Pure haploinsufficiency for Dravet syndrome Na_V1.1 (SCN1A) sodium channel truncating mutations

Abstract

SUMMARYPurpose: Dravet syndrome (DS), a devastating epileptic encephalopathy, is mostly caused by mutations of the SCN1A gene, coding for the voltage-gated Na + channel Na V 1.1 a subunit. About 50% of SCN1A DS mutations truncate Na V 1.1, possibly causing complete loss of its function. However, it has not been investigated yet if Na V 1.1 truncated mutants are dominant negative, if they impair expression or function of wild-type channels, as it has been shown for truncated mutants of other proteins (e.g., Ca V channels). We studied the effect of two DS truncated Na V 1.1 mutants, R222* and R1234*, on coexpressed wildtype Na + channels. Methods: We engineered R222* or R1234* in the human cDNA of Na V 1.1 (hNa V 1.1) and studied their effect on coexpressed wild-type hNa V 1.1, hNa V 1.2 or hNa V 1.3 cotransfecting tsA-201 cells, and on hNa V 1.6 transfecting an human embryonic kidney (HEK) cell line stably expressing this channel. We also studied hippocampal neurons dissociated from Na V 1.1 knockout (KO) mice, an animal model of DS expressing a truncated Na V 1.1 channel.Key Findings: We found no modifications of current amplitude coexpressing the truncated mutants with hNa V 1.1, hNa V 1.2, or hNa V 1.3, but a 30% reduction coexpressing them with hNa V 1.6. However, we showed that also coexpression of functional full-length hNa V 1.1 caused a similar reduction. Therefore, this effect should not be involved in the pathomechanism of DS. Some gating properties of hNa V 1.1, hNa V 1.3, and hNa V 1.6 were modified, but recordings of hippocampal neurons dissociated from Na V 1.1 KO mice did not show any significant modifications of these properties. Therefore, Na V 1.1 truncated mutants are not dominant negative, consistent with haploinsufficiency as the cause of DS. Significance: We have better clarified the pathomechanism of DS, pointed out an important difference between pathogenic truncated Ca V 2.1 mutants and hNa V 1.1 ones, and shown that hNa V 1.6 expression can be reduced in physiologic conditions by coexpression of hNa V 1.1. Moreover, our data may provide useful information for the development of therapeutic approaches.

Nonfunctional Na V 1.1 familial hemiplegic migraine mutant transformed into gain of function by partial rescue of folding defects

“…S2) (10, 25). We studied the properties of WT and L1649Q currents in neurons by using channels in which we engineered the mutation F383S, which confers resistance to the specific blocker TTX (15). In these conditions, the currents covery from a 150-ms inactivating pulse at the indicated potentials: −110 mV, τ REC -WT = 2.1 ± 0.1 ms (n = 5), τ REC -L1649Q = 1.0 ± 0.1 ms (n = 6); −100 mV, τ REC -WT = 2.8 ± 0.2 ms (n = 5), τ REC -L1649Q = 1.3 ± 0.1 ms (n = 5); −90 mV, τ REC -WT = 3.98 ± 0.04 ms (n = 7), τ REC -L1649Q = 1.45 ± 0.14 ms (n = 8); −80 mV τ REC -WT = 6.6 ± 1.6 ms (n = 5), τ REC-L1649Q = 2.5 ± 0.2 ms (n = 5); −70 mV, τ REC -WT =13.1 ± 3.5 ms (n = 4), τ REC -L1649Q = 4.6 ± 0.5 ms (n = 5); −60 mV, τ REC -L1649Q = 9.3 ± 1.2 ms (n = 5); P < 0.01 or <0.05 for all of the potentials.…”

“…The results of most studies suggest that epileptogenic Na V 1.1 mutations cause variable degrees of loss of function of Na V 1.1, leading to reduced Na + current and excitability in GABAergic neurons, and resulting in decreased inhibition in neuronal networks (10)(11)(12)(13)(14). The most severe phenotypes (e.g., Dravet syndrome, an extremely severe epileptic encephalopathy) are in general caused by mutations that induce complete Na V 1.1 loss of function, leading to haploinsufficiency (15). Thus, it has been hypothesized that a more severe loss of function would cause more severe epilepsy (8).…”

Specific deletion of Na V 1.1 sodium channels in inhibitory interneurons causes seizures and premature death in a mouse model of Dravet syndrome

“…Mutations in the poreforming subunits of Na V 1.1 channels cause multiple types of epilepsy (8,9). Heterozygous missense and truncation mutations in the Scn1a gene encoding Na V 1.1 channels lead to haploinsufficiency of the channel function and cause Dravet syndrome (DS, also known as severe myoclonic epilepsy of infancy) (10)(11)(12). This disease begins with infantile-onset febrile seizures at 6-9 mo of age and progresses to intractable afebrile, generalized seizures in later years.…”

The Effect of Scn1A Mutations on Patient-Derived Gabaergic Neurons: What Are the Implications For Future Dravet Syndrome Therapeutics?

“…This has significantly accelerated research into this condition, more specifically by using murine DS models, as well as our understanding of DS pathogenesis [7,8]. It is currently known that many DS mutations in SCN1A cause a significant deterioration in Na V 1.1 function (i.e., producing a nonfunctional channel) [9]; haploinsufficiency of this channel may be the primary mechanism [10]. A functional decline in the GABAergic interneurons, where Na V 1.1 is primarily expressed in the mouse neocortex and hippocampus, may cause the major symptoms of DS [7,8]; and a functional decline in Purkinje neurons, where Na V 1.1 is also strongly expressed, may cause ataxia [11].…”