Molecular and Cellular Context Influences SCN8A Variant Function

Abstract

ABSTRACTPathogenic variants inSCN8A, which encodes the voltage-gated sodium (NaV) channel NaV1.6, are associated with neurodevelopmental disorders including epileptic encephalopathy. Previous approaches to determineSCN8Avariant function may be confounded by the use of a neonatal-expressed alternatively spliced isoform of NaV1.6 (NaV1.6N), and engineered mutations to render the channel tetrodotoxin (TTX) resistant. In this study, we investigated the impact ofSCN8Aalternative splicing on variant function by comparing the functional attributes of 15 variants expressed in two developmentally regulated splice isoforms (NaV1.6N, NaV1.6A). We employed automated patch clamp recording to enhance throughput, and developed a novel neuronal cell line (ND7/LoNav) with low levels of endogenous NaVcurrent to obviate the need for TTX-resistance mutations. Expression of NaV1.6N or NaV1.6A in ND7/LoNav cells generated NaVcurrents that differed significantly in voltage-dependence of activation and inactivation. TTX-resistant versions of both isoforms exhibited significant functional differences compared to the corresponding wild-type (WT) channels. We demonstrated that many of the 15 disease-associated variants studied exhibited isoform-dependent functional effects, and that many of the studiedSCN8Avariants exhibited functional properties that were not easily classified as either gain- or loss-of-function. Our work illustrates the value of considering molecular and cellular context when investigatingSCN8Avariants.