The pathogenic p.N1662D SCN2A mutation reveals an essential molecular interaction for Nav1.2 channel inactivation

Mutations in the SCN2A gene encoding the Nav1.2 sodium channel can lead to neurodevelopmental disorders. We studied the N1662D variant associated with severe early-onset developmental and epileptic encephalopathy (DEE). The N1662D mutation almost completely prevented fast inactivation without affecting activation. The comparison of wild-type and N1662D channel structures suggested that the ambifunctional hydrogen bond formation between residues N1662 and Q1494 is essential for fast inactivation. Fast inactivation could also be prevented with engineered Q1494A or Q1494L Nav1.2 channel variants, whereas Q1494E or Q1494K variants resulted in incomplete inactivation and persistent current. Molecular dynamics simulations revealed a reduced affinity of the hydrophobic IFM-motif to its receptor site with N1662D and Q1494L variants relative to wild-type. These results demonstrate that the interactions between N1662 and Q1494 underpin the stability and the orientation of the inactivation gate and are essential for the development of fast inactivation. Six DEE-associated Nav1.2 variants, with mutations mapped to channel segments known to be implicated in fast inactivation were also evaluated. Remarkably, the L1657P variant also prevented fast inactivation and produced biophysical characteristics similar to N1662D, whereas the M1501V, M1501T, F1651C, P1658S, and A1659V variants resulted in biophysical properties that were consistent with gain-of-function and enhanced action potential firing of hybrid neurons in dynamic action potential clamp experiments. Paradoxically, low densities of N1662D or L1657P currents potentiated action potential firing, whereas increased densities resulted in sustained depolarization. The contribution of non-inactivating Nav1.2 channels to neuronal excitability may constitute a novel cellular mechanism in the pathogenesis of SCN2A-related DEE. SIGNIFICANCE STATEMENTSCN2A gene-related early-onset developmental and epileptic encephalopathy (EO-DEE) is a rare and severe disorder that manifests in early infancy and childhood. SCN2A mutations affecting the fast inactivation gating mechanism can cause altered voltage dependence and incomplete inactivation of the encoded Nav1.2 channel, leading to abnormal neuronal excitability. In this biophysical and clinical study of neuronal Nav1.2 variants, we identified amino acid residues that are critical for the stability and orientation of the inactivation gate during fast inactivation. Mutations of these residues prevent fast inactivation and may lead to EO-DEE via a novel pathophysiological mechanism. The results provide novel structural insights into the molecular mechanism of Nav1.2 channel fast inactivation and inform treatment strategies for SCN2A-related EO-DEE.