Sodium Channel Inactivation Gate Research Articles

Novel deletion mutation in the cardiac sodium channel inactivation gate causes long QT syndrome

Disturbances in cardiac sodium channel function are associated with inherited arrhythmia susceptibility. Mutations in SCN5A, which encodes the cardiac sodium channel (NaV1.5), cause congenital long QT syndrome type 3 (LQT3), Brugada syndrome (BrS) and a variety of cardiac conduction disorders (CCD) [1,2]. These disorders have can have complex genotype-phenotype relationships [3,4]. Here we report the clinical features of an LQTS family segregating a novel amino acid deletion mutation (N1472del) in SCN5A that produces a unique pattern of biophysical disturbances consistent with the clinical phenotype.

Vulnerability in Simulated Ischemic Ventricular Transmural Tissues

Vulnerability is an effective index to evaluate increased risk for unidirectional conduction block and reentry in hearts. Recent reports in animal experiments have indicated an opposite characteristics of the vulnerability in normal and ischemic transmural tissues. In order to clarify the differences and to investigate the mechanisms, a computer simulation method was used in this study to investigate the vulnerability relative to the premature pacing sites in normal and ischemic transmural tissues. Endo-, mid- and epi-cardial myocytes incorporating different severities of ischemia were developed across a tissue strand. The sodium channel inactivation gating variable h was calculated to provide the degree of sodium current recovery preceding the premature pacing. In the normal tissue, the measured vulnerable window was demonstrated to be wider by delivering an endocardial premature beat than that by applying an epicardial premature pacing. On the contrary, during ischemia the epicardium showed a wider vulnerable window than the endocardium. The results illustrated that during ischemia h decreased with accumulation of [K⁺]o, and action potential duration dispersion was obviously altered due to anoxia. In contrast, the elevated [K⁺]o was suggested to play an important role in the difference of the location-dependent vulnerability in normal and ischemic tissues.

Structural Basis for Calcium Modulation of the Cardiac Sodium Channel

Voltage gated sodium channels drive the upstroke of the cardiac action potential and then undergo a fast inactivation process which is essential for the termination of their contribution to excitability and for the maintenance of cardiac rhythms. The degree to which the channel opens and inactivates relies on many cellular factors. Increased cytoplasmic calcium, for instance, results in a rightward shifted steady-state inactivation relationship for the cardiac Nav1.5 isoform, resulting in more channels being available to contribute to the action potential. We have previously shown using isothermal titration calorimetry (ITC) that Ca2+ /calmodulin can bind the sodium channel inactivation gate comprised of the cytoplasmic linker between domains III and IV in a calcium dependent manner (Kd at 1mM free [Ca2+]=3uM). To further examine this interaction in greater detail, we have determined a high-resolution (1.35A) crystal structure of the rigid portion of the two proteins in complex. This new structure confirms our previous findings that tyrosine 1494 acts as a major interaction point with the calmodulin C-lobe. With this structural data in hand we have designed channel mutations that increase (Kd=600nM) or decrease (approximately Kd=30uM) the affinity of calmodulin to the inactivation gate. We then analyzed the effect of these novel mutations on channel gating via patch clamp electrophysiology to further investigate the mechanisms of calcium regulation of the cardiac sodium channel. We account for our observations obtained from ITC and patch clamp experiments with a kinetic model describing calcium modulation of cardiac sodium channels. Our results suggest that the inactivation gate is a key molecular determinant in the regulation of cardiac sodium channels by calcium.

A Double Tyrosine Motif in the Cardiac Sodium Channel Domain III-IV Linker Couples Calcium-dependent Calmodulin Binding to Inactivation Gating

Voltage-gated sodium channels maintain the electrical cadence and stability of neurons and muscle cells by selectively controlling the transmembrane passage of their namesake ion. The degree to which these channels contribute to cellular excitability can be managed therapeutically or fine-tuned by endogenous ligands. Intracellular calcium, for instance, modulates sodium channel inactivation, the process by which sodium conductance is negatively regulated. We explored the molecular basis for this effect by investigating the interaction between the ubiquitous calcium binding protein calmodulin (CaM) and the putative sodium channel inactivation gate composed of the cytosolic linker between homologous channel domains III and IV (DIII-IV). Experiments using isothermal titration calorimetry show that CaM binds to a novel double tyrosine motif in the center of the DIII-IV linker in a calcium-dependent manner, N-terminal to a region previously reported to be a CaM binding site. An alanine scan of aromatic residues in recombinant DIII-DIV linker peptides shows that whereas multiple side chains contribute to CaM binding, two tyrosines (Tyr(1494) and Tyr(1495)) play a crucial role in binding the CaM C-lobe. The functional relevance of these observations was then ascertained through electrophysiological measurement of sodium channel inactivation gating in the presence and absence of calcium. Experiments on patch-clamped transfected tsA201 cells show that only the Y1494A mutation of the five sites tested renders sodium channel steady-state inactivation insensitive to cytosolic calcium. The results demonstrate that calcium-dependent calmodulin binding to the sodium channel inactivation gate double tyrosine motif is required for calcium regulation of the cardiac sodium channel.

Modulation of Sodium Channel Inactivation Gating by a Novel Lactam: Implications for Seizure Suppression in Chronic Limbic Epilepsy

Epilepsy remains a devastating neurological disorder associated with recurrent, unprovoked, spontaneous epileptic seizures. Current treatments involve seizure suppression using antiepileptic drugs (AEDs); however, many patients remain refractory to current treatments or suffer serious side effects. In view of this continued need for more effective and safer AEDs, we have designed a novel compound, 3-hydroxy-3-(4-methoxyphenyl)-1-methyl-1,3-dihydro-indol-2-one (YWI92), based on a lactam structural class, and evaluated its modulation of human neuronal sodium channel isoform (hNa(v))1.2 currents and hippocampal neuron action potential firing. Furthermore, we have tested its AED activity using a chronic and acute rat seizure model. In a similar manner to lamotrigine, a clinically used AED, YWI92 exhibited tonic block of hNa(v)1.2 channels and caused a hyperpolarizing shift in the steady-state inactivation curve when using a 30-s inactivating prepulse. YWI92 also delayed the time constants of channel repriming after a 30-s inactivating prepulse and exhibited use-dependent block at 20-Hz stimulation frequency. In membrane excitability experiments, YWI92 inhibited burst firing in CA1 neurons of animals with temporal lobe epilepsy at concentrations that had little effect on CA1 neurons from control animals. These actions on neuronal activity translated into AED activity in the maximal electroshock acute seizure model (ED(50) = 22.96 mg/kg), and importantly, in a chronic temporal lobe epilepsy model, in which the mean number of seizures was reduced. Notably, YWI92 exhibited no sedative/ataxic side effects at concentrations up to 500 mg/kg. In summary, greater affinity for inactivated sodium channels, particularly after long depolarizing prepulses, may be important for both anticonvulsant activity and drug tolerability.

Molecular Modeling and Dynamics of the Sodium Channel Inactivation Gate

The intracellular linker L III-IV of voltage-gated sodium channels is known to be involved in their mechanism of inactivation. Its primary sequence is well conserved in sodium channels from different tissues and species. However, the role of charged residues in this region, first thought to play an important role in inactivation, has not been well identified, whereas the IFM triad (I1488-M1490) has been characterized as the crucial element for inactivation. In this work, we constructed theoretical models and performed molecular dynamics simulations, exploring the role of L III-IV-charged residues in the presence of a polar/nonpolar planar interface represented by a dielectric discontinuity. From structural predictions, two α-helical segments are proposed. Moreover, from dynamics simulations, a time-conserved motif is detected and shown to play a relevant role in guiding the inactivation particle toward its receptor site.

Helix-stabilizing effects of the pentapeptide KIFMK and its related peptides on the sodium channel inactivation gate peptides.

We have previously found by NMR and CD spectroscopic studies that the helical content of the sodium channel inactivation gate-related peptide (Ac-GGQDIFMTEEQK-NH2; MP-1A) in 80% trifluoroethanol solutions was increased by adding a pentapeptide, KIFMK. In order to study in further detail whether the presence of the IFM motif and the two lysine residues is a prerequisite for stabilizing the helical conformation, we examined interactions between various oligopeptides (RIFMR, KIFMTK, KIQMK, KAFAK, KIIIK) and MP-1A and its related peptides; that is, MP-2A in which Phe was replaced by Gln, MP-1MMA in which Thr was replaced by Met, MP-1TA in which Thr was removed from MP-1A, and MP-1A' in which L-Phe was replaced by D-Phe. It was found that the IFM motif was absolutely necessary in both the oligopeptide and the inactivation gate peptide. This finding means that hydrophobic interactions are operative between KIFMK and MP-1A. In contrast, KIFMK destabilized the helical structure of MP-1MMA, MP-1TA, and MP-1A', showing that the conformation around the IFM motif in the inactivation gate peptides is an important factor. It was concluded that the IFM motif and the two Lys residues are a prerequisite for effectively stabilizing the alpha-helix of MP-1A.

Structural study of the sodium channel inactivation gate peptide including an isoleucine-phenylalanine-methionine motif and its analogous peptide (phenylalanine/glutamine) in trifluoroethanol solutions and SDS micelles

In order to gain insight into the gating mechanisms of Na+ channels, in particular their inactivation mechanisms, we studied the structures of the Na+ channel inactivation gate related peptide which includes the IFM (Ile-Phe-Met) motif (Ac-KKKFGGQDIFMTEEQKK-NH2; K1480-K1496 in rat brain type-IIA Na+ channels, MP-3A) and its F/Q(Gln) substituted one (MP-4A) in trifluoroethanol (TFE) solutions and sodium dodecyl sulfate (SDS) micelles using circular dichroism (CD) and 1H-NMR spectroscopies. Based on observed nuclear Overhauser effect constraints, three-dimensional structures of MP-3A and MP-4A were determined using simulated annealing molecular dynamics/ energy minimization calculations. In TFE solutions, no appreciable differences in the structure were observed using either CD or NMR spectra. In SDS micelles, however, the two peptides exhibited definitely different structures from each other. It was found that in MP-3A, residues 11488 and T1491 were spatially proximate with each other owing to hydrogen bonding between the amide proton of 11488 and the hydroxyl oxygen atom of T1491, whereas in MP-4A, F/Q substitution separated them owing to conformational changes. The solvent-accessible surfaces calculated for the structures of MP-3A and MP-4A showed that the former has a smoother interaction surface to the hydrophobic docking site than the latter. In conclusion, the conformational changes, as well as decreased hydrophobicity around the IFM motif owing to the F/Q mutation, may be one reason why F1489Q mutated channels cannot inactivate almost completely.

Interactions between local anesthetics and Na+ channel inactivation gate peptides in phosphatidylserine suspensions as studied by 1H-NMR spectroscopy.

Interactions between local anesthetics and a sodium channel inactivation gate peptide (Ac-GGQDIFMTEEQK-NH2, MP-1A), which was dissected from the cytoplasmic linker between domains III and IV of the sodium channel alpha-subunit (G1484-K1495 in rat brain type IIA), have been studied by 1H-NMR spectroscopy. Changes in 1H-NMR chemical shifts of the aromatic proton resonances of dibucaine (pH 7.0) and lidocaine (pH 6.0 and 9.0) in phosphatidylserine (PS) suspensions were observed. The effects of substitution of glutamine (F1489Q; MP-2A) or D-phenylalanine (MP-1A') for L-phenylalanine (F1489) in MP-1A and the effects of substitution of neutral amino acid residues for the corresponding acidic amino acid residues (D1487N, MP-1NA; E1492Q, MP-IQEA; E1493Q, MP-IEQA) in MP-1A, on the aromatic 1H-NMR chemical shift changes of dibucaine and lidocaine were also investigated. From these results it was concluded that: the aromatic ring of phenylalanine of MP-1A and the aromatic ring of the cationic form of dibucaine or lidocaine are interacting by pi-pi stacking; the tertiary amine nitrogen of dibucaine is interacting electrostatically with D1487, whereas that of lidocaine is interacting with E1492.

1H-NMR and Circular Dichroism Spectroscopic Studies on Changes in Secondary Structures of the Sodium Channel Inactivation Gate Peptides as Caused by the Pentapeptide KIFMK

The pentapeptide KIFMK, which contains three clustered hydrophobic amino acid residues of isoleucine, phenylalanine, and methionine (IFM) in the sodium channel inactivation gate on the cytoplasmic linker between domains III and IV (III-IV linker), is known to restore fast inactivation to the mutant sodium channels having a defective inactivation gate or to accelerate the inactivation of the wild-type sodium channels. To investigate the docking site of KIFMK and to clarify the mechanisms for restoring the fast inactivation, we have studied the interactions between KIFMK and the fragment peptide in the III-IV linker GGQDIFMTEEQK (MP-1A; G1484-K1495 in rat brain IIA) by one- and two-dimensional 1H-NMR and circular dichroism (CD) spectroscopies. KIFMK was found to increase the helical content of MP-1A in 80% trifluoroethanol (TFE) solution by ∼11%. A pentapeptide, KIFMT, which can restore inactivation but less effectively than KIFMK, also increased the helical content of MP-1A, but to a lesser extent (∼6%) than did KIFMK. In contrast, KDIFMTK, which is ineffective in restoring inactivation, decreased the helical content (∼−4%). Furthermore, we studied the interactions between KIFMK and modified peptides from MP-1A, that is, MP-1NA (D1487N), MP-1QEA (E1492Q), or MP-1EQA (E1493Q). The KIFMK was found to increase the helical content of MP-1EQA to an extent nearly identical to that of MP-1A, whereas it was found to decrease those of MP-1NA and MP-1QEA. These findings mean that KIFMK, by allowing each of the Lys residues to interact with D1487 and E1492, respectively, stabilized the helical structure of the III-IV linker around the IFM residues. This helix-stabilizing effect of KIFMK on the III-IV linker may restore and/or accelerate fast inactivation to the sodium channels having a defective inactivation gate or to wild-type sodium channels.

Phenotypic characterization of a novel long-QT syndrome mutation (R1623Q) in the cardiac sodium channel.

A heritable form of the long-QT syndrome (LQT3) has been linked to mutations in the cardiac sodium channel gene (SCN5A). Recently, a sporadic SCN5A mutation was identified in a Japanese girl afflicted with the long-QT syndrome. In contrast to the heritable mutations, this externally positioned domain IV, S4 mutation (R1623Q) neutralized a charged residue that is critically involved in activation-inactivation coupling. We have characterized the R1623Q mutation in the human cardiac sodium channel (hH1) using both whole-cell and single-channel recordings. In contrast to the autosomal dominant LQT3 mutations, R1623Q increased the probability of long openings and caused early reopenings, producing a threefold prolongation of sodium current decay. Lidocaine restored rapid decay of the R1623Q macroscopic current. The R1623Q mutation produces inactivation gating defects that differ mechanistically from those caused by LQT3 mutations. These findings provide a biophysical explanation for this severe long-QT phenotype and extend our understanding of the mechanistic role of the S4 segment in cardiac sodium channel inactivation gating and class I antiarrhythmic drug action.

Demyelination as a test for a mobile Na channel modulator in frog node of Ranvier

We found previously that the external surface of frog skeletal muscle fibers can be irreversibly modified by treatment with the amino group-specific reagent, trinitrobenzene sulfonic acid (TNBS). Reaction of the muscle membrane with TNBS permanently shifts the potential dependence of the sodium channel inactivation gating process, h infinity, to more hyperpolarized potentials. The experiments presented here show nearly identical effects on the sodium currents of voltage-clamped frog node of Ranvier in the presence of TNBS. In contrast to the results in muscle, in myelinated nerve the voltage dependence of sodium-channel inactivation returns rapidly to control values following a brief exposure to TNBS. We have used partial demyelination to test the hypothesis that recovery of the normal voltage dependence for h infinity following TNBS treatment is due to lateral diffusion of reacted groups away from the sodium channels in the node. We find that increasing the membrane area exposed to TNBS by partial demyelination greatly slows reversal of the TNBS effects. This result suggests that a modifiable membrane component that affects sodium channel gating is mobile in the plane of the membrane and can rapidly diffuse between nodal and internodal regions.