Enhancement Of Slow Inactivation Research Articles

Effect of phenytoin on sodium conductances in rat hippocampal CA1 pyramidal neurons.

The antiepileptic drug phenytoin (PHT) is thought to reduce the excitability of neural tissue by stabilizing sodium channels (NaV) in inactivated states. It has been suggested the fast-inactivated state (IF) is the main target, although slow inactivation (IS) has also been implicated. Other studies on local anesthetics with similar effects on sodium channels have implicated the NaV voltage sensor interactions. In this study, we reexamined the effect of PHT in both equilibrium and dynamic transitions between fast and slower forms of inactivation in rat hippocampal CA1 pyramidal neurons. The effects of PHT were observed on fast and slow inactivation processes, as well as on another identified "intermediate" inactivation process. The effect of enzymatic removal of IF was also studied, as well as effects on the residual persistent sodium current (INaP). A computational model based on a gating charge interaction was derived that reproduced a range of PHT effects on NaV equilibrium and state transitions. No effect of PHT on IF was observed; rather, PHT appeared to facilitate the occupancy of other closed states, either through enhancement of slow inactivation or through formation of analogous drug-bound states. The overall significance of these observations is that our data are inconsistent with the commonly held view that the archetypal NaV channel inhibitor PHT stabilizes fast inactivation states, and we demonstrate that conventional slow activation "IS" and the more recently identified intermediate-duration inactivation process "II" are the primary functional targets of PHT. In addition, we show that the traditional explanatory frameworks based on the "modulated receptor hypothesis" can be substituted by simple, physiologically plausible interactions with voltage sensors. Additionally, INaP was not preferentially inhibited compared with peak INa at short latencies (50 ms) by PHT.

Update on the role of eslicarbazepine acetate in the treatment of partial-onset epilepsy

Eslicarbazepine acetate (ESL) is a once daily new third generation antiepileptic drug that shares the basic chemical structure of carbamazepine and oxcarbazepine – a dibenzazepine nucleus with the 5-carboxamide substituent, but is structurally different at the 10,11-position. ESL is a pro-drug metabolized to its major active metabolite eslicarbazepine. Despite the fact that the exact mechanism of action has not been fully elucidated, it is thought to involve inhibition of voltage-gated sodium channels (VGSC). ESL inhibits sodium currents in a voltage-dependent way by an interaction predominantly with the inactivated state of the VGSC, thus selectively reducing the activity of rapidly firing (epileptic) neurons. ESL reduces VGSC availability through enhancement of slow inactivation. In Phase III studies, adjunctive therapy with ESL 800 or 1,200 mg/day leads to a significant decrease in the seizure frequency in adults with refractory partial onset epilepsy. Based on these results, ESL has been approved in Europe (by the European Medicines Agency) and in the United States (by the US Food and Drug Administration) as add-on therapy. Data on efficacy and safety have been confirmed by 1-year extension and real life observational studies. Recently, based on results from two randomized, double-blind, historical control Phase III trials, ESL received US Food and Drug Administration approval also as a monotherapy for patients with partial onset epilepsy. In the pediatric setting, encouraging results have been obtained suggesting its potential role in the management of epileptic children. Overall ESL was generally well tolerated. The most common adverse events were dizziness, somnolence, headache, nausea, diplopia, and vomiting. Adverse events can be minimized by appropriate titration. In conclusion, ESL seems to overcome some drawbacks of the previous antiepileptic drugs, suggesting a major role of ESL in the management of focal onset epilepsy for both new onset and refractory cases, either as monotherapy or as adjunctive treatment.

Eslicarbazepine acetate for the treatment of focal epilepsy: an update on its proposed mechanisms of action.

Eslicarbazepine acetate (ESL) is a once daily antiepileptic drug (AED) approved by the European Medicines Agency (EMA), the Food and Drug Administration (FDA) and Health Canada as an adjunctive therapy in adults with partial-onset seizures (POS). In humans and in relevant animal laboratory species, ESL undergoes extensive first pass hydrolysis to its major active metabolite eslicarbazepine that represents ∼95% of circulating active moieties. ESL and eslicarbazepine showed anticonvulsant activity in animal models. ESL may not only suppress seizure activity but may also inhibit the generation of a hyperexcitable network. Data reviewed here suggest that ESL and eslicarbazepine demonstrated the following in animal models: (1) the selectivity of interaction with the inactive state of the voltage-gated sodium channel (VGSC), (2) reduction in VGSC availability through enhancement of slow inactivation, instead of alteration of fast inactivation of VGSC, (3) the failure to cause a paradoxical upregulation of persistent Na+ current (INaP), and (4) the reduction in firing frequencies of excitatory neurons in dissociated hippocampal cells from patients with epilepsy who were pharmacoresistant to carbamazepine (CBZ). In addition, eslicarbazepine effectively inhibited high- and low-affinity hCaV3.2 inward currents with greater affinity than CBZ. These preclinical findings may suggest the potential for antiepileptogenic effects; furthermore, the lack of effect upon KV7.2 outward currents may translate into a reduced potential for eslicarbazepine to facilitate repetitive firing.

ShakerIR and Kv1.5 mutant channels with enhanced slow inactivation also exhibit K+ o-dependent resting inactivation

Previous studies have shown that in N-type inactivation-removed Shaker (ShakerIR) channels, the T449K and T449A mutations result in enhanced slow inactivation. These mutant channels also show a loss of conductance in 0 mM K⁺ o that was attributed to an inactivation process occurring from the closed, resting state and which we refer to as resting inactivation. Similar behavior has also been observed in the Kv1.5 H463G mutant channel. To date, the time courses for the onset of and recovery from resting inactivation have been unknown, but a comparison of the kinetics for resting inactivation induced at -80 mV and slow inactivation evoked at +50 mV may provide information on whether these two processes are mechanistically related. Here, we present an analysis of the time courses for the onset of and recovery from [K⁺]o-dependent resting inactivation and depolarization-induced inactivation of these mutant channels. Despite the enhancement of slow inactivation in the ShakerIR T449K, T449A, and Kv1.5 H463G mutants, the time constant for slow inactivation at +50 mV (τ inact) was relatively insensitive to the increases or decreases of [K(+)]o, confirming that accelerated inactivation from the open state does not underlie the loss of conductance in 0 mM K⁺. For all three mutants, the time constant for resting inactivation (τ RI), induced by exposure to 0 mM K⁺ o solution at -80 mV, was at least an order of magnitude larger than τ inact. On the other hand, the time course of recovery at -80 mV of each mutant from 0 mM K(+) o-induced resting inactivation was the same as that from depolarization-induced slow inactivation. This latter result suggests that the 0 mM K⁺ o-induced resting inactivation of these mutant ShakerIR and Kv1.5 channels is mechanistically related to slow inactivation.

Impaired slow inactivation due to a polymorphism and substitutions of Ser-906 in the II-III loop of the human Nav1.4 channel.

The loop connecting domains II and III of the sodium channel alpha-subunit is not known to have a major effect on channel gating. Recently mutations in the II-III loop of various sodium channel isoforms have been reported to cause channelopathies suggesting the functional importance of this region. In the II-III loop of the skeletal muscle isoform Na(v)1.4, we found a Ser-to-Thr substitution at position 906 in 5% of patients with dyskalemic periodic paralysis but also in 4% of healthy human individuals. To investigate whether this position is important for channel gating, we characterized the following amino acids at 906 by whole-cell patch-clamp experiments: Gln, Ser, Thr, Cys, Pro, Val, ordered according to their hydrophobicity. All substitutions mainly affected slow inactivation. For example, Gln caused a +13-mV right-shift of the steady-state slow inactivation curve, and entry into slow inactivation was 6 times slower compared with Ser, leading to a destabilization of the slow inactivated state; in contrast, Val, at the other end of the hydrophobicity spectrum, shifted the steady-state slow inactivation curve by -6 mV and slowed the recovery from the slow inactivated state threefold compared with Ser, resulting in an enhancement of slow inactivation. Recovery from the slow inactivated state was also slowed by Pro, Cys and Thr. Our results suggest that (1) a benign polymorphism affects channel function, (2) the II-III loop is important for slow inactivation, and (3) the effects on slow inactivation may depend on the hydrophobicity of the residue at position 906.

Enhanced inactivation and acceleration of activation of the sodium channel associated with epilepsy in man.

Generalized epilepsy with febrile seizures-plus (GEFS+) is a benign Mendelian syndrome characterized by childhood-onset febrile and afebrile seizures. Three point mutations within two voltage-gated sodium channel genes have been identified so far: in GEFS+ type 1 a mutation in the beta1-subunit gene SCN1B, and in GEFS+ type 2 two mutations within the neuronal alpha-subunit gene SCN1A. Functional expression of the SCN1B and one of the SCN1A mutations revealed defects in fast channel inactivation which are in line with previous findings on myotonia causing mutations in SCN4A, the skeletal muscle sodium channel alpha-subunit gene, all showing an impaired fast inactivation. We now studied the second GEFS+ mutation (T875M in SCN1A), using the highly homologous SCN4A gene (mutation T685M). Unexpectedly, the experiments revealed a pronounced enhancement of both fast and slow inactivation and a defect of channel activation for T685M compared to wild-type channels. Steady-state fast and slow inactivation curves were shifted in the hyperpolarizing direction, entry into slow inactivation was threefold accelerated, recovery from slow inactivation was slowed by threefold and the time course of activation was slightly but significantly accelerated. In contrast to other disease-causing mutations in SCN1A, SCN1B and SCN4A, the only mechanism that could explain hyperexcitability of the cell membrane would be the acceleration of activation. Because the enhancement of slow inactivation was the most obvious alteration in gating found for T685M, this might be the disease-causing mechanism for that mutation. In this case, the occurrence of epileptic seizures could be explained by a decrease of excitability of inhibitory neurons.

The Human Skeletal Muscle Na Channel Mutation R669H Associated with Hypokalemic Periodic Paralysis Enhances Slow Inactivation

Missense mutations of the human skeletal muscle voltage-gated Na channel (hSkM1) underlie a variety of diseases, including hyperkalemic periodic paralysis (HyperPP), paramyotonia congenita, and potassium-aggravated myotonia. Another disorder of sarcolemmal excitability, hypokalemic periodic paralysis (HypoPP), which is usually caused by missense mutations of the S4 voltage sensors of the L-type Ca channel, was associated recently in one family with a mutation in the outermost arginine of the IIS4 voltage sensor (R669H) of hSkM1 (Bulman et al., 1999). Intriguingly, an arginine-to-histidine mutation at the homologous position in the L-type Ca(2+) channel (R528H) is a common cause of HypoPP. We have studied the gating properties of the hSkM1-R669H mutant Na channel experimentally in human embryonic kidney cells and found that it has no significant effects on activation or fast inactivation but does cause an enhancement of slow inactivation. R669H channels exhibit an approximately 10 mV hyperpolarized shift in the voltage dependence of slow inactivation and a twofold to fivefold prolongation of recovery after prolonged depolarization. In contrast, slow inactivation is often disrupted in HyperPP-associated Na channel mutants. These results demonstrate that, in R669H-associated HypoPP, enhanced slow inactivation does not preclude, and may contribute to, prolonged attacks of weakness and add support to previous evidence implicating the IIS4 voltage sensor in slow-inactivation gating.