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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.

Voltage-gated sodium channel Na

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.

Background: Taxane-induced peripheral neuropathy (TIPN) is the most devastating survivorship issue for patients receiving therapy. Dose reductions due to TIPN in the curative setting lead to inferior outcomes for African American patients, as prior research has shown that this group is more susceptible to developing severe neuropathy. The mechanistic underpinnings of TIPN, however, have not been entirely elucidated. While it would be appealing to use primary tissue to study the development of TIPN, procuring nerves from patients is not realistically feasible, as nerve biopsies are painful and may result in permanent damage. Therefore, our laboratory has investigated paclitaxel-induced neuronal morphological and molecular changes using an ex vivo model of human-induced pluripotent stem cell (iPSC)-derived neurons.Methods: iPSCs are undifferentiated and endlessly dividing cells that can be generated from a patient’s somatic cells, such as peripheral blood mononuclear cells (PBMCs). We successfully reprogrammed PBMCs into iPSCs using the Erythroid Progenitor Reprograming Kit (STEMCell TechnologiesTM); pluripotency was verified by flow cytometry analysis. iPSCs were then induced into neurons using a differentiation protocol that bypasses the neural progenitor stage and uses selected small-molecule modulators of key signaling pathways (SMAD, Notch, FGFR1 inhibition and Wnt activation). Results: Flow cytometry analysis revealed expression of core pluripotency transcription factors Nanog, Oct3/4 and Sox2 in iPSCs overlaps with commercially purchased pluripotent cell line UCSD064i-20-2. Trilineage differentiation of iPSCs was confirmed with immunofluorescent imaging with germ-layer-specific markers; Sox17 and ExoA2 for ectoderm, Nestin and Pax6 for mesoderm, and Ncam and Brachyury for endoderm. Sensory neuron markers, β-III tubulin and Peripherin were applied to stain the cells for the maturity of iPSC-derived neurons. Patch-clamp electrophysiology and calcitonin gene-related peptide (CGRP) release data supported functionality of the induced neurons and provided insight into the timing for which downstream assays could be performed (week 4 post-induction). We have also performed a cell viability assay and fluorescence-activated cell sorting (FACS) using four cell-surface markers (CD184, CD44, CD15, and CD24) to select a neuronal population. At least 70% of the cells were viable in the isolated neuron population. Conclusion: We have found that these iPSC-derived neurons recapitulate mature neuronal phenotypes and demonstrate functionality. Thus, this represents a patient derived ex vivo neuronal model to investigate the molecular mechanisms of clinical TIPN. Citation Format: Geneva Cunningham, Erica Cantor, Xi Wu, Fei Shen, Guanglong Jiang, Santosh Philips, Casey Bales, Yucheng Xiao, Theodore R. Cummins, Jill C. Fehrenbacher, Bryan P. Schneider. Modeling taxane-induced peripheral neuropathy ex vivo using patient-derived neurons [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-07-09.

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 thePossumtransgenic 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 β4 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 β4 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 STATEMENTThis 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.

The falling-film flow characteristics significantly influence the heat and mass transfer process in the horizontal tube absorber. This study experimentally investigates the effect of ultrasonic field on the flow mode transition and thickness distribution of the liquid film on the horizontal tube. The water-calcium chloride (CaCl2/water) was selected as the working fluid. The effects of the factors, such as the tube diameter, tube spacing, solution concentration, ultrasonic power, sound source distance and ultrasonic incidence angle, on the hydrodynamics of the liquid flow were analyzed. The results show that the mode transition Reynolds number increases with the increasing ultrasonic power, but reduces comparing to that without ultrasound. Moreover, in the column mode, the fluctuation range of the film thickness decreases with the increase of the ultrasound power; and the ultrasonic field help increase the average film thickness outside the tube and enhance the uniformity of liquid film.

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 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 (Cys(1169), Cys(1170), and Cys(1978)) were identified. These sites differentially modulate distinct Nav1.6 properties. Interestingly, Cys(1978) is exclusive to Nav1.6 among all Nav isoforms and is evolutionally conserved in Nav1.6 among most species. Cys(1978) S-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.

Purpose Diabetes leads to the downregulation of the retinal Kir4.1 channels and Muller cell dysfunction. The insulin receptor substrate-1 (IRS-1) is a critical regulator of insulin signaling in Muller 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 Muller cells. The rat Muller 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 Muller cells and the dysfunctional signaling mediated by IRS-1 may be detrimental to Kir4.1.

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 Navβ4 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.

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.

Voltage-gated sodium channels (VGSCs) in peripheral sensory neurons play a critical role in transmitting information regarding noxious stimuli from the periphery to the central nervous system. Multiple VGSCs are expressed in nociceptive neurons. Nociceptive neurons express fast, slow, and persistent VGSCs that can exhibit diverse pharmacology. It is proposed that these isoforms exhibit unique properties that are specifically tuned for the failsafe transmission of nociceptive information in spite of tissue damage. Genetic mutations in several isoforms can cause both loss of pain sensation and severe neuropathic pain sensations. Aberrant pain sensations associated with inherited and acquired pain conditions can be difficult to treat, but it is hoped that a detailed understanding of the structure and function of sensory neuronal VGSCs will aid the development of targeted isoform-specific inhibitors. This chapter reviews what is known about VGSCs that play specialized roles in pain transmission.

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.

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. However, to fully exploit the MP-ICP platforms for concurrent topography and chemical sensing, a comprehensive characterization of the analytical performance of this newly-developed platform is needed. In this report, a series of experiments were performed to further our understanding of the system and to answer some fundamental questions, in preparation for future developments of this approach. First, we constructed MP-ICPs that contained different types of ion channels including TRPV1 and BK channels to establish the generalizability of the methods. Next, we proved the capability of the MP-ICP platforms in single ion channel activity measurements, and demonstrated that the channel behaviors can be faithfully obtained with this approach. In addition, we studied the interplay between the SICM barrel and the ICP barrel. 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 was performed and site-specific molecular/ionic flux sensing was demonstrated at single-ion-channel level, which showed that the MP-ICP platform can be used to quantify local molecular/ionic concentrations.

We prepared 13 derivatives of N-(biphenyl-4′-yl)methyl (R)-2-acetamido-3-methoxypropionamide that differed in type and placement of a R-substituent in the terminal aryl unit. We demonstrated that the R-substituent impacted the compound’s whole animal and cellular pharmacological activities. In rodents, select compounds exhibited excellent anticonvulsant activities and protective indices (PI = TD50/ED50) that compared favorably with clinical antiseizure drugs. Compounds with a polar, aprotic R-substituent potently promoted Na+ channel slow inactivation and displayed frequency (use) inhibition of Na+ currents at low micromolar concentrations. The possible advantage of affecting these two pathways to decrease neurological hyperexcitability is discussed.

Voltage-gated sodium channels are critical determinants of nerve and muscle excitability. Although numerous toxins and small molecules target sodium channels, identifying the mechanisms of action is challenging. Here we used gating-pore currents selectively generated in each of the voltage-sensors from the four α-subunit domains (DI–DIV) to monitor the activity of individual voltage-sensors and to investigate the molecular determinants of sodium channel pharmacology. The tarantula toxin huwentoxin-IV (HWTX-IV), which inhibits sodium channel current, exclusively enhanced inward gating-pore currents through the DII voltage-sensor. By contrast, the tarantula toxin ProTx-II, which also inhibits sodium channel currents, altered the gating-pore currents in multiple voltage-sensors in a complex manner. Thus, whereas HWTX-IV inhibits central-pore currents by selectively trapping the DII voltage-sensor in the resting configuration, ProTx-II seems to inhibit central-pore currents by differentially altering the configuration of multiple voltage-sensors. The sea anemone toxin anthopleurin B, which impairs open-channel inactivation, exclusively enhanced inward gating-pore currents through the DIV voltage-sensor. This indicates that trapping the DIV voltage-sensor in the resting configuration selectively impairs open-channel inactivation. Furthermore, these data indicate that although activation of all four voltage-sensors is not required for central-pore current generation, activation of the DII voltage-sensor is crucial. Overall, our data demonstrate that gating-pore currents can determine the mechanism of action for sodium channel gating modifiers with high precision. We propose this approach could be adapted to identify the molecular mechanisms of action for gating modifiers of various voltage-gated ion channels.

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 Navβ4 (likely the endogenous open channel blocker in sensory neurons) exhibited differential effects on resurgent currents, suggesting that specific regions within FHF2A and Navβ4 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.

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.

Over 150 mutations in theSCN2Agene, 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 Navβ4 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 inXenopusoocytes 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.

Bite Me! As unpleasant as it is, pain serves a purpose, to alert the body to potential damage. This protective function may explain why few predators have evolved resistance to the painful venoms used as a defense by their prey. The grasshopper mouse, however, is insensitive to one of the most painful stings in the animal kingdom—that of the bark scorpion. Rowe et al. (p. 441 ; see the Perspective by Lewin ) now show that grasshopper mice use the toxins present in the scorpion venom to block voltage-gated pain transmission, temporarily reducing their sensitivity to nonvenom-induced pain. Thus, grasshopper mice use the scorpion's painful defense to their advantage and have evolved a mechanism that allows for reduction of pain sensitivity only when it is needed.

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.

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., Cardiac voltage-gated sodium channels (Nav1.5) are crucial regulators of heart electric activity. Here the authors show that palmitoylation, a process of lipid modification of cysteine residues, modulates Nav1.5 function and affects cardiomyocyte excitability, representing a potential target in treating cardiac diseases.

Key points • We investigated how the F1486 deletion LQT3 mutation impairs the functional properties of the human cardiac voltage-gated sodium channel (hNav1.5) and alters action potential firing. • Voltage-clamp recordings from HEK 293 cells and cardiomyocytes expressing recombinant channels demonstrated that the F1486del mutation reduces peak current density but also impairs inactivation and increases late current density. • Current-clamp recordings from cardiomyocytes indicated that the increase in late current density would result in prolonged action potential duration and this was confirmed using computer simulations. • The deletion of F1486 abolished the ability of lidocaine to stabilize the inactivated state and eliminated the high-affinity binding of lidocaine to inactivated channels. • Our data show that the hNav1.5-F1486del mutation has complex functional consequences and indicate that knowledge of the specific molecular defect is critical when developing potential treatments for individuals with prolonged QT intervals. Abstract The deletion of phenylalanine 1486 (F1486del) in the human cardiac voltage-gated sodium channel (hNav1.5) is associated with fatal long QT (LQT) syndrome. In this study we determined how F1486del impairs the functional properties of hNav1.5 and alters action potential firing in heterologous expression systems (human embryonic kidney (HEK) 293 cells) and their native cardiomyocyte background. Cells expressing hNav1.5-F1486del exhibited a loss-of-function alteration, reflected by an 80% reduction of peak current density, and several gain-of-function alterations, including reduced channel inactivation, enlarged window current, substantial augmentation of persistent late sodium current and an increase in ramp current. We also observed substantial action potential duration (APD) prolongation and prominent early afterdepolarizations (EADs) in neonatal cardiomyocytes expressing the F1486del channels, as well as in computer simulations of myocyte activity. In addition, lidocaine sensitivity was dramatically reduced, which probably contributed to the poor therapeutic outcome observed in the patient carrying the hNav1.5-F1486del mutation. Therefore, despite the significant reduction in peak current density, the F1486del mutation also leads to substantial gain-of-function alterations that are sufficient to cause APD prolongation and EADs, the predominant characteristic of LQTs. These data demonstrate that hNav1.5 mutations can have complex functional consequences and highlight the importance of identifying the specific molecular defect when evaluating potential treatments for individuals with prolonged QT intervals.

Huwentoxin-I (HWTX-I) is a 33-residue peptide isolated from the venom of Ornithoctonus huwena and could inhibit TTX-sensitive voltage-gated sodium channels and N-type calcium channels in mammalian dorsal root ganglion (DRG) neurons. However, the effects of HWTX-I on mammalian central neuronal and insect sodium channel subtypes remain unknown. In this study, we found that HWTX-I potently inhibited sodium channels in rat hippocampal and cockroach dorsal unpaired median (DUM) neurons with the IC(50) values of 66.1±5.2 and 4.80±0.58nM, respectively. Taken together with our previous work on DRG neurons (IC(50)≈55nM), the order of sodium channel sensitivity to HWTX-I inhibition was insect central DUM≫mammalian peripheral>mammalian central neurons. HWTX-I exhibited no effect on the steady-state activation and inactivation of sodium channels in rat hippocampal and cockroach DUM neurons.

Alternative splicing is known to alter pharmacological sensitivities, kinetics, channel distribution under pathological conditions, and developmental regulation of VGSCs. Mutations that alter channel properties in Na(V)1.7 have been genetically implicated in patients with bouts of extreme pain classified as inherited erythromelalgia (IEM) or paroxysmal extreme pain disorder (PEPD). Furthermore, patients with IEM or PEPD report differential age onsets. A recent study reported that alternative splicing of Na(V)1.7 exon 5 affects ramp current properties. Since IEM and PEPD mutations also alter Na(V)1.7 ramp current properties we speculated that alternative splicing might impact the functional consequences of IEM or PEPD mutations. We compared the effects alternative splicing has on the biophysical properties of Na(V)1.7 wild-type, IEM (I136V) and PEPD (I1461T) channels. Our major findings demonstrate that although the 5A splice variant of the IEM channel had no functional impact, the 5A splice variant of the PEPD channel significantly hyperpolarized the activation curve, slowed deactivation and closed-state inactivation, shifted the ramp current activation to more hyperpolarized potentials, and increased ramp current amplitude. We hypothesize a D1/S3-S4 charged residue difference between the 5N (Asn) and the 5A (Asp) variants within the coding region of exon 5 may contribute to shifts in channel activation and deactivation. Taken together, the additive effects observed on ramp currents from exon 5 splicing and the PEPD mutation (I1461T) are likely to impact the disease phenotype and may offer insight into how alternative splicing may affect specific intramolecular interactions critical for voltage-dependent gating.

Our previous work demonstrated that huwentoxin-IV was an inhibitor cystine knot peptide from Chinese tarantula Ornithoctonus huwena venom that blocked tetrodotoxin-sensitive voltage-gated sodium channels from mammalian sensory neurons [Peng, K., Shu, Q., Liu, Z., Liang, S., 2002. Function and solution structure of huwentoxin-IV, a potent neuronal tetrodotoxin (TTX)-sensitive sodium channel antagonist from Chinese bird spider Selenocosmia huwena. J. Biol. Chem. 277(49), 47564-47571]. However, the actions of the neurotoxin on central neuronal sodium channels remain unknown. In this study, we chemically synthesized native huwentoxin-IV and found that sodium channel isoforms from rat hippocampus neurons were also sensitive to native and synthetic toxins, but the toxin-binding affinity (IC(50) approximately 0.4 microM) was 12-fold lower than to peripheral isoforms. The blockade by huwentoxin-IV could be reversed by strong depolarization due to the dissociation of toxin-channel complex as observed for receptor site 3 toxins. Moreover, small unilamellar vesicle-binding assays showed that in contrast to ProTx-II from the tarantula Thrixopelma pruriens, huwentoxin-IV almost lacked the ability to partition into the negatively charged and neutral phospholipid bilayer of artificial membranes. These findings indicated that huwentoxin-IV was a sodium channel antagonist preferentially targeting peripheral isoforms via a mechanism quite different from ProTx-II.

Jingzhaotoxin-III (JZTX-III) is a peptide toxin isolated from the venom of the Chinese spider Chilobrachys jingzhao that inhibits Nav channels of rat cardiac myocytes by modifying voltage-dependent gating and also binds to Kv2.1 channel (Kd = 0.43 microM) with an action model similar to that of hanatoxin1 and SGTx1. The solution structure of JZTX-III was determined by (1)H 2D NMR method. The toxin adopts an ICK motif composed of three beta-strands connected by four turns. Structural comparison of JZTX-III with those of other ICK motif peptides shows that they all adopt a conserved surface profile, a hydrophobic patch surrounded by charged residues, which might be the crucial site for voltage-gating ion channel inhibition. Furthermore, the similar action model of JZTX-III affecting both Kv and Nav channels implies that JZTX-III recognized a conserved receptor within the voltage sensing domains, which is similar to that of hanatoxin1 binding to both Kv and Cav channels.

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.

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, Navβ4, is also enriched in the axon initial segment of fast-spiking GABAergic neurons. The C-terminal tail of Navβ4 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 Navβ4 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 Navβ4. 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 Navβ4 peptide. Although the Navβ4 peptide substantially increased the rate of recovery from apparent inactivation, Navβ4 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.

We have isolated a cardiotoxin, denoted jingzhaotoxin-III (JZTX-III), from the venom of the Chinese spider Chilobrachys jingzhao. The toxin contains 36 residues stabilized by three intracellular disulfide bridges (I-IV, II-V, and III-VI), assigned by a chemical strategy of partial reduction and sequence analysis. Cloned and sequenced using 3'-rapid amplification of cDNA ends and 5'-rapid amplification of cDNA ends, the full-length cDNA encoded a 63-residue precursor of JZTX-III. Different from other spider peptides, it contains an uncommon endoproteolytic site (-X-Ser-) anterior to mature protein and the intervening regions of 5 residues, which is the smallest in spider toxin cDNAs identified to date. Under whole cell recording, JZTX-III showed no effects on voltage-gated sodium channels (VGSCs) or calcium channels in dorsal root ganglion neurons, whereas it significantly inhibited tetrodotoxin-resistant VGSCs with an IC(50) value of 0.38 microm in rat cardiac myocytes. Different from scorpion beta-toxins, it caused a 10-mV depolarizing shift in the channel activation threshold. The binding site for JZTX-III on VGSCs is further suggested to be site 4 with a simple competitive assay, which at 10 microm eliminated the slowing currents induced by Buthus martensi Karsch I (BMK-I, scorpion alpha-like toxin) completely. JZTX-III shows higher selectivity for VGSC isoforms than other spider toxins affecting VGSCs, and the toxin hopefully represents an important ligand for discriminating cardiac VGSC subtype.

Hainantoxin-I is a novel peptide toxin, purified from the venom of the Chinese bird spider Selenocosmia hainana (=Ornithoctonus hainana). It includes 33 amino acid residues with a disulfide linkage of I–IV, II–V and III–VI, assigned by partial reduction and sequence analysis. Under two-electrode voltage-clamp conditions, hainantoxin-I can block rNav1.2/β1 and the insect sodium channel para/tipE expressed in Xenopus laevis oocytes with IC50 values of 68±6 μM and 4.3±0.3 μM respectively. The three-dimensional solution structure of hainantoxin-I belongs to the inhibitor cystine knot structural family determined by two-dimensional 1H nuclear magnetic resonance techniques. Structural comparison of hainantoxin-I with those of other toxins suggests that the combination of the charged residues and a vicinal hydrophobic patch should be responsible for ligand binding. This is the first report of an insect sodium channel blocker from spider venom and it provides useful information for the structure–function relationship studies of insect sodium channels.

Hainantoxin-III and hainantoxin-IV, isolated from the venom of the Chinese bird spider Seleconosmia hainana , are neurotoxic peptides composed of 33–35 residues with three disulfide bonds. Using whole-cell patch-clamp technique, we investigated their action on ionic channels of adult rat dorsal root ganglion neurons. It was found that the two toxins did not affect Ca 2+ channels (both high voltage activated and low voltage activated types) nor tetrodotoxin-resistant voltage-gated Na + channels (VGSCs). However, hainantoxin-III and hainantoxin-IV strongly depressed the amplitude of tetrodotoxin-sensitive Na + currents with IC 50 values of 1.1 and 44.6 nM, respectively. Both hainantoxin-III (1 nM) and hainantoxin-IV (50 nM) caused a hyperpolarizing shift of about 10 mV in the voltage midpoint of steady-state Na + channel inactivation, but they showed difference in the reprime kinetics of VGSCs: hainantoxin-III significantly decreased the recovery rate from inactivation at a prepulse potential of −80 mV while hainantoxin-IV did not do. It is interesting to note that similar to huwentoxin-IV, the two hainantoxins did not affect the activation and inactivation kinetics of Na + currents and at a concentration of 1 μM they completely inhibited the slowing inactivation currents induced by BMK-I (toxin I from the scorpion Buthus martensi Karsch), a scorpion α-like toxin. The results indicate that hainantoxin-III and hainantoxin-IV are novel spider toxins and affect the mammal neural Na + channels through a mechanism quite different from other spider toxins targeting the neural receptor site 3, such as δ-aractoxins and μ-agatoxins.

A neurotoxin, named hainantoxin-IV, was purified from the venom of the spider Selenocosmia hainana. The amino acid sequence was determined by Edman degradation, revealing it to be a 35-residue polypeptide amidated at its C terminal and including three disulfide bridges: Cys2-Cys17, Cys9-Cys24, and Cys16-Cys31 assigned by partial reduction and sequence analysis. Hainantoxin-IV shares 80% sequence identity with huwentoxin-IV from the spider S. huwena, a potent antagonist that acts at site 1 on tetrodotoxin-sensitive (TTX-S) sodium channels, suggesting that hainantoxin-IV adopts an inhibitor cystine knot structural motif like huwentoin-IV. Under whole-cell voltage-clamp conditions, this toxin has no effect on tetrodotoxin-resistant voltage-gated sodium channels in adult rat dorsal root ganglion neurons, while it blocks TTX-S sodium channels in a manner similar to huwentoxin-IV, and the actions of both toxins on sodium currents are very similar to that of tetrodotoxin. Thus, they define a new family of spider toxins affecting sodium channels.

Voltage-gated sodium channel (NaV) trafficking is incompletely understood. Post-translational modifications of NaVs and/or auxiliary subunits and protein-protein interactions have been posited as NaV-trafficking mechanisms. Here, we tested if modification of the axonal collapsin response mediator protein 2 (CRMP2) by a small ubiquitin-like modifier (SUMO) could affect NaV trafficking; CRMP2 alters the extent of NaV slow inactivation conferred by the anti-epileptic (R)-lacosamide, implying NaV-CRMP2 functional coupling. Expression of a CRMP2 SUMOylation-incompetent mutant (CRMP2-K374A) in neuronal model catecholamine A differentiated (CAD) cells did not alter lacosamide-induced NaV slow inactivation compared with CAD cells expressing wild type CRMP2. Like wild type CRMP2, CRMP2-K374A expressed robustly in CAD cells. Neurite outgrowth, a canonical CRMP2 function, was moderately reduced by the mutation but was still significantly higher than enhanced GFP-transfected cortical neurons. Notably, huwentoxin-IV-sensitive NaV1.7 currents, which predominate in CAD cells, were significantly reduced in CAD cells expressing CRMP2-K374A. Increasing deSUMOylation with sentrin/SUMO-specific protease SENP1 or SENP2 in wild type CRMP2-expressing CAD cells decreased NaV1.7 currents. Consistent with a reduction in current density, biotinylation revealed a significant reduction in surface NaV1.7 levels in CAD cells expressing CRMP2-K374A; surface NaV1.7 expression was also decreased by SENP1 + SENP2 overexpression. Currents in HEK293 cells stably expressing NaV1.7 were reduced by CRMP2-K374A in a manner dependent on the E2-conjugating enzyme Ubc9. No decrement in current density was observed in HEK293 cells co-expressing CRMP2-K374A and NaV1.1 or NaV1.3. Diminution of sodium currents, largely NaV1.7, was recapitulated in sensory neurons expressing CRMP2-K374A. Our study elucidates a novel regulatory mechanism that utilizes CRMP2 SUMOylation to choreograph NaV1.7 trafficking.