Teresa K. Aman

The beta subunits of voltage-gated Na channels (Scnxb) regulate the gating of pore-forming alpha subunits, as well as their trafficking and localization. In heterologous expression systems, beta1, beta2, and beta3 subunits influence inactivation and persistent current in different ways. To test how the beta4 protein regulates Na channel gating, we transfected beta4 into HEK (human embryonic kidney) cells stably expressing Na(V)1.1. Unlike a free peptide with a sequence from the beta4 cytoplasmic domain, the full-length beta4 protein did not block open channels. Instead, beta4 expression favored open states by shifting activation curves negative, decreasing the slope of the inactivation curve, and increasing the percentage of noninactivating current. Consequently, persistent current tripled in amplitude. Expression of beta1 or chimeric subunits including the beta1 extracellular domain, however, favored inactivation. Coexpressing Na(V)1.1 and beta4 with beta1 produced tiny persistent currents, indicating that beta1 overcomes the effects of beta4 in heterotrimeric channels. In contrast, beta1(C121W), which contains an extracellular epilepsy-associated mutation, did not counteract the destabilization of inactivation by beta4 and also required unusually large depolarizations for channel opening. In cultured hippocampal neurons transfected with beta4, persistent current was slightly but significantly increased. Moreover, in beta4-expressing neurons from Scn1b and Scn1b/Scn2b null mice, entry into inactivated states was slowed. These data suggest that beta1 and beta4 have antagonistic roles, the former favoring inactivation, and the latter favoring activation. Because increased Na channel availability may facilitate action potential firing, these results suggest a mechanism for seizure susceptibility of both mice and humans with disrupted beta1 subunits.

The Scn8a gene encodes the voltage-gated Na channel alpha subunit Na(V)1.6, which is widely expressed throughout the nervous system. Global null mutations that eliminate Scn8a in all cells result in severe motor dysfunction and premature death, precluding analysis of the physiological role of Na(V)1.6 in different neuronal types. To test the effect of cerebellar Na(V)1.6 on motor coordination in mice, we used the Cre-lox system to eliminate Scn8a expression exclusively in Purkinje neurons (Purkinje KO) and/or granule neurons (granule KO). Whereas granule KO mice had only minor behavioral defects, adult Purkinje KO mice exhibited ataxia, tremor, and impaired coordination. These disorders were exacerbated in double mutants lacking Scn8a in both Purkinje and granule cells (double KO). In Purkinje cells isolated from adult Purkinje KO and double KO but not granule KO mice, the ratio of resurgent-to-transient tetrodotoxin- (TTX)-sensitive Na current amplitudes decreased from approximately 15 to approximately 5%. In cerebellar slices, Purkinje cell spontaneous and maximal firing rates were reduced 10-fold and twofold relative to control in Purkinje KO and double KO but not granule KO mice. Additionally, short-term plasticity of high-frequency parallel fiber EPSCs was altered relative to control in Purkinje KO and double KO but not granule KO mice. These data suggest that the specialized kinetics of Purkinje Na channels depend directly on Scn8a expression. The loss of these channels leads to a decrease in Purkinje cell firing rates as well as a modification of the synaptic properties of afferent parallel fibers, with the ultimate consequence of disrupting motor behavior.

The NMDA-type glutamate receptor (NMDAR) is essential for synaptogenesis, synaptic plasticity, and higher cognitive function. Emerging evidence indicates that NMDAR Ca(2+) permeability is under the control of cAMP/protein kinase A (PKA) signaling. Whereas the functional impact of PKA on NMDAR-dependent Ca(2+) signaling is well established, the molecular target remains unknown. Here we identify serine residue 1166 (Ser1166) in the carboxy-terminal tail of the NMDAR subunit GluN2B to be a direct molecular and functional target of PKA phosphorylation critical to NMDAR-dependent Ca(2+) permeation and Ca(2+) signaling in spines. Activation of β-adrenergic and D1/D5-dopamine receptors induces Ser1166 phosphorylation. Loss of this single phosphorylation site abolishes PKA-dependent potentiation of NMDAR Ca(2+) permeation, synaptic currents, and Ca(2+) rises in dendritic spines. We further show that adverse experience in the form of forced swim, but not exposure to fox urine, elicits striking phosphorylation of Ser1166 in vivo, indicating differential impact of different forms of stress. Our data identify a novel molecular and functional target of PKA essential to NMDAR-mediated Ca(2+) signaling at synapses and regulated by the emotional response to stress.