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Scorpion β-toxins affect the activation of voltage-sensitive sodium channels (NaChs). Although these toxins have been instrumental in the study of channel gating and architecture, little is known about their active sites. By using an efficient system for the production of recombinant toxins, we analyzed by point mutagenesis the entire surface of the β-toxin, Bj-xtrIT, an anti-insect selective excitatory toxin from the scorpion Buthotus judaicus. Each toxin mutant was purified and analyzed using toxicity and binding assays, as well as by circular dichroism spectroscopy to discern the differences among mutations that caused structural changes and those that specifically affected bioactivity. This analysis highlighted a functional discontinuous surface of 1405 Ų, which was composed of a number of non-polar and three charged amino acids clustered around the main α-helical motif and the C-tail. Among the charged residues, Glu30 is a center of a putative "hot spot" in the toxin-receptor binding-interface and is shielded from bulk solvent by a hydrophobic "gasket" (Tyr26and Val34). Comparison of the Bj-xtrIT structure with that of other fi-toxins that are active on mammals suggests that the hot spot and an adjacent non-polar region are spatially conserved. These results highlight for the first time structural elements that constitute a putative "pharmacophore" involved in the interaction of β-toxins with receptor site-4 on NaChs. Furthermore, the unique structure of the C-terminal region most likely determines the specificity of excitatory toxins for insect NaChs. [ABSTRACT FROM AUTHOR]

Activation of voltage-gated sodium (Nav) channels initiates and propagates action potentials in electrically excitable cells. β-Scorpion toxins, including toxin IV from Centruroides suffusus suffusus (CssIV), enhance activation of Nav channels. CssIV stabilizes the voltage sensor in domain II in its activated state via a voltage-sensor trapping mechanism. Amino acid residues required for the action of CssIV have been identified in the S1-S2 and S3-S4 extracellular loops of domain II. The extracellular loops of domain III are also involved in toxin action, but individual amino acid residues have not been identified. We used site-directed mutagenesis and voltage clamp recording to investigate amino acid residues of domain III that are involved in CssIV action. In the IIISS2-S6 loop, five substitutions at four positions altered voltage-sensor trapping by CssIVE15A. Three substitutions (E1438A, D1445A, and D1445Y) markedly decreased voltage-sensor trapping, whereas the other two substitutions (N1436G and L1439A) increased voltage-sensor trapping. These bidirectional effects suggest that residues in IIISS2-S6 make both positive and negative interactions with CssIV. N1436G enhanced voltage-sensor trapping via increased binding affinity to the resting state, whereas L1439A increased voltage-sensor trapping efficacy. Based on these results, a three-dimensional model of the toxin-channel interaction was developed using the Rosetta modeling method. These data provide additional molecular insight into the voltage-sensor trapping mechanism of toxin action and define a three-point interaction site for β-scorpion toxins on Nav channels. Binding of α- and β-scorpion toxins to two distinct, pseudo-symmetrically organized receptor sites onNav channels acts synergistically to modify channel gating and paralyze prey. [ABSTRACT FROM AUTHOR]

Importance: National implementation of rapid trio genome sequencing (rtGS) in a clinical acute setting is essential to ensure advanced and equitable care for ill neonates., Objective: To evaluate the feasibility, diagnostic efficacy, and clinical utility of rtGS in neonatal intensive care units (NICUs) throughout Israel., Design, Setting, and Participants: This prospective, public health care-based, multicenter cohort study was conducted from October 2021 to December 2022 with the Community Genetics Department of the Israeli Ministry of Health and all Israeli medical genetics institutes (n = 18) and NICUs (n = 25). Critically ill neonates suspected of having a genetic etiology were offered rtGS. All sequencing, analysis, and interpretation of data were performed in a central genomics center at Tel-Aviv Sourasky Medical Center. Rapid results were expected within 10 days. A secondary analysis report, issued within 60 days, focused mainly on cases with negative rapid results and actionable secondary findings. Pathogenic, likely pathogenic, and highly suspected variants of unknown significance (VUS) were reported., Main Outcomes and Measures: Diagnostic rate, including highly suspected disease-causing VUS, and turnaround time for rapid results. Clinical utility was assessed via questionnaires circulated to treating neonatologists., Results: A total of 130 neonates across Israel (70 [54%] male; 60 [46%] female) met inclusion criteria and were recruited. Mean (SD) age at enrollment was 12 (13) days. Mean (SD) turnaround time for rapid report was 7 (3) days. Diagnostic efficacy was 50% (65 of 130) for disease-causing variants, 11% (14 of 130) for VUS suspected to be causative, and 1 novel gene candidate (1%). Disease-causing variants included 12 chromosomal and 52 monogenic disorders as well as 1 neonate with uniparental disomy. Overall, the response rate for clinical utility questionnaires was 82% (107 of 130). Among respondents, genomic testing led to a change in medical management for 24 neonates (22%). Results led to immediate precision medicine for 6 of 65 diagnosed infants (9%), an additional 2 (3%) received palliative care, and 2 (3%) were transferred to nursing homes., Conclusions and Relevance: In this national cohort study, rtGS in critically ill neonates was feasible and diagnostically beneficial in a public health care setting. This study is a prerequisite for implementation of rtGS for ill neonates into routine care and may aid in design of similar studies in other public health care systems.

Purpose: KLHL20 is part of a CUL3-RING E3 ubiquitin ligase involved in protein ubiquitination. KLHL20 functions as the substrate adaptor that recognizes substrates and mediates the transfer of ubiquitin to the substrates. Although KLHL20 regulates neurite outgrowth and synaptic development in animal models, a role in human neurodevelopment has not yet been described. We report on a neurodevelopmental disorder caused by de novo missense variants in KLHL20., Methods: Patients were ascertained by the investigators through Matchmaker Exchange. Phenotyping of patients with de novo missense variants in KLHL20 was performed., Results: We studied 14 patients with de novo missense variants in KLHL20, delineating a genetic syndrome with patients having mild to severe intellectual disability, febrile seizures or epilepsy, autism spectrum disorder, hyperactivity, and subtle dysmorphic facial features. We observed a recurrent de novo missense variant in 11 patients (NM_014458.4:c.1069G>A p.[Gly357Arg]). The recurrent missense and the 3 other missense variants all clustered in the Kelch-type β-propeller domain of the KLHL20 protein, which shapes the substrate binding surface., Conclusion: Our findings implicate KLHL20 in a neurodevelopmental disorder characterized by intellectual disability, febrile seizures or epilepsy, autism spectrum disorder, and hyperactivity., Competing Interests: Conflict of Interest M.M.M. and R.E.S. are employees of GeneDx, Inc. All other authors declare no conflict interest., (Copyright © 2022 American College of Medical Genetics and Genomics. Published by Elsevier Inc. All rights reserved.)

Experimental limitations such as optical loss and noise have prevented entanglement-enhanced measurements from demonstrating a significant quantum advantage in sensitivity. Holland-Burnett entangled states can mitigate these limitations and still present a quantum advantage in sensitivity. Here we model a fiber-based Mach-Zehnder interferometer with internal loss, detector efficiency, and external phase noise and without pure entanglement. This model features a practical fiber source that transforms the two-mode squeezed vacuum (TMSV) into Holland-Burnett entangled states. We predict that a phase sensitivity 28% beyond the shot noise limit is feasible with current technology. Simultaneously, a TMSV source can provide about 25 times more photon flux than other entangled sources. This system will make fiber-based quantum-enhanced sensing accessible and practical for remote sensing and probing photosensitive materials.

Mice use ultrasonic vocalizations (USVs) to convey a variety of socially relevant information. These vocalizations are affected by the sex, age, strain, and emotional state of the emitter and can thus be used to characterize it. Current tools used to detect and analyze murine USVs rely on user input and image processing algorithms to identify USVs, therefore requiring ideal recording environments. More recent tools which utilize convolutional neural networks models to identify vocalization segments perform well above the latter but do not exploit the sequential structure of audio vocalizations. On the other hand, human voice recognition models were made explicitly for audio processing; they incorporate the advantages of CNN models in recurrent models that allow them to capture the sequential nature of the audio. Here we describe the HybridMouse software: an audio analysis tool that combines convolutional (CNN) and recurrent (RNN) neural networks for automatically identifying, labeling, and extracting recorded USVs. Following training on manually labeled audio files recorded in various experimental conditions, HybridMouse outperformed the most commonly used benchmark model utilizing deep-learning tools in accuracy and precision. Moreover, it does not require user input and produces reliable detection and analysis of USVs recorded under harsh experimental conditions. We suggest that HybrideMouse will enhance the analysis of murine USVs and facilitate their use in scientific research., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Goussha, Bar, Netser, Cohen, Hel-Or and Wagner.)

Background: Contemporary models of cognitive behavioral therapy (CBT) for social anxiety disorder (SAD) emphasize emotion dysregulation as a core impairment whose reduction may play a causal role in psychotherapy. The current study examined changes in use of emotion regulation strategies as possible mechanisms of change in CBT for SAD. Specifically, we examined changes in expressive suppression and cognitive reappraisal during CBT and whether these changes predict treatment outcome., Methods: Patients (n = 34; 13 females; Mean age = 28.36 (6.97)) were allocated to 16-20 sessions of CBT. An electrocortical measure of emotion regulation and a clinician-rated measure of SAD were administered monthly. Self-report measures of emotion regulation and social anxiety were administered weekly. Multilevel models were used to examine changes in emotion regulation during treatment and cross-lagged associations between emotion regulation and anxiety., Results: CBT led to decreased suppression frequency, increased reappraisal self-efficacy, and decreased unpleasantness for SAD-related pictures (ps < .05). At post-treatment, patients were equivalent to healthy controls in terms of suppression frequency and subjective reactivity to SAD-related stimuli. Gains were maintained at 3-months follow-up. Decreases in suppression frequency and electrocortical reactivity to SAD-related pictures predicted lower subsequent anxiety but not the other way around (ps < .05). Lower anxiety predicted greater subsequent increases in reappraisal self-efficacy., Limitations: The lack of a control group precludes conclusions regarding mechanisms specificity., Conclusions: Decreased frequency of suppression is a potential mechanism of change in CBT for SAD., (Copyright © 2020. Published by Elsevier B.V.)

Majewski Osteodysplastic Primordial Dwarfism type II (MOPDII) is a form of dwarfism associated with severe microcephaly, characteristic skeletal findings, distinct dysmorphic features and increased risk for cerebral infarctions. The condition is caused by bi-allelic loss-of-function variants in the gene PCNT. Here we describe the identification of a novel founder pathogenic variant c.3465-1G > A observed in carriers from multiple Druze villages in Northern Israel. RNA studies show that the variant results in activation of a cryptic splice site causing a coding frameshift. The study was triggered by the diagnosis of a single child with MOPDII and emphasizes the advantages of applying next generation sequencing technologies in community genetics and the importance of establishing population-specific sequencing databases., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Purpose: Sifrim-Hitz-Weiss syndrome (SIHIWES) is a recently described multisystemic neurodevelopmental disorder caused by de novo variants inCHD4. In this study, we investigated the clinical spectrum of the disorder, genotype-phenotype correlations, and the effect of different missense variants on CHD4 function., Methods: We collected clinical and molecular data from 32 individuals with mostly de novo variants in CHD4, identified through next-generation sequencing. We performed adenosine triphosphate (ATP) hydrolysis and nucleosome remodeling assays on variants from five different CHD4 domains., Results: The majority of participants had global developmental delay, mild to moderate intellectual disability, brain anomalies, congenital heart defects, and dysmorphic features. Macrocephaly was a frequent but not universal finding. Additional common abnormalities included hypogonadism in males, skeletal and limb anomalies, hearing impairment, and ophthalmic abnormalities. The majority of variants were nontruncating and affected the SNF2-like region of the protein. We did not identify genotype-phenotype correlations based on the type or location of variants. Alterations in ATP hydrolysis and chromatin remodeling activities were observed in variants from different domains., Conclusion: The CHD4-related syndrome is a multisystemic neurodevelopmental disorder. Missense substitutions in different protein domains alter CHD4 function in a variant-specific manner, but result in a similar phenotype in humans.

PTPN23 is a His-domain protein-tyrosine phosphatase implicated in ciliogenesis, the endosomal sorting complex required for transport (ESCRT) pathway, and RNA splicing. Until recently, no defined human phenotype had been associated with alterations in this gene. We identified and report a cohort of seven patients with either homozygous or compound heterozygous rare deleterious variants in PTPN23. Combined with four patients previously reported, a total of 11 patients with this disorder have now been identified. We expand the phenotypic and variation spectrum associated with defects in this gene. Patients have strong phenotypic overlap, suggesting a defined autosomal recessive syndrome caused by reduced function of PTPN23. Shared characteristics of affected individuals include developmental delay, brain abnormalities (mainly ventriculomegaly and/or brain atrophy), intellectual disability, spasticity, language disorder, microcephaly, optic atrophy, and seizures. We observe a broad range of variants across patients that are likely strongly reducing the expression or disrupting the function of the protein. However, we do not observe any patients with an allele combination predicted to result in complete loss of function of PTPN23, as this is likely incompatible with life, consistent with reported embryonic lethality in the mouse. None of the observed or reported variants are recurrent, although some have been identified in homozygosis in patients from consanguineous populations. This study expands the phenotypic and molecular spectrum of PTPN23 associated disease and identifies major shared features among patients affected with this disorder, while providing additional support to the important role of PTPN23 in human nervous and visual system development and function.

Using trio exome sequencing, we identified de novo heterozygous missense variants in PAK1 in four unrelated individuals with intellectual disability, macrocephaly and seizures. PAK1 encodes the p21-activated kinase, a major driver of neuronal development in humans and other organisms. In normal neurons, PAK1 dimers reside in a trans-inhibited conformation, where each autoinhibitory domain covers the kinase domain of the other monomer. Upon GTPase binding via CDC42 or RAC1, the PAK1 dimers dissociate and become activated. All identified variants are located within or close to the autoinhibitory switch domain that is necessary for trans-inhibition of resting PAK1 dimers. Protein modelling supports a model of reduced ability of regular autoinhibition, suggesting a gain of function mechanism for the identified missense variants. Alleviated dissociation into monomers, autophosphorylation and activation of PAK1 influences the actin dynamics of neurite outgrowth. Based on our clinical and genetic data, as well as the role of PAK1 in brain development, we suggest that gain of function pathogenic de novo missense variants in PAK1 lead to moderate-to-severe intellectual disability, macrocephaly caused by the presence of megalencephaly and ventriculomegaly, (febrile) seizures and autism-like behaviour., (© The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain.)

Background: C-reactive protein (CRP) blood level is associated with clinical outcomes of several diseases. However, the independent predictive role of CRP in the heterogeneous population of patients admitted to internal medicine wards is not known., Objectives: To determine whether single CRP levels at admission independently predicts clinical outcome and flow of patients in general medicine wards., Methods: This study comprised 275 patients (50.5% female) with a mean age of 68.25 ± 17.0 years, hospitalized with acute disease in a general internal medicine ward. The association between admission CRP levels and clinical outcomes including mortality, the need for mechanical ventilation, duration of hospitalization, and re-admission within 6 months was determined., Results: A significant association was found between CRP increments of 80 mg/L and risk for the major clinical outcomes measured. The mortality odds ratio (OR) was 1.89 (95% confidence interval (95%CI, 1.37-2.61, P < 0.001), mechanical ventilation OR 1.67 (95%CI, 1.10-2.34, P = 0.006), re-admission within 6 months OR 2.29 (95%CI, 1.66-3.15 P < 0.001), and prolonged hospitalization >7 days OR 2.09 (95%CI, 1.59-2.74, P < 0.001). Lower increments of10 mg/L in CRP levels were associated with these outcomes although with lower ORs. Using a stepwise regression model for admission CRP levels resulted in area under the receiver operating characteristics curves between 0.70 and 0.76 for these outcomes., Conclusions: A single admission CRP blood level is independently associated with major parameters of clinical outcomes in acute care patients hospitalized in internal medicine wards.

Herein, we describe two members of one family who presented with recurrent episodes of hepatic failure, cerebellar ataxia, peripheral neuropathy, and short stature. Liver transplantation was considered. Whole-exome sequencing (Trio) revealed a synonymous variant in exon 4 of SCYL1:c.459C>T p. (Gly153Gly), which did not appear to affect the protein sequence. Computational prediction analysis suggested that this modification could alter the SCYL1 mRNA splicing processing to create a premature termination codon. The SCYL1 mRNAs in our patient's lymphocytes were analyzed and aberrant splicing was found. Molecular analysis of family members identified the parents as heterozygous recessive carriers and the proband as well as an affected aunt as homozygous. Evidently, harmless synonymous variants in the SCYL1 gene can damage gene splicing and hence the expression. We confirmed that the pathogenicity of this variant in the SCYL1 gene was associated with spinocerebellar ataxia, autosomal recessive 21 (SCAR21). Other reported cases (accept one) of liver failure found in the SCYL1 variants resolved during childhood, therefore orthotropic liver transplantation was no longer appropriate.

No studies have compared face-to-face cognitive-behavioral therapy (CBT) and attention bias modification (ABM) for social anxiety disorder (SAD) and their purported mechanisms. We asked: 1) Is CBT more effective than ABM? and 2) Are changes in attentional biases and cognitions temporally related to symptom change? Forty-three patients were randomly assigned to 8 sessions of ABM or up to 20 sessions of individual CBT. Intent-to-treat results revealed that CBT was superior to ABM in response rates and on symptom measures at endpoint, but not on other measures. No differences were found on measures in rates of change between CBT and ABM. Frequency of negative cognitions changed in both groups and negative beliefs changed only in CBT. Attentional bias did not change in either group. Cognitive changes bidirectionally correlated with symptom change in cross-lagged analyses in CBT, but not in ABM, suggesting a reciprocal relationship. Trial-level bias away from negative faces was simultaneously related to symptom change in ABM only. Results suggest that CBT is superior to ABM when administered at their typical doses, but raise questions given the similar rates of change. In addition, results support theories of cognitive change and raise questions about changes in attentional biases in CBT., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Objective: The molecular diagnosis of extreme forms of obesity, in which accurate detection of both copy number variations (CNVs) and point mutations, is crucial for an optimal care of the patients and genetic counseling for their families. Whole-exome sequencing (WES) has benefited considerably this molecular diagnosis, but its poor ability to detect CNVs remains a major limitation. We aimed to develop a method (CoDE-seq) enabling the accurate detection of both CNVs and point mutations in one step., Methods: CoDE-seq is based on an augmented WES method, using probes distributed uniformly throughout the genome. CoDE-seq was validated in 40 patients for whom chromosomal DNA microarray was available. CNVs and mutations were assessed in 82 children/young adults with suspected Mendelian obesity and/or intellectual disability and in their parents when available (n total  = 145)., Results: CoDE-seq not only detected all of the 97 CNVs identified by chromosomal DNA microarrays but also found 84 additional CNVs, due to a better resolution. When compared to CoDE-seq and chromosomal DNA microarrays, WES failed to detect 37% and 14% of CNVs, respectively. In the 82 patients, a likely molecular diagnosis was achieved in >30% of the patients. Half of the genetic diagnoses were explained by CNVs while the other half by mutations., Conclusions: CoDE-seq has proven cost-efficient and highly effective as it avoids the sequential genetic screening approaches currently used in clinical practice for the accurate detection of CNVs and point mutations., (Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.)

SPG45 is a rare form of autosomal recessive spastic paraplegia associated with mental retardation. Detailed phenotyping and mutation analysis was undertaken in three individuals with SPG45 from a consanguineous family of Arab Muslim origin. Using whole-exome sequencing, we identified a novel homozygous missense mutation in NT5C2 (c.1379T>C; p.Leu460Pro). Our data expand the molecular basis of SPG45, adding the first missense mutation to the current database of nonsense, frameshift, and splice site mutations. NT5C2 mutations seem to have a broad clinical spectrum and should be sought in patients manifesting either as uncomplicated or complicated HSP., (© 2017 Wiley Periodicals, Inc.)

Introduction: Whole exome sequencing is a diagnostic approach for the identification of molecular etiology in patients with suspected monogenic diseases. In this article we report on our experience with whole-exome sequencing (WES) of DNA samples taken from patients referred for genetic evaluation due to suspected undiagnosed genetic conditions., Methods: Exome enrichment was achieved by Nextera Rapid Capture Expanded Exome Kit. Whole-exome sequencing was performed on Illumina HiSeq 2500. Potentially damaging rare variants were selected for familial cosegregation analysis., Results: A total of 39 patients presenting a wide range of phenotypes suspected to have a genetic cause were sent to WES. Approximately 80% were children with neurological phenotypes. Variations having a high probability of being causative were identified in 20 families, achieving a 51.3% molecular diagnostic rate. Among these, 7 exhibited autosomal dominant disease, 12 autosomal recessive diseases and one X-linked disease; 28% of the patients (11/39) were found to carry a novel mutation located in previously reported genes. Novel mutations located in genes not known to be associated with genetic disease were identified in 23% of the patients (9/39)., Conclusions: Whole exome sequencing identified the underlying genetic cause in more than half of the patients referred for evaluation in the genetics clinic at the tertiary hospital. These data demonstrate the utility of WES as a powerful tool for effective diagnostics of monogenic genetic diseases.

Context: Primary ovarian insufficiency (POI) is caused by ovarian follicle depletion or follicle dysfunction, characterized by amenorrhea with elevated gonadotropin levels. The disorder presents as absence of normal progression of puberty., Objective: To elucidate the cause of ovarian dysfunction in a family with POI., Design: We performed whole-exome sequencing in 2 affected individuals. To evaluate whether DNA double-strand break (DSB) repair activities are altered in biallelic mutation carriers, we applied an enhanced green fluorescent protein-based assay for the detection of specific DSB repair pathways in blood-derived cells., Setting: Diagnoses were made at the Pediatric Endocrine Clinic, Clalit Health Services, Sharon-Shomron District, Israel. Genetic counseling and sample collection were performed at the Pediatric Genetics Unit, Schneider Children's Medical Center Israel, Petah Tikva, Israel., Patients and Intervention: Two sisters born to consanguineous parents of Israeli Muslim Arab ancestry presented with a lack of normal progression of puberty, high gonadotropin levels, and hypoplastic or absent ovaries on ultrasound. Blood samples for DNA extraction were obtained from all family members., Main Outcome Measure: Exome analysis to elucidate the cause of POI in 2 affected sisters., Results: Analysis revealed a stop-gain homozygous mutation in the SPIDR gene (KIAA0146) c.839G>A, p.W280*. This mutation altered SPIDR activity in homologous recombination, resulting in the accumulation of 53BP1-labeled DSBs postionizing radiation and γH2AX-labeled damage during unperturbed growth., Conclusions: SPIDR is important for ovarian function in humans. A biallelic mutation in this gene may be associated with ovarian dysgenesis in cases of autosomal recessive inheritance., (Copyright © 2017 by the Endocrine Society)

Inherited optic neuropathies are a heterogeneous group of disorders characterized by mild to severe visual loss, colour vision deficit, central or paracentral visual field defects and optic disc pallor. Optic atrophies can be classified into isolated or non-syndromic and syndromic forms. While multiple modes of inheritance have been reported, autosomal dominant optic atrophy and mitochondrial inherited Leber's hereditary optic neuropathy are the most common forms. Optic atrophy type 1, caused by mutations in the OPA1 gene is believed to be the most common hereditary optic neuropathy, and most patients inherit a mutation from an affected parent. In this study we used whole-exome sequencing to investigate the genetic aetiology in a patient affected with isolated optic atrophy. Since the proband was the only affected individual in his extended family, and was a product of consanguineous marriage, homozygosity mapping followed by whole-exome sequencing were pursued. Exome results identified a novel de novo OPA1 mutation in the proband. We conclude, that though de novo OPA1 mutations are uncommon, testing of common optic atrophy-associated genes such as mitochondrial mutations and OPA1 gene sequencing should be performed first in single individuals presenting with optic neuropathy, even when dominant inheritance is not apparent.

The ability of the brain to adapt to environmental demands implies that neurons can change throughout life. The extent to which single neurons actually change remains largely unstudied, however. To evaluate how functional properties of single neurons change over time, we devised a way to perform in vivo time-lapse electrophysiological recordings from the exact same neuron. We monitored the contralateral and ipsilateral sensory-evoked spiking activity of individual L2/3 neurons from the somatosensory cortex of mice. At the end of the first recording session, we electroporated the neuron with a DNA plasmid to drive GFP expression. Then, 2 wk later, we visually guided a recording electrode in vivo to the GFP-expressing neuron for the second time. We found that contralateral and ipsilateral evoked responses (i.e., probability to respond, latency, and preference), and spontaneous activity of individual L2/3 pyramidal neurons are stable under control conditions, but that this stability could be rapidly disrupted. Contralateral whisker deprivation induced robust changes in sensory-evoked response profiles of single neurons. Our experiments provide a framework for studying the stability and plasticity of single neurons over long time scales using electrophysiology.

Scorpion β-toxin 4 from Centruroides suffusus suffusus (Css4) enhances the activation of voltage-gated sodium channels through a voltage sensor trapping mechanism by binding the activated state of the voltage sensor in domain II and stabilizing it in its activated conformation. Here we describe the antagonist and partial agonist properties of a mutant derivative of this toxin. Substitution of seven different amino acid residues for Glu(15) in Css4 yielded toxin derivatives with both increased and decreased affinities for binding to neurotoxin receptor site 4 on sodium channels. Css4(E15R) is unique among this set of mutants in that it retained nearly normal binding affinity but lost its functional activity for modification of sodium channel gating in our standard electrophysiological assay for voltage sensor trapping. More detailed analysis of the functional effects of Css4(E15R) revealed weak voltage sensor trapping activity, which was very rapidly reversed upon repolarization and therefore was not observed in our standard assay of toxin effects. This partial agonist activity of Css4(E15R) is observed clearly in voltage sensor trapping assays with brief (5 ms) repolarization between the conditioning prepulse and the test pulse. The effects of Css4(E15R) are fit well by a three-step model of toxin action involving concentration-dependent toxin binding to its receptor site followed by depolarization-dependent activation of the voltage sensor and subsequent voltage sensor trapping. Because it is a partial agonist with much reduced efficacy for voltage sensor trapping, Css4(E15R) can antagonize the effects of wild-type Css4 on sodium channel activation and can prevent paralysis by Css4 when injected into mice. Our results define the first partial agonist and antagonist activities for scorpion toxins and open new avenues of research toward better understanding of the structure-function relationships for toxin action on sodium channel voltage sensors and toward potential toxin-based therapeutics to prevent lethality from scorpion envenomation.

The scorpion alpha-toxin Lqh2 (from Leiurus quinquestriatus hebraeus) is active at various mammalian voltage-gated sodium channels (Na(v)s) and is inactive at insect Na(v)s. To resolve the molecular basis of this preference we used the following strategy: 1) Lqh2 was expressed in recombinant form and key residues important for activity at the rat brain channel rNa(v)1.2a were identified by mutagenesis. These residues form a bipartite functional surface made of a conserved "core domain" (residues of the loops connecting the secondary structure elements of the molecule core), and a variable "NC domain" (five-residue turn and the C-tail) as was reported for other scorpion alpha-toxins. 2) The functional role of the two domains was validated by their stepwise construction on the similar scaffold of the anti-insect toxin LqhalphaIT. Analysis of the activity of the intermediate constructs highlighted the critical role of Phe(15) of the core domain in toxin potency at rNa(v)1.2a, and has suggested that the shape of the NC-domain is important for toxin efficacy. 3) Based on these findings and by comparison with other scorpion alpha-toxins we were able to eliminate the activity of Lqh2 at rNa(v)1.4 (skeletal muscle), hNa(v)1.5 (cardiac), and rNa(v)1.6 channels, with no hindrance of its activity at Na(v)1.1-1.3. These results suggest that by employing a similar approach the design of further target-selective sodium channel modifiers is imminent.

The bioactive surface of scorpion beta-toxins that interact with receptor site-4 at voltage-gated sodium channels is constituted of residues of the conserved betaalphabetabeta core and the C-tail. In an attempt to evaluate the extent by which residues of the toxin core contribute to bioactivity, the anti-insect and anti-mammalian beta-toxins Bj-xtrIT and Css4 were truncated at their N and C termini, resulting in miniature peptides composed essentially of the core secondary structure motives. The truncated beta-toxins (DeltaDeltaBj-xtrIT and DeltaDeltaCss4) were non-toxic and did not compete with the parental toxins on binding at receptor site-4. Surprisingly, DeltaDeltaBj-xtrIT and DeltaDeltaCss4 were capable of modulating in an allosteric manner the binding and effects of site-3 scorpion alpha-toxins in a way reminiscent of that of brevetoxins, which bind at receptor site-5. While reducing the binding and effect of the scorpion alpha-toxin Lqh2 at mammalian sodium channels, they enhanced the binding and effect of LqhalphaIT at insect sodium channels. Co-application of DeltaDeltaBj-xtrIT or DeltaDeltaCss4 with brevetoxin abolished the brevetoxin effect, although they did not compete in binding. These results denote a novel surface at DeltaDeltaBj-xtrIT and DeltaDeltaCss4 capable of interaction with sodium channels at a site other than sites 3, 4, or 5, which prior to the truncation was masked by the bioactive surface that interacts with receptor site-4. The disclosure of this hidden surface at both beta-toxins may be viewed as an exercise in "reverse evolution," providing a clue as to their evolution from a smaller ancestor of similar scaffold.

The affinity of scorpion alpha-toxins for various voltage-gated sodium channels (Na(v)s) differs considerably despite similar structures and activities. It has been proposed that key bioactive residues of the five-residue-turn (residues 8-12) and the C-tail form the NC domain, whose topology is dictated by a cis or trans peptide-bond conformation between residues 9 and 10, which correlates with the potency on insect or mammalian Na(v)s. We examined this hypothesis using Lqh3, an alpha-like toxin from Leiurus quinquestriatus hebraeus that is highly active in insects and mammalian brain. Lqh3 exhibits slower association kinetics to Na(v)s compared with other alpha-toxins and its binding to insect Na(v)s is pH-dependent. Mutagenesis of Lqh3 revealed a bi-partite bioactive surface, composed of the Core and NC domains, as found in other alpha-toxins. Yet, substitutions at the five-residue turn and stabilization of the 9-10 bond in the cis conformation did not affect the activity. However, substitution of hydrogen-bond donors/acceptors at the NC domain reduced the pH-dependency of toxin binding, while retaining its high potency at Drosophila Na(v)s expressed in Xenopus oocytes. Based on these results and the conformational flexibility and rearrangement of intramolecular hydrogen-bonds at the NC domain, evident from the known solution structure, we suggest that acidic pH or specific mutations at the NC domain favor toxin conformations with high affinity for the receptor by stabilizing the bound toxin-receptor complex. Moreover, the C-tail flexibility may account for the slower association rates and suggests a novel mechanism of dynamic conformer selection during toxin binding, enabling alpha-like toxins to affect a broad range of Na(v)s.

Scorpion alpha-toxins are similar in their mode of action and three-dimensional structure but differ considerably in affinity for various voltage-gated sodium channels (NaChs). To clarify the molecular basis of the high potency of the alpha-toxin LqhalphaIT (from Leiurus quinquestriatus hebraeus) for insect NaChs, we identified by mutagenesis the key residues important for activity. We have found that the functional surface is composed of two distinct domains: a conserved "Core-domain" formed by residues of the loops connecting the secondary structure elements of the molecule core and a variable "NC-domain" formed by a five-residue turn (residues 8-12) and a C-terminal segment (residues 56-64). We further analyzed the role of these domains in toxin activity on insects by their stepwise construction onto the scaffold of the anti-mammalian alpha-toxin, Aah2 (from Androctonus australis hector). The chimera harboring both domains, Aah2(LqhalphaIT(face)), was as active to insects as LqhalphaIT. Structure determination of Aah2(LqhalphaIT(face)) by x-ray crystallography revealed that the NC-domain deviates from that of Aah2 and forms an extended protrusion off the molecule core as appears in LqhalphaIT. Notably, such a protrusion is observed in all alpha-toxins active on insects. Altogether, the division of the functional surface into two domains and the unique configuration of the NC-domain illuminate the molecular basis of alpha-toxin specificity for insects and suggest a putative binding mechanism to insect NaChs.

Gating modifiers constitute a large group of polypeptide toxins that interact with the voltage-sensing module of ion channels. Among them, scorpion beta-toxins induce a negative shift in the voltage dependence of sodium channel activation. To explain their effect, a "voltage sensor trapping" model has been proposed in which the voltage sensor of domain-II (DIIS4) is trapped in an outward, activated position by a prebound beta-toxin upon membrane depolarization. Whereas toxin effect on channel activation was enhanced upon neutralization of the two outermost arginines in DIIS4, toxin residues involved in sensor trapping have not been identified. Using the scorpion excitatory beta-toxin, Bj-xtrIT, we found two conserved acidic residues, Glu15 and Glu30, mandatory for toxin action. Whereas mutagenesis of Glu30 affected both toxicity and binding affinity, substitutions E15A/F abolished activity but had minor effects on binding. Complete uncoupling of activity from binding was obtained with mutant E15R, acting as an efficient antagonist of Bj-xtrIT. On the basis of the voltage sensor trapping model and our results, we propose that Glu15 interacts with the emerging gating charges of DIIS4 upon membrane depolarization. Conserved acidic residues found in a variety of gating modifiers from scorpions and spiders may interact similarly with the voltage sensor.

Scorpion beta-toxins affect the activation of voltage-sensitive sodium channels (NaChs). Although these toxins have been instrumental in the study of channel gating and architecture, little is known about their active sites. By using an efficient system for the production of recombinant toxins, we analyzed by point mutagenesis the entire surface of the beta-toxin, Bj-xtrIT, an anti-insect selective excitatory toxin from the scorpion Buthotus judaicus. Each toxin mutant was purified and analyzed using toxicity and binding assays, as well as by circular dichroism spectroscopy to discern the differences among mutations that caused structural changes and those that specifically affected bioactivity. This analysis highlighted a functional discontinuous surface of 1405 A(2), which was composed of a number of non-polar and three charged amino acids clustered around the main alpha-helical motif and the C-tail. Among the charged residues, Glu(30) is a center of a putative "hot spot" in the toxin-receptor binding-interface and is shielded from bulk solvent by a hydrophobic "gasket" (Tyr(26) and Val(34)). Comparison of the Bj-xtrIT structure with that of other beta-toxins that are active on mammals suggests that the hot spot and an adjacent non-polar region are spatially conserved. These results highlight for the first time structural elements that constitute a putative "pharmacophore" involved in the interaction of beta-toxins with receptor site-4 on NaChs. Furthermore, the unique structure of the C-terminal region most likely determines the specificity of excitatory toxins for insect NaChs.