The novel persistent sodium current inhibitor PRAX-562 has potent anticonvulsant activity with improved protective index relative to standard of care sodium channel blockers.

OBJECTIVE: This study investigates the effects of PRAX-562 on sodium current (INa ), intrinsic neuronal excitability, and protection from evoked seizures to determine whether a preferential persistent INa inhibitor would exhibit improved preclinical efficacy and tolerability compared to two standard voltage-gated sodium channel (NaV ) blockers. METHODS: Inhibition of INa  was characterized using patch clamp analysis. The effect on intrinsic excitability was measured using evoked action potentials recorded from hippocampal CA1 pyramidal neurons in mouse brain slices. Anticonvulsant activity was evaluated using the maximal electroshock seizure (MES) model, and tolerability was assessed by measuring spontaneous locomotor activity (sLMA). RESULTS: PRAX-562 potently and preferentially inhibited persistent INa induced by ATX-II or the SCN8A mutation N1768D (half-maximal inhibitory concentration [IC50 ] = 141 and 75 nmol·L-1 , respectively) relative to peak INa tonic/resting block (60× preference). PRAX-562 also exhibited potent use-dependent block (31× preference to tonic block). This profile is considerably different from standard NaV blockers, including carbamazepine (CBZ; persistent INa IC50 = 77 500 nmol·L-1 , preference ratios of 30× [tonic block], less use-dependent block observed at various frequencies). In contrast to CBZ, PRAX-562 reduced neuronal intrinsic excitability with only a minor reduction in action potential amplitude. PRAX-562 (10 mg/kg po) completely prevented evoked seizures without affecting sLMA (MES unbound brain half-maximal efficacious concentration = 4.3 nmol·L-1 , sLMA half-maximal tolerated concentration = 69.7 nmol·L-1 , protective index [PI] = 16×). In contrast, CBZ and lamotrigine (LTG) had PIs of approximately 5.5×, with significant overlap between doses that were anticonvulsant and that reduced locomotor activity. SIGNIFICANCE: PRAX-562 demonstrated robust preclinical anticonvulsant activity similar to CBZ but improved compared to LTG. PRAX-562 exhibited significantly improved preclinical tolerability compared with standard NaV blockers (CBZ and LTG), potentially due to the preference for persistent INa . Preferential targeting of persistent INa may represent a differentiated therapeutic option for diseases of hyperexcitability, where standard NaV blockers have demonstrated efficacy but poor tolerability.

Discovery of the First Orally Available, Selective KNa1.1 Inhibitor: In Vitro and In Vivo Activity of an Oxadiazole Series.

The gene KCNT1 encodes the sodium-activated potassium channel KNa1.1 (Slack, Slo2.2). Variants in the KCNT1 gene induce a gain-of-function (GoF) phenotype in ionic currents and cause a spectrum of intractable neurological disorders in infants and children, including epilepsy of infancy with migrating focal seizures (EIMFS) and autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). Effective treatment options for KCNT1-related disease are absent, and novel therapies are urgently required. We describe the development of a novel class of oxadiazole KNa1.1 inhibitors, leading to the discovery of compound 31 that reduced seizures and interictal spikes in a mouse model of KCNT1 GoF.

Eleclazine exhibits enhanced selectivity for long QT syndrome type 3-associated late Na+ current.

BACKGROUND: Eleclazine (GS-6615) is a sodium channel blocker designed to improve the selectivity for cardiac late Na+ current (INa) over peak INa. OBJECTIVES: The goals of this study were to investigate the inhibition of late INa by eleclazine using a sample of long QT syndrome type 3 (LQT3) and overlap LQT3/Brugada syndrome mutant channels; to compare the apparent binding rates for eleclazine with those for other class 1 antiarrhythmic agents; and to investigate the binding site. METHODS: Wild-type human cardiac voltage-gated sodium channel (hNaV1.5) and 21 previously reported variants were studied using patch clamp recordings from a heterologous expression system. RESULTS: Eleclazine inhibited anemone toxin II-enhanced late INa from wild-type hNaV1.5 with a drug concentration that causes 50% block of 0.62 ± 0.12 µM (84-fold selectivity over peak INa). The drug concentration that causes 50% block of eleclazine to inhibit the enhanced late INa from LQT3 mutant channels ranged from 0.33 to 1.7 µM. At predicted therapeutic concentrations, eleclazine and ranolazine inhibited peak INa to a similar degree as assessed with 4 overlap LQT3/Brugada syndrome mutations. Eleclazine was found to interact with hNaV1.5 significantly faster than ranolazine and 6 other class 1 antiarrhythmic agents. Engineered mutations (F1760A/Y1767A) located within the local anesthetic binding site decreased the inhibition of late INa and peak INa by eleclazine. CONCLUSION: At predicted therapeutic concentrations, eleclazine elicits potent inhibition of late INa across a cohort of NaV1.5 mutant channels. These properties are consistent with a class 1b antiarrhythmic agent that associates with unusually rapid binding/unbinding rates.

Early somatic mosaicism is a rare cause of long-QT syndrome.

Somatic mosaicism, the occurrence and propagation of genetic variation in cell lineages after fertilization, is increasingly recognized to play a causal role in a variety of human diseases. We investigated the case of life-threatening arrhythmia in a 10-day-old infant with long QT syndrome (LQTS). Rapid genome sequencing suggested a variant in the sodium channel NaV1.5 encoded by SCN5A, NM_000335:c.5284G > T predicting p.(V1762L), but read depth was insufficient to be diagnostic. Exome sequencing of the trio confirmed read ratios inconsistent with Mendelian inheritance only in the proband. Genotyping of single circulating leukocytes demonstrated the mutation in the genomes of 8% of patient cells, and RNA sequencing of cardiac tissue from the infant confirmed the expression of the mutant allele at mosaic ratios. Heterologous expression of the mutant channel revealed significantly delayed sodium current with a dominant negative effect. To investigate the mechanism by which mosaicism might cause arrhythmia, we built a finite element simulation model incorporating Purkinje fiber activation. This model confirmed the pathogenic consequences of cardiac cellular mosaicism and, under the presenting conditions of this case, recapitulated 2:1 AV block and arrhythmia. To investigate the extent to which mosaicism might explain undiagnosed arrhythmia, we studied 7,500 affected probands undergoing commercial gene-panel testing. Four individuals with pathogenic variants arising from early somatic mutation events were found. Here we establish cardiac mosaicism as a causal mechanism for LQTS and present methods by which the general phenomenon, likely to be relevant for all genetic diseases, can be detected through single-cell analysis and next-generation sequencing.

The novel late Na+ current inhibitor, GS-6615 (eleclazine) and its anti-arrhythmic effects in rabbit isolated heart preparations.

BACKGROUND AND PURPOSE: Enhanced late Na+ current (late INa ) in the myocardium is pro-arrhythmic. Inhibition of this current is a promising strategy to stabilize ventricular repolarization and suppress arrhythmias. Here, we describe GS-6615, a selective inhibitor of late INa , already in clinical development for the treatment of long QT syndrome 3 (LQT3). EXPERIMENTAL APPROACH: The effects of GS-6615 to inhibit late INa , versus other ion currents to shorten the ventricular action potential duration (APD), monophasic APD (MAPD) and QT interval, and decrease to the incidence of ventricular arrhythmias was determined in rabbit cardiac preparations. To mimic the electrical phenotype of LQT3, late INa was increased using the sea anemone toxin (ATX-II). KEY RESULTS: GS-6615 inhibited ATX-II enhanced late INa in ventricular myocytes (IC50  = 0.7 µM), shortened the ATX-II induced prolongation of APD, MAPD, QT interval, and decreased spatiotemporal dispersion of repolarization and ventricular arrhythmias. Inhibition by GS-6615 of ATX-II enhanced late INa was strongly correlated with shortening of myocyte APD and isolated heart MAPD (R2  = 0.94 and 0.98 respectively). In contrast to flecainide, GS-6615 had the minimal effects on peak INa . GS-6615 did not decrease the maximal upstroke velocity of the action potential (Vmax) nor widen QRS intervals. CONCLUSIONS AND IMPLICATIONS: GS-6615 was a selective inhibitor of late INa , stabilizes the ventricular repolarization and suppresses arrhythmias in a model of LQT3. The concentrations at which the electrophysiological effects of GS-6615 were observed are comparable to plasma levels associated with QTc shortening in patients with LQT3, indicating that these effects are clinically relevant.

Eleclazine, a new selective cardiac late sodium current inhibitor, confers concurrent protection against autonomically induced atrial premature beats, repolarization alternans and heterogeneity, and atrial fibrillation in an intact porcine model.

BACKGROUND: The cardiac late sodium current (INa) has been increasingly implicated in the initiation of atrial fibrillation (AF). Eleclazine (formerly known as GS-6615) is a new selective late INa inhibitor and is undergoing clinical testing for the treatment of cardiac arrhythmias. OBJECTIVE: We tested whether late INa inhibition by eleclazine confers protection against atrial premature beats (APBs) and AF. METHODS: In closed-chest anesthetized Yorkshire pigs, epinephrine (2.0 µg/kg, intravenous, bolus over 1 minute) was administered alone to induce APBs (n = 6) or in combination with intrapericardial acetylcholine (0.5-4 mL of 12.5 mM solution) to induce spontaneous AF (n = 11). Effects of eleclazine (0.3 and 0.9 mg/kg, intravenous, over 15 minutes) on APBs and AF were determined. RESULTS: Epinephrine-induced APBs were reduced >3-fold (P < .04) after eleclazine (0.9 mg/kg) infusion. The combined administration of epinephrine and acetylcholine resulted in AF in all animals tested, which was invariably preceded by APBs. Eleclazine pretreatment suppressed AF in all 7 animals in at least 1 test episode during the 60- to 150-minute observation period (P = .04). The plasma eleclazine level at 120 minutes was 828 ± 45.8 nM, within exposure range evaluated clinically. Eleclazine shortened ventricular QT and atrial PTa intervals by 7% (P < .001 for both) and reduced atrial repolarization alternans (P = .003) and heterogeneity (P = .021) without attenuation of the inotropic response to catecholamine (P = .56). The drug inhibited the enhanced late INa of single atrial myocytes with a potency of 736 ± 67 nM. CONCLUSION: Selective cardiac late INa inhibition with eleclazine suppresses autonomically mediated atrial repolarization alternans and heterogeneity, APBs, and AF in an intact porcine model.

Mutations in SCN10A are responsible for a large fraction of cases of Brugada syndrome.

BACKGROUND: BrS is an inherited sudden cardiac death syndrome. Less than 35% of BrS probands have genetically identified pathogenic variants. Recent evidence has implicated SCN10A, a neuronal sodium channel gene encoding Nav1.8, in the electrical function of the heart. OBJECTIVES: The purpose of this study was to test the hypothesis that SCN10A variants contribute to the development of Brugada syndrome (BrS). METHODS: Clinical analysis and direct sequencing of BrS susceptibility genes were performed for 150 probands and family members as well as >200 healthy controls. Expression and coimmunoprecipitation studies were performed to functionally characterize the putative pathogenic mutations. RESULTS: We identified 17 SCN10A mutations in 25 probands (20 male and 5 female); 23 of the 25 probands (92.0%) displayed overlapping phenotypes. SCN10A mutations were found in 16.7% of BrS probands, approaching our yield for SCN5A mutations (20.1%). Patients with BrS who had SCN10A mutations were more symptomatic and displayed significantly longer PR and QRS intervals compared with SCN10A-negative BrS probands. The majority of mutations localized to the transmembrane-spanning regions. Heterologous coexpression of wild-type (WT) SCN10A with WT-SCN5A in HEK cells caused a near doubling of sodium channel current compared with WT-SCN5A alone. In contrast, coexpression of SCN10A mutants (R14L and R1268Q) with WT-SCN5A caused a 79.4% and 84.4% reduction in sodium channel current, respectively. The coimmunoprecipitation studies provided evidence for the coassociation of Nav1.8 and Nav1.5 in the plasma membrane. CONCLUSIONS: Our study identified SCN10A as a major susceptibility gene for BrS, thus greatly enhancing our ability to genotype and risk stratify probands and family members.

Blockade of Na+ channels in pancreatic α-cells has antidiabetic effects.

Pancreatic α-cells express voltage-gated Na(+) channels (NaChs), which support the generation of electrical activity leading to an increase in intracellular calcium, and cause exocytosis of glucagon. Ranolazine, a NaCh blocker, is approved for treatment of angina. In addition to its antianginal effects, ranolazine has been shown to reduce HbA1c levels in patients with type 2 diabetes mellitus and coronary artery disease; however, the mechanism behind its antidiabetic effect has been unclear. We tested the hypothesis that ranolazine exerts its antidiabetic effects by inhibiting glucagon release via blockade of NaChs in the pancreatic α-cells. Our data show that ranolazine, via blockade of NaChs in pancreatic α-cells, inhibits their electrical activity and reduces glucagon release. We found that glucagon release in human pancreatic islets is mediated by the Nav1.3 isoform. In animal models of diabetes, ranolazine and a more selective NaCh blocker (GS-458967) lowered postprandial and basal glucagon levels, which were associated with a reduction in hyperglycemia, confirming that glucose-lowering effects of ranolazine are due to the blockade of NaChs. This mechanism of action is unique in that no other approved antidiabetic drugs act via this mechanism, and raises the prospect that selective Nav1.3 blockers may constitute a novel approach for the treatment of diabetes.

Ranolazine reduces neuronal excitability by interacting with inactivated states of brain sodium channels.

Ranolazine is an approved drug for chronic stable angina that acts by suppressing a noninactivating current conducted by the cardiac sodium channel [persistent sodium ion current (INa)]. Ranolazine has also been shown to inhibit the increased persistent INa carried by NaV1.1 channels encoding epilepsy- and migraine-associated mutations. Here, we investigate the antiepileptic properties of ranolazine exhibited through the reduction of hippocampal neuronal excitability. At therapeutically relevant concentrations, ranolazine reduced action potential firing frequency of hippocampal neurons in response to repetitive depolarizing current injections. Similarly, using a single current injection paradigm, ranolazine required a long depolarization (4 seconds) to produce significant inhibition of excitability, which was similar to that observed for the anticonvulsants phenytoin (slowly binds to the fast-inactivated state) and lacosamide (binds to the slow-inactivated state). Ranolazine enhanced the development of fast and slow inactivation assessed with conditioning prepulses of 100, 1000, or 10,000 milliseconds. Recovery of channels from inactivated states was also slowed in the presence of ranolazine. Interestingly, the use-dependent inhibition of hippocampal neurons was dependent on the duration of the voltage step, suggesting ranolazine does not selectively affect the open state and may also interact with inactivated states. NEURON (Yale University, New Haven, CT) computational simulations predict equal inhibition of action potential generation for binding to either fast-inactivated or slow-inactivated states. Binding of ranolazine to either preopen or open states did not affect the excitability of the simulation. Ranolazine was able to significantly reduce the epileptiform activity of the neuronal cultures, suggesting possible antiepileptic activity.

Febrile temperatures unmask biophysical defects in Nav1.1 epilepsy mutations supportive of seizure initiation.

Generalized epilepsy with febrile seizures plus (GEFS+) is an early onset febrile epileptic syndrome with therapeutic responsive (a)febrile seizures continuing later in life. Dravet syndrome (DS) or severe myoclonic epilepsy of infancy has a complex phenotype including febrile generalized or hemiclonic convulsions before the age of 1, followed by intractable myoclonic, complex partial, or absence seizures. Both diseases can result from mutations in the Nav1.1 sodium channel, and initially, seizures are typically triggered by fever. We previously characterized two Nav1.1 mutants-R859H (GEFS+) and R865G (DS)-at room temperature and reported a mixture of biophysical gating defects that could not easily predict the phenotype presentation as either GEFS+ or DS. In this study, we extend the characterization of Nav1.1 wild-type, R859H, and R865G channels to physiological (37°C) and febrile (40°C) temperatures. At physiological temperature, a variety of biophysical defects were detected in both mutants, including a hyperpolarized shift in the voltage dependence of activation and a delayed recovery from fast and slow inactivation. Interestingly, at 40°C we also detected additional gating defects for both R859H and R865G mutants. The GEFS+ mutant R859H showed a loss of function in the voltage dependence of inactivation and an increased channel use-dependency at 40°C with no reduction in peak current density. The DS mutant R865G exhibited reduced peak sodium currents, enhanced entry into slow inactivation, and increased use-dependency at 40°C. Our results suggest that fever-induced temperatures exacerbate the gating defects of R859H or R865G mutants and may predispose mutation carriers to febrile seizures.

Nontruncating SCN1A mutations associated with severe myoclonic epilepsy of infancy impair cell surface expression.

Mutations in SCN1A, encoding the voltage-gated sodium channel Na(V)1.1, are the most common cause of severe myoclonic epilepsy of infancy (SMEI) or Dravet syndrome. SMEI is most often associated with premature truncations of Na(V)1.1 that cause loss of function, but nontruncating mutations also occur. We hypothesized that some nontruncating mutations might impair trafficking of Na(V)1.1 to the plasma membrane. Here we demonstrated that seven nontruncating missense or in-frame deletion mutations (L986F, delF1289, R1648C, F1661S, G1674R, and G1979E) exhibited reduced cell surface expression relative to wild type (WT) Na(V)1.1 consistent with impaired trafficking. We tested whether two commonly prescribed antiepileptic drugs (phenytoin, lamotrigine), as well as the cystic fibrosis transmembrane conductance regulator (CFTR) trafficking corrector VRT-325, could rescue cell surface and functional expression of two representative Na(V)1.1 mutants (R1648C, G1674R). Treatment of cells with phenytoin increased cell surface expression of WT-Na(V)1.1 and both mutant channels, whereas lamotrigine only increased surface expression of R1648C. VRT-325 did not alter surface expression of WT-Na(V)1.1 or mutant channels. Although phenytoin increased surface expression of G1674R, channel function was not restored, suggesting that this mutation also causes an intrinsic loss of function. Both phenytoin and lamotrigine increased functional expression of R1648C, but lamotrigine also increased persistent sodium current evoked by this mutation. Our findings indicate that certain nontruncating SCN1A mutations associated with SMEI have impaired cell surface expression and that some alleles may be amenable to pharmacological rescue of this defect. However, rescue of dysfunctional Na(V)1.1 channels to the plasma membrane could contribute to exacerbating rather than ameliorating the disease.

Nav 1.1 dysfunction in genetic epilepsy with febrile seizures-plus or Dravet syndrome.

Relatively few SCN1A mutations associated with genetic epilepsy with febrile seizures-plus (GEFS+) and Dravet syndrome (DS) have been functionally characterized. In contrast to GEFS+, many mutations detected in DS patients are predicted to have complete loss of function. However, functional consequences are not immediately apparent for DS missense mutations. Therefore, we performed a biophysical analysis of three SCN1A missense mutations (R865G, R946C and R946H) we detected in six patients with DS. Furthermore, we compared the functionality of the R865G DS mutation with that of a R859H mutation detected in a GEFS+ patient; the two mutations reside in the same voltage sensor domain of Na(v) 1.1. The four mutations were co-expressed with ß1 and ß2 subunits in tsA201 cells, and characterized using the whole-cell patch clamp technique. The two DS mutations, R946C and R946H, were nonfunctional. However, the novel voltage sensor mutants R859H (GEFS+) and R865G (DS) produced sodium current densities similar to those in wild-type channels. Both mutants had negative shifts in the voltage dependence of activation, slower recovery from inactivation, and increased persistent current. Only the GEFS+ mutant exhibited a loss of function in voltage-dependent channel availability. Our results suggest that the R859H mutation causes GEFS+ by a mixture of biophysical defects in Na(v) 1.1 gating. Interestingly, while loss of Na(v) 1.1 function is common in DS, the R865G mutation may cause DS by overall gain-of-function defects.

SCN1A splice variants exhibit divergent sensitivity to commonly used antiepileptic drugs.

PURPOSE: A common genetic variant (rs3812718) in a splice donor consensus sequence within the neuronal sodium channel gene SCN1A (encoding Na(V) 1.1) modulates the proportion of transcripts incorporating either the canonical (5A) or alternative (5N) exon 5. A pharmacogenetic association has been reported whereby increased expression of exon 5N containing Na(V) 1.1 transcripts correlated with lower required doses of phenytoin in epileptics. We tested the hypothesis that SCN1A alternative splicing affects the pharmacology of Na(V) 1.1 channels. METHODS: To directly examine biophysical and pharmacologic differences between the exon 5 splice variants, we performed whole-cell patch clamp recording of tsA201 cells transiently coexpressing either Na(V) 1.1-5A or Na(V) 1.1-5N with the ß1 and ß2 accessory subunits. We examined tonic inhibition and use-dependent inhibition of Na(V) 1.1 splice isoforms by phenytoin, carbamazepine, and lamotrigine. We also examined the effects of phenytoin and lamotrigine on channel biophysical properties and determined concentration-response relationships for both splice variants. KEY FINDINGS: We observed no significant differences in voltage dependence of activation, steady-state inactivation, and recovery from inactivation between splice variants. However, Na(V) 1.1-5N channels exhibited enhanced tonic block by phenytoin and lamotrigine compared to Na(V) 1.1-5A. In addition, Na(V) 1.1-5N exhibited enhanced use-dependent block by phenytoin and lamotrigine across a range of stimulation frequencies and concentrations. Phenytoin and lamotrigine induced shifts in steady-state inactivation and recovery from fast inactivation for both splice isoforms. No splice isoform differences were observed for channel inhibition by carbamazepine. SIGNIFICANCE: These results suggest Na(V) 1.1 channels containing exon 5N are more sensitive to the commonly used antiepileptic drugs phenytoin and lamotrigine.

Ranolazine block of human Na v 1.4 sodium channels and paramyotonia congenita mutants.

The antianginal drug ranolazine exerts voltage- and use-dependent block (UDB) of several Na+ channel isoforms, including Na(v) 1.4. We hypothesized that ranolazine will similarly inhibit the paramyotonia congenita Na(v) 1.4 gain-of-function mutations, R1448C, R1448H, and R1448P that are associated with repetitive action potential firing. Whole-cell Na+ current (I(Na)) was recorded from HEK293 cells expressing the hNa(v) 1.4 WT or R1448 mutations. At a holding potential (HP) of -140 mV, ranolazine exerted UDB (10 Hz) of WT and R1448 mutations (IC 50 = 59 - 71 µM). The potency for ranolazine UDB increased when the frequency of stimulation was raised to 30 Hz (IC 50 = 20 - 27 uM). When the HP was changed to -70 mV to mimic the resting potential of an injured skeletal muscle fibre, the potency of ranolazine to block I(Na) further increased; values of ranolazine IC 50 for block of WT, R1448C, R1448H, and R1448P were 3.8, 0.9, 6.3, and 0.9 uM, respectively. Ranolazine (30 uM) also caused a hyperpolarizing shift in the voltage-dependence of inactivation of WT and R1448 mutations. The effects of ranolazine (30 uM) to reduce I(Na) were similar (~35% I(Na) inhibition) when different conditioning pulse durations (2-20 msec) were used. Ranolazine (10 µM) suppressed the abnormal I(Na) induced by slow voltage ramps for R1448C channels. In computer simulations, 3 µM ranolazine inhibited the sustained and excessive firing of skeletal muscle action potentials that are characteristic of myotonia. Taken together, the data indicate that ranolazine interacts with the open state and stabilizes the inactivated state(s) of Na(v)1.4 channels, causes voltage- and use-dependent block of I(Na) and suppresses persistent I(Na). These data further suggest that ranolazine might be useful to reduce the sustained action potential firing seen in paramyotonia congenita.

Ranolazine selectively blocks persistent current evoked by epilepsy-associated Naν1.1 mutations.

BACKGROUND AND PURPOSE: Mutations of SCN1A, the gene encoding the pore-forming subunit of the voltage-gated sodium channel Na(V) 1.1, have been associated with a spectrum of genetic epilepsies and a familial form of migraine. Several mutant Na(V) 1.1 channels exhibit increased persistent current due to incomplete inactivation and this biophysical defect may contribute to altered neuronal excitability in these disorders. Here, we investigated the ability of ranolazine to preferentially inhibit increased persistent current evoked by mutant Na(V) 1.1 channels. EXPERIMENTAL APPROACH: Human wild-type (WT) and mutant Na(V) 1.1 channels were expressed heterologously in human tsA201 cells and whole-cell patch clamp recording was used to assess tonic and use-dependent ranolazine block. KEY RESULTS: Ranolazine (30 µM) did not affect WT Na(V) 1.1 channel current density, activation or steady-state fast inactivation but did produce mild slowing of recovery from inactivation. Ranolazine blocked persistent current with 16-fold selectivity over tonic block of peak current and 3.6-fold selectivity over use-dependent block of peak current. Similar selectivity was observed for ranolazine block of increased persistent current exhibited by Na(V) 1.1 channel mutations representing three distinct clinical syndromes, generalized epilepsy with febrile seizures plus (R1648H, T875M), severe myoclonic epilepsy of infancy (R1648C, F1661S) and familial hemiplegic migraine type 3 (L263V, Q1489K). In vitro application of achievable brain concentrations (1, 3 µM) to cells expressing R1648H channels was sufficient to suppress channel activation during slow voltage ramps, consistent with inhibition of persistent current. CONCLUSIONS AND IMPLICATIONS: Our findings support the feasibility of using selective suppression of increased persistent current as a potential new therapeutic strategy for familial neurological disorders associated with certain sodium channel mutations.

Multiplexed transposon-mediated stable gene transfer in human cells.

Generation of cultured human cells stably expressing one or more recombinant gene sequences is a widely used approach in biomedical research, biotechnology, and drug development. Conventional methods are not efficient and have severe limitations especially when engineering cells to coexpress multiple transgenes or multiprotein complexes. In this report, we harnessed the highly efficient, nonviral, and plasmid-based piggyBac transposon system to enable concurrent genomic integration of multiple independent transposons harboring distinct protein-coding DNA sequences. Flow cytometry of cell clones derived from a single multiplexed transfection demonstrated approximately 60% (three transposons) or approximately 30% (four transposons) stable coexpression of all delivered transgenes with selection for a single marker transposon. We validated multiplexed piggyBac transposon delivery by coexpressing large transgenes encoding a multisubunit neuronal voltage-gated sodium channel (SCN1A) containing a pore-forming subunit and two accessory subunits while using two additional genes for selection. Previously unobtainable robust sodium current was demonstrated through 38 passages, suitable for use on an automated high-throughput electrophysiology platform. Cotransfection of three large (up to 10.8 kb) piggyBac transposons generated a heterozygous SCN1A stable cell line expressing two separate alleles of the pore-forming subunit and two accessory subunits (total of four sodium channel subunits) with robust functional expression. We conclude that the piggyBac transposon system can be used to perform multiplexed stable gene transfer in cultured human cells, and this technology may be valuable for applications requiring concurrent expression of multiprotein complexes.

Propranolol blocks cardiac and neuronal voltage-gated sodium channels.

Propranolol is a widely used, non-selective ß-adrenergic receptor antagonist with proven efficacy in treating cardiovascular disorders and in the prevention of migraine headaches. At plasma concentrations exceeding those required for ß-adrenergic receptor inhibition, propranolol also exhibits anti-arrhythmic ("membrane stabilizing") effects that are not fully explained by ß-blockade. Previous in vitro studies suggested that propranolol may have local anesthetic effects. We directly tested the effects of propranolol on heterologously expressed recombinant human cardiac (NaV1.5) and brain (NaV1.1, NaV1.2, NaV1.3) sodium channels using whole-cell patch-clamp recording. We found that block was not stereospecific as we observed approximately equal IC50 values for tonic and use-dependent block by R-(+) and S-(-) propranolol (tonic block: R: 21.4 µM vs S: 23.6 µM; use-dependent block: R: 2.7 µM vs S: 2.6 µM). Metoprolol and nadolol did not block NaV1.5 indicating that sodium channel block is not a class effect of ß-blockers. The biophysical effects of R-(+)-propranolol on NaV1.5 and NaV1.1 resembled that of the prototypical local anesthetic lidocaine including the requirement for a critical phenylalanine residue (F1760 in NaV1.5) in the domain 4 S6 segment. Finally, we observed that brain sodium channels exhibited less sensitivity to R-(+)-propranolol than NaV1.5 channels. Our findings establish sodium channels as targets for propranolol and may help explain some beneficial effects of the drug in treating cardiac arrhythmias, and may explain certain adverse central nervous system effects.

Divergent sodium channel defects in familial hemiplegic migraine.

Familial hemiplegic migraine type 3 (FHM3) is a severe autosomal dominant migraine disorder caused by mutations in the voltage-gated sodium channel Na(V)1.1 encoded by SCN1A. We determined the functional consequences of three mutations linked to FHM3 (L263V, Q1489K, and L1649Q) in an effort to identify molecular defects that underlie this inherited migraine disorder. Only L263V and Q1489K generated quantifiable sodium currents when coexpressed in tsA201 cells with the human beta(1) and beta(2) accessory subunits. The third mutant, L1649Q, failed to generate measurable whole-cell current because of markedly reduced cell surface expression. Compared to WT-Na(V)1.1, Q1489K exhibited increased persistent current but also enhanced entry into slow inactivation as well as delayed recovery from fast and slow inactivation, thus resulting in a predominantly loss-of-function phenotype further demonstrated by a greater loss of channel availability during repetitive stimulation. In contrast, L263V exhibited gain-of-function features, including delayed entry into, as well as accelerated recovery from, fast inactivation; depolarizing shifts in the steady-state voltage dependence of fast and slow inactivation; increased persistent current; and delayed entry into slow inactivation. Notably, the two mutations (Q1489K and L1649Q) that exhibited partial or complete loss of function are linked to typical FHM, whereas the gain-of-function mutation L263V occurred in a family having both FHM and a high incidence of generalized epilepsy. We infer from these data that a complex spectrum of Na(V)1.1 defects can cause FHM3. Our results also emphasize the complex relationship between migraine and epilepsy and provide further evidence that both disorders may share common molecular mechanisms.

Impaired NaV1.2 function and reduced cell surface expression in benign familial neonatal-infantile seizures.

PURPOSE: Mutations in SCN2A, the gene encoding the brain voltage-gated sodium channel alpha-subunit Na(V)1.2, are associated with inherited epilepsies including benign familial neonatal-infantile seizures (BFNIS). Functional characterization of three BFNIS mutations was performed to identify defects in channel function that underlie this disease. METHODS: We examined three BFNIS mutations (R1319Q, L1330F, and L1563V) using whole-cell patch-clamp recording of heterologously expressed human Na(V)1.2. Membrane biotinylation was employed to examine the cell surface protein expression of the four Na(V)1.2 alleles. RESULTS: R1319Q displayed mixed effects on activation and fast inactivation gating, consistent with a net loss of channel function. L1563V exhibited impaired fast inactivation predicting a net gain of channel function. The L1330F mutation significantly decreased overall channel availability during repetitive stimulation. Patch-clamp analysis also revealed that cells expressing BFNIS mutants exhibited lower levels of sodium current compared to wild type (WT) Na(V)1.2. Biochemical experiments demonstrated that all three BFNIS mutations exhibited a significant reduction in cell surface expression compared to WT. DISCUSSION: Our findings indicate that BFNIS is associated with a range of biophysical defects accompanied by reduced levels of channel protein at the plasma membrane.

Impaired inactivation gate stabilization predicts increased persistent current for an epilepsy-associated SCN1A mutation.

Mutations in SCN1A (encoding the neuronal voltage-gated sodium channel alpha1 subunit, Na(V)1.1, or SCN1A) are associated with genetic epilepsy syndromes including generalized epilepsy with febrile seizures plus (GEFS+) and severe myoclonic epilepsy of infancy. Here, we present the formulation and use of a computational model for SCN1A to elucidate molecular mechanisms underlying the increased persistent sodium current exhibited by the GEFS+ mutant R1648H. Our model accurately reproduces all experimentally measured SCN1A whole-cell biophysical properties including biphasic whole-cell current decay, channel activation, and entry into and recovery from fast and slow inactivation. The model predicts that SCN1A open-state inactivation results from a two-step process that can be conceptualized as initial gate closure, followed by recruitment of a mechanism ("latch") to stabilize the inactivated state. Selective impairment of the second latching step results in an increase in whole-cell persistent current similar to that observed for the GEFS+ mutant R1648H. These results provide a deeper level of understanding of mutant SCN1A dysfunction in an inherited epilepsy syndrome, which will enable more precise computational studies of abnormal neuronal activity in epilepsy and may help guide new targeted therapeutic strategies.