Distinctive In Vitro Phenotypes in iPSC-Derived Neurons From Patients With Gain- and Loss-of-Function SCN2A Developmental and Epileptic Encephalopathy.

SCN2A encodes NaV1.2, an excitatory neuron voltage-gated sodium channel and a major monogenic cause of neurodevelopmental disorders, including developmental and epileptic encephalopathies (DEE) and autism. Clinical presentation and pharmocosensitivity vary with the nature of SCN2A variant dysfunction and can be divided into gain-of-function (GoF) cases with pre- or peri-natal seizures and loss-of-function (LoF) patients typically having infantile spasms after 6 months of age. We established and assessed patient induced pluripotent stem cell (iPSC) - derived neuronal models for two recurrent SCN2A DEE variants with GoF R1882Q and LoF R853Q associated with early- and late-onset DEE, respectively. Two male patient-derived iPSC isogenic pairs were differentiated using Neurogenin-2 overexpression yielding populations of cortical-like glutamatergic neurons. Functional properties were assessed using patch clamp and multielectrode array recordings and transcriptomic profiles obtained with total mRNA sequencing after 2-4 weeks in culture. At 3 weeks of differentiation, increased neuronal activity at cellular and network levels was observed for R1882Q iPSC-derived neurons. In contrast, R853Q neurons showed only subtle changes in excitability after 4 weeks and an overall reduced network activity after 7 weeks in vitro. Consistent with the reported efficacy in some GoF SCN2A patients, phenytoin (sodium channel blocker) reduced the excitability of neurons to the control levels in R1882Q neuronal cultures. Transcriptomic alterations in neurons were detected for each variant and convergent pathways suggested potential shared mechanisms underlying SCN2A DEE. In summary, patient iPSC-derived neuronal models of SCN2A GoF and LoF pathogenic variants causing DEE show specific functional and transcriptomic in vitro phenotypes.

Nav1.2 channel mutations preventing fast inactivation lead to SCN2A encephalopathy.

SCN2A gene-related early-infantile developmental and epileptic encephalopathy (EI-DEE) is a rare and severe disorder that manifests in early infancy. SCN2A mutations affecting the fast inactivation gating mechanism can result in altered voltage dependence and incomplete inactivation of the encoded neuronal Nav1.2 channel and lead to abnormal neuronal excitability. In this study, we evaluated clinical data of seven missense Nav1.2 variants associated with DEE and performed molecular dynamics simulations, patch-clamp electrophysiology, and dynamic clamp real-time neuronal modelling to elucidate the molecular and neuron-scale phenotypic consequences of the mutations. 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 Q1494 K 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 that were similar to those of N1662D, whereas the M1501 V, M1501T, F1651C, P1658S, and A1659 V 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. Our results provide novel structural insights into the molecular mechanism of Nav1.2 channel fast inactivation and inform treatment strategies for SCN2A-related EI-DEE. The contribution of non-inactivating Nav1.2 channels to neuronal excitability may constitute a distinct cellular mechanism in the pathogenesis of SCN2A-related DEE.

Generation of a stably transfected mouse embryonic stem cell line for inducible differentiation to excitatory neurons.

In vitro differentiation of stem cells into various cell lineages is valuable in developmental studies and an important source of cells for modelling physiology and pathology, particularly for complex tissues such as the brain. Conventional protocols for in vitro neuronal differentiation often suffer from complicated procedures, high variability and low reproducibility. Over the last decade, the identification of cell fate-determining transcription factors has provided new tools for cellular studies in neuroscience and enabled rapid differentiation driven by ectopic transcription factor expression. As a proneural transcription factor, Neurogenin 2 (Ngn2) expression alone is sufficient to trigger rapid and robust neurogenesis from pluripotent cells. Here, we established a stable cell line, by piggyBac (PB) transposition, that conditionally expresses Ngn2 for generation of excitatory neurons from mouse embryonic stem cells (ESCs) using an all-in-one PB construct. Our results indicate that Ngn2-induced excitatory neurons have mature and functional characteristics consistent with previous studies using conventional differentiation methods. This approach provides an all-in-one PB construct for rapid and high copy number gene delivery of dox-inducible transcription factors to induce differentiation. This approach is a valuable in vitro cell model for disease modeling, drug screening and cell therapy.

Carbogen-Induced Respiratory Acidosis Blocks Experimental Seizures by a Direct and Specific Inhibition of NaV1.2 Channels in the Axon Initial Segment of Pyramidal Neurons.

Brain pH is a critical factor for determining neuronal activity, with alkalosis increasing and acidosis reducing excitability. Acid shifts in brain pH through the breathing of carbogen (5% CO2/95% O2) reduces seizure susceptibility in animal models and patients. The molecular mechanisms underlying this seizure protection remain to be fully elucidated. Here, we demonstrate that male and female mice exposed to carbogen are fully protected from thermogenic-triggered seizures. Whole-cell patch-clamp recordings revealed that acid shifts in extracellular pH (pHo) significantly reduce action potential firing in CA1 pyramidal neurons but did not alter firing in hippocampal inhibitory interneurons. In real-time dynamic clamp experiments, acidification reduced simulated action potential firing generated in hybrid model neurons expressing the excitatory neuron predominant NaV1.2 channel. Conversely, acidification had no effect on action potential firing in hybrid model neurons expressing the interneuron predominant NaV1.1 channel. Furthermore, knockdown of Scn2a mRNA in vivo using antisense oligonucleotides reduced the protective effects of carbogen on seizure susceptibility. Both carbogen-mediated seizure protection and the reduction in CA1 pyramidal neuron action potential firing by low pHo were maintained in an Asic1a knock-out mouse ruling out this acid-sensing channel as the underlying molecular target. These data indicate that the acid-mediated reduction in excitatory neuron firing is mediated, at least in part, through the inhibition of NaV1.2 channels, whereas inhibitory neuron firing is unaffected. This reduction in pyramidal neuron excitability is the likely basis of seizure suppression caused by carbogen-mediated acidification.SIGNIFICANCE STATEMENT Brain pH has long been known to modulate neuronal excitability. Here, we confirm that brain acidification reduces seizure susceptibility in a mouse model of thermogenic seizures. Extracellular acidification reduced excitatory pyramidal neuron firing while having no effect on interneuron firing. Acidification also reduced dynamic clamp firing in cells expressing the NaV1.2 channel but not in cells expressing NaV1.1 channels. In vivo knockdown of Scn2a mRNA reduced seizure protection of acidification. In contrast, acid-mediated seizure protection was maintained in the Asic1a knock-out mouse. These data suggest NaV1.2 channel as an important target for acid-mediated seizure protection. Our results have implications on how natural variations in pH can modulate neuronal excitability and highlight potential antiseizure drug development strategies based on the NaV1.2 channel.

Progressive myoclonus epilepsies-Residual unsolved cases have marked genetic heterogeneity including dolichol-dependent protein glycosylation pathway genes.

Progressive myoclonus epilepsies (PMEs) comprise a group of clinically and genetically heterogeneous rare diseases. Over 70% of PME cases can now be molecularly solved. Known PME genes encode a variety of proteins, many involved in lysosomal and endosomal function. We performed whole-exome sequencing (WES) in 84 (78 unrelated) unsolved PME-affected individuals, with or without additional family members, to discover novel causes. We identified likely disease-causing variants in 24 out of 78 (31%) unrelated individuals, despite previous genetic analyses. The diagnostic yield was significantly higher for individuals studied as trios or families (14/28) versus singletons (10/50) (OR = 3.9, p value = 0.01, Fisher's exact test). The 24 likely solved cases of PME involved 18 genes. First, we found and functionally validated five heterozygous variants in NUS1 and DHDDS and a homozygous variant in ALG10, with no previous disease associations. All three genes are involved in dolichol-dependent protein glycosylation, a pathway not previously implicated in PME. Second, we independently validate SEMA6B as a dominant PME gene in two unrelated individuals. Third, in five families, we identified variants in established PME genes; three with intronic or copy-number changes (CLN6, GBA, NEU1) and two very rare causes (ASAH1, CERS1). Fourth, we found a group of genes usually associated with developmental and epileptic encephalopathies, but here, remarkably, presenting as PME, with or without prior developmental delay. Our systematic analysis of these cases suggests that the small residuum of unsolved cases will most likely be a collection of very rare, genetically heterogeneous etiologies.

The zebrafish grime mutant uncovers an evolutionarily conserved role for Tmem161b in the control of cardiac rhythm.

The establishment of cardiac function in the developing embryo is essential to ensure blood flow and, therefore, growth and survival of the animal. The molecular mechanisms controlling normal cardiac rhythm remain to be fully elucidated. From a forward genetic screen, we identified a unique mutant, grime, that displayed a specific cardiac arrhythmia phenotype. We show that loss-of-function mutations in tmem161b are responsible for the phenotype, identifying Tmem161b as a regulator of cardiac rhythm in zebrafish. To examine the evolutionary conservation of this function, we generated knockout mice for Tmem161b. Tmem161b knockout mice are neonatal lethal and cardiomyocytes exhibit arrhythmic calcium oscillations. Mechanistically, we find that Tmem161b is expressed at the cell membrane of excitable cells and live imaging shows it is required for action potential repolarization in the developing heart. Electrophysiology on isolated cardiomyocytes demonstrates that Tmem161b is essential to inhibit Ca2+ and K+ currents in cardiomyocytes. Importantly, Tmem161b haploinsufficiency leads to cardiac rhythm phenotypes, implicating it as a candidate gene in heritable cardiac arrhythmia. Overall, these data describe Tmem161b as a highly conserved regulator of cardiac rhythm that functions to modulate ion channel activity in zebrafish and mice.

Biophysical characterization and modelling of SCN1A gain-of-function predicts interneuron hyperexcitability and a predisposition to network instability through homeostatic plasticity.

SCN1A gain-of-function variants are associated with early onset developmental and epileptic encephalopathies (DEEs) that possess distinct clinical features compared to Dravet syndrome caused by SCN1A loss-of-function. However, it is unclear how SCN1A gain-of-function may predispose to cortical hyper-excitability and seizures. Here, we first report the clinical features of a patient carrying a de novo SCN1A variant (T162I) associated with neonatal-onset DEE, and then characterize the biophysical properties of T162I and three other SCN1A variants associated with neonatal-onset DEE (I236V) and early infantile DEE (P1345S, R1636Q). In voltage clamp experiments, three variants (T162I, P1345S and R1636Q) exhibited changes in activation and inactivation properties that enhanced window current, consistent with gain-of-function. Dynamic action potential clamp experiments utilising model neurons incorporating Nav1.1. channels supported a gain-of-function mechanism for all four variants. Here, the T162I, I236V, P1345S, and R1636Q variants exhibited higher peak firing rates relative to wild type and the T162I and R1636Q variants produced a hyperpolarized threshold and reduced neuronal rheobase. To explore the impact of these variants upon cortical excitability, we used a spiking network model containing an excitatory pyramidal cell (PC) and parvalbumin positive (PV) interneuron population. SCN1A gain-of-function was modelled by enhancing the excitability of PV interneurons and then incorporating three simple forms of homeostatic plasticity that restored pyramidal cell firing rates. We found that homeostatic plasticity mechanisms exerted differential impact upon network function, with changes to PV-to-PC and PC-to-PC synaptic strength predisposing to network instability. Overall, our findings support a role for SCN1A gain-of-function and inhibitory interneuron hyperexcitability in early onset DEE. We propose a mechanism through which homeostatic plasticity pathways can predispose to pathological excitatory activity and contribute to phenotypic variability in SCN1A disorders.

Sodium channel expression and transcript variation in the developing brain of human, Rhesus monkey, and mouse.

Genetic variation in voltage-gated sodium (NaV) channels is a significant contributor to neurodevelopmental disorders. NaV channel alpha subunits are encoded by the SCNxA family and four are predominately expressed in the brain: SCN1A, SCN2A, SCN3A, and SCN8A. Gene expression is developmentally regulated, and they are known to express functionally distinct transcript variants. Precision therapies targeting these genes and their transcript variants are currently in preclinical development, yet the developmental expression of these transcripts in the human brain is yet to be fully understood. Additionally, the functional consequences of some mutations differ depending on the studied channel isoform, suggesting differential transcript variant expression can affect disease prognoses. We characterise the expression of the four SCNxAs and their transcript variants in human, Rhesus monkey and mouse brain using publicly available RNA-sequencing data and analysis tools, demonstrating that this approach can be used to answer important biological questions of gene and transcript developmental regulation. We find that gene expression and transcript variant regulation are conserved across species at similar developmental stages and determine the developmental milestones for transcript variant expression. Our study provides a guide to researchers testing therapies and clinicians advising prognoses based on the expression of channel isoforms.

Dynamic action potential clamp predicts functional separation in mild familial and severe de novo forms of SCN2A epilepsy.

De novo variants in SCN2A developmental and epileptic encephalopathy (DEE) show distinctive genotype-phenotype correlations. The two most recurrent SCN2A variants in DEE, R1882Q and R853Q, are associated with different ages and seizure types at onset. R1882Q presents on day 1 of life with focal seizures, while infantile spasms is the dominant seizure type seen in R853Q cases, presenting at a median age of 8 months. Voltage clamp, which characterizes the functional properties of ion channels, predicted gain-of-function for R1882Q and loss-of-function for R853Q. Dynamic action potential clamp, that we implement here as a method for modeling neurophysiological consequences of a given epilepsy variant, predicted that the R1882Q variant would cause a dramatic increase in firing, whereas the R853Q variant would cause a marked reduction in action potential firing. Dynamic clamp was also able to functionally separate the L1563V variant, seen in benign familial neonatal-infantile seizures from R1882Q, seen in DEE, suggesting a diagnostic potential for this type of analysis. Overall, the study shows a strong correlation between clinical phenotype, SCN2A genotype, and functional modeling. Dynamic clamp is well positioned to impact our understanding of pathomechanisms and for development of disease mechanism-targeted therapies in genetic epilepsy.

SCN1A gain of function in early infantile encephalopathy.

OBJECTIVE: To elucidate the biophysical basis underlying the distinct and severe clinical presentation in patients with the recurrent missense SCN1A variant, p.Thr226Met. Patients with this variant show a well-defined genotype-phenotype correlation and present with developmental and early infantile epileptic encephalopathy that is far more severe than typical SCN1A Dravet syndrome. METHODS: Whole cell patch clamp and dynamic action potential clamp were used to study T226M Nav 1.1 channels expressed in mammalian cells. Computational modeling was used to explore the neuronal scale mechanisms that account for altered action potential firing. RESULTS: T226M channels exhibited hyperpolarizing shifts of the activation and inactivation curves and enhanced fast inactivation. Dynamic action potential clamp hybrid simulation showed that model neurons containing T226M conductance displayed a left shift in rheobase relative to control. At current stimulation levels that produced repetitive action potential firing in control model neurons, depolarization block and cessation of action potential firing occurred in T226M model neurons. Fully computationally simulated neuron models recapitulated the findings from dynamic action potential clamp and showed that heterozygous T226M models were also more susceptible to depolarization block. INTERPRETATION: From a biophysical perspective, the T226M mutation produces gain of function. Somewhat paradoxically, our data suggest that this gain of function would cause interneurons to more readily develop depolarization block. This "functional dominant negative" interaction would produce a more profound disinhibition than seen with haploinsufficiency that is typical of Dravet syndrome and could readily explain the more severe phenotype of patients with T226M mutation. Ann Neurol 2019;85:514-525.

Novel Missense CACNA1G Mutations Associated with Infantile-Onset Developmental and Epileptic Encephalopathy.

The CACNA1G gene encodes the low-voltage-activated Cav3.1 channel, which is expressed in various areas of the CNS, including the cerebellum. We studied two missense CACNA1G variants, p.L208P and p.L909F, and evaluated the relationships between the severity of Cav3.1 dysfunction and the clinical phenotype. The presentation was of a developmental and epileptic encephalopathy without evident cerebellar atrophy. Both patients exhibited axial hypotonia, developmental delay, and severe to profound cognitive impairment. The patient with the L909F mutation had initially refractory seizures and cerebellar ataxia, whereas the L208P patient had seizures only transiently but was overall more severely affected. In transfected mammalian cells, we determined the biophysical characteristics of L208P and L909F variants, relative to the wild-type channel and a previously reported gain-of-function Cav3.1 variant. The L208P mutation shifted the activation and inactivation curves to the hyperpolarized direction, slowed the kinetics of inactivation and deactivation, and reduced the availability of Ca2+ current during repetitive stimuli. The L909F mutation impacted channel function less severely, resulting in a hyperpolarizing shift of the activation curve and slower deactivation. These data suggest that L909F results in gain-of-function, whereas L208P exhibits mixed gain-of-function and loss-of-function effects due to opposing changes in the biophysical properties. Our study expands the clinical spectrum associated with CACNA1G mutations, corroborating further the causal association with distinct complex phenotypes.

Voltage-Gated R-Type Calcium Channel Inhibition via Human µ-, δ-, and κ-opioid Receptors Is Voltage-Independently Mediated by Gßγ Protein Subunits.

Elucidating the mechanisms that modulate calcium channels via opioid receptor activation is fundamental to our understanding of both pain perception and how opioids modulate pain. Neuronal voltage-gated N-type calcium channels (Cav2.2) are inhibited by activation of G protein-coupled opioid receptors (ORs). However, inhibition of R-type (Cav2.3) channels by µ- or κ-ORs is poorly defined and has not been reported for δ-ORs. To investigate such interactions, we coexpressed human µ-, δ-, or κ-ORs with human Cav2.3 or Cav2.2 in human embryonic kidney 293 cells and measured depolarization-activated Ba(2+) currents (IBa). Selective agonists of µ-, δ-, and κ-ORs inhibited IBa through Cav2.3 channels by 35%. Cav2.2 channels were inhibited to a similar extent by κ-ORs, but more potently (60%) via µ- and δ-ORs. Antagonists of δ- and κ-ORs potentiated IBa amplitude mediated by Cav2.3 and Cav2.2 channels. Consistent with G protein ßγ (Gßγ) interaction, modulation of Cav2.2 was primarily voltage-dependent and transiently relieved by depolarizing prepulses. In contrast, Cav2.3 modulation was voltage-independent and unaffected by depolarizing prepulses. However, Cav2.3 inhibition was sensitive to pertussis toxin and to intracellular application of guanosine 5'-[ß-thio]diphosphate trilithium salt and guanosine 5'-[γ-thio]triphosphate tetralithium salt. Coexpression of Gßγ-specific scavengers-namely, the carboxyl terminus of the G protein-coupled receptor kinase 2 or membrane-targeted myristoylated-phosducin-attenuated or abolished Cav2.3 modulation. Our study reveals the diversity of OR-mediated signaling at Cav2 channels and identifies neuronal Cav2.3 channels as potential targets for opioid analgesics. Their novel modulation is dependent on pre-existing OR activity and mediated by membrane-delimited Gßγ subunits in a voltage-independent manner.

Effects of linker sequence modifications on the structure, stability, and biological activity of a cyclic α-conotoxin.

The cyclic conotoxin analogue cVc1.1 is a promising lead molecule for the development of new treatments for neuropathic and chronic pain. The design of this peptide includes a linker sequence that joins the N and C termini together, improving peptide stability while maintaining the structure and activity of the original linear Vc1.1. The effect of linker length on the structure, activity and stability of cyclised conotoxins has been studied previously but the effect of altering the composition of the linker sequence has not been investigated. In this study, we designed three analogues of cVc1.1 with linker sequences that varied in charge, hydrophobicity and hydrogen bonding capacity and examined the effect on structure, stability, membrane permeability and biological activity. The three designed peptides were successfully synthesized using solid phase peptide synthesis approaches and had similar structures and stability compared with cVc1.1. Despite modifications in charge, hydrophobicity and hydrogen bonding potential, which are all factors that can affect membrane permeability, no changes in the ability of the peptides to pass through membranes in either PAMPA or Caco-2 cell assay were observed. Surprisingly, modification of the linker sequence was deleterious to biological activity. These results suggest the linker sequence might be a useful part of the molecule for optimization of bioactivity and not just the physiochemical properties of cVc1.1. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 864-875, 2016.

Structure-Activity Studies of Cysteine-Rich α-Conotoxins that Inhibit High-Voltage-Activated Calcium Channels via GABA(B) Receptor Activation Reveal a Minimal Functional Motif.

α-Conotoxins are disulfide-rich peptides that target nicotinic acetylcholine receptors. Recently we identified several α-conotoxins that also modulate voltage-gated calcium channels by acting as G protein-coupled GABA(B) receptor (GABA(B)R) agonists. These α-conotoxins are promising drug leads for the treatment of chronic pain. To elucidate the diversity of α-conotoxins that act through this mechanism, we synthesized and characterized a set of peptides with homology to α-conotoxins known to inhibit high voltage-activated calcium channels via GABA(B)R activation. Remarkably, all disulfide isomers of the active α-conotoxins Pu1.2 and Pn1.2, and the previously studied Vc1.1 showed similar levels of biological activity. Structure determination by NMR spectroscopy helped us identify a simplified biologically active eight residue peptide motif containing a single disulfide bond that is an excellent lead molecule for developing a new generation of analgesic peptide drugs.

Mechanisms of conotoxin inhibition of N-type (Ca(v)2.2) calcium channels.

N-type (Ca(v)2.2) voltage-gated calcium channels (VGCC) transduce electrical activity into other cellular functions, regulate calcium homeostasis and play a major role in processing pain information. Although the distribution and function of these channels vary widely among different classes of neurons, they are predominantly expressed in nerve terminals, where they control neurotransmitter release. To date, genetic and pharmacological studies have identified that high-threshold, N-type VGCCs are important for pain sensation in disease models. This suggests that N-type VGCC inhibitors or modulators could be developed into useful drugs to treat neuropathic pain. This review discusses the role of N-type (Ca(v)2.2) VGCCs in nociception and pain transmission through primary sensory dorsal root ganglion (DRG) neurons (nociceptors). It also outlines the potent and selective inhibition of N-type VGCCs by conotoxins, small disulfide-rich peptides isolated from the venom of marine cone snails. Of these conotoxins, ω-conotoxins are selective N-type VGCC antagonists that preferentially block nociception in inflammatory pain models, and allodynia and/or hyperalgesia in neuropathic pain models. Another conotoxin family, α-conotoxins, were initially proposed as competitive antagonists of muscle and neuronal nicotinic acetylcholine receptors (nAChR). Surprisingly, however, α-conotoxins Vc1.1 and RgIA, also potently inhibit N-type VGCC currents in the sensory DRG neurons of rodents and α9 nAChR knockout mice, via intracellular signaling mediated by G protein-coupled GABAB receptors. Understanding how conotoxins inhibit VGCCs is critical for developing these peptides into analgesics and may result in better pain management. This article is part of a Special Issue entitled: Calcium channels.

γ-Aminobutyric acid type B (GABAB) receptor expression is needed for inhibition of N-type (Cav2.2) calcium channels by analgesic α-conotoxins.

α-Conotoxins Vc1.1 and RgIA are small peptides isolated from the venom of marine cone snails. They have effective anti-nociceptive actions in rat models of neuropathic pain. Pharmacological studies in rodent dorsal root ganglion (DRG) show their analgesic effect is mediated by inhibition of N-type (Ca(v)2.2) calcium channels via a pathway involving γ-aminobutyric acid type B (GABA(B)) receptor. However, there is no direct demonstration that functional GABA(B) receptors are needed for inhibition of the Ca(v)2.2 channel by analgesic α-conotoxins. This study examined the effect of the GABA(B) agonist baclofen and α-conotoxins Vc1.1 and RgIA on calcium channel currents after transient knockdown of the GABA(B) receptor using RNA interference. Isolated rat DRG neurons were transfected with small interfering RNAs (siRNA) targeting GABA(B) subunits R1 and R2. Efficient knockdown of GABA(B) receptor expression at mRNA and protein levels was confirmed by quantitative real time PCR (qRT-PCR) and immunocytochemical analysis, respectively. Whole-cell patch clamp recordings conducted 2-4 days after transfection showed that inhibition of N-type calcium channels in response to baclofen, Vc1.1 and RgIA was significantly reduced in GABA(B) receptor knockdown DRG neurons. In contrast, neurons transfected with a scrambled nontargeting siRNA were indistinguishable from untransfected neurons. In the HEK 293 cell heterologous expression system, Vc1.1 and RgIA inhibition of Ca(v)2.2 channels needed functional expression of both human GABA(B) receptor subunits. Together, these results confirm that GABA(B) receptors must be activated for the modulation of N-type (Ca(v)2.2) calcium channels by analgesic α-conotoxins Vc1.1 and RgIA.

Functional correlates of clinical phenotype and severity in recurrent SCN2A variants.

In SCN2A-related disorders, there is an urgent demand to establish efficient methods for determining the gain- (GoF) or loss-of-function (LoF) character of variants, to identify suitable candidates for precision therapies. Here we classify clinical phenotypes of 179 individuals with 38 recurrent SCN2A variants as early-infantile or later-onset epilepsy, or intellectual disability/autism spectrum disorder (ID/ASD) and assess the functional impact of 13 variants using dynamic action potential clamp (DAPC) and voltage clamp. Results show that 36/38 variants are associated with only one phenotypic group (30 early-infantile, 5 later-onset, 1 ID/ASD). Unexpectedly, we revealed major differences in outcome severity between individuals with the same variant for 40% of early-infantile variants studied. DAPC was superior to voltage clamp in predicting the impact of mutations on neuronal excitability and confirmed GoF produces early-infantile phenotypes and LoF later-onset phenotypes. For one early-infantile variant, the co-expression of the α1 and ß2 subunits of the Nav1.2 channel was needed to unveil functional impact, confirming the prediction of 3D molecular modeling. Neither DAPC nor voltage clamp reliably predicted phenotypic severity of early-infantile variants. Genotype, phenotypic group and DAPC are accurate predictors of the biophysical impact of SCN2A variants, but other approaches are needed to predict severity.

Biophysical analysis of an HCN1 epilepsy variant suggests a critical role for S5 helix Met-305 in voltage sensor to pore domain coupling.

Hyperpolarization-gated, cyclic nucleotide-activated (HCN1-4) channels are inwardly rectifying cation channels that display voltage dependent activation and de-activation. Pathogenic variants in HCN1 are associated with severe developmental and epileptic encephalopathies including the de novo HCN1 M305L variant. M305 is located in the S5 domain that is implicated in coupling voltage sensor domain movement to pore opening. This variant lacks voltage-dependent activation and de-activation and displays normal cation selectivity. To elucidate the impact of the mutation on the channel structure-function relations, molecular dynamics simulations of the wild type and mutant homotetramers were compared and identified a sulphur-aromatic interaction between M305 and F389 that contributes to the coupling of the voltage-sensing domain to the pore domain. To mimic the heterozygous condition as a heterotetrameric channel assembly, Xenopus oocytes were co-injected with various ratios of wild-type and mutant subunit cRNAs and the biophysical properties of channels with different subunit stoichiometries were determined. The results showed that a single mutated subunit was sufficient to significantly disrupt the voltage dependence of activation. The functional data were qualitatively consistent with predictions of a model that assumes independent activation of the voltage sensing domains allosterically controlling the closed to open transition of the pore. Overall, the M305L mutation results in an HCN1 channel that lacks voltage dependence and facilitates excitatory cation flow at membrane potentials that would normally close the channel. Our findings provide molecular insights into HCN1 channels and reveal the structural and biophysical basis of the severe epilepsy phenotype associated with the M305L mutation.

Analgesic alpha-conotoxins Vc1.1 and Rg1A inhibit N-type calcium channels in rat sensory neurons via GABAB receptor activation.

alpha-Conotoxins Vc1.1 and Rg1A are peptides from the venom of marine Conus snails that are currently in development as a treatment for neuropathic pain. Here we report that the alpha9alpha10 nicotinic acetylcholine receptor-selective conotoxins Vc1.1 and Rg1A potently and selectively inhibit high-voltage-activated (HVA) calcium channel currents in dissociated DRG neurons in a concentration-dependent manner. The post-translationally modified peptides vc1a and [P6O]Vc1.1 were inactive, as were all other alpha-conotoxins tested. Vc1.1 inhibited the omega-conotoxin-sensitive HVA currents in DRG neurons but not those recorded from Xenopus oocytes expressing Ca(V)2.2, Ca(V)2.1, Ca(V)2.3, or Ca(V)1.2 channels. Inhibition of HVA currents by Vc1.1 was not reversed by depolarizing prepulses but was abolished by pertussis toxin (PTX), intracellular GDPbetaS, or a selective inhibitor of pp60c-src tyrosine kinase. These data indicate that Vc1.1 does not interact with N-type calcium channels directly but inhibits them via a voltage-independent mechanism involving a PTX-sensitive, G-protein-coupled receptor. Preincubation with a variety of selective receptor antagonists demonstrated that only the GABA(B) receptor antagonists, [S-(R*,R*)][-3-[[1-(3,4-dichlorophenyl)ethyl]amino]-2-hydroxy propyl]([3,4]-cyclohexylmethyl) phosphinic acid hydrochloride (2S)-3[[(1S)-1-(3,4-dichlorophenyl)-ethyl]amino-2-hydroxypropyl](phenylmethyl) phosphinic acid and phaclofen, blocked the effect of Vc1.1 and Rg1A on Ca2+ channel currents. Together, the results identify Ca(V)2.2 as a target of Vc1.1 and Rg1A, potentially mediating their analgesic actions. We propose a novel mechanism by which alpha-conotoxins Vc1.1 and Rg1A modulate native N-type (Ca(V)2.2) Ca2+ channel currents, namely acting as agonists via G-protein-coupled GABA(B) receptors.

Acute administration of fish oil inhibits triggered activity in isolated myocytes from rabbits and patients with heart failure.

BACKGROUND: Fish oil reduces sudden death in patients with prior myocardial infarction. Sudden death in heart failure may be due to triggered activity based on disturbed calcium handling. We hypothesized that superfusion with omega3-polyunsaturated fatty acids (omega3-PUFAs) from fish inhibits triggered activity in heart failure. METHODS AND RESULTS: Ventricular myocytes were isolated from explanted hearts of rabbits with volume- and pressure-overload-induced heart failure and of patients with end-stage heart failure. Membrane potentials (patch-clamp technique) and intracellular calcium (indo-1 fluorescence) were recorded after 5 minutes of superfusion with Tyrode's solution (control), omega-9 monounsaturated fatty acid oleic acid (20 micromol/L), or omega3-PUFAs (docosahexaenoic acid or eicosapentaenoic acid 20 micromol/L). omega3-PUFAs shortened the action potential at low stimulation frequencies and caused an approximately 25% decrease in diastolic and systolic calcium (all P<0.05). Subsequently, noradrenalin and rapid pacing were used to evoke triggered activity, delayed afterdepolarizations, and calcium aftertransients. omega3-PUFAs abolished triggered activity and reduced the number of delayed afterdepolarizations and calcium aftertransients compared with control and oleic acid. Omega3-PUFAs reduced action potential shortening and intracellular calcium elevation in response to noradrenalin. Results from human myocytes were in accordance with the findings obtained in rabbit myocytes. CONCLUSIONS: Superfusion with omega3-PUFAs from fish inhibits triggered arrhythmias in myocytes from rabbits and patients with heart failure by lowering intracellular calcium and reducing the response to noradrenalin.