CaMKII Inhibition Attenuates Distinct Gain-of-Function Effects Produced by Mutant Nav1.6 Channels and Reduces Neuronal Excitability.

Aberrant Nav1.6 activity can induce hyperexcitability associated with epilepsy. Gain-of-function mutations in the SCN8A gene encoding Nav1.6 are linked to epilepsy development; however, the molecular mechanisms mediating these changes are remarkably heterogeneous and may involve post-translational regulation of Nav1.6. Because calcium/calmodulin-dependent protein kinase II (CaMKII) is a powerful modulator of Nav1.6 channels, we investigated whether CaMKII modulates disease-linked Nav1.6 mutants. Whole-cell voltage clamp recordings in ND7/23 cells show that CaMKII inhibition of the epilepsy-related mutation R850Q largely recapitulates the effects previously observed for WT Nav1.6. We also characterized a rare missense variant, R639C, located within a regulatory hotspot for CaMKII modulation of Nav1.6. Prediction software algorithms and electrophysiological recordings revealed gain-of-function effects for R639C mutant channel activity, including increased sodium currents and hyperpolarized activation compared to WT Nav1.6. Importantly, the R639C mutation ablates CaMKII phosphorylation at a key regulatory site, T642, and, in contrast to WT and R850Q channels, displays a distinct response to CaMKII inhibition. Computational simulations demonstrate that modeled neurons harboring the R639C or R850Q mutations are hyperexcitable, and simulating the effects of CaMKII inhibition on Nav1.6 activity in modeled neurons differentially reduced hyperexcitability. Acute CaMKII inhibition may represent a promising mechanism to attenuate gain-of-function effects produced by Nav1.6 mutations.

Neural Activity Correlates With Behavior Effects of Anti-Seizure Drugs Efficacy Using the Zebrafish Pentylenetetrazol Seizure Model.

Approximately 30% of patients with epilepsy do not achieve adequate seizure control through current anti-seizure drugs and treatment methods. Therefore, a critical need exists to efficiently screen anti-seizure drugs to enhance our ability to tailor treatment protocols and improve patient outcomes. The zebrafish pentylenetetrazol (PTZ) seizure model has become an increasingly popular screening paradigm for novel anti-seizure compounds. However, previous research using this model was variable due to differing experimental methods. Here, we present a method that was optimized to improve reliability and reproducibility in our laboratory using this PTZ model to develop a more robust screening of anti-seizure drugs comparing behavior and neural activity. Our behavior assay, spanning 90 min using 10 mM PTZ on 7 days post fertilization zebrafish, provides a broad window to observe anti-seizure drug efficacy. To compare our method with previously published data, we tested carbamazepine, lamotrigine, and topiramate, which have been tested in previous PTZ zebrafish assays. In addition, we assessed the candidate anti-seizure compound GS967, which has not been previously tested in the zebrafish seizure model. We examined the efficacy of anti-seizure drugs by acute administration concurrent with PTZ application and by pretreatment prior to exposure with PTZ. Pretreatment permitted us to examine potential neuroprotection and determine whether treatment time affects anti-seizure drugs' responses. As independent validation of anti-seizure drugs' effects, we evaluated whether the anti-seizure drug efficacy in the behavioral assay correlated with neural activity measurements, using electroencephalogram (EEG) and calcium signaling using GCaMP. There was no significant difference in the reduction of PTZ-induced seizure behavior activity between the pretreatment groups and acute treatment groups. Acute treatment with anti-seizure drugs in the EEG and GCaMP assays from 15 to 30 min post-anti-seizure drug exposure revealed consistent results between behavioral, EEG, and GCaMP assays for two of the three anti-seizure drugs. Lamotrigine only reduced neural activity (EEG and GCaMP assays). Carbamazepine, topiramate, and GS967 reduced activity in all three assays. The findings show that EEG and GCaMP assays largely correlate with the behavior findings, helping us connect physiological and behavior responses to anti-seizure drug and better assess anti-seizure drug efficacy.

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

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.

Constitutive regulation of the glutamate/aspartate transporter EAAT1 by Calcium-Calmodulin-Dependent Protein Kinase II.

Glutamate clearance by astrocytes is an essential part of normal excitatory neurotransmission. Failure to adapt or maintain low levels of glutamate in the central nervous system is associated with multiple acute and chronic neurodegenerative diseases. The primary excitatory amino acid transporters in human astrocytes are EAAT1 and EAAT2 (GLAST and GLT-1, respectively, in rodents). While the inhibition of calcium/calmodulin-dependent kinase (CaMKII), a ubiquitously expressed serine/threonine protein kinase, results in diminished glutamate uptake in cultured primary rodent astrocytes (Ashpole et al. 2013), the molecular mechanism underlying this regulation is unknown. Here, we use a heterologous expression model to explore CaMKII regulation of EAAT1 and EAAT2. In transiently transfected HEK293T cells, pharmacological inhibition of CaMKII (using KN-93 or tat-CN21) reduces [3 H]-glutamate uptake in EAAT1 without altering EAAT2-mediated glutamate uptake. While over-expressing the Thr287Asp mutant to enhance autonomous CaMKII activity had no effect on either EAAT1 or EAAT2-mediated glutamate uptake, over-expressing a dominant-negative version of CaMKII (Asp136Asn) diminished EAAT1 glutamate uptake. SPOTS peptide arrays and recombinant glutathione S-transferase-fusion proteins of the intracellular N- and C-termini of EAAT1 identified two potential phosphorylation sites at residues Thr26 and Thr37 in the N-terminus. Introducing an Ala (a non-phospho mimetic) at Thr37 diminished EAAT1-mediated glutamate uptake, suggesting that the phosphorylation state of this residue is important for constitutive EAAT1 function. Our study is the first to identify a glutamate transporter as a direct CaMKII substrate and suggests that CaMKII signaling is a critical driver of constitutive glutamate uptake by EAAT1.