CaMKII Phosphorylation of the Voltage-Gated Sodium Channel Nav1.6 Regulates Channel Function and Neuronal Excitability

Abstract

Voltage-gated sodium channels (Navs) undergo remarkably complex modes of modulation to fine tune membrane excitability and neuronal firing properties. In neurons, the isoform Nav1.6 is highly enriched at the axon initial segment and nodes, making it critical for the initiation and propagation of neuronal impulses. Thus, Nav1.6 modulation and dysfunction may profoundly impact the input-output properties of neurons in normal and pathological conditions. Phosphorylation is a powerful and reversible mechanism that exquisitely modulates ion channels. To this end, the multifunctional calcium/calmodulin-dependent protein kinase II (CaMKII) can transduce neuronal activity through phosphorylation of diverse substrates to serve as a master regulator of neuronal function. Because Nav1.6 and CaMKII are independently linked to excitability disorders, I sought to investigate modulation of Nav1.6 function by CaMKII signaling to reveal an important mechanism underlying neuronal excitability. Multiple biochemical approaches show Nav1.6 is a novel substrate for CaMKII and reveal multi-site phosphorylation within the L1 domain; a hotspot for post-translational regulation in other Nav isoforms. Consistent with these findings, pharmacological inhibition of CaMKII reduces transient and persistent sodium currents in Purkinje neurons. Because Nav1.6 is the predominant sodium current observed in Purkinje neurons, these data suggest that Nav1.6 may be modulated through CaMKII signaling. In support of this, my studies demonstrate that CaMKII inhibition significantly attenuates Nav1.6 transient and persistent sodium currents and shifts the voltage-dependence of activation to more depolarizing potentials in heterologous cells. Interestingly, I show that these functional effects are likely mediated by CaMKII phosphorylation of Nav1.6 at S561 and T642, and that each phosphorylation site regulates distinct biophysical characteristics of the channel. These findings are further extended to investigate CaMKII modulation of disease-linked mutant Nav1.6 channels. I show that different Nav1.6 mutants display distinct responses to CaMKII modulation and reveal that acute CaMKII inhibition attenuates gain-of-function effects produced by mutant channels. Importantly, computational simulations modeling the effects of CaMKII inhibition on WT and mutant Nav1.6 channels demonstrate dramatic reductions in neuronal excitability in Purkinje and cortical pyramidal cell models. Together, these findings suggest that CaMKII modulation of Nav1.6 may be a powerful mechanism to regulate physiological and pathological neuronal excitability.