David S. Ragsdale

Voltage-gated sodium channels as therapeutic targets in epilepsy and other neurological disorders

Voltage-gated sodium channels (VGSCs) are key mediators of intrinsic neuronal and muscle excitability. Abnormal VGSC activity is central to the pathophysiology of epileptic seizures, and many of the most widely used antiepileptic drugs, including phenytoin, carbamazepine, and lamotrigine, are inhibitors of VGSC function. These antiepileptic drugs might also be efficacious in the treatment of other nervous system disorders, such as migraine, multiple sclerosis, neurodegenerative diseases, and neuropathic pain. In this Review, we summarise the structure and function of VGSCs and their involvement in the pathophysiology of several neurological disorders. We also describe the biophysical and molecular bases for the mechanisms of action of antiepileptic VGSC blockers and discuss the efficacy of these drugs in the treatment of epileptic and non-epileptic disorders. Overall, clinical and experimental data indicate that these drugs are efficacious for a range of diseases, and that the development of drugs with enhanced selectivity for specific VGSC isoforms might be an effective and novel approach for the treatment of several neurological diseases.

Sodium channels as molecular targets for antiepileptic drugs

Voltage-gated sodium channels mediate regenerative inward currents that are responsible for the initial depolarization of action potentials in brain neurons. Many of the most widely used antiepileptic drugs, as well as a number of promising new compounds suppress the abnormal neuronal excitability associated with seizures by means of complex voltage- and frequency-dependent inhibition of ionic currents through sodium channels. Over the past decade, advances in molecular biology have led to important new insights into the molecular structure of the sodium channel and have shed light on the relationship between channel structure and channel function. In this review, we examine how our current knowledge of sodium channel structure-function relationships contributes to our understanding of the action of anticonvulsant sodium channel blockers.

How do mutant Nav1.1 sodium channels cause epilepsy

Voltage-gated sodium channels comprise pore-forming alpha subunits and auxiliary beta subunits. Nine different alpha subtypes, designated Nav1.1-Nav1.9 have been identified in excitable cells. Nav1.1, 1.2 and 1.6 are major subtypes in the adult mammalian brain. More than 200 mutations in the Nav1.1 alpha subtype have been linked to inherited epilepsy syndromes, ranging in severity from the comparatively mild disorder Generalized Epilepsy with Febrile Seizures Plus to the epileptic encephalopathy Severe Myoclonic Epilepsy of Infancy. Studies using heterologous expression and functional analysis of recombinant Nav1.1 channels suggest that epilepsy mutations in Nav1.1 may cause either gain-of-function or loss-of-function effects that are consistent with either increased or decreased neuronal excitability. How these diverse effects lead to epilepsy is poorly understood. This review summarizes the data on sodium channel mutations and epilepsy and builds a case for the hypothesis that most Nav1.1 mutations have their ultimate epileptogenic effects by reducing Nav1.1-mediated whole cell sodium currents in GABAergic neurons, resulting in widespread loss of brain inhibition, an ideal background for the genesis of epileptic seizures.

Functional modulation of human brain Nav1.3 sodium channels, expressed in mammalian cells, by auxiliary β1, β2 and β3 subunits

Voltage-gated sodium channels consist of a pore-forming α subunit and two auxiliary β subunits. Excitable cells express multiple α subtypes, designated Nav1.1–Nav1.9, and three β subunits, designated β1, β2 and β3. Understanding how the different α subtypes, in combination with the various β subunits, determine sodium channel behavior is important for elucidating the molecular basis of sodium channel functional diversity. In this study, we used whole-cell electrophysiological recording to examine the properties of the human Nav1.3 α subtype, stably expressed in Chinese hamster ovary cells, and to investigate modulation of Nav1.3 function by β1, β2 and β3 subunits. In the absence of β subunits, human Nav1.3 formed channels that inactivated rapidly (τinactivation≈0.5 ms at 0 mV) and almost completely by the end of 190-ms-long depolarizations. Using an intracellular solution with aspartate as the main anion, the midpoint for channel activation was ∼−12 mV. The midpoint for inactivation, determined using 100-ms conditioning pulses, was ∼−47 mV. The time constant for repriming of inactivated channels at −80 mV was ∼6 ms. Coexpression of β1 or β3 did not affect inactivation time course or the voltage dependence of activation, but shifted the inactivation curve ∼10 mV negative, and slowed the repriming rate ca. three-fold. β2 did not affect channel properties, either by itself or in combination with β1 or β3. Nav1.3 expression is increased in damaged nociceptive peripheral afferents. This change in channel expression levels is correlated with the emergence of a rapidly inactivating and rapidly repriming sodium current, which has been proposed to contribute to the pathophysiology of neuropathic pain. The results of this study support the hypothesis that Nav1.3 may mediate this fast sodium current.

The intracellular segment of the sodium channel β1 subunit is required for its efficient association with the channel α subunit

Sodium channels consist of a pore‐forming α subunit and auxiliary β1 and β2 subunits. The subunit β1 alters the kinetics and voltage‐dependence of sodium channels expressed in Xenopus oocytes or mammalian cells. Functional modulation in oocytes depends on specific regions in the N‐terminal extracellular domain of β1, but does not require the intracellular C‐terminal domain. Functional modulation is qualitatively different in mammalian cells, and thus could involve different molecular mechanisms. As a first step toward testing this hypothesis, we examined modulation of brain NaV1.2a sodium channel α subunits expressed in Chinese hamster lung cells by a mutant β1 construct with 34 amino acids deleted from the C‐terminus. This deletion mutation did not modulate sodium channel function in this cell system. Co‐immunoprecipitation data suggest that this loss of functional modulation was caused by inefficient association of the mutant β1 with α, despite high levels of expression of the mutant protein. In Xenopus oocytes, injection of approximately 10 000 times more mutant β1 RNA was required to achieve the level of functional modulation observed with injection of full‐length β1. Together, these findings suggest that the C‐terminal cytoplasmic domain of β1 is an important determinant of β1 binding to the sodium channel α subunit in both mammalian cells and Xenopus oocytes.

Increased Persistent Sodium Currents in Rat Entorhinal Cortex Layer V Neurons in a Post–Status Epilepticus Model of Temporal Lobe Epilepsy

Summary:  Purpose: Spontaneous seizures in rats emerge several weeks after induction of status epilepticus with pharmacologic treatment or electrical stimulation, providing an animal model for human temporal lobe epilepsy. In this study, we investigated whether status epilepticus caused changes in the function of voltage‐gated sodium channels in entorhinal cortex layer V neurons, a cellular group important for the genesis of limbic seizures.

Reduced GABAB receptor subunit expression and paired-pulse depression in a genetic model of absence seizures

Neocortical networks play a major role in the genesis of generalized spike-and-wave (SW) discharges associated with absence seizures in humans and in animal models, including genetically predisposed WAG/Rij rats. Here, we tested the hypothesis that alterations in GABA(B) receptors contribute to neocortical hyperexcitability in these animals. By using Real-Time PCR we found that mRNA levels for most GABA(B(1)) subunits are diminished in epileptic WAG/Rij neocortex as compared with age-matched non-epileptic controls (NEC), whereas GABA(B(2)) mRNA is unchanged. Next, we investigated the cellular distribution of GABA(B(1)) and GABA(B(2)) subunits by confocal microscopy and discovered that GABA(B(1)) subunits fail to localize in the distal dendrites of WAG/Rij neocortical pyramidal cells. Intracellular recordings from neocortical cells in an in vitro slice preparation demonstrated reduced paired-pulse depression of pharmacologically isolated excitatory and inhibitory responses in epileptic WAG/Rij rats as compared with NECs; moreover, paired-pulse depression in NEC slices was diminished by a GABA(B) receptor antagonist to a greater extent than in WAG/Rij rats further suggesting GABA(B) receptor dysfunction. In conclusion, our data identify changes in GABA(B) receptor subunit expression and distribution along with decreased paired-pulse depression in epileptic WAG/Rij rat neocortex. We propose that these alterations may contribute to neocortical hyperexcitability and thus to SW generation in absence epilepsy.

An epilepsy mutation in the β1 subunit of the voltage-gated sodium channel results in reduced channel sensitivity to phenytoin

The antiepileptic drug phenytoin inhibits voltage-gated sodium channels. Phenytoin block is enhanced at depolarized membrane potentials and during high frequency channel activation. These properties, which are important for the clinical efficacy of the drug, depend on voltage-dependent channel gating. In this study, we examined the action of phenytoin on sodium channels, comprising a mutant auxiliary beta1 subunit (mutation C121Wbeta1), which causes the inherited epilepsy syndrome, generalized epilepsy with febrile seizures plus (GEFS+). Whole cell sodium currents in Chinese hamster ovary (CHO) cells coexpressing human Na(v)1.3 sodium channels and C121Wbeta1 exhibited altered gating properties, compared to currents in cells coexpressing Na(v)1.3 and wild type beta1. In addition mutant channels were less sensitive to inhibition by phenytoin, showing reduced tonic block at -70mV (EC(50)=26microM for C121Wbeta1 versus 11microM for wild type beta1) and less frequency-dependent inhibition in response to a 20Hz pulse train ( approximately 40% inhibition for C121Wbeta1 versus approximately 70% inhibition for wild type beta1, with 50microM phenytoin). Mutant and wild type channels did not differ in inactivated state affinity for phenytoin, suggesting that their pharmacological differences were secondary to their differences in voltage-dependent gating, rather than being caused by direct effects of the mutation on the drug receptor. Together, these data show that a sodium channel mutation responsible for epilepsy can also alter channel response to antiepileptic drugs.

Functional characterization of the D188V mutation in neuronal voltage-gated sodium channel causing generalized epilepsy with febrile seizures plus (GEFS)

Mutations in the alpha 1 subunit of the voltage-gated sodium channel (SCN1A) have been increasingly recognized as an important cause of familial epilepsy in humans. However, the functional consequences of these mutations remain largely unknown. We identified a mutation (D188V) in SCN1A segregating with generalized epilepsy with febrile seizures (GEFS) in a large kindred. Compared to wild-type sodium channels, in vitro expression of channels harboring the D188V mutation were found to be more resistant to the decline in amplitude that is normally observed over the course of high frequency pulse trains. This small change on a single aspect of channel function is compatible with an increase in membrane excitability, such as during sustained and uncontrolled neuronal discharges. These data suggest that this specific effect on sodium channel function could be a general mechanism in the pathophysiology of epilepsies caused by mutations in sodium channels in humans.

Proepileptic Influence of a Focal Vascular Lesion Affecting Entorhinal Cortex-CA3 Connections After Status Epilepticus

In limbic seizures, neuronal excitation is conveyed from the entorhinal cortex directly to CA1 and subicular regions. This phenomenon is associated with a reduced ability of CA3 to respond to entorhinal cortex inputs. Here, we describe a lesion that destroys the perforant path in CA3 after status epilepticus (SE) induced by pilocarpine injection in 8-week-old rats. Using magnetic resonance imaging, immunohistochemical, and ultrastructural analyses, we determined that this lesion develops after 30 minutes of SE and is characterized by microhemorrhages and ischemia. After a longer period of SE, the lesion invariably involves the upper blade of the dentate gyrus. Adult rats treated with subcutaneous diazepam (20 mg kg−1 for 3 days) did not develop the dentate gyrus lesion and had less frequent spontaneous recurrent seizures (p < 0.01). Young (3-week-old) rats rarely (20%) developed the CA3 lesion, and their spontaneous seizures were delayed (p < 0.01). To investigate the role of the damaged CA3 in seizure activity, we reinduced SE in adult and young epileptic rats. Using FosB/&Dgr;FosB markers, we found induction of FosB/&Dgr;FosB immunopositivity in CA3 neurons of young but not in adult rats. These experiments indicate that SE can produce a focal lesion in the perforant path that may affect the roles of the hippocampus in epileptic rats.