Pure haploinsufficiency for Dravet syndrome Na V1.1 (SCN1A) sodium channel truncating mutations

Purpose: Dravet syndrome (DS), a devastating epileptic encephalopathy, is mostly caused by mutations of the SCN1A gene, coding for the voltage-gated Na ⁺ channel Na _V1.1 α subunit. About 50% of SCN1A DS mutations truncate Na _V1.1, possibly causing complete loss of its function. However, it has not been investigated yet if Na _V1.1 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 _V1.1 mutants, R222* and R1234*, on coexpressed wild-type Na ⁺ channels. Methods: We engineered R222* or R1234* in the human cDNA of Na _V1.1 (hNa _V1.1) and studied their effect on coexpressed wild-type hNa _V1.1, hNa _V1.2 or hNa _V1.3 cotransfecting tsA-201 cells, and on hNa _V1.6 transfecting an human embryonic kidney (HEK) cell line stably expressing this channel. We also studied hippocampal neurons dissociated from Na _V1.1 knockout (KO) mice, an animal model of DS expressing a truncated Na _V1.1 channel. Key Findings: We found no modifications of current amplitude coexpressing the truncated mutants with hNa _V1.1, hNa _V1.2, or hNa _V1.3, but a 30% reduction coexpressing them with hNa _V1.6. However, we showed that also coexpression of functional full-length hNa _V1.1 caused a similar reduction. Therefore, this effect should not be involved in the pathomechanism of DS. Some gating properties of hNa _V1.1, hNa _V1.3, and hNa _V1.6 were modified, but recordings of hippocampal neurons dissociated from Na _V1.1 KO mice did not show any significant modifications of these properties. Therefore, Na _V1.1 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 _V2.1 mutants and hNa _V1.1 ones, and shown that hNa _V1.6 expression can be reduced in physiologic conditions by coexpression of hNa _V1.1. Moreover, our data may provide useful information for the development of therapeutic approaches.