Elsevier

Neurobiology of Disease

Volume 47, Issue 3, September 2012, Pages 378-384
Neurobiology of Disease

Compromised function in the Nav1.2 Dravet syndrome mutation R1312T

https://doi.org/10.1016/j.nbd.2012.05.017Get rights and content

Abstract

Ion channels, specifically voltage-gated sodium channels (Navs), are common culprits in inheritable seizure disorders. Some Nav isoforms are particularly susceptible, while others are only weakly associated with neuronal hyperexcitability. Representative of the latter group is Nav1.2 (gene name SCN2A): despite its abundance in the brain, Nav1.2-related epilepsy is rare and only few studies have been conducted as to the pathophysiological basis of Nav1.2 in neuronal hyperexcitability. We here present a detailed functional analysis of Nav1.2 mutant, R1312T, which was originally found in a child with Dravet syndrome (formerly known as severe myoclonic epilepsy of infancy or SMEI). Whole-cell voltage clamp analysis revealed clearly compromised function: the mutant channels fast- and slow-inactivated at markedly more negative potentials and recovered from fast inactivation more slowly, which resulted in a use-dependent current reduction to less than 50% of wildtype levels. We also noted a small hyperpolarizing shift in the voltage dependence of activation. Our findings expand the spectrum of abnormal Nav channel behavior in epilepsy and raise the question as to how loss-of-function in a sodium channel predominantly expressed in excitatory neurons can lead to hyperexcitability.

Introduction

Voltage-gated sodium channels (Nav1.1–Nav1.9, gene names SCN1ASCN11A) control the sodium exchange between the extracellular and the intracellular spaces. This makes them important players in neurological disorders such as epilepsy. Virtually all of the central nervous system's Nav channels have been tied to seizure-related hyperexcitability, some with hundreds of mutations (the SCN1A Infobase — http://scn1a.info, Lossin, 2009), while others appear to be only peripherally involved. Nav1.2 (gene name SCN2A) belongs to the latter group, with only a limited number of studies supporting a role of this channel in epilepsy and fewer still determining the functional consequences of possibly pathogenic Nav1.2 mutants. The connection between epilepsy and this relatively abundant neuronal Nav channel is, however, not in doubt: previous findings have tied genetic abnormalities in the SCN2A gene to milder epilepsies such as GEFS + (Sugawara et al., 2001) and BFNIS (Herlenius et al., 2007). Recent genetic analyses in our epilepsy cohort revealed that SCN2A variation is also important in more severe epilepsies, specifically Dravet syndrome, a myoclonic infantile epilepsy with poor prognosis (Shi et al., 2009). The mutation in question is a single-base substitution (c.3935G>C) in coding exon 20, which is predicted to replace a strongly conserved arginine at open reading frame position 1312 with a threonine (p. Arg1312Thr — referred to as R1312T hereafter, Fig. 1). The affected residue is part of the channel's voltage-sensing mechanism. It belongs to a group of charged amino acid residues – arginines, histidines, and lysines – that move in the membrane's electric field, causing a conformational change that opens the otherwise closed ion pore. The open state is only temporary, lasting milliseconds, as the intracellular mouth of the pore gets plugged by the linker connecting the third and fourth homologous domains of the channel (Fig. 1). This so-called fast inactivation is accompanied by a second, slower type of Nav channel closure, a less-well described phenomenon that presents itself as a reduction in the current amplitude when the membrane is depolarized for seconds at a time. Both inactivation types require hyperpolarization for their release. However, the duration of hyperpolarization needed to reprime from slow inactivation is orders of magnitude longer than what is necessary for fast inactivation recovery, which permits experimental separation of the two forms of inactivation.

To get a better understanding of the full spectrum of ion channel dysfunction in Dravet syndrome, we determined the functional consequences of the R1312T substitution using heterologous expression and patch-clamp electrophysiology. Our analyses revealed a channel whose biophysical signature is severely disturbed. The changes provide plausible grounds for the observed neurological abnormalities, surprisingly so not by excessive Nav1.2 action, but by a loss of function. The magnitude of the observed electrophysiological changes highlights p.Arg1312 as a key residue in this channel's functionality.

Section snippets

Patient history and diagnosis

The proband first came to our attention as a case of severe childhood epilepsy (Shi et al., 2009). Neither one of the parents suffered from epilepsy, nor was there a history of hyperexcitability in either side of the family. Candidate sequencing of the SCN1A (Fukuma et al., 2004, Oguni et al., 2005) and GABRG2 genes (Harkin et al., 2002) revealed no genetic alterations; multiplex ligation-dependent probe amplification (MLPA) for copy number variation was negative. Examination of the SCN2A gene,

Results

The purpose of our experiments was to determine the effects of the R1312T substitution on the biophysical behavior of neuronal Nav1.2. The residue in question locates to D3/S4, which is one of the four transmembrane helices making up the voltage sensor. Given its positive charge in this critical region, R1312 is likely important for voltage detection. This is supported by the complete conservation of the analogous residue across all human Nav isoforms (Fig. 1).

Discussion

In our analyses, four out of eight electrophysiological parameters presented a statistically significant decrease in R1312T-mediated current compared to the wildtype. These were reduced steady-state availability in fast and slow inactivation (Figs. 3C and 5B, respectively), slowed recovery from fast inactivation (Fig. 3D), and enhanced use dependence with repetitive stimulation (Fig. 4). Three additional parameters show tendencies toward less current in the mutant without reaching statistical

Conclusion

Voltage-gated neuronal Na+ channels take up a key position in excitation control and are therefore common loci for inheritable excitability disorders. In the here-presented mutation, we found very clear electrophysiological deviations that leave little doubt of this mutation's role in the associated epilepsy. Rather intriguing about our findings is the obvious involvement of slow inactivation. A growing number of Na+ channelopathies have abnormalities in this channel behavior, which raises the

Acknowledgments

The authors thank all participants and their family members for their cooperation in this study. Excellent technical assistance was provided by Akiyo Hamachi, Minako Yonetani, and Shahab Shahangian. We thank Dr. Alfred L. George, Jr., (Vanderbilt University, Nashville, TN) for his kind gifts of pIRES-CD8-hβ1 and pIRES-EGFP-hβ2 as well as for critically reading this manuscript. This work was supported in part by a Grant-in-Aid for Scientific Research (A) 21249062, a Grant-in-Aid for Challenging

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