Abstracts

Rationale:
SCN8A encodes the voltage-gated sodium channel NaV1.6 expressed at axon initial segments and nodes of Ranvier in central and peripheral neurons. Several dozen SCN8A variants have been associated with epileptic encephalopathy and related neurodevelopmental disorders. SCN8A undergoes developmentally-regulated alternative mRNA splicing resulting in distinct splice isoforms expressed either predominantly in early life or later. Because splice isoforms of other NaV channels exhibit distinct functional properties, assessing the function of SCN8A variants should consider splice isoform as a contributing factor. However, there have been no studies comparing the functional properties of SCN8A splice isoforms.
Method:
Mammalian expression plasmids of two major SCN8A splice isoforms (annotated by NCBI as variant 1 and variant 2) were constructed. SCN8A variant 1 corresponds to the splice isoform expressed predominantly during early brain development (neonatal, NaV1.6N), whereas variant 2 is the canonical isoform (adult, NaV1.6A) expressed in mature brain. To render the plasmids stable in bacteria, we inserted small introns at the exon 14-15 and 22-23 junctions.  Functional analysis of the two channel isoforms was done using a workflow that combines high efficiency electroporation of stabilized human NaV1.6 into a modified neuronal cell line with greatly attenuated endogenous Nav currents (ND7/LoNav cells), followed by automated planar patch-clamp recording. Vectors were designed to enable co-expression of untagged channel proteins with fluorescent proteins as mean for tracking successful cell transfection by flow cytometry.
Results:
Wild-type NaV1.6 isoforms electroporated into ND7/LoNav cells had transfection efficiencies of 88% and 82% for NaV1.6A and NaV1.6N, respectively. Peak current density was observed at +10 mV and was similar between isoforms (NaV1.6A: -187.4±15.3 pA/pF, n=67; NaV1.6N: -156.9±16.6 pA/pF, n=74, P=0.177). Comparing isoforms, we observed no differences in parameters reflecting the voltage-dependence of activation (NaV1.6A: -17.2±1.2 mV, n=48; NaV1.6N: V½ -14.2±1.0 mV, n=51, P=0.058), but a large and significant difference in the voltage-dependence of inactivation (NaV1.6A: V½ -56.5±1.0 mV, n=50; NaV1.6N: V½ -51.0±0.5 mV, n=54, P< 0.001). Persistent current recorded at -10 mV and measured at the end of a 50ms pulse and expressed as a percentage of peak transient current was significantly larger for NaV1.6N (10.7±1.7%, n=74) than NaV1.6A (4.4±1.1%, n=67, P=0.002) channels.
Conclusion:
Developmentally-regulated alternative mRNA splicing of SCN8A generates channel isoforms with distinct functional properties that can be assayed using automated patch clamp recording. Splice isoform may be an important factor to consider when evaluating the function of SCN8A variants, especially when considering variants associated with early onset neurodevelopment disorders.
Funding:
:NIH U54-NS108874