indirectly via calcium-sensitive pathways in a steeply inverse heart rate-dependent fashion. Taken together these results suggest that careful analysis of clinical data for carriers of these C-terminal mutations under a wide variety of conditions in which critical parameters such as heart rate and sympathetic tone can be controlled will be extremely useful in dissecting the crucial properties of these mutant channels that cause these inherited arrhythmias and the clinical conditions that are most likely to exacerbate fatal events in carriers of the specific gene defects.
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