JAK2 regulates Nav1.6 channel function via FGF14Y158 phosphorylation

BackgroundProtein interactions between voltage-gated sodium (Nav) channels and accessory proteins play an essential role in neuronal firing and plasticity. However, a surprisingly limited number of kinases have been identified as regulators of these molecular complexes. We hypothesized that numerous as-of-yet unidentified kinases indirectly regulate the Nav channel via modulation of the intracellular fibroblast growth factor 14 (FGF14), an accessory protein with numerous unexplored phosphomotifs and required for channel function in neurons.MethodsHere we present results from an in-cell high-throughput screening (HTS) against the FGF14: Nav1.6 complex using >3000 diverse compounds targeting an extensive range of signaling pathways. Regulation by top kinase targets was then explored using in vitro phosphorylation, biophysics, mass-spectrometry and patch-clamp electrophysiology.ResultsCompounds targeting Janus kinase 2 (JAK2) were over-represented among HTS hits. Phosphomotif scans supported by mass spectrometry revealed FGF14^Y158, a site previously shown to mediate both FGF14 homodimerization and interactions with Nav1.6, as a JAK2 phosphorylation site. Following inhibition of JAK2, FGF14 homodimerization increased in a manner directly inverse to FGF14:Nav1.6 complex formation, but not in the presence of the FGF14^Y158A mutant. Patch-clamp electrophysiology revealed that through Y158, JAK2 controls FGF14-dependent modulation of Nav1.6 channels. In hippocampal CA1 pyramidal neurons, the JAK2 inhibitor Fedratinib reduced firing by a mechanism that is dependent upon expression of FGF14.ConclusionsThese studies point toward a novel mechanism by which levels of JAK2 in neurons could directly influence firing and plasticity by controlling the FGF14 dimerization equilibrium, and thereby the availability of monomeric species for interaction with Nav1.6.     

Identifying a Kinase Network Regulating FGF14:Nav1.6 Complex Assembly Using Split-Luciferase Complementation

Kinases play fundamental roles in the brain. Through complex signaling pathways, kinases regulate the strength of protein:protein interactions (PPI) influencing cell cycle, signal transduction, and electrical activity of neurons. Changes induced by kinases on neuronal excitability, synaptic plasticity and brain connectivity are linked to complex brain disorders, but the molecular mechanisms underlying these cellular events remain for the most part elusive. To further our understanding of brain disease, new methods for rapidly surveying kinase pathways in the cellular context are needed. The bioluminescence-based luciferase complementation assay (LCA) is a powerful, versatile toolkit for the exploration of PPI. LCA relies on the complementation of two firefly luciferase protein fragments that are functionally reconstituted into the full luciferase enzyme by two interacting binding partners. Here, we applied LCA in live cells to assay 12 kinase pathways as regulators of the PPI complex formed by the voltage-gated sodium channel, Nav1.6, a transmembrane ion channel that elicits the action potential in neurons and mediates synaptic transmission, and its multivalent accessory protein, the fibroblast growth factor 14 (FGF14). Through extensive dose-dependent validations of structurally-diverse kinase inhibitors and hierarchical clustering, we identified the PI3K/Akt pathway, the cell-cycle regulator Wee1 kinase, and protein kinase C (PKC) as prospective regulatory nodes of neuronal excitability through modulation of the FGF14:Nav1.6 complex. Ingenuity Pathway Analysis shows convergence of these pathways on glycogen synthase kinase 3 (GSK3) and functional assays demonstrate that inhibition of GSK3 impairs excitability of hippocampal neurons. This combined approach provides a versatile toolkit for rapidly surveying PPI signaling, allowing the discovery of new modular pathways centered on GSK3 that might be the basis for functional alterations between the normal and diseased brain.     

The Fibroblast Growth Factor 14·Voltage-gated Sodium Channel Complex Is a New Target of Glycogen Synthase Kinase 3 (GSK3)

The FGF14 protein controls biophysical properties and subcellular distribution of neuronal voltage-gated Na⁺ (Nav) channels through direct binding to the channel C terminus. To gain insights into the dynamic regulation of this protein/protein interaction complex, we employed the split luciferase complementation assay to screen a small molecule library of kinase inhibitors against the FGF14·Nav1.6 channel complex and identified inhibitors of GSK3 as hits. Through a combination of a luminescence-based counter-screening, co-immunoprecipitation, patch clamp electrophysiology, and quantitative confocal immunofluorescence, we demonstrate that inhibition of GSK3 reduces the assembly of the FGF14·Nav channel complex, modifies FGF14-dependent regulation of Na⁺ currents, and induces dissociation and subcellular redistribution of the native FGF14·Nav channel complex in hippocampal neurons. These results further emphasize the role of FGF14 as a critical component of the Nav channel macromolecular complex, providing evidence for a novel GSK3-dependent signaling pathway that might control excitability through specific protein/protein interactions.     

Amyloid Precursor Protein Enhances Nav1.6 Sodium Channel Cell Surface Expression

Amyloid precursor protein (APP) is commonly associated with Alzheimer disease, but its physiological function remains unknown. Nav1.6 is a key determinant of neuronal excitability in vivo. Because mouse models of gain of function and loss of function of APP and Nav1.6 share some similar phenotypes, we hypothesized that APP might be a candidate molecule for sodium channel modulation. Here we report that APP colocalized and interacted with Nav1.6 in mouse cortical neurons. Knocking down APP decreased Nav1.6 sodium channel currents and cell surface expression. APP-induced increases in Nav1.6 cell surface expression were G_o protein-dependent, enhanced by a constitutively active G_o protein mutant, and blocked by a dominant negative G_o protein mutant. APP also regulated JNK activity in a G_o protein-dependent manner. JNK inhibition attenuated increases in cell surface expression of Nav1.6 sodium channels induced by overexpression of APP. JNK, in turn, phosphorylated APP. Nav1.6 sodium channel surface expression was increased by T668E and decreased by T668A, mutations of APP695 mimicking and preventing Thr-668 phosphorylation, respectively. Phosphorylation of APP695 at Thr-668 enhanced its interaction with Nav1.6. Therefore, we show that APP enhances Nav1.6 sodium channel cell surface expression through a G_o-coupled JNK pathway.     

Use-dependent potentiation of the Nav1.6 sodium channel

Nav1.2 and Nav1.6 are two voltage-gated sodium channel isoforms that are abundant in the adult central nervous system. These channels are expressed in different cells and localized in different neuronal regions, which may reflect functional specialization. To examine this possibility, we compared the properties of Nav1.2 and Nav1.6 in response to a rapid series of repetitive depolarizations. Currents through Nav1.6 coexpressed with beta1 demonstrated use-dependent potentiation during a rapid train of depolarizations. This potentiation was in contrast to the use-dependent decrease in current for Nav1.2 with beta1. The voltage dependence of potentiation correlated with the voltage dependence of activation, and it still occurred when fast inactivation was removed by mutation. Rapid stimulation accelerated a slow phase of activation in the Nav1.6 channel that had fast inactivation removed, resulting in faster channel activation. Although the Nav1.2 channel with fast inactivation removed also demonstrated slightly faster activation, that channel showed very pronounced slow inactivation compared to Nav1.6. These results indicate that potentiation of Nav1.6 sodium currents results from faster channel activation, and that this effect is masked by slow inactivation in Nav1.2. The data suggest that Nav1.6 might be more resistant to inactivation, which might be helpful for high-frequency firing at nodes of Ranvier compared to Nav1.2.     

Addition of positive charges at the C-terminal peptide region of CssII, a mammalian scorpion peptide toxin, improves its affinity for sodium channels Nav1.6

CssII is a β-scorpion peptide that modifies preferentially sodium currents of the voltage-dependent Na⁺ channel (Nav) sub-type 1.6. Previously, we have found that the C-terminal amidation of CssII increases its affinity for Nav, which opens at more negative potentials in the presence of CssII. Although C-terminal amidation in vitro conditions is possible, five CssII peptide toxin variants with C-terminal residues modified were heterogously expressed (rN66S, rN66H, rN66R, r[T64R/N66S] and r[T64R/N66R], in which r stands for recombinant, the capital letters to the amino acid residues and the numbers indicate the position of the given residue into the primary sequence of the toxin) and correctly folded. A secondary structure prediction of CssII agrees with the experimental secondary structure obtained by circular dichroism; so all bacterial expressed neurotoxin variants maintained the typical α/β secondary structure motif of most Na⁺ channel scorpion toxins. The electrophysiological properties of all recombinant variants were examined, and it was found that substitutions of threonine (T) and asparagine (N) at the C-terminal region for arginine (R) (r[T64R/N66R]) increase their affinity for Nav1.6. Although, the molecular interactions involved in this mechanism are still not clearly determined, there is experimental evidence supporting the suspicion that incorporation of basic charged amino acid residues at the C-terminal tail of a group of α-scorpion toxin was favored by natural selection.     

Nav1.3 and FGF14 are primary determinants of the TTX-sensitive sodium current in mouse adrenal chromaffin cells

Adrenal chromaffin cells (CCs) in rodents express rapidly inactivating, tetrodotoxin (TTX)-sensitive sodium channels. The resulting current has generally been attributed to Nav1.7, although a possible role for Nav1.3 has also been suggested. Nav channels in rat CCs rapidly inactivate via two independent pathways which differ in their time course of recovery. One subpopulation recovers with time constants similar to traditional fast inactivation and the other ∼10-fold slower, but both pathways can act within a single homogenous population of channels. Here, we use Nav1.3 KO mice to probe the properties and molecular components of Nav current in CCs. We find that the absence of Nav1.3 abolishes all Nav current in about half of CCs examined, while a small, fast inactivating Nav current is still observed in the rest. To probe possible molecular components underlying slow recovery from inactivation, we used mice null for fibroblast growth factor homology factor 14 (FGF14). In these cells, the slow component of recovery from fast inactivation is completely absent in most CCs, with no change in the time constant of fast recovery. The use dependence of Nav current reduction during trains of stimuli in WT cells is completely abolished in FGF14 KO mice, directly demonstrating a role for slow recovery from inactivation in determining Nav current availability. Our results indicate that FGF14-mediated inactivation is the major determinant defining use-dependent changes in Nav availability in CCs. These results establish that Nav1.3, like other Nav isoforms, can also partner with FGF subunits, strongly regulating Nav channel function.     

Proteomic analysis of native cerebellar iFGF14 complexes

Intracellular Fibroblast Growth Factor 14 (iFGF14) and the other intracellular FGFs (iFGF11-13) regulate the properties and densities of voltage-gated neuronal and cardiac Na⁺ (Nav) channels. Recent studies have demonstrated that the iFGFs can also regulate native voltage-gated Ca²⁺ (Cav) channels. In the present study, a mass spectrometry (MS)-based proteomic approach was used to identify the components of native cerebellar iFGF14 complexes. Using an anti-iFGF14 antibody, native iFGF14 complexes were immunoprecipitated from wild type adult mouse cerebellum. Parallel control experiments were performed on cerebellar proteins isolated from mice (Fgf14^?/?) harboring a targeted disruption of the Fgf14 locus. MS analyses of immunoprecipitated proteins demonstrated that the vast majority of proteins identified in native cerebellar iFGF14 complexes are Nav channel pore-forming (α) subunits or proteins previously reported to interact with Nav α subunits. In contrast, no Cav channel α or accessory subunits were revealed in cerebellar iFGF14 immunoprecipitates. Additional experiments were completed using an anti-PanNav antibody to immunoprecipitate Nav channel complexes from wild type and Fgf14^?/? mouse cerebellum. Western blot and MS analyses revealed that the loss of iFGF14 does not measurably affect the protein composition or the relative abundance of Nav channel interacting proteins in native adult mouse cerebellar Nav channel complexes.     

Quantitative Proteomics Reveals Protein–Protein Interactions with Fibroblast Growth Factor 12 as a Component of the Voltage-Gated Sodium Channel 1.2 (Nav1.2) Macromolecular Complex in Mammalian Brain

Voltage-gated sodium channels (Nav1.1–Nav1.9) are responsible for the initiation and propagation of action potentials in neurons, controlling firing patterns, synaptic transmission and plasticity of the brain circuit. Yet, it is the protein–protein interactions of the macromolecular complex that exert diverse modulatory actions on the channel, dictating its ultimate functional outcome. Despite the fundamental role of Nav channels in the brain, information on its proteome is still lacking. Here we used affinity purification from crude membrane extracts of whole brain followed by quantitative high-resolution mass spectrometry to resolve the identity of Nav1.2 protein interactors. Of the identified putative protein interactors, fibroblast growth factor 12 (FGF12), a member of the nonsecreted intracellular FGF family, exhibited 30-fold enrichment in Nav1.2 purifications compared with other identified proteins. Using confocal microscopy, we visualized native FGF12 in the brain tissue and confirmed that FGF12 forms a complex with Nav1.2 channels at the axonal initial segment, the subcellular specialized domain of neurons required for action potential initiation. Co-immunoprecipitation studies in a heterologous expression system validate Nav1.2 and FGF12 as interactors, whereas patch-clamp electrophysiology reveals that FGF12 acts synergistically with CaMKII, a known kinase regulator of Nav channels, to modulate Nav1.2-encoded currents. In the presence of CaMKII inhibitors we found that FGF12 produces a bidirectional shift in the voltage-dependence of activation (more depolarized) and the steady-state inactivation (more hyperpolarized) of Nav1.2, increasing the channel availability. Although providing the first characterization of the Nav1.2 CNS proteome, we identify FGF12 as a new functionally relevant interactor. Our studies will provide invaluable information to parse out the molecular determinant underlying neuronal excitability and plasticity, and extending the relevance of iFGFs signaling in the normal and diseased brain.Voltage-gated sodium channels (Nav)¹ are transmembrane proteins consisting of a pore-forming α subunit (Nav1.1-Nav1.9) and one or more accessory β-subunits (β₁–β₄) (1–3). Predominately clustered at the axonal initial segment (AIS), the α subunit alone is necessary and sufficient for channel assembly and the initiation and propagation of action potentials following membrane depolarization (4). Although the α subunit is functional on its own, it is the transient and stable protein–protein interactions that modulate subcellular trafficking, compartmentalization, functional expression, and fine-tune the channel biophysical properties (5–9). Thus, the Nav channel and the protein constituents that comprise the protein–protein interaction network are all part of a macromolecular complex that modulates the spatiotemporal dynamics of neuronal input and output playing a critical role in synaptic transmission, signal integration, and neuronal plasticity. Perturbations in this protein–protein interaction network can lead to deficits in neuronal excitability, and eventually neurodegeneration and cell death (10–15).Given the relevance of these interactions for the native channel activity and its overall role in controlling brain circuits, it is increasingly important to uncover these associations. Antibody-based affinity purification (AP) combined with mass spectrometry (MS) is widely used for the enrichment and analysis of target proteins and constituents of their protein–protein interactions as it can be performed at near physiological conditions and preserves post-translational modifications relevant to protein complex organization (16–19). Differential mass spectrometry provides an unbiased method for the efficient, MS-based measurement of relative protein fold changes across multiple complex biological samples. This technology has been successfully applied to a number of ion channels (20–26), but—to the best of our knowledge—not to the study of any member of the Nav channel family. Using a target-directed AP approach combined with quantitative MS, we identified proteins constituting the putative interactome of Nav1.2, one of three dominant Nav channel isoforms in the mammalian brain, from native tissue (1, 2, 4, 8). Among these putative interactors, the fibroblast growth factor 12 (FGF12), a member of the intracellular FGF family (5, 13, 14), stood out as one of the most abundant coprecipitating proteins with ∼30-fold enrichment over other interactors. With a combination of confocal microscopy in brain tissue, reconstitution of the interactor in a heterologous systems and electrophysiological assays, we provide validation for FGF12 as a bona fide relevant component of the Nav1.2 proteome and a modulator of Nav1.2-encoded currents. Altogether, the identified channel/protein interaction between FGF12 and Nav1.2 provides new insights for structural and functional interpretation of neuronal excitability, synaptic transmission, and plasticity in the normal and diseased brain.     

In silico detection of binding mode of J-superfamily conotoxin pl14a with Kv1.6 channel

A novel conotoxin pl14a containing 25 amino acid residues with an amidated C-terminus from vermivorous cone snail, Conus planorbis belongs to J-conotoxin superfamily and this is the first conotoxin, which inhibits both nicotinic acetylcholine receptor subtypes and Kv1.6 channel. We have attempted through bioinformatics approaches to elucidate the extent of specificity of pl14a towards Kv1 channel subtypes (Kv1.1-Kv1.6). Our work provides rationale for the relatively high specificity and binding mode of pl14a to Kv1.6 channel. The pl14a peptide contains two types of structural elements, namely the putative dyad (Lys18 and Tyr19) and basic residue ring constituted of arginine residues. We have carried out in silico docking studies so as to assess the contribution of one or combination of both structural elements of pl14a in blocking of Kv1.6 channel. For this purpose, we have built by homology modelling, the theoretical 3D structure of Kv1.6 channel based on the available crystal structure of mammalian shaker Kv1.2 channel. Docking studies suggest that positively charged residues ring may be involved in the blocking mechanism of Kv1.6 channel. The models suggest that the peptide interacts with negatively charged extracellular loops and pore-mouth of the potassium channel and blocks the channel by covering the pore as a lid, akin to previously proposed blocking mechanism of kappaM-conotoxin RIIIK from Conus radiatus to Tsha1 potassium channel. The newly detected pharmacophore for pl14a interacting with Kv1.6 channel provides a pointer to experimental work to validate the observations made here. Based on differences in the number and distribution of the positively-charged residues in other conopeptides from the J-superfamily, we hypothesize different selectivity profiles against subtypes of the potassium channels for these conopeptides.     

天麻素通过下调Nav1.6通道表达缓解糖尿病神经病理性疼痛的研究

目的:探究天麻素对Ⅱ型糖尿病神经病理性痛的镇痛作用以及天麻素对背根神经节Nav1.6通道的表达调控作用。方法:将60只雄性SD大鼠随机分为空白对照组、糖尿病组和天麻素处理组(10 mg·kg^-1·d^-1)。通过高脂饮食喂养4周,低剂量腹腔注射STZ(30 mg·kg^-1)的方法构建Ⅱ型糖尿病神经病理性痛大鼠模型,利用痛行为学检测观察各组大鼠的机械刺激足缩反应阈值变化,采用免疫荧光组织化学及Western blot方法观察各组大鼠背根神经节上Nav1.6通道的表达变化。结果:与空白对照组相比,糖尿病模型大鼠出现显著的机械刺激疼痛阈值下降(P<0.05),且模型组大鼠背根神经节神经元上的Nav1.6通道表达上调(P<0.05)。与糖尿病组相比,连续腹腔注射天麻素3天、7天、14天后,模型动物的疼痛明显缓解(P<0.05),另外天麻素可以翻转背根神经节上Nav1.6通道的高表达(P<0.05)。结论:天麻素可能通过降低Nav1.6通道的表达来缓解Ⅱ型糖尿病神经病理性疼痛,从而为天麻素缓解糖尿病神经病理性疼痛提供新的理论依据。     

Matrix Metalloproteinase (MMP) Proteolysis of the Extracellular Loop of Voltage-gated Sodium Channels and Potential Alterations in Pain Signaling

Congenital insensitivity to pain (CIP) or congenital analgesia is a rare monogenic hereditary condition. This disorder is characterized by the inability to perceive any form of pain. Nonsense mutations in Nav.1.7, the main pain signaling voltage-gated sodium channel, lead to its truncations and, consequently, to the inactivation of the channel functionality. However, a non-truncating homozygously inherited missense mutation in a Bedouin family with CIP (Nav1.7-R907Q) has also been reported. Based on our currently acquired in-depth knowledge of matrix metalloproteinase (MMP) cleavage preferences, we developed the specialized software that predicts the presence of the MMP cleavage sites in the peptide sequences. According to our in silico predictions, the peptide sequence of the exposed extracellular unstructured region linking the S5–S6 transmembrane segments in the DII domain of the human Nav1.7 sodium channel is highly sensitive to MMP-9 proteolysis. Intriguingly, the CIP R907Q mutation overlaps with the predicted MMP-9 cleavage site sequence. Using MMP-9 proteolysis of the wild-type, CIP, and control peptides followed by mass spectrometry of the digests, we demonstrated that the mutant sequence is severalfold more sensitive to MMP-9 proteolysis relative to the wild type. Because of the substantial level of sequence homology among sodium channels, our data also implicate MMP proteolysis in regulating the cell surface levels of the Nav1.7, Nav1.6, and Nav1.8 channels, but not Nav1.9. It is likely that the aberrantly accelerated MMP-9 proteolysis during neurogenesis is a biochemical rational for the functional inactivation in Nav1.7 and that the enhanced cleavage of the Nav1.7-R907Q mutant is a cause of CIP in the Bedouin family.     

AaHIV a sodium channel scorpion toxin inhibits the proliferation of DU145 prostate cancer cells

Prostate cancer is the most highly diagnosed cancer in men worldwide. It is characterized by high proliferation, great invasion and metastatic potential. Sodium channel subtypes have been identified as highly expressed in different prostate cancer cell lines. In this study, we have screened the negatively charged fractions of Androctonus australis (Aa) scorpion venom to identify active peptides on DU145 prostate cancer cells proliferation. The most active compound was identified to be the sodium channel peptide AaHIV with an IC₅₀ value of 15 μM. At this concentration, AaHIV had low effect on the adhesion of DU145 cells to fibronectin. When compared to other Na⁺ channel Aa toxins, AaHIV was found to be 2 times more active than AaHI and AaHII on DU145 cells proliferation and slightly less active than AaHII on their adhesion. The three peptides are inactive on DU145 cells migration. AaHIV was found to be 16 times more active than veratridine, asteroidal alkaloid from plants of the lily family widely used as a sodium channel activator. Electrophysiological experiments showed that the AaHIV toxin activates Nav1.6 channel, suggesting that this sodium channel subtype is implicated in the proliferation of DU145 prostate cancer cells.     

Design,synthesis and evaluation of potent and selective inhibitors of mono-(ADP-ribosyl)transferases PARP10 and PARP14

A series of diaryl ethers were designed and synthesized to discern the structure activity relationships against the two closely related mono-(ADP-ribosyl)transferases PARP10 and PARP14. Structure activity studies identified 8b as a sub-micromolar inhibitor of PARP10 with?～15-fold selectivity over PARP14. In addition, 8k and 8m were discovered to have sub-micromolar potency against PARP14 and demonstrated moderate selectivity over PARP10. A crystal structure of the complex of PARP14 and 8b shows binding of the compound in a novel hydrophobic pocket and explains both potency and selectivity over other PARP family members. In addition, 8b, 8k and 8m also demonstrate selectivity over PARP1. Together, this study identified novel, potent and metabolically stable derivatives to use as chemical probes for these biologically interesting therapeutic targets.     

Discovery of novel 4-phenyl-2-(pyrrolidinyl)nicotinamide derivatives as potent Nav1.1 activators

The voltage-gated sodium channel, Na_v1.1, is predominantly expressed in parvalbumin-positive fast spiking interneurons and has been genetically linked to Dravet syndrome. Starting from a high throughput screening hit isoxazole derivative 5, modifications of 5 via combinations of IonWorks and Q-patch assays successfully identified the nicotinamide derivative 4. Its increasing decay time constant (tau) of Na_v1.1 currents at 0.03?μM along with significant selectivity against Na_v1.2, Na_v1.5, and Na_v1.6 and acceptable brain exposure in mice was observed. Compound 4 is a promising Na_v1.1 activator that can be used to analyze pathophysiological functions of the Na_v1.1 channel towards treating various central nervous system diseases.     

Discovery of a novel allosteric inhibitor scaffold for polyadenosine-diphosphate-ribose polymerase 14 (PARP14) macrodomain 2

The polyadenosine-diphosphate-ribose polymerase 14 (PARP14) has been implicated in DNA damage response pathways for homologous recombination. PARP14 contains three (ADP ribose binding) macrodomains (MD) whose exact contribution to overall PARP14 function in pathology remains unclear. A medium throughput screen led to the identification of N-(2(-9H-carbazol-1-yl)phenyl)acetamide (GeA-69, 1) as a novel allosteric PARP14 MD2 (second MD of PARP14) inhibitor. We herein report medicinal chemistry around this novel chemotype to afford a sub-micromolar PARP14 MD2 inhibitor. This chemical series provides a novel starting point for further development of PARP14 chemical probes.     

Analysis of the Structural and Molecular Basis of Voltage-sensitive Sodium Channel Inhibition by the Spider Toxin Huwentoxin-IV (μ-TRTX-Hh2a)

Voltage-gated sodium channels (VGSCs) are essential to the normal function of the vertebrate nervous system. Aberrant function of VGSCs underlies a variety of disorders, including epilepsy, arrhythmia, and pain. A large number of animal toxins target these ion channels and may have significant therapeutic potential. Most of these toxins, however, have not been characterized in detail. Here, by combining patch clamp electrophysiology and radioligand binding studies with peptide mutagenesis, NMR structure determination, and molecular modeling, we have revealed key molecular determinants of the interaction between the tarantula toxin huwentoxin-IV and two VGSC isoforms, Nav1.7 and Nav1.2. Nine huwentoxin-IV residues (F6A, P11A, D14A, L22A, S25A, W30A, K32A, Y33A, and I35A) were important for block of Nav1.7 and Nav1.2. Importantly, molecular dynamics simulations and NMR studies indicated that folding was normal for several key mutants, suggesting that these amino acids probably make specific interactions with sodium channel residues. Additionally, we identified several amino acids (F6A, K18A, R26A, and K27A) that are involved in isoform-specific VGSC interactions. Our structural and functional data were used to model the docking of huwentoxin-IV into the domain II voltage sensor of Nav1.7. The model predicts that a hydrophobic patch composed of Trp-30 and Phe-6, along with the basic Lys-32 residue, docks into a groove formed by the Nav1.7 S1-S2 and S3-S4 loops. These results provide new insight into the structural and molecular basis of sodium channel block by huwentoxin-IV and may provide a basis for the rational design of toxin-based peptides with improved VGSC potency and/or selectivity.     

Discovery of novel purine nucleoside derivatives as phosphodiesterase 2 (PDE2) inhibitors: Structure-based virtual screening,optimization and biological evaluation

Phosphodiesterase 2 (PDE2) has received much attention for the potential treatment of the central nervous system (CNS) disorders and pulmonary hypertension. Herein, we identified that clofarabine (4), an FDA-approved drug, displayed potential PDE2 inhibitory activity (IC₅₀?=?3.12?±?0.67?μM) by structure-based virtual screening and bioassay. Considering the potential therapeutic benefit of PDE2, a series of purine nucleoside derivatives based on the structure and binding mode of 4 were designed, synthesized and evaluated, which led to the discovery of the best compound 14e with a significant improvement of inhibitory potency (IC₅₀?=?0.32?±?0.04?μM). Further molecular docking and molecular dynamic (MD) simulations studies revealed that 5′-benzyl group of 14e could interact with the unique hydrophobic pocket of PDE2 by forming extra van der Waals interactions with hydrophobic residues such as Leu770, Thr768, Thr805 and Leu809, which might contribute to its enhancement of PDE2 inhibition. These potential compounds reported in this article and the valuable structure-activity relationships (SARs) might bring significant instruction for further development of potent PDE2 inhibitors.     

A Yeast BiFC-seq Method for Genome-wide Interactome Mapping

Genome-wide physical protein–protein interaction (PPI) mapping remains a major challenge for current technologies. Here, we reported a high-efficiency BiFC-seq method, yeast-enhanced green fluorescent protein-based bimolecular fluorescence complementation (yEGFP-BiFC) coupled with next-generation DNA sequencing, for interactome mapping. We first applied yEGFP-BiFC method to systematically investigate an intraviral network of the Ebola virus. Two-thirds (9/14) of known interactions of EBOV were recaptured, and five novel interactions were discovered. Next, we used the BiFC-seq method to map the interactome of the tumor protein p53. We identified 97 interactors of p53, more than three-quarters of which were novel. Furthermore, in a more complex background, we screened potential interactors by pooling two BiFC libraries together and revealed a network of 229 interactions among 205 proteins. These results show that BiFC-seq is a highly sensitive, rapid, and economical method for genome-wide interactome mapping.     

Knockdown of Nav1.6a Na+ channels affects zebrafish motoneuron development

In addition to rapid signaling, electrical activity provides important cues to developing neurons. Electrical activity relies on the function of several different types of voltage-gated ion channels. Whereas voltage-gated Ca2+ channel activity regulates several aspects of neuronal differentiation, much less is known about developmental roles of voltage-gated Na+ channels, essential mediators of electrical signaling. Here, we focus on the zebrafish Na+ channel isotype, Nav1.6a, which is encoded by the scn8a gene. A restricted set of spinal neurons, including dorsal sensory Rohon-Beard cells, two motoneuron subtypes with different axonal trajectories, express scn8a during embryonic development. CaP, an early born primary motoneuron subtype with ventrally projecting axons expresses scn8a, as does a class of secondary motoneurons with axons that project dorsally. To test for developmental roles of scn8a, we knocked down Nav1.6a protein using antisense morpholinos. Na+ channel protein and current amplitudes were reduced in neurons that express scn8a. Furthermore, Nav1.6a knockdown altered axonal morphologies of some but not all motoneurons. Dorsally projecting secondary motoneurons express scn8a and displayed delayed axonal outgrowth. By contrast, CaP axons developed normally, despite expression of the gene. Surprisingly, ventrally projecting secondary motoneurons, a population in which scn8a was not detected, displayed aberrant axonal morphologies. Mosaic analysis indicated that effects on ventrally projecting secondary motoneurons were non cell-autonomous. Thus, voltage-gated Na+ channels play cell-autonomous and non cell-autonomous roles during neuronal development.