Position-dependent effects of SCN2A premature stop codons on neuronal excitability and behavior

SCN2A encodes the voltage-gated sodium channel Na _V 1.2, a key determinant of spike initiation and propagation in glutamatergic neurons. Premature termination codons are often assumed to produce uniform haploinsufficiency via nonsense-mediated decay, yet whether distinct SCN2A premature stop codons yield equivalent molecular, cellular, and behavioral outcomes remains unknown. We generated two mouse lines carrying patient mutations- Scn2a ^Y84X/+ (p.Tyr84UAA; early coding sequence) and Scn2a ^R1627X/+ (p.Arg1627UGA; terminal coding exon)-on a C57BL/6J background. Allele-specific expression was quantified by targeted next-generation sequencing of whole-brain reverse transcribed cDNA. Na _V 1.2 protein was measured in half-brain lysates by automated western blot and ex vivo whole-cell recordings were obtained from layer 5b pyramidal-tract neurons in medial prefrontal cortex. A panel of behavioral assays assessed locomotion/exploration, motor learning, anxiety-like behavior, sociability, sensorimotor gating, and seizure susceptibility. Allele-specific RNA handling diverged by position: mRNA carrying Y84X engaged partial nonsense-mediated decay, whereas R1627X transcripts were at allelic balance. Despite this difference in RNA fate, Na _V 1.2 protein was comparably reduced in both lines. Electrophysiologically, both premature termination codon mutations slowed the action-potential upstroke, with a larger decrement in Scn2a ^Y84X/+ than in Scn2a ^R1627X/+ . Spike threshold was depolarized only in Scn2a ^Y84X/+ , whereas Scn2a ^R1627X/+ remained similar to wild type. Frequency-current relations showed reduced firing at near-rheobase inputs in both mutants, with responses approaching wild type at stronger currents. Behaviorally, locomotion, sociability, and sensorimotor gating were preserved. Both lines exhibited increased grooming-consistent with restrictive, repetitive behavior; Scn2a ^Y84X/+ alone showed greater exploration in the elevated-risk context and a male-predominant deficit in rotarod learning. In maximal electroshock testing, mortality was lower in both lines without differences in seizure threshold or severity. Our results show that distinct SCN2A premature termination codons are not equivalent to one another, nor to a uniform haploinsufficient state. An early, nonsense-mediated decay-competent premature stop codon (Y84X) and a terminal-exon one (R1627X) produce partially overlapping yet allele-specific effects on neuronal excitability and behavior. These findings establish premature termination codon position as a determinant of phenotype, supporting allele-tailored mechanistic studies and therapeutic strategies.