Complex biophysical changes and reduced neuronal firing in an SCN8A variant associated with developmental delay and epilepsy

Summary Background Mutations in the SCN8A gene, encoding the voltage-gated sodium channel Na V 1.6, lead to various neurodevelopmental disorders. The SCN8A p.(Gly1625Arg) mutation (Na V 1.6 G1625R ) was identified in a patient diagnosed with developmental epileptic encephalopathy (DEE), presenting with moderate epilepsy and severe developmental delay. Methods We performed biophysical and neurophysiological characterizations of Na V 1.6 G1625R in Neuro-2a cells and cultured hippocampal neurons, followed by computational modeling to determine the impact of its heterozygous expression on cortical neuron function. Findings Voltage-clamp analyses of Na V 1.6 G1625R demonstrated a heterogeneous mixture of gain-and loss-of-function properties, including reduced current amplitudes, a marked increase in the time constant of fast voltage-dependent inactivation and a depolarizing shift in the voltage dependence of inactivation. Recordings in transfected cultured neurons showed that these intricate biophysical properties had a minor effect on neuronal excitability when firing relayed on both endogenous and transfected Na V channels. Conversely, there was a marked reduction in the number of action potentials when firing was driven by the transfected mutant Na V 1.6 channels. Computational modeling of mature cortical neurons further revealed a mild reduction in neuronal firing when mimicking the patients’ heterozygous Na V 1.6 G1625R expression. Structural modeling of Na V 1.6 G1625R and a double-mutant cycle analysis suggested the possible formation of pathophysiologically-relevant cation-π interaction between R1625 and F1588, affecting the voltage dependence of inactivation. Interpretation Our analyses demonstrate a complex combination of gain and loss-of-function changes resulting in an overall mild reduction in neuronal firing, related to a perturbed interaction network within the voltage sensor domain. Funding ISF, DFG, BMBF, The Hartwell Foundation, ICRF, ISCA Research in context Evidence before this study Mutations in the SCN8A gene, encoding the voltage-gated sodium channel Na V 1.6, result in multiple neurodevelopmental syndromes ranging from benign epilepsy to developmental delay without epilepsy or developmental epileptic encephalopathy (DEE). Recent studies established that most DEE-causing SCN8A mutations result in a gain of function effect. However, several SCN8A mutations that diverge from this pattern were described. Added value of this study We performed a multi-tiered study of the SCN8A p.(Gly1625Arg) variant (Na V 1.6 G1625R ), identified in a patient with atypical DEE presentation, featuring moderate epilepsy that is well controlled by the sodium channel blocker Oxcarbazepine, along with profound stagnated developmental delay. This variant is positioned within the S4 segment of domain IV, a critical region for Na V 1.6 function, where pathogenic variants were shown to cause either a loss or gain of channel function, but often with mixed biophysical alterations. Our biophysical characterization of Na V 1.6 G1625R in Neuro-2a cells demonstrated complex gain-and loss-of-function properties, cumulating to reduced firing in cultured hippocampal neurons and computational modeling of mature cortical neurons, demonstrating an overall loss-of-function effect. Implications of all the available evidence Our results indicate the necessity for combined biophysical and neuronal characterization of individual SCN8A variants, especially those presenting with complex biophysical changes or atypical clinical presentation. Moreover, while sodium channel blockers are the recommended treatment for SCN8A variants associated with gain-of-function, additional considerations may be needed for DEE-causing variants that are associated with mild loss-of-function.