Tao, E. | Search bioRxiv

bioRxiv2023

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.

biophysics↗

bioRxiv2023

A binding site for phosphoinositide modulation of voltage-gated sodium channels described by multiscale simulations

Voltage gated sodium channels (Nav) are membrane proteins which open to facilitate the inward flux of sodium ions into excitable cells. In response to stimuli, Nav channels transition from the resting, closed state to an open, conductive state, before rapidly inactivating. Dysregulation of this functional cycle due to mutations causes diseases including epilepsy, pain conditions and cardiac disorders, making Nav channels a significant pharmacological target. Phosphoinositides are important lipid cofactors for ion channel function. The phosphoinositide PI(4,5)P2 decreases Nav1.4 activity by increasing the difficulty of channel opening, accelerating fast inactivation and slowing recovery from fast inactivation. Using multiscale molecular dynamics simulations, we show that PI(4,5)P2 binds stably to inactivated Nav at a conserved site within the DIV S4-S5 linker, which couples the voltage sensing domain (VSD) to the pore. As the Nav C-terminal domain is proposed to also bind here during recovery from inactivation, we hypothesise that PI(4,5)P2 prolongs inactivation by competitively binding to this site. In atomistic simulations, PI(4,5)P2 reduces the mobility of both the DIV S4-S5 linker and the DIII-IV linker, responsible for fast inactivation, slowing the conformational changes required for the channel to recover to the resting state. We further show that in a resting state Nav model, phosphoinositides bind to VSD gating charges, which may anchor them and impede VSD activation. Our results provide a mechanism by which phosphoinositides alter the voltage dependence of activation and the rate of recovery from inactivation, an important step for the development of novel therapies to treat Nav-related diseases. SignificanceVoltage-gated sodium channels form pores in the membrane to mediate electrical activity in nerve and muscle cells. They play critical roles throughout the human body and their dysfunction leads to diseases including epilepsy, cardiac arrhythmias and pain disorders. Membrane lipids called phosphoinositides have recently been shown to reduce the activity of a voltage-gated sodium channel, but the molecular basis of this mechanism is not known. Here we use simulations to reveal where these lipids bind to the channels and how they reduce channel activity by making it harder for the pores to open and slower to subsequently recover to the closed resting state.

biophysics↗