Selective gamma-ketoaldehyde scavengers protect Nav1.5 from oxidant-induced inactivation.

The cardiac sodium channel (SCN5A, Na(V)1.5) is a key determinant of electrical impulse conduction in cardiac tissue. Acute myocardial infarction leads to diminished sodium channel availability, both because of decreased channel expression and because of greater inactivation of channels already present. Myocardial infarction leads to significant increases in reactive oxygen species and their downstream effectors including lipoxidation products. The effects of reactive oxygen species on Na(V)1.5 function in whole hearts can be modeled in cultured myocytes, where oxidants shift the availability curve of I(Na) to hyperpolarized potentials, decreasing cardiac sodium current at the normal activation threshold. We recently examined potential mediators of the oxidant-induced inactivation and found that one specific lipoxidation product, the isoketals, recapitulated the effects of oxidant on sodium currents. Isoketals are highly reactive gamma-ketoaldehydes formed by the peroxidation of arachidonic acid that covalently modify the lysine residues of proteins. We now confirm that exposure to oxidants induces lipoxidative modification of Na(V)1.5 and that the selective isoketal scavengers block voltage-dependent changes in sodium current by the oxidant tert-butylhydroperoxide, both in cells heterologously expressing Na(V)1.5 and in a mouse cardiac myocyte cell line (HL-1). Thus, inhibition of this lipoxidative modification pathway is sufficient to protect the sodium channel from oxidant induced inactivation and suggests the potential use of isoketal scavengers as novel therapeutics to prevent arrhythmogenesis during myocardial infarction.

Development of a large-scale de-identified DNA biobank to enable personalized medicine.

Our objective was to develop a DNA biobank linked to phenotypic data derived from an electronic medical record (EMR) system. An "opt-out" model was implemented after significant review and revision. The plan included (i) development and maintenance of a de-identified mirror image of the EMR, namely, the "synthetic derivative" (SD) and (ii) DNA extracted from discarded blood samples and linked to the SD. Surveys of patients indicated general acceptance of the concept, with only a minority ( approximately 5%) opposing it. As a result, mechanisms to facilitate opt-out included publicity and revision of a standard "consent to treatment" form. Algorithms for sample handling and procedures for de-identification were developed and validated in order to ensure acceptable error rates (<0.3 and <0.1%, respectively). The rate of sample accrual is 700-900 samples/week. The advantages of this approach are the rate of sample acquisition and the diversity of phenotypes based on EMRs.

Molecular basis of isolated cardiac conduction disease.

Cardiac conduction disorders are among the most common rhythm disturbances causing disability in millions of people worldwide and necessitating pacemaker implantation. Isolated cardiac conduction disease (ICCD) can affect various regions within the heart, and therefore the clinical features also vary from case to case. Typically, it is characterized by progressive alteration of cardiac conduction through the atrioventricular node, His-Purkinje system, with right or left bundle branch block and QRS widening. In some instances, the disorder may progress to complete atrioventricular block, with syncope and even death. While the role of genetic factors in conduction disease has been suggested as early as the 1970s, it was only recently that specific genetic loci have been reported. Multiple mutations in the gene encoding for the cardiac voltage-gated sodium channel (SCN5A), which plays a fundamental role in the initiation, propagation, and maintenance of normal cardiac rhythm, have been linked to conduction disease, allowing for genotype-phenotype correlation. The electrophysiological characterization of heterologously expressed mutant Na+ channels has revealed gating defects that consistently lead to a loss of channel function. However, studies have also revealed significant overlap between aberrant rhythm phenotypes, and single mutations have been identified that evoke multiple distinct rhythm disorders with common gating lesions. These new insights highlight the complexities involved in linking single mutations, ion-channel behavior, and cardiac rhythm but suggest that interplay between multiple factors could underlie the manifestation of the disease phenotype.

Inherited sodium channelopathies: novel therapeutic and proarrhythmic molecular mechanisms.

Voltage-gated sodium (Na) channels, transmembrane proteins that produce the ionic current responsible for the rapid upstroke of the cardiac action potential, are key elements required for rapid conduction through the myocardium and maintenance of the cardiac rhythm. The exquisite sensitivity of the cardiac rhythm to Na channel function is manifest in the proarrhythmic complications of "antiarrhythmic" Na channel blockade in patients with myocardial ischemia. More recently, studies of inherited single amino acid substitutions in Na channels have unveiled a remarkable array of cardiac rhythm disturbances, as well as surprising pharmacologic sensitivities. Hence, the sodium channelopathies are providing new molecular insights into mechanisms whereby altered ion channel behavior precipitates cardiac arrhythmias.

Gating-dependent mechanisms for flecainide action in SCN5A-linked arrhythmia syndromes.

BACKGROUND: Mutations in the cardiac sodium (Na) channel gene (SCN5A) give rise to the congenital long-QT syndrome (LQT3) and the Brugada syndrome. Na channel blockade by antiarrhythmic drugs improves the QT interval prolongation in LQT3 but worsens the Brugada syndrome ST-segment elevation. Although Na channel blockade has been proposed as a treatment for LQT3, flecainide also evokes "Brugada-like" ST-segment elevation in LQT3 patients. Here, we examine how Na channel inactivation gating defects in LQT3 and Brugada syndrome elicit proarrhythmic sensitivity to flecainide. METHODS AND RESULTS: We measured whole-cell Na current (I(Na)) from tsA-201 cells transfected with DeltaKPQ, a LQT3 mutation, and 1795insD, a mutation that provokes both the LQT3 and Brugada syndromes. The 1795insD and DeltaKPQ channels both exhibited modified inactivation gating (from the closed state), thus potentiating tonic I(Na) block. Flecainide (1 micromol/L) tonic block was only 16.8+/-3.0% for wild type but was 58.0+/-6.0% for 1795insD (P<0.01) and 39.4+/-8.0% (P<0.05) for DeltaKPQ. In addition, the 1795insD mutation delayed recovery from inactivation by enhancing intermediate inactivation, with a 4-fold delay in recovery from use-dependent flecainide block. CONCLUSIONS: We have linked 2 inactivation gating defects ("closed-state" fast inactivation and intermediate inactivation) to flecainide sensitivity in patients carrying LQT3 and Brugada syndrome mutations. These results provide a mechanistic rationale for predicting proarrhythmic sensitivity to flecainide based on the identification of specific SCN5A inactivation gating defects.

A novel extracellular calcium sensing mechanism in voltage-gated potassium ion channels.

Potassium (K(+)) channels influence neurotransmitter release, burst firing rate activity, pacing, and critical dampening of neuronal circuits. Internal and external factors that further modify K(+) channel function permit fine-tuning of neuronal circuits. Human ether-à-go-go-related gene (HERG) K(+) channels are unusually sensitive to external calcium concentration ([Ca(2+)](o)). Small changes in [Ca(2+)](o) shift the voltage dependence of channel activation to more positive membrane potentials, an effect that cannot be explained by nonspecific surface charge screening or channel pore block. The HERG-calcium concentration-response relationship spans the physiological range for [Ca(2+)](o). The modulatory actions of calcium are attributable to differences in the Ca(2+) affinity between rested and activated channels. Adjacent extracellular, negatively charged amino acids (E518 and E519) near the S4 voltage sensor influence both channel gating and Ca(2+) dependence. Neutralization of these charges had distinct effects on channel gating and calcium sensitivity. A change in the degree of energetic coupling between these amino acids on transition from closed to activated channel states reveals movement in this region during channel gating and defines a molecular mechanism for protein state-dependent ligand interactions. The results suggest a novel extracellular [Ca(2+)](o) sensing mechanism coupled to allosteric changes in channel gating and a mechanism for fine-tuning cell repolarization.

A sodium-channel mutation causes isolated cardiac conduction disease.

Cardiac conduction disorders slow the heart rhythm and cause disability in millions of people worldwide. Inherited mutations in SCN5A, the gene encoding the human cardiac sodium (Na+) channel, have been associated with rapid heart rhythms that occur suddenly and are life-threatening; however, a chief function of the Na+ channel is to initiate cardiac impulse conduction. Here we provide the first functional characterization of an SCN5A mutation that causes a sustained, isolated conduction defect with pathological slowing of the cardiac rhythm. By analysing the SCN5A coding region, we have identified a single mutation in five affected family members; this mutation results in the substitution of cysteine 514 for glycine (G514C) in the channel protein. Biophysical characterization of the mutant channel shows that there are abnormalities in voltage-dependent 'gating' behaviour that can be partially corrected by dexamethasone, consistent with the salutary effects of glucocorticoids on the clinical phenotype. Computational analysis predicts that the gating defects of G514C selectively slow myocardial conduction, but do not provoke the rapid cardiac arrhythmias associated previously with SCN5A mutations.

The cardiac sodium channel: gating function and molecular pharmacology.

Cardiac sodium (Na) channels are dynamic molecules that undergo rapid structural changes in response to the changing electrical field in the myocardium. Inherited mutations in SCN5A, the gene encoding the cardiac Na channel, provoke life-threatening cardiac arrhythmias, often by modifying these voltage-dependent conformational changes. These disorders (i.e. the long QT syndrome and Brugada syndrome) may serve as valuable models for understanding the mechanistic linkages between Na channel dysfunction and cardiac arrhythmias in more common, acquired conditions such as cardiac ischemia. In addition, the balance between therapeutic and adverse effects from Na channel blockade by antiarrhythmic compounds may be shifted by subtle alterations in Na channel function. This review examines recent studies that tie key loci in the Na channel primary sequence to its dynamic function, while examining the emerging themes linking Na channel structure, function, and pharmacology to inherited and acquired disorders of cardiac excitability.

Probing the interaction between inactivation gating and Dd-sotalol block of HERG.

Potassium channels encoded by HERG underlie I:(Kr), a sensitive target for most class III antiarrhythmic drugs, including methanesulfonanilides such as Dd-sotalol. Recently it was shown that these drugs are trapped in the channel as it closes during hyperpolarization. At the same time, HERG channels rapidly open and inactivate when depolarized, and methanesulfonanilide block is known to develop in a use-dependent manner, suggesting a potential role for inactivation in drug binding. However, the role of HERG inactivation in class III drug action is uncertain: pore mutations that remove inactivation reduce block, yet many of these mutations also modify the channel permeation properties and could alter drug affinity through gating-independent mechanisms. In the present study, we identify a definitive role for inactivation gating in Dd-sotalol block of HERG, using interventions complementary to mutagenesis. These interventions (addition of extracellular Cd(2+), removal of extracellular Na(+)) modify the voltage dependence of inactivation but not activation. In normal extracellular solutions, block of HERG current by 300 micromol/L Dd-sotalol reached 80% after a 10-minute period of repetitive depolarization to +20 mV. Maneuvers that impeded steady-state inactivation also reduced Dd-sotalol block of HERG: 100 micromol/L Cd(2+) reduced steady-state block to 55% at +20 mV (P:<0.05); removing extracellular Na(+) reduced block to 44% (P:<0.05). An inactivation-disabling mutation (G628C-S631C) reduced Dd-sotalol block to only 11% (P:<0.05 versus wild type). However, increasing the rate of channel inactivation by depolarizing to +60 mV reduced Dd-sotalol block to 49% (P:<0.05 versus +20 mV), suggesting that the drug does not primarily bind to the inactivated state. Coexpression of MiRP1 with HERG had no effect on inactivation gating and did not modify Dd-sotalol block. We postulate that Dd-sotalol accesses its receptor in the open pore, and the drug-receptor interaction is then stabilized by inactivation. Whereas deactivation traps the bound methanesulfonanilide during hyperpolarization, we propose that HERG inactivation stabilizes the drug-receptor interaction during membrane depolarization.

A structural rearrangement in the sodium channel pore linked to slow inactivation and use dependence.

Voltage-gated sodium (Na(+)) channels are a fundamental target for modulating excitability in neuronal and muscle cells. When depolarized, Na(+) channels may gradually enter long-lived, slow-inactivated conformational states, causing a cumulative loss of function. Although the structural motifs that underlie transient, depolarization-induced Na(+) channel conformational states are increasingly recognized, the conformational changes responsible for more sustained forms of inactivation are unresolved. Recent studies have shown that slow inactivation components exhibiting a range of kinetic behavior (from tens of milliseconds to seconds) are modified by mutations in the outer pore P-segments. We examined the state-dependent accessibility of an engineered cysteine in the domain III, P-segment (F1236C; rat skeletal muscle) to methanethiosulfonate-ethylammonium (MTSEA) using whole-cell current recordings in HEK 293 cells. F1236C was reactive with MTSEA applied from outside, but not inside the cell, and modification was markedly increased by depolarization. Depolarized F1236C channels exhibited both intermediate (I(M); tau approximately 30 ms) and slower (I(S); tau approximately 2 s) kinetic components of slow inactivation. Trains of brief, 5-ms depolarizations, which did not induce slow inactivation, produced more rapid modification than did longer (100 ms or 6 s) pulse widths, suggesting both the I(M) and I(S) kinetic components inhibit depolarization-induced MTSEA accessibility of the cysteine side chain. Lidocaine inhibited the depolarization-dependent sulfhydryl modification induced by sustained (100 ms) depolarizations, but not by brief (5 ms) depolarizations. We conclude that competing forces influence the depolarization-dependent modification of the cysteine side chain: conformational changes associated with brief periods of depolarization enhance accessibility, whereas slow inactivation tends to inhibit the side chain accessibility. The findings suggest that slow Na(+) channel inactivation and use-dependent lidocaine action are linked to a structural rearrangement in the outer pore.

Enhanced Na(+) channel intermediate inactivation in Brugada syndrome.

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.

Two distinct congenital arrhythmias evoked by a multidysfunctional Na(+) channel.

The congenital long-QT syndrome (LQT3) and the Brugada syndrome are distinct, life-threatening rhythm disorders linked to autosomal dominant mutations in SCN5A, the gene encoding the human cardiac Na(+) channel. It is believed that these two syndromes result from opposite molecular effects: LQT3 mutations induce a gain of function, whereas Brugada syndrome mutations reduce Na(+) channel function. Paradoxically, an inherited C-terminal SCN5A mutation causes affected individuals to manifest electrocardiographic features of both syndromes: QT-interval prolongation (LQT3) at slow heart rates and distinctive ST-segment elevations (Brugada syndrome) with exercise. In the present study, we show that the insertion of the amino acid 1795insD has opposite effects on two distinct kinetic components of Na(+) channel gating (fast and slow inactivation) that render unique, simultaneous effects on cardiac excitability. The mutation disrupts fast inactivation, causing sustained Na(+) current throughout the action potential plateau and prolonging cardiac repolarization at slow heart rates. At the same time, 1795insD augments slow inactivation, delaying recovery of Na(+) channel availability between stimuli and reducing the Na(+) current at rapid heart rates. Our findings reveal a novel molecular mechanism for the Brugada syndrome and identify a new dual mechanism whereby single SCN5A mutations may evoke multiple cardiac arrhythmia syndromes by influencing diverse components of Na(+) channel gating function. The full text of this article is available at http://www.circresaha.org.

Lidocaine induces a slow inactivated state in rat skeletal muscle sodium channels.

1. Local anaesthetics such as lidocaine (lignocaine) interact with sodium channels in a manner that is exquisitely sensitive to the voltage-dependent conformational state of the ion channel. When depolarized in the presence of lidocaine, sodium channels assume a long-lived quiescent state. Although studies over the last decade have localized the lidocaine receptor to the inner aspect of the aqueous pore, the mechanistic basis of depolarization-induced 'use-dependent' lidocaine block remains uncertain. 2. Recent studies have shown that lowering the extracellular Na+ concentration ([Na+]o) and mutations in the sodium channel outer P-loop modulate occupancy of a quiescent 'slow' inactivated state with intermediate kinetics (termed IM) that involves structural rearrangements in the outer pore. 3. Site-directed mutagenesis and ion-replacement experiments were performed using voltage-clamped Xenopus oocytes and cultured (HEK-293) cells expressing wild-type and mutant rat skeletal muscle (mu1) sodium channels. 4. Our results show that lowering [Na+]o potentiates use-dependent lidocaine block. The effect of [Na+]o is maintained despite a III-IV linker mutation that partially disrupts fast inactivation (F1304Q). In contrast, the effect of lowering [Na+]o on lidocaine block is reduced by a P-loop mutation (W402A) that limits occupancy of IM. 5. Our findings are consistent with a simple allosteric model where lidocaine binding induces channels to occupy a native slow inactivated state that is inhibited by [Na+]o.

A revised view of cardiac sodium channel "blockade" in the long-QT syndrome.

Mutations in SCN5A, encoding the cardiac sodium (Na) channel, are linked to a form of the congenital long-QT syndrome (LQT3) that provokes lethal ventricular arrhythmias. These autosomal dominant mutations disrupt Na channel function, inhibiting channel inactivation, thereby causing a sustained ionic current that delays cardiac repolarization. Sodium channel-blocking antiarrhythmics, such as lidocaine, potently inhibit this pathologic Na current (I(Na)) and are being evaluated in patients with LQT3. The mechanism underlying this effect is unknown, although high-affinity "block" of the open Na channel pore has been proposed. Here we report that a recently identified LQT3 mutation (R1623Q) imparts unusual lidocaine sensitivity to the Na channel that is attributable to its altered functional behavior. Studies of lidocaine on individual R1623Q single-channel openings indicate that the open-time distribution is not changed, indicating the drug does not block the open pore as proposed previously. Rather, the mutant channels have a propensity to inactivate without ever opening ("closed-state inactivation"), and lidocaine augments this gating behavior. An allosteric gating model incorporating closed-state inactivation recapitulates the effects of lidocaine on pathologic I(Na). These findings explain the unusual drug sensitivity of R1623Q and provide a general and unanticipated mechanism for understanding how Na channel-blocking agents may suppress the pathologic, sustained Na current induced by LQT3 mutations.

Calmodulin kinase determines calcium-dependent facilitation of L-type calcium channels.

A dynamic positive feedback mechanism, known as 'facilitation', augments L-type calcium-ion currents (ICa) in response to increased intracellular Ca2+ concentrations. The Ca2+-binding protein calmodulin (CaM) has been implicated in facilitation, but the single-channel signature and the signalling events underlying Ca2+/CaM-dependent facilitation are unknown. Here we show that the Ca2+/CaM-dependent protein kinase II (CaMK) is necessary and possibly sufficient for ICa facilitation. CaMK induces a channel-gating mode that is characterized by frequent, long openings of L-type Ca2+ channels. We conclude that CaMK-mediated phosphorylation is an essential signalling event in triggering Ca2+/CaM-dependent ICa facilitation.

Isoform-specific lidocaine block of sodium channels explained by differences in gating.

When depolarized from typical resting membrane potentials (V(rest) approximately -90 mV), cardiac sodium (Na) currents are more sensitive to local anesthetics than brain or skeletal muscle Na currents. When expressed in Xenopus oocytes, lidocaine block of hH1 (human cardiac) Na current greatly exceeded that of mu1 (rat skeletal muscle) at membrane potentials near V(rest), whereas hyperpolarization to -140 mV equalized block of the two isoforms. Because the isoform-specific tonic block roughly parallels the drug-free voltage dependence of channel availability, isoform differences in the voltage dependence of fast inactivation could underlie the differences in block. However, after a brief (50 ms) depolarizing pulse, recovery from lidocaine block is similar for the two isoforms despite marked kinetic differences in drug-free recovery, suggesting that differences in fast inactivation cannot entirely explain the isoform difference in lidocaine action. Given the strong coupling between fast inactivation and other gating processes linked to depolarization (activation, slow inactivation), we considered the possibility that isoform differences in lidocaine block are explained by differences in these other gating processes. In whole-cell recordings from HEK-293 cells, the voltage dependence of hH1 current activation was approximately 20 mV more negative than that of mu1. Because activation and closed-state inactivation are positively coupled, these differences in activation were sufficient to shift hH1 availability to more negative membrane potentials. A mutant channel with enhanced closed-state inactivation gating (mu1-R1441C) exhibited increased lidocaine sensitivity, emphasizing the importance of closed-state inactivation in lidocaine action. Moreover, when the depolarization was prolonged to 1 s, recovery from a "slow" inactivated state with intermediate kinetics (I(M)) was fourfold longer in hH1 than in mu1, and recovery from lidocaine block in hH1 was similarly delayed relative to mu1. We propose that gating processes coupled to fast inactivation (activation and slow inactivation) are the key determinants of isoform-specific local anesthetic action.

Enhancement of HERG K+ currents by Cd2+ destabilization of the inactivated state.

We have studied the functional effects of extracellular Cd(2+) on human ether-a-go-go-related gene (HERG) encoded K(+) channels. Low concentrations (10-200 microM) of extracellular Cd(2+) increased outward currents through HERG channels; 200 microM Cd(2+) more than doubled HERG currents and altered current kinetics. Cd(2+) concentrations up to 200 microM did not change the voltage dependence of channel activation, but shifted the voltage dependence of inactivation to more depolarized membrane potentials. Cd(2+) concentrations >or=500 microM shifted the voltage dependence of channel activation to more positive potentials. These results are consistent with a somewhat specific ability of Cd(2+) to destabilize the inactivated state. We tested the hypothesis that channel inactivation is essential for Cd(2+)-induced increases in HERG K(+) currents, using a double point mutation (G628C/S631C) that diminishes HERG inactivation (Smith, P. L., T. Baukrowitz, and G. Yellen. 1996. Nature (Lond.). 379:833-836). This inactivation-removed mutant is insensitive to low concentrations of Cd(2+). Thus, Cd(2+) had two distinct effects on HERG K(+) channels. Low concentrations of Cd(2+) caused relatively selective effects on inactivation, resulting in a reduction of the apparent rectification of the channel and thereby increasing HERG K(+) currents. Higher Cd(2+) concentrations affected activation gating as well, possibly by a surface charge screening mechanism or by association with a lower affinity site.