Molecular Basis of an Inherited Epilepsy

Division of Genetic Medicine, Center for Molecualr Neurosciences, Vanderbilt University, Nashville, TN 37232, USA.
Neuron (Impact Factor: 15.05). 07/2002; 34(6):877-84. DOI: 10.1016/S0896-6273(02)00714-6
Source: PubMed


Epilepsy is a common neurological condition that reflects neuronal hyperexcitability arising from largely unknown cellular and molecular mechanisms. In generalized epilepsy with febrile seizures plus, an autosomal dominant epilepsy syndrome, mutations in three genes coding for voltage-gated sodium channel alpha or beta1 subunits (SCN1A, SCN2A, SCN1B) and one GABA receptor subunit gene (GABRG2) have been identified. Here, we characterize the functional effects of three mutations in the human neuronal sodium channel alpha subunit SCN1A by heterologous expression with its known accessory subunits, beta1 and beta2, in cultured mammalian cells. SCN1A mutations alter channel inactivation, resulting in persistent inward sodium current. This gain-of-function abnormality will likely enhance excitability of neuronal membranes by causing prolonged membrane depolarization, a plausible underlying biophysical mechanism responsible for this inherited human epilepsy.

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Available from: Christoph Lossin,
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    • "The cDNA genes encoding a TTX-and HWTX-IV-sensitive Nav1.5 (C373Y/R800D/S802E) and auxibiliary subunit β 1 were cloned from human and subcloned into the pcDNA3.1 vector (Nav1.5) and an internal ribosome entry site vector (β1) (Lossin et al., 2002). All mutations inserted into the sodium channel cDNA gene were constructed using the QuickChange II XL Site-Directed Mutagenesis kit according to the manufacture's instruction. "
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    ABSTRACT: Voltage gated sodium channels are critical determinants of nerve and muscle excitability. While numerous toxins and small molecules target sodium channels, identifying the mechanisms of action is challenging. Here we used gating pore currents selectively generated in each of the voltage-sensors from the four alpha-subunit domains (DI-DIV) to monitor the activity of individual voltage-sensors and to investigate the molecular determinants of sodium channel pharmacology. The tarantula toxin HWTX-IV, which inhibits sodium channel current, exclusively enhanced inward gating pore currents through the DII voltage-sensor. By contrast, the tarantula toxin ProTx-II, which also inhibits sodium channel currents, altered the gating pore currents in multiple voltage-sensors in a complex manner. Thus while HWTX-IV inhibits central pore currents by selectively trapping the DII voltage-sensor in the resting configuration, ProTx-II seems to inhibit central pore currents by differentially altering the configuration of multiple voltage-sensors. The sea anemone toxin anthopleurin B, which impairs open-channel inactivation, exclusively enhanced inward gating pore currents through the DIV voltage-sensor. This indicates that trapping the DIV voltage-sensor in the resting configuration selectively impairs open-channel inactivation. Furthermore, these data indicate that while activation of all four voltage-sensors is not required for central pore current generation, activation of the DII voltage-sensor is crucial. Overall, our data demonstrate that gating pore currents can determine the mechanism of action for sodium channel gating modifiers with high precision. We propose this approach could be adapted to identify the molecular mechanisms of action for gating modifiers of various voltage gated ion channels.
    Molecular pharmacology 06/2014; 86(2). DOI:10.1124/mol.114.092338 · 4.13 Impact Factor
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    • "All culture media and supplements were obtained from BioWhittaker. Transient transfections were performed with Lipofectamine (Invitrogen) using in total 6 µg of Na v 1.1, 1, and 2 pDNAs in a ratio of 10:1:1, as described previously by Lossin et al. (2002). Cells that expressed Figure 1. "
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    ABSTRACT: Generalized epilepsy with febrile seizures plus (GEFS+) is an early onset febrile epileptic syndrome with therapeutic responsive (a)febrile seizures continuing later in life. Dravet syndrome (DS) or severe myoclonic epilepsy of infancy has a complex phenotype including febrile generalized or hemiclonic convulsions before the age of 1, followed by intractable myoclonic, complex partial, or absence seizures. Both diseases can result from mutations in the Nav1.1 sodium channel, and initially, seizures are typically triggered by fever. We previously characterized two Nav1.1 mutants-R859H (GEFS+) and R865G (DS)-at room temperature and reported a mixture of biophysical gating defects that could not easily predict the phenotype presentation as either GEFS+ or DS. In this study, we extend the characterization of Nav1.1 wild-type, R859H, and R865G channels to physiological (37°C) and febrile (40°C) temperatures. At physiological temperature, a variety of biophysical defects were detected in both mutants, including a hyperpolarized shift in the voltage dependence of activation and a delayed recovery from fast and slow inactivation. Interestingly, at 40°C we also detected additional gating defects for both R859H and R865G mutants. The GEFS+ mutant R859H showed a loss of function in the voltage dependence of inactivation and an increased channel use-dependency at 40°C with no reduction in peak current density. The DS mutant R865G exhibited reduced peak sodium currents, enhanced entry into slow inactivation, and increased use-dependency at 40°C. Our results suggest that fever-induced temperatures exacerbate the gating defects of R859H or R865G mutants and may predispose mutation carriers to febrile seizures.
    The Journal of General Physiology 12/2013; 142(6):641-53. DOI:10.1085/jgp.201311042 · 4.79 Impact Factor
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    • "examined the rat Scn1a (R1648H) mutation in Xenopus oocytes and found less use-dependant inactivation and accelerated recovery from inactivation, exactly the opposite of what was found in the in vivo studies. Lossin et al. (2002) studied the human SCN1A (R1648H) mutation in tsA201 cells and found concordance with the in vivo studies for recovery from inactivation but discordance for increases in persistent sodium current, which were only seen in the in vitro study. However, recording persistent current is difficult in neurons , as acknowledged by the authors (Tang et al., 2009), and may depend on space clamp issues and the contamination of the total current by other sodium channels that would reduce the relative contribution of persistent current. "
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    ABSTRACT: Voltage-gated sodium channels (VGSCs) are integral membrane proteins. They are essential for normal neurologic function and are, currently, the most common recognized cause of genetic epilepsy. This review summarizes the neurobiology of VGSCs, their association with different epilepsy syndromes, and the ways in which we can experimentally interrogate their function. The most important sodium channel subunit of relevance to epilepsy is SCN1A, in which over 650 genetic variants have been discovered. SCN1A mutations are associated with a variety of epilepsy syndromes; the more severe syndromes are associated with truncation or complete loss of function of the protein. SCN2A is another important subtype associated with epilepsy syndromes, across a range of severe and less severe epilepsies. This subtype is localized primarily to excitatory neurons, and mutations have a range of functional effects on the channel. SCN8A is the other main adult subtype found in the brain and has recently emerged as an epilepsy gene, with the first human mutation discovered in a severe epilepsy syndrome. Mutations in the accessory β subunits, thought to modulate trafficking and function of the α subunits, have also been associated with epilepsy. Genome sequencing is continuing to become more affordable, and as such, the amount of incoming genetic data is continuing to increase. Current experimental approaches have struggled to keep pace with functional analysis of these mutations, and it has proved difficult to build associations between disease severity and the precise effect on channel function. These mutations have been interrogated with a range of experimental approaches, from in vitro, in vivo, to in silico. In vitro techniques will prove useful to scan mutations on a larger scale, particularly with the advance of high-throughput automated patch-clamp techniques. In vivo models enable investigation of mutation in the context of whole brains with connected networks and more closely model the human condition. In silico models can help us incorporate the impact of multiple genetic factors and investigate epistatic interactions and beyond.
    Epilepsia 08/2012; 53(11). DOI:10.1111/j.1528-1167.2012.03631.x · 4.57 Impact Factor
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