A Novel Missense Mutation,I890T,in the Pore Region of Cardiac Sodium Channel Causes Brugada Syndrome

Brugada syndrome (BrS) is a life-threatening, inherited arrhythmogenic syndrome associated with autosomal dominant mutations in SCN5A, the gene encoding the cardiac Na⁺ channel alpha subunit (Na_v1.5). The aim of this work was to characterize the functional alterations caused by a novel SCN5A mutation, I890T, and thus establish whether this mutation is associated with BrS. The mutation was identified by direct sequencing of SCN5A from the proband’s DNA. Wild-type (WT) or I890T Na_v1.5 channels were heterologously expressed in human embryonic kidney cells. Sodium currents were studied using standard whole cell patch-clamp protocols and immunodetection experiments were performed using an antibody against human Na_v1.5 channel. A marked decrease in current density was observed in cells expressing the I890T channel (from −52.0±6.5 pA/pF, n = 15 to −35.9±3.4 pA/pF, n = 22, at −20 mV, WT and I890T, respectively). Moreover, a positive shift of the activation curve was identified (V _1/2 = −32.0±0.3 mV, n = 18, and −27.3±0.3 mV, n = 22, WT and I890T, respectively). No changes between WT and I890T currents were observed in steady-state inactivation, time course of inactivation, slow inactivation or recovery from inactivation parameters. Cell surface protein biotinylation analyses confirmed that Na_v1.5 channel membrane expression levels were similar in WT and I890T cells. In summary, our data reveal that the I890T mutation, located within the pore of Na_v1.5, causes an evident loss-of-function of the channel. Thus, the BrS phenotype observed in the proband is most likely due to this mutation.     

A novel mutation in the SCN5A gene is associated with Brugada syndrome

Brugada syndrome (BS) is an inherited cardiac disorder associated with a high risk of sudden cardiac death and is caused by mutations in the SCN5A gene encoding the cardiac sodium channel alpha-subunit (Na(v)1.5). The aim of this study was to identify the genetic cause of familial BS and characterize the electrophysiological properties of a novel SCN5A mutation (W1191X). Four families and one patient with BS were screened for SCN5A mutations by PCR and direct sequencing. Wild-type (WT) and mutant Na(v)1.5 channels were expressed in tsA201 cells, and the sodium currents (I(Na)) were analyzed using the whole-cell patch-clamp technique. A novel mutation, W1191X, was identified in a family with BS. Expression of the WT or the mutant channel (Na(v)1.5/W1191X) co-transfected with the beta(1)-subunit in tsA201 cells resulted in a loss of function of Na(v)1.5 channels. While voltage-clamp recordings of the WT channel showed a distinct acceleration of Na(v)1.5 activation and fast inactivation kinetics, the Na(v)1.5/W1191X mutant failed to generate any currents. Co-expression of the WT channel and the mutant channel resulted in a 50% reduction in I(Na). No effect on activation and inactivation were observed with this heterozygous expression. The W1191X mutation is associated with BS and resulted in the loss of function of the cardiac sodium channel.     

Electrophysiological Characteristics of a SCN5A Voltage Sensors Mutation R1629Q Associated With Brugada Syndrome

Brugada syndrome (BrS) is an inherited arrhythmogenic syndrome leading to sudden cardiac death, partially associated with autosomal dominant mutations in SCN5A, which encodes the cardiac sodium channel alpha-subunit (Na_v1.5). To date some SCN5A mutations related with BrS have been identified in voltage sensor of Na_v1.5. Here, we describe a dominant missense mutation (R1629Q) localized in the fourth segment of domain IV region (DIV-S4) in a Chinese Han family. The mutation was identified by direct sequencing of SCN5A from the proband’s DNA. Co-expression of Wild-type (WT) or R1629Q Na_v1.5 channel and hβ1 subunit were achieved in human embryonic kidney cells by transient transfection. Sodium currents were recorded using whole cell patch-clamp protocols. No significant changes between WT and R1629Q currents were observed in current density or steady-state activation. However, hyperpolarized shift of steady–state inactivation curve was identified in cells expressing R1629Q channel (WT: V_1/2 = -81.1 ± 1.3 mV, n = 13; R1629Q: V_1/2 = -101.7 ± 1.2 mV, n = 18). Moreover, R1629Q channel showed enhanced intermediate inactivation and prolonged recovery time from inactivation. In summary, this study reveals that R1629Q mutation causes a distinct loss-of-function of the channel due to alter its electrophysiological characteristics, and facilitates our understanding of biophysical mechanisms of BrS.     

Functional Characterization of a Novel Frameshift Mutation in the C-terminus of the Nav1.5 Channel Underlying a Brugada Syndrome with Variable Expression in a Spanish Family

Introduction

We functionally analyzed a frameshift mutation in the SCN5A gene encoding cardiac Na⁺ channels (Nav1.5) found in a proband with repeated episodes of ventricular fibrillation who presented bradycardia and paroxysmal atrial fibrillation. Seven relatives also carry the mutation and showed a Brugada syndrome with an incomplete and variable expression. The mutation (p.D1816VfsX7) resulted in a severe truncation (201 residues) of the Nav1.5 C-terminus.

Methods and Results

Wild-type (WT) and mutated Nav1.5 channels together with hNavβ1 were expressed in CHO cells and currents were recorded at room temperature using the whole-cell patch-clamp. Expression of p.D1816VfsX7 alone resulted in a marked reduction (≈90%) in peak Na⁺ current density compared with WT channels. Peak current density generated by p.D1816VfsX7+WT was ≈50% of that generated by WT channels. p.D1816VfsX7 positively shifted activation and inactivation curves, leading to a significant reduction of the window current. The mutation accelerated current activation and reactivation kinetics and increased the fraction of channels developing slow inactivation with prolonged depolarizations. However, late I_Na was not modified by the mutation. p.D1816VfsX7 produced a marked reduction of channel trafficking toward the membrane that was not restored by decreasing incubation temperature during cell culture or by incubation with 300 μM mexiletine and 5 mM 4-phenylbutirate.

Conclusion

Despite a severe truncation of the C-terminus, the resulting mutated channels generate currents, albeit with reduced amplitude and altered biophysical properties, confirming the key role of the C-terminal domain in the expression and function of the cardiac Na⁺ channel.     

Distinct functional defect of three novel Brugada syndrome related cardiac sodium channel mutations

The Brugada syndrome is characterized by ST segment elevation in the right precodial leads V1-V3 on surface ECG accompanied by episodes of ventricular fibrillation causing syncope or even sudden death. The molecular and cellular mechanisms that lead to Brugada syndrome are not yet completely understood. However, SCN5A is the most well known responsible gene that causes Brugada syndrome. Until now, more than a hundred mutations in SCN5A responsible for Brugada syndrome have been described. Functional studies of some of the mutations have been performed and show that a reduction of human cardiac sodium current accounts for the pathogenesis of Brugada syndrome. Here we reported three novel SCN5A mutations identified in patients with Brugada syndrome in Taiwan (p.I848fs, p.R965C, and p.1876insM). Their electrophysiological properties were altered by patch clamp analysis. The p.I848fs mutant generated no sodium current. The p.R965C and p.1876insM mutants produced channels with steady state inactivation shifted to a more negative potential (9.4 mV and 8.5 mV respectively), and slower recovery from inactivation. Besides, the steady state activation of p.1876insM was altered and was shifted to a more positive potential (7.69 mV). In conclusion, the SCN5A channel defect related to Brugada syndrome might be diverse but all resulted in a decrease of sodium current.     

Characterization of the novel heterozygous SCN5A genetic variant Y739D associated with Brugada syndrome

Genetic variants in SCN5A gene were identified in patients with various arrhythmogenic conditions including Brugada syndrome. Despite significant progress of last decades in studying the molecular mechanism of arrhythmia-associated SCN5A mutations, the understanding of relationship between genetics, electrophysiological consequences and clinical phenotype is lacking. We have found a novel genetic variant Y739D in the SCN5A-encoded sodium channel Na_v1.5 of a male patient with Brugada syndrome (BrS). The objective of the study was to characterize the biophysical properties of Na_v1.5-Y739D and provide possible explanation of the phenotype observed in the patient. The WT and Y739D channels were heterologously expressed in the HEK-293T cells and the whole-cell sodium currents were recorded. Substitution Y739D reduced the sodium current density by 47 ± 2% at ?20 mV, positively shifted voltage-dependent activation, accelerated both fast and slow inactivation, and decelerated recovery from the slow inactivation. The Y739D loss-of-function phenotype likely causes the BrS manifestation. In the hNa_v1.5 homology models, which are based on the cryo-EM structure of rat Na_v1.5 channel, Y739 in the extracellular loop IIS1-S2 forms H-bonds with K1381 and E1435 and pi-cation contacts with K1397 (all in loop IIIS5-P1). In contrast, Y739D accepts H-bonds from K1397 and Y1434. Substantially different contacts of Y739 and Y739D with loop IIIS5-P1 would differently transmit allosteric signals from VSD-II to the fast-inactivation gate at the N-end of helix IIIS5 and slow-inactivation gate at the C-end of helix IIIP1. This may underlie the atomic mechanism of the Y739D channel dysfunction.     

Biophysical phenotypes of SCN5A mutations causing long QT and Brugada syndromes

Long QT and Brugada syndromes are two hereditary cardiac diseases. Brugada syndrome has so far been associated with only one gene, SCN5A, which encodes the cardiac sodium channel. However, in long QT syndrome (LQTS) at least six genes, including the SCN5A, are implicated. The substitution (D1790G) causes LQTS and the insertion (D1795) induces both LQTS and Brugada syndromes in carrier patients. hH1/insD1795 and hH1/D1790G mutant channels were expressed in the tsA201 human cell line and characterized using the patch clamp technique in whole-cell configuration. Our data revealed a persistent inward sodium current of about 6% at -30 mV for both D1790G and insD1795, and a reduction of 62% of channel expression for the insD1795. Moreover, a shift of steady-state inactivation curve in both mutants was also observed. Our findings uphold the idea that LQT3 is related to a persistent sodium current whereas reduction in the expression level of cardiac sodium channels is one of the biophysical characteristics of Brugada syndrome.     

Partial expression defect for the SCN5A missense mutation G1406R depends on splice variant background Q1077 and rescue by mexiletine

Mutations in the cardiac Na(+) channel gene SCN5A cause loss of function and underlie arrhythmia syndromes. SCN5A in humans has two splice variants, one lacking a glutamine at position 1077 (Q1077del) and one containing Q1077. We investigated the effect of splice variant background on loss of function and rescue for G1406R, a mutation reported to cause Brugada syndrome. Mutant and wild-type (WT) channels in both backgrounds were transfected into HEK-293 cells and incubated for up to 72 h with and without mexiletine. At 8 h, neither current nor cell surface expression was observed for the mutant in either background, but both were present in WT channels. At 24 h, small (<10% compared with WT) currents were noted and accompanied by cell surface expression. At 48 h, current density was approximately 40% of WT channels for the mutant in the Q1077del variant background but remained at <10% of WT channels in Q1077. Current levels were stable by 72 h. Coexpression with beta(1)- or beta(3)-subunits or insertion of the polymorphism H558R in the background did not significantly affect current expression. Mexiletine restored current density of the mutant channel in both backgrounds to nearly WT levels. The mutant channels also showed a negative shift in inactivation, slower recovery, and enhanced slow inactivation, consistent with a loss of function phenotype. These data show that a trafficking defect may be partial and time dependent and may differ with the splice variant background. Also, expression defects and gating abnormalities may contribute to loss of function for the same mutation.     

A Heterozygous Deletion Mutation in the Cardiac Sodium Channel Gene SCN5A with Loss- and Gain-of-Function Characteristics Manifests as Isolated Conduction Disease,without Signs of Brugada or Long QT Syndrome

Background

The SCN5A gene encodes for the α-subunit of the cardiac sodium channel Na_V1.5, which is responsible for the rapid upstroke of the cardiac action potential. Mutations in this gene may lead to multiple life-threatening disorders of cardiac rhythm or are linked to structural cardiac defects. Here, we characterized a large family with a mutation in SCN5A presenting with an atrioventricular conduction disease and absence of Brugada syndrome.

Method and Results

In a large family with a high incidence of sudden cardiac deaths, a heterozygous SCN5A mutation (p.1493delK) with an autosomal dominant inheritance has been identified. Mutation carriers were devoid of any cardiac structural changes. Typical ECG findings were an increased P-wave duration, an AV-block I° and a prolonged QRS duration with an intraventricular conduction delay and no signs for Brugada syndrome. HEK293 cells transfected with 1493delK showed strongly (5-fold) reduced Na⁺ currents with altered inactivation kinetics compared to wild-type channels. Immunocytochemical staining demonstrated strongly decreased expression of SCN5A 1493delK in the sarcolemma consistent with an intracellular trafficking defect and thereby a loss-of-function. In addition, SCN5A 1493delK channels that reached cell membrane showed gain-of-function aspects (slowing of the fast inactivation, reduction in the relative fraction of channels that fast inactivate, hastening of the recovery from inactivation).

Conclusion

In a large family, congregation of a heterozygous SCN5A gene mutation (p.1493delK) predisposes for conduction slowing without evidence for Brugada syndrome due to a predominantly trafficking defect that reduces Na⁺ current and depolarization force.     

Accelerated inactivation in a mutant Na(+) channel associated with idiopathic ventricular fibrillation

Idiopathic ventricular fibrillation (IVF) can cause sudden death in both adults and children. One form of IVF (Brugada syndrome), characterized by S-T segment elevation (STE) in the electrocardiogram, has been linked to mutations of SCN5A, the gene encoding the voltage-gated cardiac Na(+) channel. A missense mutation of SCN5A that substitutes glutamine for leucine at codon 567 (L567Q, in the cytoplasmic linker between domains I and II) is identified with sudden infant death and Brugada syndrome in one family. However, neither the functional effect of the L567Q mutation nor the molecular mechanism underlying the pathogenicity of the mutation is known. Patch-clamp analysis of L567Q channels expressed in human embryonic kidney cells revealed a marked acceleration and a negative shift in the voltage dependence of inactivation. Unlike other Brugada mutations, this phenotype was expressed independently of temperature or auxiliary beta(1)-subunits. These results support a proposed linkage between Brugada syndrome and some instances of sudden infant death and the hypothesis that reduced Na(+) conductance is the primary cause of IVF with STE.     

Clinical aspects and physiopathology of Brugada syndrome: review of current concepts

Brugada syndrome (BS) is an inherited cardiac disorder characterized by typical electrocardiographic patterns of ST segment elevation in the precordial leads, right bundle branch block, fast polymorphic ventricular tachycardia in patients without any structural heart disease, and a high risk of sudden cardiac death. The incidence of BS is high in male vs. female (i.e., 8-10/1: male/female). The disorder is caused by mutations in the SCN5A gene encoding Nav1.5, the cardiac sodium channel, which is the only gene in which mutations were found to cause the disease. Mutations in SCN5A associated with the BS phenotype usually result in a loss of channel function by a reduction in Na+ currents. We review the clinical aspects, risk stratification, and therapeutic management of this important syndrome.     

A Novel LQT-3 Mutation Disrupts an Inactivation Gate Complex with Distinct Rate-Dependent Phenotypic Consequences

Inherited mutations of SCN5A, the gene that encodes Nav1.5, the alpha subunit of the principle voltage-gated Na+ channel in the heart, cause congenital Long QT Syndrome variant 3 (LQT-3) by perturbation of channel inactivation. LQT-3 mutations induce small, but aberrant, inward current that prolongs the ventricular action potential and subjects mutation carriers to arrhythmia risk dictated in part by the biophysical consequences of the mutations. Most previously investigated LQT-3 mutations are associated with increased arrhythmia risk during rest or sleep. Here we report a novel LQT-3 mutation discovered in a pediatric proband diagnosed with LQTS but who experienced cardiac events during periods of mild exercise as well as rest. The mutation, which changes a single amino acid (S1904L) in the Nav1.5 carboxy terminal domain, disrupts the channel inactivation gate complex and promotes late Na+ channel currents, not by promoting a bursting mode of gating, but by increasing the propensity of the channel to reopen during prolonged depolarization. Incorporating a modified version of the Markov model of the Nav1.5 channel into a mathematical model of the human ventricular action potential predicts that the biophysical consequences of the S1904L mutation result in action potential prolongation that is seen for all heart rates but, in contrast to other previously-investigated LQT-3 mutant channels, is most pronounced at fast rates resulting in a drastic reduction in the cells ability to adapt APD to heart rate.     

Channel openings are necessary but not sufficient for use-dependent block of cardiac Na(+) channels by flecainide: evidence from the analysis of disease-linked mutations

Na(+) channel blockers such as flecainide have found renewed usefulness in the diagnosis and treatment of two clinical syndromes arising from inherited mutations in SCN5A, the gene encoding the alpha subunit of the cardiac voltage-gated Na(+) channel. The Brugada syndrome (BrS) and the LQT-3 variant of the Long QT syndrome are caused by disease-linked SCN5A mutations that act to change functional and pharmacological properties of the channel. Here we have explored a set of SCN5A mutations linked both to BrS and LQT-3 to determine what disease-modified channel properties underlie distinct responses to the Na(+) channel blocker flecainide. We focused on flecainide block that develops with repetitive channel activity, so-called use-dependent block (UDB). Our results indicate that mutation-induced changes in the voltage-dependence of channel availability (inactivation) may act as determinants of flecainide block. The data further indicate that UDB by flecainide requires channel opening, but is not likely due to open channel block. Rather, flecainide appears to interact with inactivation states that follow depolarization-induced channel opening, and mutation-induced changes in channel inactivation will alter flecainide block independent of the disease to which the mutation is linked. Analysis of flecainide block of mutant channels linked to these rare disorders has provided novel insight into the molecular determinants of drug action.     

Enhanced Slow Inactivation of the Human Skeletal Muscle Sodium Channel Causing Normokalemic Periodic Paralysis

Normokalemic periodic paralysis (normoPP) is a type of skeletal muscle function disorder which is characterized by paralysis attack with concomitant normal serum potassium level. We previously reported that R675Q mutation of human skeletal muscle voltage-gated sodium channel α subunit (SCN4A) may be the novel mutation which caused normoPP in Chinese families. However, it is still not clear how this mutation affects the SCN4A channel function. In this study, we used patch-clamp recording to study the function of wild type (WT) and R675Q mutant of SCN4A channels expressed in human embryonic kidney (HEK293) cells. We found that R675Q mutation did not affect the voltage dependence of sodium channel activation. The fast inactivation was also not significantly affected by R675Q mutation. However, R675Q mutation of SCN4A channels exhibited an 11.1 mV hyperpolarized shift in the voltage dependence of slow inactivation and significantly prolonged the recovery from prolonged inactivation state. Our results thus indicate that SCN4A was functionally affected by R675Q mutation, suggesting a possible reason for causing normoPP in Chinese patients.     

A novel SCN5A mutation associated with idiopathic ventricular fibrillation without typical ECG findings of Brugada syndrome

Mutations in the human cardiac Na+ channel alpha subunit gene (SCN5A) are responsible for Brugada syndrome, an idiopathic ventricular fibrillation (IVF) subgroup characterized by right bundle branch block and ST elevation on an electrocardiogram (ECG). However, the molecular basis of IVF in subgroups lacking these ECG findings has not been elucidated. We performed genetic screenings of Japanese IVF patients and found a novel SCN5A missense mutation (S1710L) in one symptomatic IVF patient that did not exhibit the typical Brugada ECG. Heterologously expressed S1710L channels showed marked acceleration in the current decay together with a large hyperpolarizing shift of steady-state inactivation and depolarizing shift of activation. These findings suggest that SCN5A is one of the responsible genes for IVF patients who do not show typical ECG manifestations of the Brugada syndrome.     

Inherited Brugada and long QT-3 syndrome mutations of a single residue of the cardiac sodium channel confer distinct channel and clinical phenotypes.

Defects of the SCN5A gene encoding the cardiac sodium channel alpha-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.     

SCN5A mutation (T1620M) causing Brugada syndrome exhibits different phenotypes when expressed in Xenopus oocytes and mammalian cells

Brugada syndrome is a hereditary cardiac disease causing abnormal ST segment elevation in the ECG, right bundle branch block, ventricular fibrillation and sudden death. In this study we characterized a new mutation in the SCN5A gene (T1620M), causing the Brugada syndrome. The mutated channels were expressed in both Xenopus leavis oocytes and in mammalian tsA201 cells with and without the beta-subunit and studied using the patch clamp technique. Opposite phenotypes were observed depending on the expression system. T1620M mutation led to a faster recovery from inactivation and a shift of steady-state inactivation to more positive voltages when expressed in Xenopus oocytes. However, using the mammalian expression system no effect on steady-state inactivation was observed, but this mutation led to a slower recovery from inactivation. Our finding supports the idea that the slower recovery from inactivation of the cardiac sodium channels seen in our mammalian expression system could decrease the density of sodium channels during the cardiac cycle explaining the in vivo arrhythmogenesis in patients with Brugada syndrome.     

The congenital long QT syndrome Type 3: An update

Congenital long QT syndrome type 3 (LQT3) is the third in frequency compared to the 15 forms known currently of congenital long QT syndrome (LQTS). Cardiac events are less frequent in LQT3 when compared with LQT1 and LQT2, but more likely to be lethal; the likelihood of dying during a cardiac event is 20% in families with an LQT3 mutation and 4% with either an LQT1 or an LQT2 mutation. LQT3 is consequence of mutation of gene SCN5A which codes for the Nav1.5 Na⁺ channel α-subunit and electrocardiographically characterized by a tendency to bradycardia related to age, prolonged QT/QTc interval (mean QTc value 478 ± 52 ms), accentuated QT dispersion consequence of prolonged ST segment, late onset of T wave and frequent prominent U wave because of longer repolarization of the M cell across left ventricular wall.     

Sodium channel kinetic changes that produce Brugada syndrome or progressive cardiac conduction system disease

Some mutations of the sodium channel gene Na(V1.5) are multifunctional, causing combinations of LQTS, Brugada syndrome and progressive cardiac conduction system disease (PCCD). The combination of Brugada syndrome and PCCD is uncommon, although they both result from a reduction in the sodium current. We hypothesize that slow conduction is sufficient to cause S-T segment elevation and undertook a combined experimental and theoretical study to determine whether conduction slowing alone can produce the Brugada phenotype. Deletion of lysine 1479 in one of two positively charged clusters in the III/IV inter-domain linker causes both syndromes. We have examined the functional effects of this mutation using heterologous expression of the wild-type and mutant sodium channel in HEK-293-EBNA cells. We show that DeltaK1479 shifts the potential of half-activation, V(1/2m), to more positive potentials (V(1/2m) = -36.8 +/- 0.8 and -24.5 +/- 1.3 mV for the wild-type and DeltaK1479 mutant respectively, n = 11, 10). The depolarizing shift increases the extent of depolarization required for activation. The potential of half-inactivation, V(1/2h), is also shifted to more positive potentials (V(1/2h) = -85 +/- 1.1 and -79.4 +/- 1.2 mV for wild-type and DeltaK1479 mutant respectively), increasing the fraction of channels available for activation. These shifts are quantitatively the same as a mutation that produces PCCD only, G514C. We incorporated experimentally derived parameters into a model of the cardiac action potential and its propagation in a one dimensional cable (simulating endo-, mid-myocardial and epicardial regions). The simulations show that action potential and ECG changes consistent with Brugada syndrome may result from conduction slowing alone; marked repolarization heterogeneity is not required. The findings also suggest how Brugada syndrome and PCCD which both result from loss of sodium channel function are sometimes present alone and at other times in combination.     

Size matters: Erythromelalgia mutation S241T in Nav1.7 alters channel gating

The Nav1.7 sodium channel is preferentially expressed in most nociceptive dorsal root ganglion neurons and in sympathetic neurons. Inherited erythromelalgia (IEM, also known as erythermalgia), an autosomal dominant neuropathy characterized by burning pain in the extremities in response to mild warmth, has been linked to mutations in Nav1.7. Recently, a substitution of Ser-241 by threonine (S241T) in the domain I S4-S5 linker of Nav1.7 was identified in a family with IEM. To investigate the possible causative role of this mutation in the pathophysiology of IEM, we used whole-cell voltage-clamp analysis to study the effects of S241T on Nav1.7 gating in HEK293 cells. We found a hyperpolarizing shift of activation midpoint by 8.4 mV, an accelerated time to peak, slowing of deactivation, and an increase in the current in response to small, slow depolarizations. Additionally, S241T produced an enhancement of slow inactivation, shifting the midpoint by -12.3 mV. Because serine and threonine have similar biochemical properties, the S241T substitution suggested that the size of the side chain at this position affected channel gating. To test this hypothesis, we investigated the effect of S241A and S241L substitutions on the gating properties of Nav1.7. Although S241A did not alter the properties of the channel, S241L mimicked the effects of S241T. We conclude that the linker between S4 and S5 in domain I of Nav1.7 modulates gating of this channel, and that a larger side chain at position 241 interferes with its gating mechanisms.