Molecular and cellular context influences SCN8A variant function.

Area of Science:

• Neuroscience
• Genetics
• Molecular Biology

Background:

• Pathogenic variants in SCN8A, encoding the NaV1.6 voltage-gated sodium channel, are associated with neurodevelopmental disorders like developmental and epileptic encephalopathy.
• Previous functional studies of SCN8A variants may be limited by the use of neonatal splice isoforms and engineered TTX-resistance mutations.

Purpose of the Study:

• To investigate the impact of SCN8A alternative splicing on the functional attributes of disease-associated variants.
• To compare the function of 15 SCN8A variants across two developmentally regulated NaV1.6 splice isoforms (NaV1.6N and NaV1.6A).

Main Methods:

• Utilized automated patch clamp recording for high-throughput analysis.
• Developed a neuronal cell line (ND7/LoNav) with low endogenous sodium channel current to avoid TTX-resistance mutations.
• Expressed NaV1.6N and NaV1.6A isoforms with and without TTX-resistance mutations in ND7/LoNav cells.

Main Results:

• Expressed NaV1.6N and NaV1.6A isoforms showed subtle but significant differences in voltage-dependent activation and inactivation.
• TTX-resistant variants displayed distinct functional differences compared to wild-type (WT) channels.
• Many disease-associated SCN8A variants exhibited isoform-dependent functional effects.
• Variant function was often not clearly classifiable as gain- or loss-of-function.

Conclusions:

• Alternative splicing of SCN8A significantly impacts the functional consequences of pathogenic variants.
• Investigating SCN8A variants requires consideration of both molecular (isoform) and cellular context.
• The functional classification of SCN8A variants may be more complex than simple gain- or loss-of-function.