Functional reciprocity between Na ⁺ channel Na _v 1.6 and β1 subunits in the coordinated regulation of excitability and neurite outgrowth

Abstract

Voltage-gated Na + channel (VGSC) β1 subunits regulate cell-cell adhesion and channel activity in vitro. We previously showed that β1 promotes neurite outgrowth in cerebellar granule neurons (CGNs) via homophilic cell adhesion, fyn kinase, and contactin. Here we demonstrate that β1-mediated neurite outgrowth requires Na + current (I Na ) mediated by Na v 1.6. In addition, β1 is required for highfrequency action potential firing. Transient I Na is unchanged in Scn1b (β1) null CGNs; however, the resurgent I Na , thought to underlie high-frequency firing in Na v 1.6-expressing cerebellar neurons, is reduced. The proportion of axon initial segments (AIS) expressing Na v 1.6 is reduced in Scn1b null cerebellar neurons. In place of Na v 1.6 at the AIS, we observed an increase in Na v 1.1, whereas Na v 1.2 was unchanged. This indicates that β1 is required for normal localization of Na v 1.6 at the AIS during the postnatal developmental switch to Na v 1.6-mediated high-frequency firing. In agreement with this, β1 is normally expressed with α subunits at the AIS of P14 CGNs. We propose reciprocity of function between β1 and Na v 1.6 such that β1-mediated neurite outgrowth requires Na v 1.6-mediated I Na , and Na v 1.6 localization and consequent high-frequency firing require β1. We conclude that VGSC subunits function in macromolecular signaling complexes regulating both neuronal excitability and migration during cerebellar development.V oltage-gated Na + channels (VGSCs), composed of one poreforming α subunit and two β subunits (1), are responsible for initiation and conduction of action potentials (APs) (2). Of the nine α subunits (3), Na v 1.1, Na v 1.2, and Na v 1.6 are found in the postnatal CNS (4, 5) where they display developmentally regulated expression patterns in specialized neuronal subcellular domains. For example, Na v 1.1 and Na v 1.2 are replaced during postnatal development at the axon initial segment (AIS) and nodes of Ranvier by Na v 1.6. Scn8a null mice display motor dysfunction, ataxia, and lethality by postnatal day (P) 21, suggesting that this developmental switch to Na v 1.6 expression in brain is critical (6, 7). Scn8a null retinal ganglion neurons display impaired excitability, demonstrating that Na v 1.6 is vital for high-frequency firing (8-10). Thus, VGSC-driven neuronal activity is important for proper CNS development, although the underlying mechanism(s) are not well understood.VGSC β1 subunits are multifunctional molecules that modulate channel kinetics and gating, regulate channel cell surface expression, and participate in cell-cell adhesion in vitro (11). Scn1b (β1) null mice are ataxic, experience spontaneous seizures, and exhibit a prolonged cardiac QT interval, demonstrating that β1 modulates electrical excitability in vivo (12, 13). Consistent with this, human mutations in SCN1B result in epilepsy and arrhythmia (14-21). As a member of the Ig superfamily of cell adhesion molecules (CAMs), β1 mediates cellular aggregation, cytoskeletal recruitment, and extracellular matrix interactio...

Abnormal neuronal patterning occurs during early postnatal brain development of Scn1b -null mice and precedes hyperexcitability

“…Fasciculation and pathfinding of corticospinal axons and CGNs is disrupted in null mice when assayed at P14 (12). In addition, the excitability of null CGNs is impaired as a result of reduced Na v 1.6 AIS expression (13). Similar to ref.…”

“…In zebrafish, scn1bb morphants show abnormal spinal neuron axons and olfactory nerve defasciculation (14). β1-mediated neurite outgrowth and β1-mediated VGSC gating reciprocally modulate each other (13). Together, these results suggest that Scn1b is required for the establishment of normal electrical activity in the brain.…”

“…12 and 14 and the present study). Disruption of the functional reciprocity between Scn1b-mediated migration and Scn1b-mediated VGSC gating may be an important factor in excitability-related disorders (13).…”

“…Procedures for tissue preparation, antibody labeling, Nissl staining, and immunohistochemistry were described previously (13,35). Additional details are given in SI Materials and Methods.…”

“…However, CA3 action potentials have higher peak voltage and amplitude, suggesting that β1/β1B may regulate VGSCs on neuronal processes rather than on the soma. VGSC expression and localization are altered in Scn1b-null hippocampus (11) and cerebellar AIS (13). P14 Scn1b-null mice exhibit aberrant neuronal pathfinding and defasciculation in the corticospinal tract and cerebellum (12).…”