CaMKII enhances voltage-gated sodium channel Nav1.6 activity and neuronal excitability

Abstract

Nav1.6 is the primary voltage-gated sodium channel isoform expressed in mature axon initial segments and nodes, making it critical for initiation and propagation of neuronal impulses. Thus, Nav1.6 modulation and dysfunction may have profound effects on input-output properties of neurons in normal and pathological conditions. Phosphorylation is a powerful and reversible mechanism regulating ion channel function. Because Nav1.6 and the multifunctional Ca²⁺/CaM-dependent protein kinase II (CaMKII) are independently linked to excitability disorders, we sought to investigate modulation of Nav1.6 function by CaMKII signaling. We show that inhibition of CaMKII, a Ser/Thr protein kinase associated with excitability, synaptic plasticity, and excitability disorders, with the CaMKII-specific peptide inhibitor CN21 reduces transient and persistent currents in Nav1.6-expressing Purkinje neurons by 87%. Using whole-cell voltage clamp of Nav1.6, we show that CaMKII inhibition in ND7/23 and HEK293 cells significantly reduces transient and persistent currents by 72% and produces a 5.8-mV depolarizing shift in the voltage dependence of activation. Immobilized peptide arrays and nanoflow LC-electrospray ionization/MS of Nav1.6 reveal potential sites of CaMKII phosphorylation, specifically Ser-561 and Ser-641/Thr-642 within the first intracellular loop of the channel. Using site-directed mutagenesis to test multiple potential sites of phosphorylation, we show that Ala substitutions of Ser-561 and Ser-641/Thr-642 recapitulate the depolarizing shift in activation and reduction in current density. Computational simulations to model effects of CaMKII inhibition on Nav1.6 function demonstrate dramatic reductions in spontaneous and evoked action potentials in a Purkinje cell model, suggesting that CaMKII modulation of Nav1.6 may be a powerful mechanism to regulate neuronal excitability.

Keywords: Ca2+/calmodulin-dependent protein kinase II (CaMKII); CaMKII; Nav1.6; electrophysiology; phosphoproteomics; phosphorylation; post-translational modification (PTM); sodium channel.