Abstract: AIMS: In the type 3 long QT syndrome (LQT3), shortening of the QT interval by overdrive pacing is used to prevent life-threatening arrhythmias. However, it is unclear whether accelerated heart rate induced by beta-adrenergic agents produces similar effects on the late sodium current (I(Na)) to those by overdrive pacing therapy. We analyzed the beta-adrenergic-like effects of protein kinase A and fluoride on I(Na) in R1623Q mutant channels. MAIN METHODS: cDNA encoding either wild-type (WT) or R1623Q mutant of hNa(v)1.5 was stably transfected into HEK293 cells. I(Na) was recorded using a whole-cell patch-clamp technique at 23 degrees C. KEY FINDINGS: In R1623Q channels, 2 mM pCPT-AMP and 120 mM fluoride significantly delayed macroscopic current decay and increased relative amplitude of the late I(Na) in a time-dependent manner. Modulations of peak I(Na) gating kinetics (activation, inactivation, recovery from inactivation) by fluoride were similar in WT and R1623Q channels. The effects of fluoride were almost completely abolished by concomitant dialysis with a protein kinase inhibitor. We also compared the effect of pacing with that of beta-adrenergic stimulation by analyzing the frequency-dependence of the late I(Na). Fluoride augmented frequency-dependent reduction of the late I(Na), which was due to preferential delay of recovery of late I(Na). However, the increase in late I(Na) by fluoride at steady-state was more potent than the frequency-dependent reduction of late I(Na). SIGNIFICANCE: Different basic mechanisms participate in the QT interval shortening by pacing and beta-adrenergic stimulation in the LQT3.
Abstract: Mutations in the cardiac sodium channel gene SCN5A are responsible for a spectrum of hereditary arrhythmias, including type-3 long QT syndrome (LQT3), Brugada syndrome (BrS), conduction disturbance and sinus node dysfunction. These syndromes were originally regarded as independent entities with distinct clinical manifestations and biophysical properties, but recent evidence shows considerable clinical overlap, implying a new disease entity known as an overlap syndrome of cardiac sodium channelopathy. Class IC sodium-channel blockers often induced the BrS phenotype in some patients with LQT3, confirming the clinical overlap of LQT3 and BrS. It also raises a concern about the safety of the class IC drug and questions about the determinants of overlap. Here, an overview is given of current knowledge on the clinical features, prevalence, and molecular and biophysical mechanisms underlying overlap syndrome to gain more insight into this complex issue and generate better therapeutic strategies for patient management.
Abstract: Sudden infant death syndrome (SIDS) is multifactorial and may result from the interaction of a number of environmental, genetic, and developmental factors. We studied three major genes causing long QT syndrome in 42 Japanese SIDS victims and found five mutations, KCNQ1-K598R, KCNH2-T895M, SCN5A-F532C, SCN5A-G1084S, and SCN5A-F1705S, in four cases; one case had both KCNH2-T895M and SCN5A-G1084S. All mutations were novel except for SCN5A-F532C, which was previously detected in an arrhythmic patient. Heterologous expression study revealed significant changes in channel properties of KCNH2-T895M, SCN5A-G1084S, and SCN5A-F1705S, but did not in KCNQ1-K598R and SCN5A-F532C. Our data suggests that nearly 10% of SIDS victims in Japan have mutations of the cardiac ion channel genes similar to in other countries.
Abstract: Phenotypic overlap of type 3 long QT syndrome (LQT3) with Brugada syndrome (BrS) is observed in some carriers of mutations in the Na channel SCN5A. While this overlap is important for patient management, the clinical features, prevalence, and mechanisms underlying such overlap have not been fully elucidated. To investigate the basis for this overlap, we genotyped a cohort of 44 LQT3 families of multiple ethnicities from 7 referral centers and found a high prevalence of the E1784K mutation in SCN5A. Of 41 E1784K carriers, 93% had LQT3, 22% had BrS, and 39% had sinus node dysfunction. Heterologously expressed E1784K channels showed a 15.0-mV negative shift in the voltage dependence of Na channel inactivation and a 7.5-fold increase in flecainide affinity for resting-state channels, properties also seen with other LQT3 mutations associated with a mixed clinical phenotype. Furthermore, these properties were absent in Na channels harboring the T1304M mutation, which is associated with LQT3 without a mixed clinical phenotype. These results suggest that a negative shift of steady-state Na channel inactivation and enhanced tonic block by class IC drugs represent common biophysical mechanisms underlying the phenotypic overlap of LQT3 and BrS and further indicate that class IC drugs should be avoided in patients with Na channels displaying these behaviors.
Abstract: Left ventricular noncompaction (LVNC) is a genetically heterogenous disorder. Mutations in the human cardiac sodium channel alpha-subunit gene (SCN5A) are involved in the pathophysiology of cardiac arrhythmias and cardiomyopathies. This study was performed to compare the frequency of SCN5A variants in LVNC patients with or without arrhythmias, and to investigate the relationship between variants and disease severity. DNA was isolated from the peripheral blood of 62 Japanese probands with LVNC, comprising 17 familial cases and 45 sporadic cases. Blood samples were screened for variants in SCN5A using single-strand conformational polymorphism analysis (SSCP) and DNA sequencing. Seven variants, rs6599230:G > A, c.453C > T, c.1141-3C > A, rs1805124:A > G (p.H558R), rs1805125:C > T (p.P1090L), c.3996C > T, and rs1805126:T > C were identified in 7 familial and 12 sporadic cases. The frequency of SCN5A variants was significantly higher in the patients with arrhythmias than those without (50% vs 7%: P = 0.0003), suggesting these variants represent a risk factor for arrhythmia and supporting the hypothesis that genes encoding ion channels are involved in LVNC pathophysiology. The LVNC patients with heart failure also had high occurrence of SCN5A variants, suggesting the presence of SCN5A variants and/or arrhythmias increase the severity of LVNC.
Abstract: BACKGROUND: A trafficking defect of mutant cardiac Na-channels (SCN5A) has been implicated in Brugada syndrome. Although R1232W polymorphism and T1620M mutation by themselves have little effect on Na-channel function, their combination has been reported to disrupt membrane trafficking, resulting in a non-functioning Na channel. METHODS AND RESULTS: Contrary to previous findings, patch-clamp recordings of heterologously expressed R1232W/T1620M showed robust Na currents and confocal microscopy exhibited predominant expression in the plasma membrane, similar to the wild-type channel. CONCLUSIONS: It is unlikely that an intragenic interaction between R1232W and T1620M of SCN5A causes a trafficking defect leading to a non-functioning Na channel.
Abstract: BACKGROUND: An association between Brugada syndrome and neurally mediated syncope has been described. Although mutations in SCN5A have been identified in Brugada syndrome, the genetic link between Brugada syndrome and neurally mediated syncope has not been determined. OBJECTIVES: The purpose of the study was to clinically and genetically characterize a man with recurrent syncope that originally was diagnosed as neurally mediated syncope at age 8 years but subsequently manifested as Brugada syndrome at age 17 years. METHODS: The proband underwent clinical examination, which included head-up tilt test, sodium channel provocation test, and electrophysiologic study. Genetic screening of SCN5A was performed for the proband and his family members. The biophysical properties of a mutant SCN5A channel in a heterologous expression system were studied using whole-cell, patch clamp technique. RESULTS: The proband showed positive head-up tilt test, coved-type ST elevation recorded from the third intercostal space, and positive pilsicainide provocation test. Ventricular fibrillation was inducible at programmed electrical stimulation, consistent with characteristics of both Brugada syndrome and neurally mediated syncope. A novel nonsense SCN5A mutation (Q55X) was identified in the proband, his mother, and his asymptomatic brother. The heterologously expressed mutant channel was nonfunctional. CONCLUSION: We genetically determined an SCN5A mutation in a patient showing the combined phenotype of neurally mediated syncope and Brugada syndrome. Neurally mediated syncope and Brugada syndrome may share, at least in part, a common pathophysiologic mechanism.
Abstract: Both tetrodotoxin-sensitive (TTX-S) and TTX-resistant (TTX-R) voltage-dependent Na+ channels are expressed in the human neuroblastoma cell line NB-1, but a gene encoding the TTX-R Na+ channel has not been identified. In this study, we have cloned cDNA encoding the alpha subunit of the TTX-R Na+ channel in NB-1 cells and designated it hNbR1. The longest open reading frame of hNbR1 (accession no. AB158469) encodes 2016 amino acid residues. Sequence analysis has indicated that hNbR1 is highly homologous with human cardiac Nav1.5/SCN5A with > 99% amino acid identity. The presence of a cysteine residue (Cys373) in the pore-loop region of domain I is consistent with the supposition that hNbR1 is resistant to TTX. Analysis of the genomic sequence of SCN5A revealed a new exon encoding S3 and S4 of domain I (exon 6A). In addition, an alternative splicing variant, lacking exon 18, that encodes 54 amino acids in the intracellular loop between domains II and III was found (hNbR1-2; accession no. AB158470). Na+ currents in human embryonic kidney cells (HEK293) transfected with hNbR1 or hNbR1-2 showed electrophysiological properties similar to those for TTX-R I(Na) in NB-1 cells. The IC50 for the TTX block was approximately 8 microM in both variants. These results suggest that SCN5A has a newly identified exon for alternative splicing and is more widely expressed than previously thought.
Abstract: OBJECTIVE: In the type 3 long QT syndrome (LQT3), arrhythmia events tend to occur at rest or during sleep. One of the mutations, R1623Q, is located in the voltage sensor of the cardiac sodium channel (hH1), and patients with R1623Q mutation have been also reported to show bradycardia-dependent cardiac events. Although the mutant channel has been characterized by inactivation gating defects, the intrinsic mechanism(s) that might explain why arrhythmia attack is most prevalent at slower heart rates has not been investigated. METHODS: cDNA encoding either the wild-type or the R1623Q mutant of hH1 was stably transfected into HEK293 cells. I(Na) was recorded using a whole-cell patch-clamp technique at 23 degrees C. RESULTS: A train of 50 depolarizing pulses from holding potentials (-120 and -80 mV) to -20 mV or a train of 50 action potential waveforms was applied at different frequencies. When using a rectangular waveform voltage clamp protocol, rate-dependent reduction of I(Na) was holding voltage-dependent but was not different between peak I(Na) and late I(Na). However, using the action potential clamp, preferential rate-dependent reduction of the phase 3 I(Na) was obvious as compared with peak I(Na). The discrepancy in the rate-dependent reduction between protocols was attributed to accelerated recovery from inactivation under non-equilibrium condition. CONCLUSION: The rate dependency of phase 3 I(Na) under non-equilibrium gating is a novel mechanism to explain the enhanced rate-dependent QT-shortening in LQT3 patients. Our findings are important for genotype-phenotype correlations in LQT3 mutants as well as for understanding the function of S4 segment of domain IV region in the cardiac Na(+) channel.
Abstract: BACKGROUND: Congenital atrial standstill has been linked to SCN5A. Incomplete penetrance observed in atrial standstill has been attributed in part to the digenic inheritance of polymorphisms in the atrial-specific gap junction connexin 40 (Cx40) in conjunction with an SCN5A mutation. OBJECTIVES: The purpose of this study was to determine the clinical and biophysical characteristics of a novel SCN5A mutation identified in a family with atrial standstill. METHODS: Family members of an apparently sporadic case of atrial standstill underwent genetic screening of SCN5A and atrial-specific genes including Cx40. Biophysical properties of the wild-type (WT) and mutant SCN5A channels in a heterologous expression system were studied using the whole-cell patch clamp technique. RESULTS: The novel SCN5A mutation L212P was identified in the proband (age 11 years) and his father. The father was in normal sinus rhythm. The proband had no P waves on surface ECG, and his right atrium could not be captured by pacing. The recombinant L212P Na channel showed a large hyperpolarizing shift in both the voltage dependence of activation (WT: -48.1 +/- 0.9 mV; L212P: -63.5 +/- 1.5 mV; P < .001) and inactivation (WT: -86.6 +/- 0.9 mV; L212P: -95.6 +/- 0.8 mV; P < .001) and delayed recovery from inactivation. Further screenings for genetic variations that might mitigate L212P dysfunction revealed that the proband, but not his father, carries Cx40 polymorphisms inherited from his asymptomatic mother. CONCLUSION: These results suggest that genetic defects in SCN5A most likely underlie atrial standstill. Coinheritance of Cx40 polymorphisms is a possible genetic factor that modifies the clinical manifestation of this inherited arrhythmia.
Abstract: OBJECTIVES: We carried out a complete screening of the SCN5A gene in 38 Japanese patients with Brugada syndrome to investigate the genotype-phenotype relationship. BACKGROUND: The gene SCN5A encodes the pore-forming alpha-subunit of voltage-gated cardiac sodium (Na) channel, which plays an important role in heart excitation/contraction. Mutations of SCN5A have been identified in 15% of patients with Brugada syndrome. METHODS: In 38 unrelated patients with clinically diagnosed Brugada syndrome, we screened for SCN5A gene mutations using denaturing high-performance liquid chromatography and direct sequencing, and conducted a functional assay for identified mutations using whole-cell patch-clamp in heterologous expression system. RESULTS: Four heterozygous mutations were identified (T187I, D356N, K1578fs/52, and R1623X) in 4 of the 38 patients. All of them had bradyarrhythmic complications: three with sick sinus syndrome (SSS) and the other (D356N) with paroxysmal complete atrioventricular block. SCN5A-linked Brugada patients were associated with a higher incidence of bradyarrhythmia (4 of 4) than non-SCN5A-linked Brugada patients (2 of 34). Families with T187I and K1578fs/52 had widespread penetrance of SSS. Notably, the patient with K1578fs/52, who had been diagnosed as having familial SSS without any clinical signs of Brugada syndrome, showed a Brugada-type ST-segment elevation after intravenous administration of pilsicainide and programmed electrical stimulation-induced ventricular tachycardia. All of the mutations encoded non-functional Na channels, and thus were suggested to cause impulse propagation defect underlying bradyarrhythmias. CONCLUSIONS: Our findings suggest that loss-of-function SCN5A mutations resulting in Brugada syndrome are distinguished by profound bradyarrhythmias.
Abstract: OBJECTIVES: The purpose of this study was to identify risk markers in patients with Brugada syndrome. BACKGROUND: Patients with Brugada syndrome who experience syncope or aborted sudden death are at high risk for recurrent lethal arrhythmias. The prognosis and therapeutic approaches in asymptomatic individuals with a Brugada-type ECG (asymptomatic Brugada syndrome) are controversial. METHODS: We genetically screened 30 asymptomatic probands (29 men and 1 woman; mean age 47.1 years) exhibiting a spontaneous Brugada-type ECG. Family members of patients with Brugada syndrome were excluded from the study. RESULTS: Twenty-nine of 30 patients (96.7%) remained symptom-free for at least 3 years. One patient (case 1) with a family history of sudden death died suddenly during sleep. Ventricular fibrillation was induced by programmed electrical stimulation in 14 of 18 subjects (78%), but none of these 18 subjects developed spontaneous ventricular arrhythmias. Genetic screening failed to identify SCN5A mutations in most cases but demonstrated a novel double missense mutation (K1527R and A1569P) located on the same allele in another asymptomatic subject (case 2). Heterologously expressed mutant Na channels exhibited a negative shift of steady-state inactivation (9.2 mV) and enhanced slow inactivation, suggesting this individual harbors a subclinical channel dysfunction compatible with symptomatic Brugada syndrome. CONCLUSIONS: Asymptomatic individuals with a Brugada-type ECG generally have a better prognosis than their symptomatic counterparts, but a subgroup of these individuals may have a poor prognosis. Severe Na channel dysfunction as a result of SCN5A mutations may not be sufficient to cause symptoms or arrhythmias in patients with Brugada syndrome, suggesting unknown factors or modifier genes influence arrhythmogenesis.
Abstract: BACKGROUND: Azimilide reportedly blocks Na(+) channels, although its mechanism remains unclear. METHODS AND RESULTS: The kinetic properties of the azimilide block of the wild-type human Na(+) channels (WT: hH1) and mutant DeltaKPQ Na(+) channels (DeltaKPQ) expressed in COS7 cells were investigated using the whole-cell patch clamp technique and a Markovian state model. Azimilide induced tonic block of WT currents by shifting the h infinity curve in the hyperpolarizing direction and caused phasic block of WT currents with intermediate recovery time constant. The peak and steady-state DeltaKPQ currents were blocked by azimilide, although with only a slight shift in the h infinity curve. The phasic block of peak and steady-state DeltaKPQ currents by azimilide was significantly larger than the blocking of the peak WT current. The affinity of azimilide predicted by a Markovian state model was higher for both the activated state (Kd(A) =1.4 micromol/L), and the inactivated state (Kd(I) =1.4 micromol/L), of WT Na(+) channels than that for the resting state (Kd(R) =102.6 micromol/L). CONCLUSIONS: These experimental and simulation studies suggest that azimilide blocks the human cardiac Na(+) channel in both the activated and inactivated states.
Abstract: Mutations, exclusively missense, of voltage-gated sodium channel alpha subunit type 1 (SCN1A) and type 2 (SCN2A) genes were reported in patients with idiopathic epilepsy: generalized epilepsy with febrile seizures plus. Nonsense and frameshift mutations of SCN1A, by contrast, were identified in intractable epilepsy: severe myoclonic epilepsy in infancy (SMEI). Here we describe a first nonsense mutation of SCN2A in a patient with intractable epilepsy and severe mental decline. The phenotype is similar to SMEI but distinct because of partial epilepsy, delayed onset (1 year 7 months), and absence of temperature sensitivity. A mutational analysis revealed that the patient had a heterozygous de novo nonsense mutation R102X of SCN2A. Patch-clamp analysis of Na(v)1.2 wild-type channels and the R102X mutant protein coexpressed in human embryonic kidney 293 cells showed that the truncated mutant protein shifted the voltage dependence of inactivation of wild-type channels in the hyperpolarizing direction. Analysis of the subcellular localization of R102X truncated protein suggested that its dominant negative effect could arise from direct or indirect cytoskeletal interactions of the mutant protein. Haploinsufficiency of Na(v)1.2 protein is one plausible explanation for the pathology of this patient; however, our biophysical findings suggest that the R102X truncated protein exerts a dominant negative effect leading to the patient's intractable epilepsy.
Abstract: Mutations in the cardiac Na+ channel gene SCN5A are responsible for multiple lethal ventricular arrhythmias including Brugada syndrome and congenital long QT syndrome. Here we report a case of Brugada syndrome with ST elevation in the right precordial and inferior leads accompanied by atrial standstill and spontaneous ventricular fibrillation. Atrial standstill and J wave elevation were provoked by procainamide. Genetic analysis revealed a missense mutation (R367H) in SCN5A. The resultant mutant Na+ channel was nonfunctional when expressed heterologously in Xenopus oocytes. Our study suggests that genetic defects in SCN5A may be associated with atrial standstill in combination with ventricular arrhythmias.
Abstract: Gating properties of Na(+) channels are the critical determinants for the state-dependent block by class I antiarrhythmic drugs; however, recent site-directed mutagenesis studies have shown that the Na(+) channel selectivity filter region controls drug access to and dissociation from the binding site. To validate these observations, we have exploited a naturally occurring cardiac Na(+) channel mutation, S1710L, located next to the putative selectivity filter residue of domain 4, and evaluated the pharmacological properties to mexiletine using whole-cell, patch-clamp recordings. Consistent with the large negative shift of steady-state inactivation and the enhanced slow inactivation, the S1710L channel showed greater mexiletine tonic block than wild-type (WT) channel. In contradiction, S1710L showed attenuated use-dependent block by mexiletine and accelerated recovery from block, suggesting that the drug escape though the external access path is facilitated. Extracellularly applied QX-314, a membrane-impermeant derivative of lidocaine, elicited significantly enhanced tonic block in S1710L similar to mexiletine. However, recovery from internally applied QX-314 was accelerated by 4.4-fold in S1710L compared with WT. These results suggest that the drug access to and dissociation from the binding site through the hydrophilic path are substantially altered. Moreover, K(+) permeability was 1.9-fold increased in S1710L, verifying that the mutated residue is located in the ion-conducting pore. We propose that the Na(+) channel selectivity filter region is a structural determinant for the antiarrhythmic drug sensitivity in addition to gating properties of the indigenous Na(+) channels that govern the state-dependent drug block.
Abstract: BACKGROUND: Tissue factor (TF), expressed in endothelial cells (ECs) and enriched in human atherosclerotic lesions, acts as a critical initiator of blood coagulation in acute coronary syndrome. Basic fibroblast growth factor (bFGF) induces the proliferation and migration of ECs and plays a role in angiogenesis and restoration of endothelial integrity. As TF is implicated in angiogenesis, we studied the effect of bFGF on TF gene and protein expression. Methods: Human umbilical vein ECs (HUVECs) were exposed to bFGF. TF mRNA was assessed by Northern blot and TF protein was assessed by Western blot. TF promoter activity was assessed by transient transfection assay and transcription factor was identified by electro mobility shift assay. RESULTS: bFGF increased TF mRNA and protein expression in HUVECs. Increased TF mRNA was attenuated by inhibition of extracellular signal-regulated kinase kinase in human ECV304 cells. Transient transfection assays of the human TF promoter-luciferase construct (-786/+121 bp) demonstrated that bFGF induced transcription was dependent on the elements within the -197 to -176 bp relative to the transcription start site of the human TF gene. This region contains NF-kappaB like binding site. Electro mobility shift assay showed that bFGF increased nuclear translocation or DNA binding of NF-kappaB transcription factor to TF promoter. Nucleotide substitution to disrupt NF-kappaB like site reduced bFGF stimulated promoter activity. Fenofibric acid, an agonist ligand for the peroxisome proliferator activated receptor-alpha, reduced basal and bFGF stimulated TF expression. CONCLUSIONS: These results indicate that bFGF may increase TF production in ECs through activation of transcription at NF-kappaB binding site, and control coagulation in vessel walls. Fibrate can inhibit TF expression and therefore reduce the thrombogenecity of human atherosclerotic lesions.
Abstract: BACKGROUND: The authors reported a mutation, P1158S, of the human skeletal muscle sodium channel gene (SCN4A) in a family with cold-induced hypokalemic periodic paralysis (hypoKPP) and myotonia. OBJECTIVE: To identify mechanisms of temperature dependency in this channelopathy. METHODS: Using the amphotericin B perforated patch clamp method, sodium currents were recorded at 22 and 32 degrees C from the wild-type (WT) and P1158S mutant SCN4A expressed in tsA201 cells. Computer simulation was performed, incorporating the gating parameters of the P1158S mutant SCN4A. RESULTS: P1158S mutant SCN4A exhibited hyperpolarizing shifts in voltage dependence of both activation and inactivation curves at a cold temperature and a slower rate of inactivation than the WT. Computer simulation reproduced the abnormal skeletal muscle electrical activities of both paralysis at a low potassium concentration in the cold and myotonia at a normal potassium concentration. CONCLUSIONS: Both paralysis and myotonia are attributable to the biophysical properties of the SCN4A mutation associated with hypoKPP. This is the first report of an SCN4A mutation that exhibits temperature-dependent shifts of voltage dependence in sodium channel gating.
Abstract: The present study investigated the protective effects of L-cysteine on the oxidation-induced blockade of Na+ channel a-subunits, hH1 (cardiac) and hSkM1 (skeletal), expressed in COS7 cells. Na+ currents were recorded by the whole-cell patch clamp technique (n = 3-7). L-cysteine alone blocked hH1 and hSkM1 in a dose-dependent manner, with saturating L-cysteine block at 3,000 micromol/L. Hg2+, a potent sulfhydryl oxidizing agent, blocked hH1 with a time to 50% inhibition (Time50%) of 20s. Preperfusion of COS7 cells with 100 micromol/L L-cysteine significantly slowed the Hg2+ block of hH1 (Time50% = 179 s). L-cysteine did not prevent Hg2+ block of hSkM1 (Time50% = 37s) or the C373Y hH1 mutant (Time50% = 43s). As for other sulfo-amino acids, homocysteine prevented the Hg2+ block of hH1, with the Time50% (70s) being significantly smaller than that of L-cysteine, whereas methionine did not prevent the Hg2+ block of hH1. L-cysteine did not prevent the Cd2+ block of hH1. These results indicate that L-cysteine selectively acts on heart-specific Cys373 in the P-loop region of hH1 to prevent Cys373 from the oxidation-induced sulfur-Hg-sulfur bridge formation.
Abstract: OBJECTIVE: Loss of Na(+) channel function has been implicated in idiopathic ventricular fibrillation (IVF) and Brugada syndrome. We have studied the biophysical properties of an IVF mutation (S1710L) that exhibited an unusual clinical phenotype: rate-dependent bundle branch block without manifestation of Brugada-type ECG pattern. METHODS: The mutant S1710L channels were expressed in mammalian cells and their gating properties, studied using whole-cell patch clamp techniques, were compared with wild-type (WT) and a Brugada syndrome mutant channel T1620M. RESULTS: The S1710L channel exhibited significantly faster macroscopic current decay than WT or T1620M. In addition, S1710L showed a negative shift in the voltage-dependence of fast inactivation and slower recovery from fast inactivation than in WT or T1620M. In addition to the alterations in fast inactivation most commonly observed in Brugada syndrome mutations, S1710L exhibited marked enhancement in slow inactivation and a large positive shift of activation that potentially decreases conduction velocity. CONCLUSIONS: These functional abnormalities may be responsible for the overlapping clinical phenotypes associated with Brugada syndrome and the cardiac conduction defect, a novel cardiac Na(+) channelopathy.
Abstract: The effects of moricizine on Na+ channel currents (INa) were investigated in guinea-pig atrial myocytes and its effects on INa in ventricular myocytes and on cloned hH1 current were compared using the whole-cell, patch-clamp technique. Moricizine induced the tonic block of INa with the apparent dissociation constant (Kd,app) of 6.3 microM at -100 mV and 99.3 microM at -140 mV. Moricizine at 30 microM shifted the h infinity curve to the hyperpolarizing direction by 8.6 +/- 2.4 mV. Moricizine also produced the phasic block of INa, which was enhanced with the increase in the duration of train pulses, and was more prominent with a holding potential (HP) of -100 mV than with an HP of -140 mV. The onset block of INa induced by moricizine during depolarization to -20 mV was continuously increased with increasing the pulse duration, and was enhanced at the less negative HP. The slower component of recovery of the moricizine-induced INa block was relatively slow, with a time constant of 4.2 +/- 2.0 s at -100 mV and 3.0 +/- 1.2 s at -140 mV. Since moricizine induced the tonic block of ventricular INa with Kd,app of 3.1 +/- 0.8 microM at HP = -100 mV and 30.2 +/- 6.8 microM at HP = -140 mV, and cloned hH1 with Kd,app of 3.0 +/- 0.5 microM at HP = -100 mV and 22.0 +/- 3.2 microM at HP = -140 mV, respectively, either ventricular INa or cloned hH1 had significantly higher sensitivity to moricizine than atrial INa. The h infinity curve of ventricular INa was shifted by 10.5 +/- 3.5 mV by 3 microM moricizine and that of hH1 was shifted by 5.0 +/- 2.3 mV by 30 microM moricizine. From the modulated receptor theory, we have estimated the dissociation constants for the resting and inactivated state to be 99.3 and 1.2 microM in atrial myocytes, 30 and 0.17 microM in ventricular myocytes, and 22 and 0.2 microM in cloned hH1, respectively. We conclude that moricizine has a higher affinity for the inactivated Na+ channel than for the resting state channel in atrial myocytes, and moricizine showed the significant atrioventricular difference of moricizine block on INa. Moricizine would exert an antiarrhythmic action on atrial myocytes, as well as on ventricular myocytes, by blocking Na+ channels with a high affinity to the inactivated state and a slow dissociation kinetics.
Abstract: BACKGROUND: Subclinical mutations in genes associated with the congenital long-QT syndromes (LQTS) have been suggested as a risk factor for drug-induced LQTS and accompanying life-threatening arrhythmias. Recent studies have identified genetic variants of the cardiac K+ channel genes predisposing affected individuals to acquired LQTS. We have identified a novel Na+ channel mutation in an individual who exhibited drug-induced LQTS. METHODS AND RESULTS: An elderly Japanese woman with documented QT prolongation and torsade de pointes during treatment with the prokinetic drug cisapride underwent mutational analysis of LQTS-related genes. A novel missense mutation (L1825P) was identified within the C-terminus region of the cardiac Na+ channel (SCN5A). The L1825P channel heterologously expressed in tsA-201 cells showed Na+ current with slow decay and a prominent tetrodotoxin-sensitive noninactivating component, similar to the gain-of-function phenotype most commonly observed for SCN5A-associated congenital LQTS (LQT3). In addition, L1825P exhibited loss of function Na+ channel features characteristic of Brugada syndrome. Peak Na+ current density observed in cells expressing L1825P was significantly diminished, and the voltage dependence of activation and inactivation was shifted toward more positive and negative potentials, respectively. CONCLUSIONS: This study demonstrates that subclinical mutations in the LQTS-related gene SCN5A may predispose certain individuals to drug-induced cardiac arrhythmias.
Abstract: L-type Ca(2+) channels are heteromultimeric and finely tuned by auxiliary subunits in different tissues and regions. Among auxiliary subunits, beta subunit has been shown to play important roles in many functional aspects of Ca(2+) channel. Rat heart was reported to specifically express beta(2a) subunit. However, the slow inactivation rates of Ca(2+) currents recorded from recombinant Ca(2+) channels with the beta(2a) subunit, and the reported inability to detect beta(2a) subunit in rabbit heart by reverse transcription-PCR analysis raise the possibility of the existence of other beta subunits. We cloned a splice variant of beta(2) subunit from rat heart, using rapid amplification of cDNA 5' ends. The splice variant is highly similar to human beta(2c) subunit that was cloned from human ventricle. Northern blot analysis detected the rat beta(2c) subunit abundantly in rat heart and brain. The deduced amino acid sequence of the beta(2c) subunit was different from that of the beta(2a) subunit only in the N-terminal region. When the beta(2c) subunit was expressed along with alpha(1c) and alpha(2)delta subunits in baby hamster kidney cells, the inactivation rates were comparable with those from native cardiac myocytes, although those with the beta(2a) subunit were slow. Taken together, these observations suggest that the beta(2c) subunit is a functional beta(2) subunit expressed in heart and that the short N-terminal region plays a major role in modifying inactivation kinetics.
Abstract: BACKGROUND: Mutations in the gene encoding the human cardiac Na(+) channel alpha-subunit (hH1) are responsible for chromosome 3-linked congenital long-QT syndrome (LQT3) and idiopathic ventricular fibrillation (IVF). An auxiliary beta(1)-subunit, widely expressed in excitable tissues, shifts the voltage dependence of steady-state inactivation toward more negative potentials and restores normal gating kinetics of brain and skeletal muscle Na(+) channels expressed in Xenopus oocytes but has little if any functional effect on the cardiac isoform. Here, we characterize the altered effects of a human beta(1)-subunit (hbeta(1)) on the heterologously expressed hH1 mutation (T1620M) previously associated with IVF. METHODS AND RESULTS: When expressed alone in Xenopus oocytes, T1620M exhibited no persistent currents, in contrast to the LQT3 mutant channels, but the midpoint of steady-state inactivation (V(1/2)) was significantly shifted toward more positive potentials than for wild-type hH1. Coexpression of hbeta(1) did not significantly alter current decay or recovery from inactivation of wild-type hH1; however, it further shifted the V(1/2) and accelerated the recovery from inactivation of T1620M. Oocyte macropatch analysis revealed that the activation kinetics of T1620M were normal. CONCLUSIONS: It is suggested that coexpression of hbeta(1) exposes a more severe functional defect that results in a greater overlap in the relationship between channel inactivation and activation (window current) in T1620M, which is proposed to be a potential pathophysiological mechanism of IVF in vivo. One possible explanation for our finding is an altered alpha-/beta(1)-subunit association in the mutant.
Abstract: The congenital long QT syndrome is an inherited disorder characterized by a delay in cardiac repolarization, leading to lethal cardiac arrhythmias such as torsade de pointes. One form of this disease involves mutations in the voltage-dependent cardiac Na(+) channel, which includes an in-frame deletion of three amino acids (Lys-1505, Pro-1506, and Gln-1507; DeltaKPQ). The potential for selective suppression of the mutant was examined by heterologous expression of DeltaKPQ-Na(+) channels in Chinese hamster fibroblast cells via single-channel recording. In a single-channel cell-attached patch study, DeltaKPQ-Na(+) channels yielded currents that peaked at approximately 1 ms after voltage steps to 0 mV with aberrant late currents, which were composed of burst and isolated openings. The affinity of certain anesthetics (pilsicainide and lidocaine) to the late currents of the mutant channels was examined. It was revealed that 1) pilsicainide (1 microM), an open channel blocker of voltage-dependent Na(+) channels, remarkably decreased the late currents primarily by the shortening of burst duration without suppressing the initial peak current; and 2) lidocaine (1 microM), an inactivated channel blocker, decreased the late currents primarily by the suppression of isolated channel openings. Because the late currents in DeltaKPQ mutants are mainly composed of the burst openings, we conclude that pilsicainide is capable of selectively blocking the late currents in the mutant Na(+) channels that show dominant abnormal burst openings such as in DeltaKPQ mutants.
Abstract: 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.
Abstract: Divalent mercury (Hg(2+)) blocked human skeletal Na(+) channels (hSkM1) in a stable dose-dependent manner (K(d) = 0.96 microM) in the absence of reducing agent. Dithiothreitol (DTT) significantly prevented Hg(2+) block of hSkM1, and Hg(2+) block was also readily reversed by DTT. Both thimerosal and 2,2'-dithiodipyridine had little effect on hSkM1; however, pretreatment with thimerosal attenuated Hg(2+) block of hSkM1. Y401C+E758C rat skeletal muscle Na(+) channels (mu1) that form a disulfide bond spontaneously between two cysteines at the 401 and 758 positions showed a significantly lower sensitivity to Hg(2+) (K(d) = 18 microM). However, Y401C+E758C mu1 after reduction with DTT had a significantly higher sensitivity to Hg(2+) (K(d) = 0.36 microM) than wild-type hSkM1. Mutants C753Amu1 (K(d) = 8.47 microM) or C1521A mu1 (K(d) = 8.63 microM) exhibited significantly lower sensitivity to Hg(2+) than did wild-type hSkM1, suggesting that these two conserved cysteinyl residues of the P-loop region may play an important role in the Hg(2+) block of the hSkM1 isoform. The heart Na(+) channel (hH1) was significantly more sensitive to low-dose Hg(2+) (K(d) = 0.43 microM) than was hSkM1. The C373Y hH1 mutant exhibited higher resistance (K(d) = 1.12 microM) to Hg(2+) than did wild-type hH1. In summary, Hg(2+) probably inhibits the muscle Na(+) channels at more than one cysteinyl residue in the Na(+) channel P-loop region. Hg(2+) exhibits a lower K(d) value (<1. 23 microM) for inhibition by forming a sulfur-Hg-sulfur bridge, as compared to reaction at a single cysteinyl residue with a higher K(d) value (>8.47 microM) by forming sulfur-Hg(+) covalently. The heart Na(+) channel isoform with more than two cysteinyl residues in the P-loop region exhibits an extremely high sensitivity (K(d) < 0. 43 microM) to Hg(+), accounting for heart-specific high sensitivity to the divalent mercury.
Abstract: Brugada syndrome is an inherited cardiac disease that causes sudden death related to idiopathic ventricular fibrillation in a structurally normal heart. The disease is characterized by ST-segment elevation in the right precordial ECG leads and is frequently accompanied by an apparent right bundle-branch block. The biophysical properties of the SCN5A mutation T1620M associated with Brugada syndrome were examined for defects in intermediate inactivation (I:(M)), a gating process in Na(+) channels with kinetic features intermediate between fast and slow inactivation. Cultured mammalian cells expressing T1620M Na(+) channels in the presence of the human beta(1) subunit exhibit enhanced intermediate inactivation at both 22 degrees C and 32 degrees C compared with wild-type recombinant human heart Na(+) channels (WT-hH1). Our findings support the hypothesis that Brugada syndrome is caused, in part, by functionally reduced Na(+) current in the myocardium due to an increased proportion of Na(+) channels that enter the I:(M) state. This phenomenon may contribute significantly to arrhythmogenesis in patients with Brugada syndrome. The full text of this article is available at http://www.circresaha.org.
Abstract: Skeletal and heart muscle excitability is based upon the pool of available sodium channels as determined by both fast and slow inactivation. Slow inactivation in hH1 sodium channels significantly differs from slow inactivation in hSkM1. The beta(1)-subunit modulates fast inactivation in human skeletal sodium channels (hSkM1) but has little effect on fast inactivation in human cardiac sodium channels (hH1). The role of the beta(1)-subunit in sodium channel slow inactivation is still unknown. We used the macropatch technique on Xenopus oocytes to study hSkM1 and hH1 slow inactivation with and without beta(1)-subunit coexpression. Our results indicate that the beta(1)-subunit is partly responsible for differences in steady-state slow inactivation between hSkM1 and hH1 channels. We also studied a sodium channel chimera, in which P-loops from each domain in hSkM1 sodium channels were replaced with corresponding regions from hH1. Our results show that these chimeras exhibit hH1-like properties of steady-state slow inactivation. These data suggest that P-loops are structural determinants of sodium channel slow inactivation, and that the beta(1)-subunit modulates slow inactivation in hSkM1 but not hH1. Changes in slow inactivation time constants in sodium channels coexpressed with the beta(1)-subunit indicate possible interactions among the beta(1)-subunit, P-loops, and the slow inactivation gate in sodium channels.
Abstract: The inhibitory effects of amlodipine besilate (CAS 11470-99-6) on the native Na+ current (INa) and cloned human cardiac Na+ channel alpha subunit (hH1) were studied by whole cell patch clamp techniques. Amlodipine produced tonic block of INa in a concentration- and holding potential (HP)-dependent manner with hyperpolarization of H infinity. Amlodipine produced phasic blockade of INa, which was dependent on HP and pulse duration. Amlodipine produced tonic blockade of hH1 in a concentration-dependent manner with 1 : 1 stoichiometry, and phasic blockade of hH1 which was dependent on the pulse duration. Amlodipine blocked INa in a voltage- and frequency-dependent manner via affinity to the resting as well as inactivated conformations of the alpha subunit.
Abstract: Mutations in a human cardiac Na+ channel gene (SCN5A) are responsible for chromosome 3-linked congenital long QT syndrome (LQT3). Here we characterized a de novo missense mutation (R1623Q, S4 segment of domain 4) identified in an infant Japanese girl with a severe form of LQT3. When expressed in oocytes, mutant Na+ channels exhibited only minor abnormalities in channel activation, but in contrast to three previously characterized LQT3 mutations, had significantly delayed macroscopic inactivation. Single channel analysis revealed that R1623Q channels have significantly prolonged open times with bursting behavior, suggesting a novel mechanism of pathophysiology in Na+ channel-linked long QT syndrome.
Abstract: The congenital long QT syndrome (LQTS) is an inherited disorder characterized by a delay in cardiac cellular repolarization leading to cardiac arrhythmias and sudden death often in young people. One form of the disease (LQT3) involves mutations in the voltage-gated cardiac sodium channel. The potential for targeted suppression of the LQT defect was explored by heterologous expression of mutant channels in cultured human cells. Kinetic and steady state analysis revealed an enhanced apparent affinity for the predominantly charged, primary amine compound, mexiletine. The affinity of the mutant channels in the inactivated state was similar to the wild type (WT) channels (IC50 approximately 15-20 microM), but the late-opening channels were inhibited at significantly lower concentrations (IC50 = 2-3 microM) causing a preferential suppression of the late openings. The targeting of the defective behavior of the mutant channels has important implications for therapeutic intervention in this disease. The results provide insights for the selective suppression of the mutant phenotype by very low concentrations of drug and indicate that mexiletine equally suppresses the defect in all three known LQT3 mutants.
Abstract: Voltage-gated Na+ channels are essential for the normal electrical excitability of neuronal and striated muscle membranes. Distinct isoforms of the Na+ channel alpha-subunit have been identified by molecular cloning, and their functional attributes have been defined by heterologous expression coupled with electrophysiological recording. Two closely related Na+ channel alpha-subunit isoforms, hH1 (human heart) and hSkM1 (human skeletal muscle), exhibit differences in their inactivation properties and in their response to the coexpressed beta 1-subunit. To localize regions that contribute to inactivation and to beta 1-subunit response, we have exploited these functional differences by studying chimeric channels composed of segments from both hH1 and hSkM1. Chimeras in which one or more of the cytoplasmic interdomain regions (ID1-2, ID2-3, and ID3-4) were exchanged between hH1 and hSkM1 exhibit inactivation properties identical with the background channel isoform, suggesting that these regions are not sufficient to cause gating differences. In contrast, inactivation properties of chimeras composed of approximately equal halves of the two channel isoforms were intermediate between hH1 and hSkM1. Furthermore, the response to the coexpressed beta 1-subunit was dependent on structures located in the carboxy-terminal half of the alpha-subunit, although domains D3, D4, and the carboxy terminal are not singularly responsible for this effect. These data indicate that inactivation differences between hH1 and hSkM1 are determined by multiple alpha-subunit domains.
Abstract: Recombinant brain, skeletal muscle, and heart voltage-gated Na+ channel alpha subunits differ in their functional responses to an accessory beta 1 subunit when coexpressed in Xenopus oocytes. We exploited the distinct beta 1 subunit responses observed for the human heart (hH1) and human skeletal muscle (hSkM1) isoforms to identify determinants of this response. Chimeric alpha subunits were constructed by exchanging the S5-S6 interhelical loops of each domain between hH1 and hSkM1 and then examined for effects on inactivation induced by coexpressed beta 1 subunit in oocytes. Substitution of single S5-S6 loops in either domain 1 (D1/S5-S6) or domain 4 (D4/S5-S6) of hSkM1 by the corresponding segments of hH1 produced channels that exhibited an attenuated response to coexpressed beta 1 subunit. Substitutions of both D1/S5-S6 and D4/S5-S6 in hSkM1 by the corresponding loops from hH1 completely abolished the effects of the beta 1 subunit on inactivation. The reciprocal chimera in which both D1/S5-S6 and D4/S5-S6 from hSkM1 were transplanted into hH1 exhibited significant beta 1 responsiveness (accelerated inactivation). The region within D4/S5-S6 that conferred beta 1 responsiveness was determined to reside primarily within an extracellular loop between the putative pore-forming segment SS2 and D4/S6. Gating modulation was also demonstrated using a chimeric beta subunit consisting of the extracellular domains of beta 1 and the transmembrane and C-terminal domains of the rat brain beta 2 subunit. These results suggest that the D1/S5-S6 and D4/S5-S6 loops in the alpha subunit and the extracellular domain of the beta 1 subunit are important determinants of the beta 1 subunit-induced gating modulation in Na+ channels.
Abstract: In the congenital long-QT syndrome, prolongation of the cardiac action potential occurs by an unknown mechanism and predisposes individuals to syncope and sudden death as a result of ventricular arrhythmias. Genetic heterogeneity has been demonstrated for autosomal dominant long-QT syndrome by the identification of multiple distinct loci, and associated mutations in two candidate genes have recently been reported. One form of hereditary long QT (LQT3) has been linked to a mutation in the gene encoding the human heart voltage-gated sodium-channel alpha-subunit (SCN5A on chromosome 3p21). Here we characterize this mutation using heterologous expression of recombinant human heart sodium channels. Mutant channels show a sustained inward current during membrane depolarization. Single-channel recordings indicate that mutant channels fluctuate between normal and non-inactivating gating modes. Persistent inward sodium current explains prolongation of cardiac action potentials, and provides a molecular mechanism for this form of congenital long-QT syndrome.
Abstract: Voltage-gated Na+ channels are heteromeric proteins consisting of alpha and beta subunits. Although alpha subunits alone are sufficient to encode functional channels, beta 1 subunits appear to modulate the kinetics of inactivation. We have used a cross-species reverse transcriptase polymerase chain reaction approach to isolate cDNAs encoding a Na+ channel beta 1 subunit from human heart and skeletal muscle. The deduced amino acid sequence of the human beta 1 subunit exhibits 96% identity with the rat brain beta 1 subunit. Human beta 1 mRNA transcripts are abundantly expressed in skeletal muscle, heart, and brain. Genomic Southern blot hybridization experiments suggest that a single gene located on chromosome 19 encodes the human beta 1 subunit that is expressed in all three of these tissues. Co-expression of the human beta 1 subunit with the recombinant human skeletal muscle alpha subunit (hSkM1) in Xenopus oocytes results in Na+ currents that inactivate rapidly. In contrast, the human beta 1 subunit has no effect on the function of the tetrodotoxin-insensitive human heart Na+ channel (hH1). These findings indicate that the human beta 1 subunit is widely expressed but does not functionally modify all Na+ channel isoforms.
Abstract: Arachidonate 12- and 15-lipoxygenase (LO) products are generated in experimental glomerulonephritis. 15-S-HETE (a 15-LO product) and lipoxins (interaction products between 5-LO and either 12-LO or 15-LO) counteract the proinflammatory actions of leukotrienes. IL-4 has been shown to up-regulate 15-LO gene expression in human leukocytes. Based on homology with human 15-LO, we cloned a 0.76 kbp fragment of a rat LO cDNA from leukocytes stimulated by interleukin-4 (IL-4). The deduced amino acid sequence shows 71.0% and 60.1% homology to human 15-LO and 12-LO, respectively, and 100% homology to a recently cloned "leukocyte type" rat 12-lipoxygenase enzyme, which possesses significant 15-lipoxygenase activity (heretofore referred to as "12/15-LO"). A deletion mutant was utilized to generate internal standard cRNA in quantitative PCR assays. Glomerular 12/15-LO mRNA increased significantly over controls 24 and 48 hours after NTS injection, then decreased at 72 hours. RNA from NTS glomeruli contained higher levels of 12/15-LO mRNA than that from unstimulated peripheral leukocytes, suggesting that 12/15-LO transcription is up-regulated locally in native and/or infiltrating glomerular cells. Glomerular IL-4 mRNA increased markedly 16 hours post-NTS, and was then reduced, suggesting a potential role for T cell-derived IL-4 in directing the expression of 12/15-LO during glomerulonephritis. This represents the first demonstration of tandem regulated in vivo gene expression for a lymphokine (IL-4) and a lipoxygenase, both of which promote counter-inflammatory influences in immune complex-mediated injury.
Abstract: Voltage-gated sodium (Na+) channels are essential for the generation and propagation of action potentials in striated muscle and neuronal tissues. Biochemically, Na+ channels consist of a large alpha subunit and one or two smaller beta subunits. The alpha subunit alone can exhibit all of the functional attributes of a voltage-gated Na+ channel, but requires a beta 1 subunit for normal inactivation kinetics. While genetic mutations in the skeletal muscle Na+ channel alpha-subunit gene can cause human disease, it is not known whether hereditary defects in the beta 1 subunit underlie any inherited syndromes. To help explore this further, we have carried out an analysis of the detailed structure of the human beta 1 subunit gene (SCN1B) including the delineation of intron-exon boundaries by genomic DNA cloning and sequence analysis. The complete coding region of SCN1B is found in approximately 9.0 kb of genomic DNA and consists of five exons (72 to 749 bp) and four introns (90 bp to 5.5 kb). Using a 15.9-kb genomic SCN1B clone, we assigned the gene to the long arm of chromosome 19 (19q13.1-q13.2) by fluorescence in situ hybridization. An intragenic polymorphic (TTA)n repeat that is positioned between two tandem Alu repetitive sequences was also characterized. The (TTA)n repeat exhibits 5 distinct alleles and a heterozygosity index of 0.59. This information should be useful in evaluating SCN1B as a candidate gene for hereditary disorders affecting membrane excitability.
Abstract: Recombinant sodium channel alpha subunits expressed in Xenopus oocytes display an anomalously slow rate of inactivation that arises from channels that predominantly exist in a slow gating mode [1,2]. Co-expression of Na+ channel beta 1 subunit with the human skeletal muscle Na+ channel alpha subunit increases the Na+ current and induces normal gating behavior in Xenopus laevis oocytes. The effects of the beta 1 subunit can be explained by an allosterically induced conformational switch of the alpha subunit protein that occurs upon binding the beta 1 subunit. This binding alters the free energy barriers separating distinct conformational states of the channel. The results illustrate a fundamental modulation of ion channel gating at the molecular level, and specifically demonstrate the importance of the beta 1 subunit for gating mode changes of Na+ channels.
Abstract: Transcripts homologous to the rat brain sodium channel beta subunit (beta 1) are prominently expressed in both innervated and denervated adult skeletal muscle and in heart, but not in neonatal skeletal or cardiac muscle. Regulation of beta 1 mRNA expression closely parallels that of SkM1 alpha during development, after denervation in adult muscle, and in primary muscle culture, but does not follow SkM2 expression under any condition examined. In oocytes, beta 1 interacts functionally with SkM1 to modulate the abnormally slow inactivation kinetics observed with this alpha subunit expressed alone. We conclude that a common beta 1 subunit is expressed in skeletal muscle, heart, and brain and that in skeletal muscle, this subunit is specifically associated with the SkM1, rather than the SkM2, sodium channel isoform.
Abstract: The isoprostanes are nonenzymatically generated prostanoids synthesized in vivo in humans and rats through reactions catalyzed by free oxygen radicals. 8-Epi-prostaglandin F2 alpha (8-epi-PGF2 alpha), an F2-isoprostane, is a potent smooth muscle constrictor. A thromboxane A2 (TxA2) receptor antagonist, SQ 29548, blocks renal vasoconstriction during 8-epi-PGF2 alpha administration in rats. With the use of cultured rat aortic smooth muscle cells, we found specific binding sites for [3H]SQ 29548 and for [125I]BOP, a TxA2 agonist. Both ligands were displaced from these binding sites by 8-epi-PGF2 alpha, although with significantly lesser potency than nonlabeled SQ 29548, I-BOP, or U-46619, a TxA2 agonist. In contrast, 8-epi-PGF2 alpha stimulated inositol 1,4,5-trisphosphate production and DNA synthesis in these cells with significantly greater potency than any TxA2 agonist, effects only partially inhibited by SQ 29548. In human TxA2 receptor cDNA-transfected cells, competition by 8-epi-PGF2 alpha for specific [3H]SQ 29548 binding was negligible. Thus 8-epi-PGF2 alpha probably exerts its biological actions in vascular smooth muscle through activation of receptor sites related to but distinct from TxA2 receptors. The existence of such binding sites suggests novel avenues for investigation into the biology of TxA2 and of free radical-mediated tissue injury.
Abstract: The mRNA level of the type-1 angiotensin II receptor (AT1) was down-regulated by angiotensin II in cultured rat glomerular mesangial cells. The effect was maximum with 1 microM AII at 6 h, sensitive to cycloheximide, and specific to AT1 since this phenomenon was blocked by DuP753, an AT1 antagonist, but not by type-2 antagonist PD123319. Dibutyryl cAMP, forskolin, and cholera toxin also caused AT1 down-regulation. These effects were not altered by either the protein kinase A inhibitor H-8 or cycloheximide. Calcium ionophore A23187, pertussis toxin, protein kinase C inhibitor staurosporine, or prolonged incubation with phorbol ester were without effect. These results suggest that there are at least two pathways to down-regulate AT1 mRNA; one way is an angiotensin II-induced, protein kinase C-independent, and cycloheximide-sensitive pathway and the other is an angiotensin II-independent, cAMP-induced, and cycloheximide-insensitive pathway.
Abstract: We isolated a cDNA encoding rat leukotriene A4 (LTA4) hydrolase from mesangial cells by the polymerase chain reaction according to the human amino acid sequence. The deduced amino acid sequence shows that rat LTA4 hydrolase is a 609 amino acid protein with an Mr 69 kDa. Comparison of human LTA4 hydrolase revealed 93% homology, and include zinc-binding motifs of aminopeptidases. COS-7 cells transfected with the cDNA revealed substantial LTA4 hydrolase activity, and their activities were abolished by preincubation with captopril, representing the first reported cDNA expression of recombinant enzyme in mammalian cells. RNA blot analysis indicated that LTA4 hydrolase was expressed in glomerular endothelial, epithelial and mesangial cells.
Abstract: In order to determine whether phosphoinositide metabolism is altered in hypertensive cardiac hypertrophy, phospholipase C (PLC) and protein kinase C activities were measured in hearts from 4- and 20-week-old spontaneously hypertensive rats (SHR) and age-matched, normotensive Wistar-Kyoto rats (WKY). PLC activities were assayed using phosphatidylinositol (PI) and phosphatidylinositol-4,5-bisphosphate (PIP2) as substrates to assess the substrate specificity. PI-hydrolyzing PLC activity (PI-PLC) was predominantly located in the cytosol, and its activity was similar in both strains. Membrane-bound PIP2-hydrolyzing PLC activity (PIP2-PLC) was significantly lower in 20-week-old SHR than in WKY, but there was no significant difference in soluble PIP2-PLC. Protein kinase C activity was significantly elevated in 20-week-old SHR and Ca2(+)-phospholipid-dependent phosphorylation was observed in the proteins of molecular weight 26, 32, 43, and 95 KDa. In 4-week-old prehypertensive SHR, there were no significant differences in PI-PLC, PIP2-PLC, or protein kinase C activities as compared with age-matched WKY. These data demonstrated that protein kinase C and membrane-bound PIP2-PLC are altered during the period of hypertension development. These alterations may have important roles in the development or maintenance of hypertensive cardiac hypertrophy in SHR.
Abstract: Phosphatidylinositol-specific phospholipase C (PI-PLC) was characterized in rat myocardium, and the effect of hypoxia on its activity was investigated. It had a substrate specificity toward phosphatidylinositol (PI) and was predominantly located in cytosol. Its optimal pH was 7.4 and it required 5 mM of Ca2+ for maximum activity, but did not hydrolyze phosphatidylcholine (PC), phosphatidylethanolamine (PE), or phosphatidylserine (PS). Vmax and Km were 51.5 nmol/mg/min, and 231 microM, respectively. Sodium deoxycholate increased its activity at a concentration of 0.05%, while Triton X-100 inhibited its activity at any concentrations examined. PI-PLC was partially purified 260 fold over the crude cytosol, with ammonium sulphate fractionation, DEAE-cellulose, Sephadex G-100, Hydroxylapatite, and Sephadex G-150 column chromatographies. In order to elucidate the biochemical function of myocardial PI-PLC in hypoxia, PI-PLC along with phospholipase A2 (PLA2) was investigated in N2 gas-saturated buffer up to for 24 hours. The activity of PI-PLC did not change during the first 2 hours, and then gradually attenuated. Substrate specificity or subcellular localization of PI-PLC unchanged during 24 hour 9 of hypoxia. PLA2 was predominantly located in microsome and had a substrate specificity toward PE in normoxic state. In hypoxia, on the other hand, it hydrolyzed PC besides PE and was activated on and after 2 hours of hypoxic incubation. PI-PLC did not seem to contribute in releasing arachidonate from membrane lipid-bilayers during myocardial ischemia. But some biochemical mechanism suggested to inhibit its activity protecting the abrupt cell damage.
