Peripherally targeted analgesia via AAV-mediated sensory neuron-specific inhibition of multiple pronociceptive sodium channels.

This study reports that targeting intrinsically disordered regions of the voltage-gated sodium channel 1.7 (NaV1.7) protein facilitates discovery of sodium channel inhibitory peptide aptamers (NaViPA) for adeno-associated virus-mediated (AAV-mediated), sensory neuron-specific analgesia. A multipronged inhibition of INa1.7, INa1.6, INa1.3, and INa1.1 - but not INa1.5 and INa1.8 - was found for a prototype and named NaViPA1, which was derived from the NaV1.7 intracellular loop 1, and is conserved among the TTXs NaV subtypes. NaViPA1 expression in primary sensory neurons (PSNs) of dorsal root ganglia (DRG) produced significant inhibition of TTXs INa but not TTXr INa. DRG injection of AAV6-encoded NaViPA1 significantly attenuated evoked and spontaneous pain behaviors in both male and female rats with neuropathic pain induced by tibial nerve injury (TNI). Whole-cell current clamp of the PSNs showed that NaViPA1 expression normalized PSN excitability in TNI rats, suggesting that NaViPA1 attenuated pain by reversal of injury-induced neuronal hypersensitivity. IHC revealed efficient NaViPA1 expression restricted in PSNs and their central and peripheral terminals, indicating PSN-restricted AAV biodistribution. Inhibition of sodium channels by NaViPA1 was replicated in the human iPSC-derived sensory neurons. These results summate that NaViPA1 is a promising analgesic lead that, combined with AAV-mediated PSN-specific block of multiple TTXs NaVs, has potential as a peripheral nerve-restricted analgesic therapeutic.

Programming the dynamic range of nanobiosensors with engineering poly-adenine-mediated spherical nucleic acid.

Spherical nucleic acid (SNA) conjugates consisting of gold cores functionalized with a densely packed DNA shells are of great significance in the field of medical detection and intracellular imaging. Especially, poly adenine (polyA)-mediated SNAs can improve the controllability and reproducibility of DNA assembly on the nanointerface, showing the tunable hybridization ability. However, due to the physics of single-site binding, the biosensor based on SNA usually exhibits a dynamic range spanning a fixed 81-fold change in target concentration, which limits its application in disease monitoring. To address this problem, we report a tri-block DNA-based approach to assemble SNA for nucleic acid detection based on structure-switching mechanism with programmable dynamic range. The tri-block DNA is a FAM-labeled stem-loop structure, which contains three blocks: polyA block as an anchoring block for tunable surface density, stem block with different GC base pair content for varying the structure stability, and the fixed loop block for target recognition. We find that varying the polyA block, the reaction temperature, and the GC base pair, SNA shows different target binding affinity and detection limit but with normally 81-fold dynamic range. We can extend the dynamic range to 1000-fold by using the combination of two SNAs with different affinity, and narrow the dynamic range to 5-fold by sequestration mechanism. Furthermore, the tunable SNA enables sensitive detection of mRNA in cells. Given its tunable dynamic range, such nanobiosensor based on SNA offers new possibility for various biomedical and clinical applications.

Deep learning for denoising in a Mueller matrix microscope.

The Mueller matrix microscope is a powerful tool for characterizing the microstructural features of a complex biological sample. Performance of a Mueller matrix microscope usually relies on two major specifications: measurement accuracy and acquisition time, which may conflict with each other but both contribute to the complexity and expenses of the apparatus. In this paper, we report a learning-based method to improve both specifications of a Mueller matrix microscope using a rotating polarizer and a rotating waveplate polarization state generator. Low noise data from long acquisition time are used as the ground truth. A modified U-Net structured network incorporating channel attention effectively reduces the noise in lower quality Mueller matrix images obtained with much shorter acquisition time. The experimental results show that using high quality Mueller matrix data as ground truth, such a learning-based method can achieve both high measurement accuracy and short acquisition time in polarization imaging.

Structural and Functional Characterization of a Novel Scorpion Toxin that Inhibits NaV1.8 via Interactions With the DI Voltage Sensor and DII Pore Module.

Voltage-gated sodium channel NaV1.8 regulates transmission of pain signals to the brain. While NaV1.8 has the potential to serve as a drug target, the molecular mechanisms that shape NaV1.8 gating are not completely understood, particularly mechanisms that couple activation to inactivation. Interactions between toxin producing animals and their predators provide a novel approach for investigating NaV structure-function relationships. Arizona bark scorpions produce Na+ channel toxins that initiate pain signaling. However, in predatory grasshopper mice, toxins inhibit NaV1.8 currents and block pain signals. A screen of synthetic peptide toxins predicted from bark scorpion venom showed that peptide NaTx36 inhibited Na+ current recorded from a recombinant grasshopper mouse NaV1.8 channel (OtNaV1.8). Toxin NaTx36 hyperpolarized OtNaV1.8 activation, steady-state fast inactivation, and slow inactivation. Mutagenesis revealed that the first gating charge in the domain I (DI) S4 voltage sensor and an acidic amino acid (E) in the DII SS2 - S6 pore loop are critical for the inhibitory effects of NaTx36. Computational modeling showed that a DI S1 - S2 asparagine (N) stabilizes the NaTx36 - OtNaV1.8 complex while residues in the DI S3 - S4 linker and S4 voltage sensor form electrostatic interactions that allow a toxin glutamine (Q) to contact the first S4 gating charge. Surprisingly, the models predicted that NaTx36 contacts amino acids in the DII S5 - SS1 pore loop instead of the SS2 - S6 loop; the DII SS2 - S6 loop motif (QVSE) alters the conformation of the DII S5 - SS1 pore loop, enhancing allosteric interactions between toxin and the DII S5 - SS1 pore loop. Few toxins have been identified that modify NaV1.8 gating. Moreover, few toxins have been described that modify sodium channel gating via the DI S4 voltage sensor. Thus, NaTx36 and OtNaV1.8 provide tools for investigating the structure-activity relationship between channel activation and inactivation gating, and the connection to alternative pain phenotypes.

A-type FHFs mediate resurgent currents through TTX-resistant voltage-gated sodium channels.

Resurgent currents (INaR) produced by voltage-gated sodium channels are required for many neurons to maintain high-frequency firing and contribute to neuronal hyperexcitability and disease pathophysiology. Here, we show, for the first time, that INaR can be reconstituted in a heterologous system by coexpression of sodium channel α-subunits and A-type fibroblast growth factor homologous factors (FHFs). Specifically, A-type FHFs induces INaR from Nav1.8, Nav1.9 tetrodotoxin (TTX)-resistant neuronal channels, and, to a lesser extent, neuronal Nav1.7 and cardiac Nav1.5 channels. Moreover, we identified the N-terminus of FHF as the critical molecule responsible for A-type FHFs-mediated INaR. Among the FHFs, FHF4A is the most important isoform for mediating Nav1.8 and Nav1.9 INaR. In nociceptive sensory neurons, FHF4A knockdown significantly reduces INaR amplitude and the percentage of neurons that generate INaR, substantially suppressing excitability. Thus, our work reveals a novel molecular mechanism underlying TTX-resistant INaR generation and provides important potential targets for pain treatment.

Identification and Characterization of Novel Proteins from Arizona Bark Scorpion Venom That Inhibit Nav1.8, a Voltage-Gated Sodium Channel Regulator of Pain Signaling.

The voltage-gated sodium channel Nav1.8 is linked to neuropathic and inflammatory pain, highlighting the potential to serve as a drug target. However, the biophysical mechanisms that regulate Nav1.8 activation and inactivation gating are not completely understood. Progress has been hindered by a lack of biochemical tools for examining Nav1.8 gating mechanisms. Arizona bark scorpion (Centruroides sculpturatus) venom proteins inhibit Nav1.8 and block pain in grasshopper mice (Onychomys torridus). These proteins provide tools for examining Nav1.8 structure-activity relationships. To identify proteins that inhibit Nav1.8 activity, venom samples were fractioned using liquid chromatography (reversed-phase and ion exchange). A recombinant Nav1.8 clone expressed in ND7/23 cells was used to identify subfractions that inhibited Nav1.8 Na+ current. Mass-spectrometry-based bottom-up proteomic analyses identified unique peptides from inhibitory subfractions. A search of the peptides against the AZ bark scorpion venom gland transcriptome revealed four novel proteins between 40 and 60% conserved with venom proteins from scorpions in four genera (Centruroides, Parabuthus, Androctonus, and Tityus). Ranging from 63 to 82 amino acids, each primary structure includes eight cysteines and a "CXCE" motif, where X = an aromatic residue (tryptophan, tyrosine, or phenylalanine). Electrophysiology data demonstrated that the inhibitory effects of bioactive subfractions can be removed by hyperpolarizing the channels, suggesting that proteins may function as gating modifiers as opposed to pore blockers.

Time-Frequency Distribution Map-Based Convolutional Neural Network (CNN) Model for Underwater Pipeline Leakage Detection Using Acoustic Signals.

Detection technology of underwater pipeline leakage plays an important role in the subsea production system. In this paper, a new method based on the acoustic leak signal collected by a hydrophone is proposed to detect pipeline leakage in the subsea production system. Through the pipeline leakage test, it is found that the radiation noise is a continuous spectrum of the medium and high-frequency noise. Both the increase in pipe pressure and the diameter of the leak hole will narrow the spectral structure and shift the spectrum center towards the low frequencies. Under the same condition, the pipe pressure has a greater impact on the noise; every 0.05 MPa increase in the pressure, the radiation sound pressure level increases by 6-7 dB. The time-frequency images were obtained by processing the acoustic signals using the Ensemble Empirical Mode Decomposition (EEMD) and Hilbert-Huang transform (HHT), and fed into a two-layer Convolutional Neural Network (CNN) for leakage detection. The results show that CNN can correctly identify the degree of pipeline leakage. Hence, the proposed method provides a new approach for the detection of pipeline leakage in underwater engineering applications.

S-Palmitoylation of the sodium channel Nav1.6 regulates its activity and neuronal excitability.

S-Palmitoylation is a reversible post-translational lipid modification that dynamically regulates protein functions. Voltage-gated sodium channels are subjected to S-palmitoylation and exhibit altered functions in different S-palmitoylation states. Our aim was to investigate whether and how S-palmitoylation regulates Nav1.6 channel function and to identify S-palmitoylation sites that can potentially be pharmacologically targeted. Acyl-biotin exchange assay showed that Nav1.6 is modified by S-palmitoylation in the mouse brain and in a Nav1.6 stable HEK 293 cell line. Using whole-cell voltage clamp, we discovered that enhancing S-palmitoylation with palmitic acid increases Nav1.6 current, whereas blocking S-palmitoylation with 2-bromopalmitate reduces Nav1.6 current and shifts the steady-state inactivation in the hyperpolarizing direction. Three S-palmitoylation sites (Cys1169, Cys1170, and Cys1978) were identified. These sites differentially modulate distinct Nav1.6 properties. Interestingly, Cys1978 is exclusive to Nav1.6 among all Nav isoforms and is evolutionally conserved in Nav1.6 among most species. Cys1978S-palmitoylation regulates current amplitude uniquely in Nav1.6. Furthermore, we showed that eliminating S-palmitoylation at specific sites alters Nav1.6-mediated excitability in dorsal root ganglion neurons. Therefore, our study reveals S-palmitoylation as a potential isoform-specific mechanism to modulate Nav activity and neuronal excitability in physiological and diseased conditions.

Resurgent and Gating Pore Currents Induced by De Novo SCN2A Epilepsy Mutations.

Over 150 mutations in the SCN2A gene, which encodes the neuronal Nav1.2 protein, have been implicated in human epilepsy cases. Of these, R1882Q and R853Q are two of the most commonly reported mutations. This study utilized voltage-clamp electrophysiology to characterize the biophysical effects of the R1882Q and R853Q mutations on the hNav1.2 channel, including their effects on resurgent current and gating pore current, which are not typically investigated in the study of Nav1.2 channel mutations. HEK cells transiently transfected with DNA encoding either wild-type (WT) or mutant hNav1.2 revealed that the R1882Q mutation induced a gain-of-function phenotype, including slowed fast inactivation, depolarization of the voltage dependence of inactivation, and increased persistent current. In this model system, the R853Q mutation primarily produced loss-of-function effects, including reduced transient current amplitude and density, hyperpolarization of the voltage dependence of inactivation, and decreased persistent current. The presence of a Navß4 peptide (KKLITFILKKTREK-OH) in the pipette solution induced resurgent currents, which were increased by the R1882Q mutation and decreased by the R853Q mutation. Further study of the R853Q mutation in Xenopus oocytes indicated a reduced surface expression and revealed a robust gating pore current at negative membrane potentials, a function absent in the WT channel. This not only shows that different epileptogenic point mutations in hNav1.2 have distinct biophysical effects on the channel, but also illustrates that individual mutations can have complex consequences that are difficult to identify using conventional analyses. Distinct mutations may, therefore, require tailored pharmacotherapies in order to eliminate seizures.

The Diurnal Rhythm of Insulin Receptor Substrate-1 (IRS-1) and Kir4.1 in Diabetes: Implications for a Clock Gene Bmal1.

Purpose: Diabetes leads to the downregulation of the retinal Kir4.1 channels and Müller cell dysfunction. The insulin receptor substrate-1 (IRS-1) is a critical regulator of insulin signaling in Müller cells. Circadian rhythms play an integral role in normal physiology; however, diabetes leads to a circadian dysrhythmia. We hypothesize that diabetes will result in a circadian dysrhythmia of IRS-1 and Kir4.1 and disturbed clock gene function will have a critical role in regulating Kir4.1 channels. Methods: We assessed a diurnal rhythm of retinal IRS-1 and Kir4.1 in db/db mice. The Kir4.1 function was evaluated using a whole-cell recording of Müller cells. The rat Müller cells (rMC-1) were used to undertake in vitro studies using a siRNA. Results: The IRS-1 exhibited a diurnal rhythm in control mice; however, with diabetes, this natural rhythm was lost. The Kir4.1 levels peaked and troughed at times similar to the IRS-1 rhythm. The IRS-1 silencing in the rMC-1 led to a decrease in Kir4.1 and BMAL1. The insulin treatment of retinal explants upregulated Kir4.1 possibly via upregulation of BMAL1 and phosphorylation of IRS-1 and Akt-1. Conclusions: Our studies highlight that IRS-1, by regulating BMAL1, is an important regulator of Kir4.1 in Müller cells and the dysfunctional signaling mediated by IRS-1 may be detrimental to Kir4.1.

Increased Resurgent Sodium Currents in Nav1.8 Contribute to Nociceptive Sensory Neuron Hyperexcitability Associated with Peripheral Neuropathies.

Neuropathic pain is a significant public health challenge, yet the underlying mechanisms remain poorly understood. Painful small fiber neuropathy (SFN) may be caused by gain-of-function mutations in Nav1.8, a sodium channel subtype predominantly expressed in peripheral nociceptive neurons. However, it is not clear how Nav1.8 disease mutations induce sensory neuron hyperexcitability. Here we studied two mutations in Nav1.8 associated with hypersensitive sensory neurons: G1662S reported in painful SFN; and T790A, which underlies increased pain behaviors in the Possum transgenic mouse strain. We show that, in male DRG neurons, these mutations, which impair inactivation, significantly increase TTX-resistant resurgent sodium currents mediated by Nav1.8. The G1662S mutation doubled resurgent currents, and the T790A mutation increased them fourfold. These unusual currents are typically evoked during the repolarization phase of action potentials. We show that the T790A mutation greatly enhances DRG neuron excitability by reducing current threshold and increasing firing frequency. Interestingly, the mutation endows DRG neurons with multiple early afterdepolarizations and leads to substantial prolongation of action potential duration. In DRG neurons, siRNA knockdown of sodium channel ß4 subunits fails to significantly alter T790A current density but reduces TTX-resistant resurgent currents by 56%. Furthermore, DRG neurons expressing T790A channels exhibited reduced excitability with fewer early afterdepolarizations and narrower action potentials after ß4 knockdown. Together, our data demonstrate that open-channel block of TTX-resistant currents, enhanced by gain-of-function mutations in Nav1.8, can make major contributions to the hyperexcitability of nociceptive neurons, likely leading to altered sensory phenotypes including neuropathic pain in SFN.SIGNIFICANCE STATEMENT This work demonstrates that two disease mutations in the voltage-gated sodium channel Nav1.8 that induce nociceptor hyperexcitability increase resurgent currents. Nav1.8 is crucial for pain sensations. Because resurgent currents are evoked during action potential repolarization, they can be crucial regulators of action potential activity. Our data indicate that increased Nav1.8 resurgent currents in DRG neurons greatly prolong action potential duration and enhance repetitive firing. We propose that Nav1.8 open-channel block is a major factor in Nav1.8-associated pain mechanisms and that targeting the molecular mechanism underlying these unique resurgent currents represents a novel therapeutic target for the treatment of aberrant pain sensations.

Advanced glycation end (AGE) product modification of laminin downregulates Kir4.1 in retinal Müller cells.

Diabetic retinopathy (DR) is a major cause of adult blindness. Retinal Müller cells maintain water homeostasis and potassium concentration via inwardly rectifying Kir4.1 channels. Accumulation of advanced glycation end products (AGEs) is a major pathologic event in DR. While diabetes leads to a decrease in the Kir4.1 channels, it remains unknown whether AGEs-linked to the basement membrane (BM) affect normal Kir4.1 channels. For this study, we hypothesized that AGE-modification of laminin is detrimental to Kir4.1 channels, therefore, disrupting Müller cell function. The AGE-modified laminin-coated substrates were prepared by incubating Petri-dishes with laminin and methylglyoxal for seven days. The rat Müller cells (rMC-1) were propagated on AGE-modified laminin, and Kir4.1 expression and function were evaluated. Quantification of AGEs using ELISA revealed a dose-dependent increase in methylglyoxal-hydro-imidazolone adducts. The rMC-1 propagated on AGE-modified laminin demonstrated a decrease in Kir4.1 levels in immunofluorescence and western blot studies and a decrease in the Kir4.1 channel function. Kir4.1 decrease on AGE-modified laminin resulted in a disorganization of an actin cytoskeleton and disruption of α-dystroglycan-syntrophin-dystrophin complexes. Our studies suggest that AGE-modification of laminin is detrimental to Kir4.1 channels. By studying the role of AGEs in Kir4.1 channels we have identified a novel mechanism of Müller cell dysfunction and its subsequent involvement in DR.

Characterization of Membrane Patch-Ion Channel Probes for Scanning Ion Conductance Microscopy.

Integration of dual-barrel membrane patch-ion channel probes (MP-ICPs) to scanning ion conductance microscopy (SICM) holds promise of providing a revolutionized approach of spatially resolved chemical sensing. A series of experiments are performed to further the understanding of the system and to answer some fundamental questions, in preparation for future developments of this approach. First, MP-ICPs are constructed that contain different types of ion channels including transient receptor potential vanilloid 1 and large conductance Ca2+ -activated K+ channels to establish the generalizability of the methods. Next, the capability of the MP-ICP platforms in single ion channel activity measurements is proved. In addition, the interplay between the SICM barrel and the ICP barrel is studied. For ion channels gated by uncharged ligands, channel activity at the ICP barrel is unaffected by the SICM barrel potential; whereas for ion channels that are gated by charged ligands, enhanced channel activity can be obtained by biasing the SICM barrel at potentials with opposite polarity to the charge of the ligand molecules. Finally, a proof-of-principle experiment is performed and site-specific molecular/ionic flux sensing is demonstrated at single-ion-channel level, which show that the MP-ICP platform can be used to quantify local molecular/ionic concentrations.

FHF2 isoforms differentially regulate Nav1.6-mediated resurgent sodium currents in dorsal root ganglion neurons.

Nav1.6 and Nav1.6-mediated resurgent currents have been implicated in several pain pathologies. However, our knowledge of how fast resurgent currents are modulated in neurons is limited. Our study explored the potential regulation of Nav1.6-mediated resurgent currents by isoforms of fibroblast growth factor homologous factor 2 (FHF2) in an effort to address the gap in our knowledge. FHF2 isoforms colocalize with Nav1.6 in peripheral sensory neurons. Cell line studies suggest that these proteins differentially regulate inactivation. In particular, FHF2A mediates long-term inactivation, a mechanism proposed to compete with the open-channel blocker mechanism that mediates resurgent currents. On the other hand, FHF2B lacks the ability to mediate long-term inactivation and may delay inactivation favoring open-channel block. Based on these observations, we hypothesized that FHF2A limits resurgent currents, whereas FHF2B enhances resurgent currents. Overall, our results suggest that FHF2A negatively regulates fast resurgent current by enhancing long-term inactivation and delaying recovery. In contrast, FHF2B positively regulated resurgent current and did not alter long-term inactivation. Chimeric constructs of FHF2A and Navß4 (likely the endogenous open channel blocker in sensory neurons) exhibited differential effects on resurgent currents, suggesting that specific regions within FHF2A and Navß4 have important regulatory functions. Our data also indicate that FHFAs and FHF2B isoform expression are differentially regulated in a radicular pain model and that associated neuronal hyperexcitability is substantially attenuated by a FHFA peptide. As such, these findings suggest that FHF2A and FHF2B regulate resurgent current in sensory neurons and may contribute to hyperexcitability associated with some pain pathologies.

Interaction site for the inhibition of tarantula Jingzhaotoxin-XI on voltage-gated potassium channel Kv2.1.

Jingzhaotoxin-XI (JZTX-XI) is a 34-residue peptide from the Chinese tarantula Chilobrachys jingzhao venom that potently inhibits both voltage-gated sodium channel Nav1.5 and voltage-gated potassium channel Kv2.1. In the present study, we further showed that JZTX-XI blocked Kv2.1 currents with the IC50 value of 0.39 ± 0.06 µM. JZTX-XI significantly shifted the current-voltage (I-V) curves and normalized conductance-voltage (G-V) curves of Kv2.1 channel to more depolarized voltages. Ala-scanning mutagenesis analyses demonstrated that mutants I273A, F274A, and E277A reduced toxin binding affinity by 10-, 16-, and 18-fold, respectively, suggesting that three common residues (I273, F274, E277) in the Kv2.1 S3b segment contribute to the formation of JZTX-XI receptor site, and the acidic residue Glu at the position 277 in Kv2.1 is the most important residue for JZTX-XI sensitivity. A single replacement of E277 with Asp(D) increased toxin inhibitory activity. These results establish that JZTX-XI inhibits Kv2.1 activation by trapping the voltage sensor in the rested state through a similar mechanism to that of HaTx1, but these two toxins have small differences in the most crucial molecular determinant. Furthermore, the in-depth investigation of the subtle differences in molecular determinants may be useful for increasing our understanding of the molecular details regarding toxin-channel interactions.

Membrane patches as ion channel probes for scanning ion conductance microscopy.

We describe dual-barrel ion channel probes (ICPs), which consist of an open barrel and a barrel with a membrane patch directly excised from a donor cell. When incorporated with scanning ion conductance microscopy (SICM), the open barrel (SICM barrel) serves to measure the distance-dependent ion current for non-invasive imaging and positioning of the probe in the same fashion of traditional SICM. The second barrel with the membrane patch supports ion channels of interest and was used to investigate ion channel activities. To demonstrate robust probe control with the dual-barrel ICP-SICM probe and verify that the two barrels are independently addressable, current-distance characteristics (approach curves) were obtained with the SICM barrel and simultaneous, current-time (I-T) traces were recorded with the ICP barrel. To study the influence that the distance between ligand-gated ion channels (i.e., large conductance Ca2+-activated K+ channels/BK channels) and the ligand source (i.e., Ca2+ source) has on channel activations, ion channel activities were recorded at two fixed probe-substrate distances (Dps) with the ICP barrel. The two fixed positions were determined from approach curves acquired with the SICM barrel. One position was defined as the "In-control" position, where the probe was in close proximity to the ligand source; the second position was defined as the "Far" position, where the probe was retracted far away from the ligand source. Our results confirm that channel activities increased dramatically with respect to both open channel probability and single channel current when the probe was near the ligand source, as opposed to when the probe was far away from the ligand source.

Characterization of ion channels on subesophageal ganglion neurons from Chinese tarantula Ornithoctonus huwena: Exploring the myth of the spider insensitive to its venom.

Chinese tarantula Ornithoctonus huwena is one of the most venomous spiders distributing in the hilly areas of southern China. In this study, using whole-cell patch-clamp technique we investigated electrophysiological and pharmacological properties of ion channels from tarantula subesophageal ganglion neurons. It was found that the neurons express multiple kinds of ion channels at least including voltage-gated calcium channels, TTX-sensitive sodium channels and two types of potassium channels. They exhibit pharmacological properties similar to mammalian subtypes. Spider calcium channels were sensitive to ω-conotoxin GVIA and diltiazem, two well-known inhibitors of mammalian neuronal high-voltage-activated (HVA) subtypes. 4-Aminopyridine and tetraethylammonium could inhibit spider outward transient and delayed-rectifier potassium channels, respectively. Huwentoxin-I and huwentoxin-IV are two abundant toxic components in the venom of Ornithoctonus huwena. Interestingly, although in our previous work they inhibit HVA calcium channels and TTX-sensitive sodium channels from mammalian sensory neurons, respectively, they fail to affect the subtypes from spider neurons. Moreover, the crude venom has no effect on delayed-rectifier potassium channels and only slightly reduces transient outward potassium channels with an IC50 value of ∼51.3 mg/L. Therefore, our findings provide important evidence for ion channels from spiders having an evolution as self-defense and prey mechanism.

Cardiac sodium channel palmitoylation regulates channel availability and myocyte excitability with implications for arrhythmia generation.

Cardiac voltage-gated sodium channels (Nav1.5) play an essential role in regulating cardiac electric activity by initiating and propagating action potentials in the heart. Altered Nav1.5 function is associated with multiple cardiac diseases including long-QT3 and Brugada syndrome. Here, we show that Nav1.5 is subject to palmitoylation, a reversible post-translational lipid modification. Palmitoylation increases channel availability and late sodium current activity, leading to enhanced cardiac excitability and prolonged action potential duration. In contrast, blocking palmitoylation increases closed-state channel inactivation and reduces myocyte excitability. We identify four cysteines as possible Nav1.5 palmitoylation substrates. A mutation of one of these is associated with cardiac arrhythmia (C981F), induces a significant enhancement of channel closed-state inactivation and ablates sensitivity to depalmitoylation. Our data indicate that alterations in palmitoylation can substantially control Nav1.5 function and cardiac excitability and this form of post-translational modification is likely an important contributor to acquired and congenital arrhythmias.

Stepwise Differentiation of Retinal Ganglion Cells from Human Pluripotent Stem Cells Enables Analysis of Glaucomatous Neurodegeneration.

Human pluripotent stem cells (hPSCs), including both embryonic and induced pluripotent stem cells, possess the unique ability to readily differentiate into any cell type of the body, including cells of the retina. Although previous studies have demonstrated the ability to differentiate hPSCs to a retinal lineage, the ability to derive retinal ganglion cells (RGCs) from hPSCs has been complicated by the lack of specific markers with which to identify these cells from a pluripotent source. In the current study, the definitive identification of hPSC-derived RGCs was accomplished by their directed, stepwise differentiation through an enriched retinal progenitor intermediary, with resultant RGCs expressing a full complement of associated features and proper functional characteristics. These results served as the basis for the establishment of induced pluripotent stem cells (iPSCs) from a patient with a genetically inherited form of glaucoma, which results in damage and loss of RGCs. Patient-derived RGCs specifically exhibited a dramatic increase in apoptosis, similar to the targeted loss of RGCs in glaucoma, which was significantly rescued by the addition of candidate neuroprotective factors. Thus, the current study serves to establish a method by which to definitively acquire and identify RGCs from hPSCs and demonstrates the ability of hPSCs to serve as an effective in vitro model of disease progression. Moreover, iPSC-derived RGCs can be utilized for future drug screening approaches to identify targets for the treatment of glaucoma and other optic neuropathies. Stem Cells 2016;34:1553-1562.

Human Nav1.6 Channels Generate Larger Resurgent Currents than Human Nav1.1 Channels, but the Navß4 Peptide Does Not Protect Either Isoform from Use-Dependent Reduction.

Voltage-gated sodium channels are responsible for the initiation and propagation of action potentials (APs). Two brain isoforms, Nav1.1 and Nav1.6, have very distinct cellular and subcellular expression. Specifically, Nav1.1 is predominantly expressed in the soma and proximal axon initial segment of fast-spiking GABAergic neurons, while Nav1.6 is found at the distal axon initial segment and nodes of Ranvier of both fast-spiking GABAergic and excitatory neurons. Interestingly, an auxiliary voltage-gated sodium channel subunit, Navß4, is also enriched in the axon initial segment of fast-spiking GABAergic neurons. The C-terminal tail of Navß4 is thought to mediate resurgent sodium current, an atypical current that occurs immediately following the action potential and is predicted to enhance excitability. To better understand the contribution of Nav1.1, Nav1.6 and Navß4 to high frequency firing, we compared the properties of these two channel isoforms in the presence and absence of a peptide corresponding to part of the C-terminal tail of Navß4. We used whole-cell patch clamp recordings to examine the biophysical properties of these two channel isoforms in HEK293T cells and found several differences between human Nav1.1 and Nav1.6 currents. Nav1.1 channels exhibited slower closed-state inactivation but faster open-state inactivation than Nav1.6 channels. We also observed a greater propensity of Nav1.6 to generate resurgent currents, most likely due to its slower kinetics of open-state inactivation, compared to Nav1.1. These two isoforms also showed differential responses to slow and fast AP waveforms, which were altered by the Navß4 peptide. Although the Navß4 peptide substantially increased the rate of recovery from apparent inactivation, Navß4 peptide did not protect either channel isoform from undergoing use-dependent reduction with 10 Hz step-pulse stimulation or trains of slow or fast AP waveforms. Overall, these two channels have distinct biophysical properties that may differentially contribute to regulating neuronal excitability.