Dravet Variant SCN1AA1783V Impairs Interneuron Firing Predominantly by Altered Channel Activation

Dravet syndrome (DS) is a developmental epileptic encephalopathy mainly caused by functional NaV1.1 haploinsufficiency in inhibitory interneurons. Recently, a new conditional mouse model expressing the recurrent human p.(Ala1783Val) missense variant has become available. In this study, we provided an electrophysiological characterization of this variant in tsA201 cells, revealing both altered voltage-dependence of activation and slow inactivation without reduced sodium peak current density. Based on these data, simulated interneuron (IN) firing properties in a conductance-based single-compartment model suggested surprisingly similar firing deficits for NaV1.1A1783V and full haploinsufficiency as caused by heterozygous truncation variants. Impaired NaV1.1A1783V channel activation was predicted to have a significantly larger impact on channel function than altered slow inactivation and is therefore proposed as the main mechanism underlying IN dysfunction. The computational model was validated in cortical organotypic slice cultures derived from conditional Scn1aA1783V mice. Pan-neuronal activation of the p.Ala1783V in vitro confirmed a predicted IN firing deficit and revealed an accompanying reduction of interneuronal input resistance while demonstrating normal excitability of pyramidal neurons. Altered input resistance was fed back into the model for further refinement. Taken together these data demonstrate that primary loss of function (LOF) gating properties accompanied by altered membrane characteristics may match effects of full haploinsufficiency on the neuronal level despite maintaining physiological peak current density, thereby causing DS.

Possible Interactions of Extracellular Loop IVP2-S6 With Voltage-Sensing Domain III in Cardiac Sodium Channel

Motion transmission from voltage sensors to inactivation gates is an important problem in the general physiology of ion channels. In a cryo-EM structure of channel hNav1.5, residues N1736 and R1739 in the extracellular loop IVP2-S6 approach glutamates E1225 and E1295, respectively, in the voltage-sensing domain III (VSD-III). ClinVar-reported variants E1230K, E1295K, and R1739W/Q and other variants in loops IVP2-S6, IIIS1-S2, and IIIS3-S4 are associated with cardiac arrhythmias, highlighting the interface between IVP2-S6 and VSD-III as a hot spot of disease mutations. Atomic mechanisms of the channel dysfunction caused by these mutations are unknown. Here, we generated mutants E1295R, R1739E, E1295R/R1739E, and N1736R, expressed them in HEK-293T cells, and explored biophysical properties. Mutation E1295R reduced steady-state fast inactivation and enhanced steady-state slow inactivation. In contrast, mutation R1739E slightly enhanced fast inactivation and attenuated slow inactivation. Characteristics of the double mutant E1295R/R1739E were rather similar to those of the wild-type channel. Mutation N1736R attenuated slow inactivation. Molecular modeling predicted salt bridging of R1739E with the outermost lysine in the activated voltage-sensing helix IIIS4. In contrast, the loss-of-function substitution E1295R repelled R1739, thus destabilizing the activated VSD-III in agreement with our data that E1295R caused a depolarizing shift of the G-V curve. In silico deactivation of VSD-III with constraint-maintained salt bridge E1295-R1739 resulted in the following changes: 1) contacts between IIIS4 and IVS5 were switched; 2) contacts of the linker-helix IIIS4-S5 with IVS5, IVS6, and fast inactivation tripeptide IFM were modified; 3) contacts of the IFM tripeptide with helices IVS5 and IVS6 were altered; 4) mobile loop IVP2-S6 shifted helix IVP2 that contributes to the slow inactivation gate and helix IVS6 that contributes to the fast inactivation gate. The likelihood of salt bridge E1295-R1739 in deactivated VSD-III is supported by Poisson–Boltzmann calculations and state-dependent energetics of loop IVP2-S6. Taken together, our results suggest that loop IVP2-S6 is involved in motion transmission from VSD-III to the inactivation gates.

Regulation and drug modulation of a voltage-gated sodium channel: Pivotal role of the S4–S5 linker in activation and slow inactivation

Voltage-gated sodium (NaV) channels control excitable cell functions. While structural investigations have revealed conformation details of different functional states, the mechanisms of both activation and slow inactivation remain unclear. Here, we identify residue T140 in the S4–S5 linker of the bacterial voltage-gated sodium channel NaChBac as critical for channel activation and drug effects on inactivation. Mutations at T140 either attenuate activation or render the channel nonfunctional. Propofol, a clinical anesthetic known to inhibit NaChBac by promoting slow inactivation, binds to a pocket between the S4–S5 linker and S6 helix in a conformation-dependent manner. Using 19F-NMR to quantify site-specific binding by saturation transfer differences (STDs), we found strong STDs in inactivated, but not activated, NaChBac. Molecular dynamics simulations show a highly dynamic pocket in the activated conformation, limiting STD buildup. In contrast, drug binding to this pocket promotes and stabilizes the inactivated states. Our results provide direct experimental evidence showing distinctly different associations between the S4–S5 linker and S6 helix in activated and inactivated states. Specifically, an exchange occurs between interaction partners T140 and N234 of the same subunit in activation, and T140 and N225 of the domain-swapped subunit in slow inactivation. The drug action on slow inactivation of prokaryotic NaV channels seems to have a mechanism similar to the recently proposed “door-wedge” action of the isoleucine-phenylalanine-methionine (IFM) motif on the fast inactivation of eukaryotic NaV channels. Elucidating this gating mechanism points to a possible direction for conformation-dependent drug development.

Dravet variant SCN1AA1783V impairs interneuron firing predominantly by altered channel activation

Dravet syndrome (DS) is a developmental epileptic encephalopathy mainly caused by functional NaV1.1 haploinsufficiency in interneurons (IN). Recently, a new conditional mouse model expressing the recurrent human p.A1783V missense variant has become available. Here we provide an electrophysiological characterization of this variant in tsA201 cells, revealing both altered voltage-dependence of activation and slow inactivation without reduced sodium peak current density. Simulating IN excitability in a Hodgkin-Huxley one-compartment model suggested surprisingly similar firing deficits for Scn1aA1783V and full haploinsufficiency as caused by heterozygous truncation variants. Impaired NaVA1783V channel activation was predicted to have a significantly larger impact on channel function than altered slow inactivation and is therefore proposed as the main mechanism underlying IN dysfunction. The computational model was validated in cortical organotypic slice cultures derived from conditional Scn1aA1783V mice. Pan-neuronal activation of the p.A1783V variant in vitro confirmed the predicted IN firing deficit while demonstrating normal excitability of pyramidal neurons. Taken together these data demonstrate that despite maintained physiological peak currents density LOF gating properties may match effects of full haploinsufficiency on neuronal level, thereby causing DS.HighlightsNaV1.1A1783V alters voltage-dependence of activation and slow inactivation while not affecting fast inactivation.Depolarizing and hyperpolarizing shifts of activation and slow inactivation curves result in combined channel loss of function (LOF).Simulations of NaV1.1A1783V interneuronal properties indicate reduced action potential firing rates comparable to full SCN1A haploinsufficiency, which is often found in Dravet syndrome.In silico modelling identifies impaired channel activation as the predominant mechanism of channel LOF.Panneuronal induction of Scn1a+/A1783V in a cortical slice culture model confirms restriction of loss of function and its restriction to interneurons.

Regulation of K+ Conductance by a Hydrogen Bond in Kv2.1, Kv2.2, and Kv1.2 Channels

The slow inactivation of voltage-gated potassium (Kv) channels plays an important role in controlling cellular excitability. Recently, the two hydrogen bonds (H-bonds) formed by W434-D447 and T439-Y445 have been reported to control the slow inactivation in Shaker potassium channels. The four residues are highly conserved among Kv channels. Our objective was to find the roles of the two H-bonds in controlling the slow inactivation of mammalian Kv2.1, Kv2.2, and Kv1.2 channels by point mutation and patch-clamp recording studies. We found that mutations of the residues equivalent to W434 and T439 in Shaker did not change the slow inactivation of the Kv2.1, Kv2.2, and Kv1.2 channels. Surprisingly, breaking of the inter-subunit H-bond formed by W366 and Y376 (Kv2.1 numbering) by various mutations resulted in the complete loss of K+ conductance of the three Kv channels. In conclusion, we found differences in the H-bonds controlling the slow inactivation of the mammalian Kv channels and Shaker channels. Our data provided the first evidence, to our knowledge, that the inter-subunit H-bond formed by W366 and Y376 plays an important role in regulating the K+ conductance of mammalian Kv2.1, Kv2.2, and Kv1.2 channels.

Differential Effects of Modified Batrachotoxins on Voltage-gated Sodium Channel Fast and Slow Inactivation

Voltage-gated sodium channels (Na_Vs), large transmembrane protein complexes responsible for the initiation and propagation of action potentials, are targets for a number of acute poisons. Many of these agents act as allosteric modulators of channel activity and serve as powerful chemical tools for understanding channel function. Batrachotoxin (BTX) is a steroidal amine derivative most commonly associated with poison dart frogs and is unique as a Na_V ligand in that it alters every property of the channel, including threshold potential of activation, inactivation, ion selectivity, and ion conduction. Structure-function studies with BTX are limited, however, by the inability to access preparative quantities of this compound from natural sources. We have addressed this problem through <i>de novo</i> synthesis of BTX, which gives access to modified toxin structures. In this report, we detail electrophysiology studies of three BTX C20-ester derivatives against recombinant Na_V subtypes (rat Na_V1.4 and human Na_V1.5). Two of these compounds, BTX-B and BTX-^cHx, are functionally equivalent to BTX, hyperpolarizing channel activation and blocking both fast and slow inactivation. BTX-yne—a C20-<i>n</i>-heptynoate ester—is a conspicuous outlier, eliminating fast but not slow inactivation. This unique property qualifies BTX-yne as the first reported Na_V modulator that separates inactivation processes. These findings are supported by functional studies with bacterial Na_Vs (BacNa_Vs) that lack a fast inactivation gate. The availability of BTX-yne should advance future efforts aimed at understanding Na_V gating mechanisms and designing allosteric regulators of Na_V activity.

Differential Effects of Modified Batrachotoxins on Voltage-gated Sodium Channel Fast and Slow Inactivation

Voltage-gated sodium channels (Na_Vs), large transmembrane protein complexes responsible for the initiation and propagation of action potentials, are targets for a number of acute poisons. Many of these agents act as allosteric modulators of channel activity and serve as powerful chemical tools for understanding channel function. Batrachotoxin (BTX) is a steroidal amine derivative most commonly associated with poison dart frogs and is unique as a Na_V ligand in that it alters every property of the channel, including threshold potential of activation, inactivation, ion selectivity, and ion conduction. Structure-function studies with BTX are limited, however, by the inability to access preparative quantities of this compound from natural sources. We have addressed this problem through <i>de novo</i> synthesis of BTX, which gives access to modified toxin structures. In this report, we detail electrophysiology studies of three BTX C20-ester derivatives against recombinant Na_V subtypes (rat Na_V1.4 and human Na_V1.5). Two of these compounds, BTX-B and BTX-^cHx, are functionally equivalent to BTX, hyperpolarizing channel activation and blocking both fast and slow inactivation. BTX-yne—a C20-<i>n</i>-heptynoate ester—is a conspicuous outlier, eliminating fast but not slow inactivation. This unique property qualifies BTX-yne as the first reported Na_V modulator that separates inactivation processes. These findings are supported by functional studies with bacterial Na_Vs (BacNa_Vs) that lack a fast inactivation gate. The availability of BTX-yne should advance future efforts aimed at understanding Na_V gating mechanisms and designing allosteric regulators of Na_V activity.