Modulation of Sodium Current in Mammalian Cells by an Epilepsy-Correlated β1-Subunit Mutation

doi:10.1006/bbrc.2002.6570

Biochemical and Biophysical Research Communications

Volume 291, Issue 4, 8 March 2002, Pages 1095-1101

https://doi.org/10.1006/bbrc.2002.6570 Get rights and content

Abstract

The syndrome of generalized epilepsy with febrile seizure plus (GEFS+) is associated with a single point mutation on the gene SCN1B that results in a substitution of the cysteine 121 with a tryptophane in the sodium channel β1-subunit protein. We have studied, in the HEK cells permanently transfected with the skeletal muscle sodium channel α-subunit (SkM1), the effects of a transient transfection of the wild type (WT) or C121W mutant β1-subunit. Coexpression of the WT β1 produces two effects on the sodium currents expressed in mammalian cells: the increase in the density of sodium channels, and the modulation of the inactivation of the sodium currents, inducing a hastening of the recovery from the inactivation. This modulation is less severe as observed when sodium channels are expressed in frog oocytes. We have observed that mutant C121W lacks this modulatory property, but maintains its property to increase the current density. Our observation suggests a possible involvement of this lack of modulation in the development of the GEFS+, providing the first hypothesis based on the observation of the functional properties of the β1-subunit C121W mutant in mammalian cells, which certainly represents a more physiological preparation, instead of in Xenopus oocytes, where the modulatory properties of the β1-subunit are artificially amplified.

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Voltage-gated sodium channel β subunits: The power outside the pore in brain development and disease
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Citation Excerpt :
Differential sialic acid modification was also found to affect β2 modulation of Nav1.5 while, in the same study, Nav1.2 was modulated regardless of α subunit sialylation status (Johnson and Bennett, 2006). The epilepsy-linked β1-C121W mutation in Scn1b results in decreased β1 subunit glycosylation and loss of its capacity to modulate VGSC properties and to traffic out of the soma to the axon in mouse neurons (Meadows et al., 2002; Tammaro et al., 2002; Kruger, O'Malley et al., 2016). This raises the possibility that control of glycosylation of β subunits, especially sialyation, may be important in subcellular targeting and α-β subunit association.
Voltage gated sodium channels (VGSCs) were first identified in terms of their role in the upstroke of the action potential. The underlying proteins were later identified as saxitoxin and scorpion toxin receptors consisting of α and β subunits. We now know that VGSCs are heterotrimeric complexes consisting of a single pore forming α subunit joined by two β subunits; a noncovalently linked β1 or β3 and a covalently linked β2 or β4 subunit. VGSC α subunits contain all the machinery necessary for channel cell surface expression, ion conduction, voltage sensing, gating, and inactivation, in one central, polytopic, transmembrane protein. VGSC β subunits are more than simple accessories to α subunits. In the more than two decades since the original cloning of β1, our knowledge of their roles in physiology and pathophysiology has expanded immensely. VGSC β subunits are multifunctional. They confer unique gating mechanisms, regulate cellular excitability, affect brain development, confer distinct channel pharmacology, and have functions that are independent of the α subunits. The vast array of functions of these proteins stems from their special station in the channelome: being the only known constituents that are cell adhesion and intra/extracellular signaling molecules in addition to being part of channel complexes. This functional trifecta and how it goes awry demonstrates the power outside the pore in ion channel signaling complexes, broadening the term channelopathy beyond defects in ion conduction.
This article is part of the Special Issue entitled ‘Channelopathies.’
Differential gene expression profiles of two excitable rat cell lines after over-expression of WT- and C121W-β1 sodium channel subunits
2015, Neuroscience
Citation Excerpt :
To prevent the loss of differentiation potential, cells were not allowed to become confluent. Constructs containing the coding sequence (CDS) of WT- or C121W-NaCh β1 subunit (Tammaro et al., 2002; Moran et al., 2003), fused with the CDS of the enhanced Blue Fluorescent Protein (eBFP) (Baroni et al., 2013, 2014) were used to transfect GH3 and H9C2 cells. Cells were transiently transfected by the calcium phosphate-DNA co-precipitation method (Bacchetti and Graham, 1977) with 6 μg of DNA and were used between 48 and 72 h after transfection.
Voltage-dependent sodium channels are membrane proteins essential for cell excitability. They are composed by a pore-forming α-subunit, encoded in mammals by up to nine different genes, and four different ancillary β-subunits. The expression pattern of the α subunit isoforms confers the distinctive functional and pharmacological properties to different excitable tissues. β-Subunits are important modulators of channel function and expression. Mutation C121W of the β1-subunit causes an autosomal dominant epileptic syndrome without cardiac symptoms. In neuroectoderm GH3 and cardiac H9C2 cells, the over-expression of β1 subunit augments α subunit mRNA and protein levels as well as sodium current density. Interestingly, the introduction of the epileptogenic C121W-β1 subunit produces additional changes in the α-subunit expression pattern of H9C2 cells, leaving unaltered the sodium channel isoform composition of GH3 cells. The challenge of the present work was to identify those genes that were differentially expressed in response to WT- or C121W-β1 subunit over-expression in the two rat cell lines under analysis. Hence, we analyzed the total mRNA extracted from control-untransfected and from WT- and C121W-β1-transfected GH3 and H9C2 cells by DNA-microarray. We found that, in agreement with their different embryonal origin, the over-expression of WT- and C121W-β1 subunits modifies the expression of different gene sets in GH3 and H9C2 cells. Focusing on the effects of the C121W mutation, we found that it causes the modification of 214 genes, most of them were down-regulated (202) in GH3 cells; on the contrary, it determined the up-regulation of only five genes in H9C2 cells. Interestingly, most genes modified by the C121W β1 subunit are involved in pivotal processes of the cell such as cellular communication and protein expression. Our results confirm the important role of the sodium channel β1 subunit in the control of NaCh gene expression, and highlight once more the tissue-specific effect of the C121W mutation.
Identification of an intra-molecular disulfide bond in the sodium channel β1-subunit
2012, Biochemical and Biophysical Research Communications
The sodium channel β1 subunit is non-covalently associated with the pore-forming α-subunits, and has been proposed to act as a modulator of channel activity, regulator of channel cell surface expression and cell adhesion molecule. Its importance is evident since mutations of the β1 subunit cause neurologic and cardiovascular disorders. The first described β1 subunit mutation is the C121W, that is related to generalized epilepsy with febrile seizures plus (GEFS+), a childhood genetic epilepsy syndrome. This mutation changed a conserved cysteine residue in position 121 into a tryptophan, putatively disrupting a disulfide bridge that should normally maintain the β1 extracellular immunoglobulin-like fold. Using the 2-D-diagonal-SDS–PAGE technique, we demonstrated the existence of this putative disulfide bridge in the Ig-like extracellular domain of the β1 subunit and its disruption in the epileptogenic C121W mutant.
Electrophysiology and beyond: Multiple roles of Na<sup>+</sup> channel β subunits in development and disease
2010, Neuroscience Letters
Voltage-gated Na⁺ channel (VGSC) β Subunits are not “auxiliary.” These multi-functional molecules not only modulate Na⁺ current (I_Na), but also function as cell adhesion molecules (CAMs)—playing roles in aggregation, migration, invasion, neurite outgrowth, and axonal fasciculation. β subunits are integral members of VGSC signaling complexes at nodes of Ranvier, axon initial segments, and cardiac intercalated disks, regulating action potential propagation through critical intermolecular and cell–cell communication events. At least in vitro, many β subunit cell adhesive functions occur both in the presence and absence of pore-forming VGSC α subunits, and in vivo β subunits are expressed in excitable as well as non-excitable cells, thus β subunits may play important functional roles on their own, in the absence of α subunits. VGSC β1 subunits are essential for life and appear to be especially important during brain development. Mutations in β subunit genes result in a variety of human neurological and cardiovascular diseases. Moreover, some cancer cells exhibit alterations in β subunit expression during metastasis. In short, these proteins, originally thought of as merely accessory to α subunits, are critical players in their own right in human health and disease. Here we discuss the role of VGSC β subunits in the nervous system.
Epileptogenic ion channel mutations: From bedside to bench and, hopefully, back again
2010, Epilepsy Research
Citation Excerpt :
Moreover, some channels have well established accessory subunits, which can be targets of mutations, for example the β1 subunit of Na+ channels or the β4 subunit of high voltage activated (HVA) Ca2+ channels (Table 1), but often their functions have not been clearly identified. For instance, Na+ channel β1 subunit can modulate gating properties and targeting of the principal α subunits, but its properties are highly variable and strictly depend on the expression system used (Isom et al., 1994; Qu et al., 2001; Meadows et al., 2002; Tammaro et al., 2002). β1 subunit mutants can cause GEFS+ or SMEI and show complete loss of function because they cannot modulate α subunits (Meisler and Kearney, 2005; Patino et al., 2009), but their effects on cell excitability are still unclear because those of wild-type β1 are not yet fully understood.
Mutations of genes coding for ion channels cause several genetically determined human epileptic syndromes. The identification of a gene variant linked to a particular disease gives important information, but it is usually necessary to perform functional studies in order to completely disclose the pathogenic mechanisms.
The functional consequences of epileptogenic mutations have been studied both in vitro and in vivo with several experimental systems, studies that have provided significant knowledge on the pathogenic mechanisms that leads to inherited human epilepsies, and possibly also on the pathogenic mechanisms of non-genetic human epilepsies due to “acquired channelopathies”. However, several open issues remain and difficulties in the interpretation of the experimental data have arisen that limit translational applications.
We will highlight the value and the limits of different approaches to the study of epileptogenic channelopathies, focussing on the importance of the experimental systems in the assessment of the functional effects of the mutations and on the possible applications of the obtained results to the clinical practice.
Electroencephalographic evidence of brainstem recruitment during scorpion envenomation
2009, NeuroToxicology
Scorpion envenomation is a public health problem in Brazil, with most severe cases occuring in children under the age of 5 years (0.6% lethality). In fact, the toxic fractions of the Tityus serrulatus scorpion venom (TSSV) have greater permeability across the BBB of weanling rats when compared to adults. Although EEG alterations have been reported in up to 75% of pediatric severe cases, the role of the CNS in envenomation morbidity is still in debate. Our working hypothesis is that the neural substrates that play a major role in morbidity generate activity undetectable from EEG scalp leads. Twenty one-day-old rats (n = 18) were injected s.c. with the deadliest toxic fraction of the TSSV, tityustoxin (TsTX; 2 × DL50 = 6 mg/kg). EEG leads were stereotaxicaly implanted in the nucleus of the solitary tract (NTS) and left parietal cortex. EEG and ECG were continuously monitored by a video EEG system until death or for a maximum period of 240 min. An experimental group pre-treated with carbamazepine (CBZ) was added in order to better access the cause–effect relationship between neural discharges and the systemic ECG alterations. High amplitude discharges in the NTS, which correlated to cardiac alterations, were recorded soon after administration of TsTX. Abnormal electrographic activity spread throughout the cortex only later in the recording. As expected, the CBZ treatment increased the latency for the first epileptiform discharge, decreased EEG/ECG alterations and increased the general survival time. In summary: peripheral scorpion toxin inoculation recruits brainstem involved in cardiovascular control and initial electrographic activity was undetectable from the cortical electrode.

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¹: Present address: Department of Pharmacy and Pharmacology; University of Bath; Bath, UK.

²: To whom correspondence and reprint requests should be addressed. Fax: (+39)-010-6475-500. E-mail: [email protected].

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Regular ArticleModulation of Sodium Current in Mammalian Cells by an Epilepsy-Correlated β1-Subunit Mutation

Abstract

Neuron

Neuron

Biophys. J.

J. Biol. Chem.

FEBS Lett.

FEBS Lett.

J. Biol. Chem.

J Biol Chem

Anal. Biochem.

Biophys. J.

Neuron

Biophys. Biochem. Res. Commun.

Structure and function of voltage-gated ion channels

Annu. Rev. Biochem.

Structure, function and expression of voltage-dependent sodium channels

Mol. Neurobiol

β3: An additional auxiliary subunit of the voltage-sensitive sodium channel that modulates channel gating with distinct kinetics

Proc. Natl. Acad. Sci. USA

Regular Article
Modulation of Sodium Current in Mammalian Cells by an Epilepsy-Correlated β1-Subunit Mutation