Theataxia3Mutation in the N-Terminal Cytoplasmic Domain of Sodium Channel Na_v1.6 Disrupts Intracellular Trafficking

Abstract

The ENU-induced neurological mutant ataxia3 was mapped to distal mouse chromosome 15. Sequencing of the positional candidate gene Scn8a encoding the sodium channel Na v 1.6 identified a TϾC transition in exon 1 resulting in the amino acid substitution p.S21P near the N terminus of the channel. The cytoplasmic N-terminal region is evolutionarily conserved but its function has not been well characterized. ataxia3 homozygotes exhibit a severe disorder that includes ataxia, tremor, and juvenile lethality. Unlike Scn8a null mice, they retain partial hindlimb function. The mutant transcript is stable but protein abundance is reduced and the mutant channel is not detected in its usual site of concentration at nodes of Ranvier. In whole-cell patch-clamp studies of transfected ND7/23 cells that were maintained at 37°C, the mutant channel did not produce sodium current, and function was not restored by coexpression of ␤1 and ␤2 subunits. However, when transfected cells were maintained at 30°C, the mutant channel generated voltage-dependent inward sodium currents with an average peak current density comparable with wild type, demonstrating recovery of channel activity. Immunohistochemistry of primary cerebellar granule cells from ataxia3 mice demonstrated that the mutant protein is retained in the cis-Golgi. This trafficking defect can account for the low level of Na v 1.6-S21P at nodes of Ranvier in vivo and at the surface of transfected cells. The data demonstrate that the cytoplasmic N-terminal domain of the sodium channel is required for anterograde transport from the Golgi complex to the plasma membrane.

Amyloid Precursor Protein Enhances Nav1.6 Sodium Channel Cell Surface Expression

“…6. It has been reported that the cytoplasmic N-terminal domain of Nav1.6 is required for anterograde transport from the Golgi complex to the plasma membrane (53) and that the microtubule-associated protein MAP1B interacts with Nav1.6 and facilitates its trafficking to the cell surface (54). It remains to be elucidated whether APP enhances the surface expression of Nav1.6 by promoting forward trafficking or by decreasing the endocytosis of sodium channels from the cell surface.…”

Characterization of a de novo SCN8A mutation in a patient with epileptic encephalopathy

“…The c.667A>G nucleotide mutation was introduced into the tetrodotoxin (TTX)-resistant derivative of the murine Nav1.6 cDNA clone Nav1.6 R as previously described (Sharkey et al, 2009). Mouse Scn8a is an appropriate model for human SCN8A , since the human and mouse proteins are 99% identical in amino acid sequence (Plummer et al, 1998), and in vivo expression of the human epileptic encephalopathy mutation N1768D in the mouse results in a similar phenotype of early onset convulsive seizures and SUDEP (Jones and Meisler, 2014)(and unpublished observations by MHM).…”

“…Channel activity was evaluated by co-transfection of the eGFP cDNA together with mutant or wildtype Nav1.6 murine cDNAs into the dorsal root ganglion (DRG) neuron-derived cell line ND7/23 as previously described (Sharkey et al, 2009; Veeramah et al, 2012). ND7/23 neuronal cells support larger Nav1.6 currents than HEK293 cells, and have been our preferred expression system for voltage-clamp experiments.…”

Cerebellum-Related Characteristics of Scn8a-Mutant Mice

“…Four spontaneous mutations of Scn8a channels in mice have been reported (Table 1) [16][17][18][19][20][21][22]). Scn8a med and Scn8a medTg are two null mutations with clinical signs of paralysis, muscle atrophy, and failure of neuromuscular transmission; such mice live for only 21-24 days.…”

“…Scn8a ataxia3 has an induced mutation that produces a phenotype resembling those of the null mutations except that partial hindlimb function is retained. Retention of the Na v 1.6 channel protein in the Golgi complex prevents its adequate transport to the cell membrane [21].…”