Search for asymptomatic carriers of SARS-CoV-2 in healthcare workers during the pandemic: a Spanish experience.

BACKGROUND: Asymptomatic carriers of SARS-CoV-2 can be a vehicle for transmission of the infection. This fact is of particular importance in the case of healthcare workers (HCWs). DESIGN: Cross-sectional study in HCWs in a medium size hospital in the South of Spain. METHODS: between April 15 and 25, 2020, naso and oropharyngeal PCR determination was performed together with IgG and IgM antibody determination by immunochromatography to the HCWs of the Costa del Sol Hospital in Marbella of the units involved in patient care with CoVID-19: Emergencies, Intensive Care and Anesthesia, Internal Medicine and Pneumology. Other units not directly involved in the care of these patients were offered to participate. On the day of sampling, a health questionnaire was answered, reporting symptoms on the same day and in the previous fourteen days. RESULTS: 498 HCWs were studied. Two individuals were detected with PCR for SARS-CoV-2 positive. Both were asymptomatic on the day of sampling, but one of them had had a CoVID-19 compatible picture in the previous two weeks and had positive IgG and IgM; therefore, only one subject was truly asymptomatic carrier (0.2%). 9 workers with positive IgG (1.8%) were detected. CONCLUSIONS: the prevalence of asymptomatic carriers among health workers of the services directly involved in the care of patients with CoVID-19 was very low in our center. This type of strategy can be one more tool in controlling the pandemic.

Suitability of the eight-item version of the Brazilian Household Food Insecurity Measurement Scale to identify risk groups: evidence from a nationwide representative sample.

OBJECTIVE: The Brazilian Household Food Insecurity Measurement Scale (EBIA) has eight general/adult items applied in all households and six additional items exclusively asked in households with children and/or adolescents (HHCA). Continuing an investigation programme on the adequacy of model-based cut-off points for EBIA, the present study aims to: (i) explore the capacity of properly stratifying HHCA according to food insecurity (FI) severity level by applying only the eight 'generic' items; and (ii) compare it against the fourteen-item scale. DESIGN: Latent class factor analysis (LCFA) models were applied to the answers to the eight general/adult items to identify latent groups corresponding to FI levels and optimal group-separating cut-off points. Analyses involved a thorough classification agreement evaluation and were performed at the national level and by macro-regions. SETTING: Data derived from the cross-sectional Brazilian National Household Sample Survey of 2013. PARTICIPANTS: A nationally representative sample of 116 543 households. RESULTS: In all households and investigated domains, LCFA detected four distinct household food (in)security groups (food security and three levels of severity of FI) and the same set of cut-off points (1/2, 4/5 and 6/7). Misclassification in the aggregate data was 0·66 % in adult-only households and 1·06 % in HHCA. Comparison of the scale reduced to eight items with the 'original' fourteen-item scale demonstrated consistency in the classification. In HHCA, the agreement between both classifications was 96·2 %. CONCLUSIONS: Results indicate the eight 'generic' items in HHCA can be reliably used when it is not possible to apply the fourteen-item scale.

Poloxamer 188 decreases membrane toxicity of mutant SOD1 and ameliorates pathology observed in SOD1 mouse model for ALS.

Here we report a gain in function for mutant (mt) superoxide dismutase I (SOD1), a cause of familial amyotrophic lateral sclerosis (FALS), wherein small soluble oligomers of mtSOD1 acquire a membrane toxicity. Phosphatidylglycerol (PG) lipid domains are selectively targeted, which could result in membrane damage or "toxic channels" becoming active in the bilayer. This PG-selective SOD1-mediated membrane toxicity is largely reversible in vitro by a widely-available FDA-approved surfactant and membrane-stabilizer P188. Treatment of G93ASOD1 transgenic mice with P188 significantly delayed symptoms onset, extended survival and decreased motoneuron death. The use of P188 or an analogue, which targets mtSOD1 misfolding-induced membrane toxicity, may provide a new direction for ALS treatment.

Nav channel binder containing a specific conjugation-site based on a low toxicity ß-scorpion toxin.

Voltage-gated sodium (Nav) channels play a key role in generating action potentials which leads to physiological signaling in excitable cells. The availability of probes for functional studies of mammalian Nav is limited. Here, by introducing two amino acid substitutions into the beta scorpion toxin Ts1, we have chemically synthesized a novel binder [S14R, W50Pra]Ts1 for Nav with high affinity, low dissociation rate and reduced toxicity while retaining the capability of conjugating Ts1 with molecules of interests for different applications. Using the fluorescent-dye conjugate, [S14R, W50Pra(Bodipy)]Ts1, we confirmed its binding to Nav1.4 through Lanthanide-based Resonance Energy Transfer. Moreover, using the gold nanoparticle conjugate, [S14R, W50Pra(AuNP)]Ts1, we were able to optically stimulate dorsal root ganglia neurons and generate action potentials with visible light via the optocapacitive effect as previously reported. [S14R, W50Pra]Ts1 is a novel probe with great potential for wider applications in Nav-related neuroscience research.

Modeling Optimal Cutoffs for the Brazilian Household Food Insecurity Measurement Scale in a Nationwide Representative Sample.

Background: This is the second part of a model-based approach to examine the suitability of the current cutoffs applied to the raw score of the Brazilian Household Food Insecurity Measurement Scale [Escala Brasileira de Insegurança Alimentar (EBIA)]. The approach allows identification of homogeneous groups who correspond to severity levels of food insecurity (FI) and, by extension, discriminant cutoffs able to accurately distinguish these groups.Objective: This study aims to examine whether the model-based approach for identifying optimal cutoffs first implemented in a local sample is replicated in a countrywide representative sample.Methods: Data were derived from the Brazilian National Household Sample Survey of 2013 (n = 116,543 households). Latent class factor analysis (LCFA) models from 2 to 5 classes were applied to the scale's items to identify the number of underlying FI latent classes. Next, identification of optimal cutoffs on the overall raw score was ascertained from these identified classes. Analyses were conducted in the aggregate data and by macroregions. Finally, model-based classifications (latent classes and groupings identified thereafter) were contrasted to the traditionally used classification.Results: LCFA identified 4 homogeneous groups with a very high degree of class separation (entropy = 0.934-0.975). The following cutoffs were identified in the aggregate data: between 1 and 2 (1/2), 5 and 6 (5/6), and 10 and 11 (10/11) in households with children and/or adolescents <18 y of age (score range: 0-14), and 1/2, between 4 and 5 (4/5), and between 6 and 7 (6/7) in adult-only households (range: 0-8). With minor variations, the same cutoffs were also identified in the macroregions. Although our findings confirm, in general, the classification currently used, the limit of 1/2 (compared with 0/1) for separating the milder from the baseline category emerged consistently in all analyses.Conclusions: Nationwide findings corroborate previous local evidence that households with an overall score of 1 are more akin to those scoring negative on all items. These results may contribute to guide experts' and policymakers' decisions on the most appropriate EBIA cutoffs.

Mechanism of functional interaction between potassium channel Kv1.3 and sodium channel NavBeta1 subunit.

The voltage-gated potassium channel subfamily A member 3 (Kv1.3) dominantly expresses on T cells and neurons. Recently, the interaction between Kv1.3 and NavBeta1 subunits has been explored through ionic current measurements, but the molecular mechanism has not been elucidated yet. We explored the functional interaction between Kv1.3 and NavBeta1 through gating current measurements using the Cut-open Oocyte Voltage Clamp (COVC) technique. We showed that the N-terminal 1-52 sequence of hKv1.3 disrupts the channel expression on the Xenopus oocyte membrane, suggesting a potential role as regulator of hKv1.3 expression in neurons and lymphocytes. Our gating currents measurements showed that NavBeta1 interacts with the voltage sensing domain (VSD) of Kv1.3 through W172 in the transmembrane segment and modifies the gating operation. The comparison between G-V and Q-V with/without NavBeta1 indicates that NavBeta1 may strengthen the coupling between hKv1.3-VSD movement and pore opening, inducing the modification of kinetics in ionic activation and deactivation.

Mapping of voltage sensor positions in resting and inactivated mammalian sodium channels by LRET.

Voltage-gated sodium channels (Navs) play crucial roles in excitable cells. Although vertebrate Nav function has been extensively studied, the detailed structural basis for voltage-dependent gating mechanisms remain obscure. We have assessed the structural changes of the Nav voltage sensor domain using lanthanide-based resonance energy transfer (LRET) between the rat skeletal muscle voltage-gated sodium channel (Nav1.4) and fluorescently labeled Nav1.4-targeting toxins. We generated donor constructs with genetically encoded lanthanide-binding tags (LBTs) inserted at the extracellular end of the S4 segment of each domain (with a single LBT per construct). Three different Bodipy-labeled, Nav1.4-targeting toxins were synthesized as acceptors: ß-scorpion toxin (Ts1)-Bodipy, KIIIA-Bodipy, and GIIIA-Bodipy analogs. Functional Nav-LBT channels expressed in Xenopus oocytes were voltage-clamped, and distinct LRET signals were obtained in the resting and slow inactivated states. Intramolecular distances computed from the LRET signals define a geometrical map of Nav1.4 with the bound toxins, and reveal voltage-dependent structural changes related to channel gating.

A Model-Based Approach to Identify Classes and Respective Cutoffs of the Brazilian Household Food Insecurity Measurement Scale.

BACKGROUND: The Brazilian Household Food Insecurity Measurement Scale (EBIA) is the main tool for assessing household food insecurity (FI) in Brazil, assisting in monitoring and improving national public policies to promote food security. Based on the sum of item scores, households have been classified into 4 levels of FI, with the use of cutoffs arising from expert discussions informed by psychometric analyses and policy considerations. OBJECTIVES: This study aimed to identify homogeneous latent groups corresponding to levels of FI, examine whether such subgroups could be defined from discriminant cutoffs applied to the overall EBIA raw score, and compare these cutoffs against those currently used. METHODS: A cross-sectional population-based study with a representative sample of 1105 households from a low-income metropolitan area of Rio de Janeiro was conducted. Latent class factor analysis (LCFA) models were applied to the answers to EBIA's items to identify homogeneous groups, obtaining the number of latent classes for FI measured by the scale. Based on this and a thorough classification agreement evaluation, optimal cutoffs for discriminating between different severity levels of FI were ascertained. Model-based grouping and the official EBIA classification cutoffs were also contrasted. RESULTS: LCFA identified 4 homogeneous groups with a very high degree of class separation (entropy = 0.906), endorsing the classification of EBIA as a 4-level measure of FI. Two sets of cutoffs were identified to separate such groups according to household type: 1/2, 5/6, and 10/11 in households with children and adolescents (score range: 0-14); and 1/2, 3/4, and 5/6 in adult-only households (score range: 0-7). CONCLUSION: Although roughly classifying EBIA as in previous studies, the current approach suggests that, in terms of raw score, households endorsing only one item of the scale would be better classified by being placed in the same stratum as those remaining negative on all items.

Elucidation of the Covalent and Tertiary Structures of Biologically Active Ts3 Toxin.

Ts3 is an alpha scorpion toxin from the venom of the Brazilian scorpion Tityus serrulatus. Ts3 binds to the domain IV voltage sensor of voltage-gated sodium channels (Nav ) and slows down their fast inactivation. The covalent structure of the Ts3 toxin is uncertain, and the structure of the folded protein molecule is unknown. Herein, we report the total chemical synthesis of four candidate Ts3 toxin protein molecules and the results of structure-activity studies that enabled us to establish the covalent structure of biologically active Ts3 toxin. We also report the synthesis of the mirror image form of the Ts3 protein molecule, and the use of racemic protein crystallography to determine the folded (tertiary) structure of biologically active Ts3 toxin by X-ray diffraction.

Probing α-3(10) transitions in a voltage-sensing S4 helix.

The S4 helix of voltage sensor domains (VSDs) transfers its gating charges across the membrane electrical field in response to changes of the membrane potential. Recent studies suggest that this process may occur via the helical conversion of the entire S4 between α and 310 conformations. Here, using LRET and FRET, we tested this hypothesis by measuring dynamic changes in the transmembrane length of S4 from engineered VSDs expressed in Xenopus oocytes. Our results suggest that the native S4 from the Ciona intestinalis voltage-sensitive phosphatase (Ci-VSP) does not exhibit extended and long-lived 310 conformations and remains mostly α-helical. Although the S4 of NavAb displays a fully extended 310 conformation in x-ray structures, its transplantation in the Ci-VSP VSD scaffold yielded similar results as the native Ci-VSP S4. Taken together, our study does not support the presence of long-lived extended α-to-310 helical conversions of the S4 in Ci-VSP associated with voltage activation.

Total chemical synthesis of biologically active fluorescent dye-labeled Ts1 toxin.

Ts1 toxin is a protein found in the venom of the Brazilian scorpion Tityus serrulatus. Ts1 binds to the domain II voltage sensor in the voltage-gated sodium channel Nav and modifies its voltage dependence. In the work reported here, we established an efficient total chemical synthesis of the Ts1 protein using modern chemical ligation methods and demonstrated that it was fully active in modifying the voltage dependence of the rat skeletal muscle voltage-gated sodium channel rNav1.4 expressed in oocytes. Total synthesis combined with click chemistry was used to label the Ts1 protein molecule with the fluorescent dyes Alexa-Fluor 488 and Bodipy. Dye-labeled Ts1 proteins retained their optical properties and bound to and modified the voltage dependence of the sodium channel Nav. Because of the highly specific binding of Ts1 toxin to Nav, successful chemical synthesis and labeling of Ts1 toxin provides an important tool for biophysical studies, histochemical studies, and opto-pharmacological studies of the Nav protein.

Nano-positioning system for structural analysis of functional homomeric proteins in multiple conformations.

Proteins may undergo multiple conformational changes required for their function. One strategy used to estimate target-site positions in unknown structural conformations involves single-pair resonance energy transfer (RET) distance measurements. However, interpretation of inter-residue distances is difficult when applied to three-dimensional structural rearrangements, especially in homomeric systems. We developed a positioning method using inverse trilateration/triangulation to map target sites within a homomeric protein in all defined states, with simultaneous functional recordings. The procedure accounts for probe diffusion to accurately determine the three-dimensional position and confidence region of lanthanide LRET donors attached to a target site (one per subunit), relative to a single fluorescent acceptor placed in a static site. As first application, the method is used to determine the position of a functional voltage-gated potassium channel's voltage sensor. Our results verify the crystal structure relaxed conformation and report on the resting and active conformations for which crystal structures are not available.

Adverse health events associated with domestic violence during pregnancy among Brazilian women.

BACKGROUND: domestic violence during pregnancy remains an unsolved and neglected social problem despite the recognised adverse physical and mental health consequences. OBJECTIVE: to examine the association between domestic violence (psychological violence and physical or sexual violence) and health problems self-reported by pregnant women. METHODS: a cross-sectional analysis from a cohort study of 1,379 pregnant women attending prenatal care in public primary care units in Campinas, São Paulo, Brazil. Data were collected by interviewing women when they enroled for prenatal care. Domestic violence and alcohol abuse were ascertained by validated questionnaires. Referred morbidities, undesirable behaviours and sociodemographic characteristics were also recorded. Univariate analyses were used to estimate prevalence and unadjusted odd ratios. Multivariate logistic regression was used to identify the independent association between psychological violence and physical or sexual violence during pregnancy and women's health outcomes. FINDINGS: psychological violence and physical or sexual violence were reported by 19.1% (n=263) and 6.5% (n=89) of the pregnant women, respectively. Psychological violence was significantly associated with obstetric problems [odds ratio (OR) 1.95; 95% confidence interval (CI) 1.39-2.73], premature rupture of membranes (OR 1.64, 95% CI 1.01-2.68), urinary tract infection (OR 1.71, 95% CI 1.19-2.42), headache (OR 1.75, 95% CI 1.25-2.40) and sexual risk behaviours (OR 2.28, 95% CI 1.18-4.41). Physical or sexual violence was significantly associated with: obstetric problems (OR 1.72, 95% CI 1.08-2.75), premature rupture of membranes (OR 2.11, 95% CI 1.14-3.88), urinary tract infection (OR 2.05, 95% CI 1.26-3.34), vaginal bleeding (OR 1.95, 95% CI 1.10-3.43) and lack of sexual desire (OR 3.67, 95% CI 2.23-6.09). CONCLUSION: domestic violence during pregnancy was associated with adverse clinical and psychological outcomes for women. These results suggest that a well-organised health-care system and trained health professionals, as well as multisectorial social support, are necessary to prevent or address the negative influence of domestic violence on women's health in Brazil.

Violência doméstica na gravidez: prevalência e fatores associados / Violence against pregnant women: prevalence and associated factors / Violencia domestica durante el embarazo: prevalencia y factores asociados

OBJETIVO: Identificar os fatores associados à violência doméstica contra gestantes. MÉTODOS: Foram entrevistadas 1.379 gestantes usuárias do Sistema Único de Saúde acompanhadas em unidades básicas de saúde no município de Campinas (SP). Foram analisadas a primeira e a segunda entrevistas de um estudo de coorte, aplicando-se questionário estruturado sobre violência doméstica validado no Brasil, de julho de 2004 a julho de 2006. Foram realizadas análise descritiva e regressão logística múltipla dos dados. RESULTADOS: Do total da amostra, 19,1 por cento (n=263) das gestantes reportaram violência psicológica e 6,5 por cento (n=89) violência física/sexual. Os fatores associados à violência psicológica foram: parceiro íntimo adolescente (p<0,019) e gestante ter presenciado agressão física antes dos 15 anos (p<0,001). Foram associados à violência física/sexual: dificuldade da gestante em comparecer às consultas de pré-natal (p<0,014), parceiro íntimo fazer uso de drogas (p<0,015) e não trabalhar (p<0,048). Os fatores associados à violência psicológica e física/sexual foram: baixa escolaridade da gestante (p<0,013 e p<0,020, respectivamente), gestante responsável pela família (p<0,001 e p=0,017, respectivamente) gestante ter sofrido agressão física na infância (p<0,029 e p<0,038, respectivamente), presença de transtorno mental comum (p<0,001) e parceiro íntimo consumir bebida alcoólica duas ou mais vezes por semana(p<0,001). CONCLUSÕES: Constataram-se altas prevalências das diferentes categorias de violência doméstica praticada pelo parceiro íntimo durante o período gestacional, assim como, com os diversos fatores a elas associados. Mecanismos apropriados para identificação e abordagem da violência doméstica na gestação são necessários, especialmente na atenção primária.

Violence against pregnant women: prevalence and associated factors.

OBJECTIVE: To identify the factors associated with domestic violence against pregnant women. METHODS: Interviews were conducted with 1,379 pregnant women undergoing antenatal care in basic health care units of the Brazilian Health System, within the municipality of Campinas (Southeastern Brazil). A structured questionnaire on domestic violence, validated in Brazil, was applied between July 2004 and July 2006. The first and second interviews in a cohort study were analyzed. Descriptive and multiple logistic regression analysis of the data were conducted. RESULTS: Psychological violence was reported by 19.1% (n=263) of the total sample of pregnant women and physical/sexual violence was reported by 6.5% (n=89) of them. The factors associated to psychological violence were: adolescent intimate partner (p<0.019) and the pregnant woman had witnessed physical aggression before she was 15 years old (p<0.001). The factors associated to physical/sexual violence were: difficulties encountered by the pregnant woman in attending her antenatal appointments (p<0.014), intimate partner uses drugs (p<0.015) and does not work (p<0.048). The factors associated to psychological and physical/ sexual violence were: low level of education of the interviewee (p<0.013 and p<0.020, respectively), the pregnant woman being responsible for the family (p<0.001 and p=0.017, respectively) pregnant woman had suffered physical aggression during childhood (p<0.029 and p<0.038, respectively), presence of common mental disorder (p<0.001) and intimate partner consumes alcoholic beverage twice or more weekly. (p<0.001). CONCLUSIONS: A high prevalence of different categories of domestic violence by an intimate partner during pregnancy was found as well as different factors associated with them. Appropriate mechanisms are necessary, particularly in primary health care, to identify and deal with domestic violence during pregnancy.

Associação entre violência doméstica na gestação e peso ao nascer ou prematuridade / The association between domestic violence during pregnancy and low birth weight or prematurity

OBJETIVO: Avaliar se a violência doméstica na gestação está associada a desfechos desfavoráveis na saúde do lactente, medidos pelo baixo peso ao nascer ou prematuridade. MÉTODO: Estudo de coorte prospectiva, realizado com gestantes que fizeram pré-natal em 10 Unidades Básicas de Saúde do município de Campinas (SP), durante os anos de 2004 a 2006. Foi utilizado questionário estruturado e validado no Brasil. As gestantes tiveram, no mínimo, duas e, no máximo, três entrevistas realizadas durante pré e pós-natal. Foi utilizada análise descritiva dos dados. O teste t de Student foi utilizado para comparar as médias do peso ao nascer e da idade gestacional entre os grupos de gestantes que sofreram, durante a atual gestação, violência doméstica e aqueles que não sofreram. A análise de regressão logística foi utilizada para verificar os fatores associados ao baixo peso ou prematuridade. RESULTADOS: Foram acompanhadas durante o período de pré-natal e pós-natal (n = 1.229) 89,1 por cento das gestantes; 10,9 por cento representam as perdas de acompanhamento, basicamente por mudança de endereço. O peso médio ao nascer foi de 3.233 g; idade gestacional foi em média 38,56 semanas. Apresentaram baixo peso ao nascer ou prematuridade 13,8 por cento dos recém-nascidos. Condições de risco para baixo peso ao nascer ou prematuridade foram: gestante ter tido recém-nascido prematuro em outra gestação (p < 0,005), ser tabagista (p < 0,001), ter tido parto por cesárea (p < 0,001), ser baixa a escolaridade do parceiro (p < 0,008). CONCLUSÃO: Neste estudo, não foi observada associação estatisticamente significativa entre violência doméstica perpetrada pelo parceiro e baixo peso ao nascer ou prematuridade.

OBJECTIVE: To investigate whether domestic violence during pregnancy is associated with unfavorable infant health outcomes, measured by low birth weight or prematurity. METHODS: This was a prospective cohort study enrolling pregnant women whose prenatal care was provided by 10 basic health units in the city of Campinas, SP, Brazil, between 2004 and 2006. A structured questionnaire was employed that had previously been validated for use in Brazil. Each mother attended a minimum of two and a maximum of three interviews during the prenatal and postnatal periods. Data were analyzed using descriptive statistics. Student's t test was used to compare means for birth weight and gestational age between mothers who had suffered domestic violence during the current pregnancy and those who had not. Logistic regression analysis was employed to identify factors associated with low birth weight or prematurity. RESULTS: During the prenatal and postnatal periods, 89.1 percent (n = 1,229) of the pregnant women were followed up, 10.9 percent being lost to follow-up, basically due to changes of address. Mean birth weight was 3,233 g; mean gestational age was 38.56 weeks. A total of 13.8 percent of the infants had low birth weight or were premature. Conditions associated with risk of low birth weight or prematurity were: mothers who had previously given birth prematurely (p < 0.005), who smoked (p < 0.001), who delivered by caesarian (p < 0.001) and whose partners had a low educational level (p < 0.008). CONCLUSIONS: In this study, no statistically significant association was observed between domestic violence perpetrated by partners and low birth weight or prematurity.

The association between domestic violence during pregnancy and low birth weight or prematurity.

OBJECTIVE: To investigate whether domestic violence during pregnancy is associated with unfavorable infant health outcomes, measured by low birth weight or prematurity. METHODS: This was a prospective cohort study enrolling pregnant women whose prenatal care was provided by 10 basic health units in the city of Campinas, SP, Brazil, between 2004 and 2006. A structured questionnaire was employed that had previously been validated for use in Brazil. Each mother attended a minimum of two and a maximum of three interviews during the prenatal and postnatal periods. Data were analyzed using descriptive statistics. Student's t test was used to compare means for birth weight and gestational age between mothers who had suffered domestic violence during the current pregnancy and those who had not. Logistic regression analysis was employed to identify factors associated with low birth weight or prematurity. RESULTS: During the prenatal and postnatal periods, 89.1% (n = 1,229) of the pregnant women were followed up, 10.9% being lost to follow-up, basically due to changes of address. Mean birth weight was 3,233 g; mean gestational age was 38.56 weeks. A total of 13.8% of the infants had low birth weight or were premature. Conditions associated with risk of low birth weight or prematurity were: mothers who had previously given birth prematurely (p < 0.005), who smoked (p < 0.001), who delivered by caesarian (p < 0.001) and whose partners had a low educational level (p < 0.008). CONCLUSIONS: In this study, no statistically significant association was observed between domestic violence perpetrated by partners and low birth weight or prematurity.

In vivo measurement of intramolecular distances using genetically encoded reporters.

The function of membrane proteins occurs in the context of the cell membrane in living cells acting in concert with various cell components such as other proteins, cofactors, etc. The understanding of the function at the molecular level requires structural techniques, but high resolution structural studies are normally obtained in vitro and in artificial membranes or detergent. Ideally the correlation of structure and function should be carried out in the native environment but most of the techniques applicable in vivo lack the high resolution necessary to track conformational changes on a molecular level. Here we report on the successful application of an improved variant of lanthanide-based resonance energy transfer a fluorescent based technique, to Shaker potassium channels expressed in live Xenopus oocytes. Lanthanide-based resonance energy transfer is particularly suitable to measure intramolecular distances with high resolution. The improvements reported in this work are mainly based on the use of two different small genetically encoded tags (the Lanthanide Binding Tag and the hexa-histidine tag), which due to their small size can be encoded at will in many positions of interest without distorting the protein's function. The technique reported here has the additional improvement that the two tags can be placed independently in contrast to previously described techniques that rely on chemical labeling procedures of thiols.

Isoflurane inhibits NaChBac, a prokaryotic voltage-gated sodium channel.

Volatile anesthetics inhibit mammalian voltage-gated Na(+) channels, an action that contributes to their presynaptic inhibition of neurotransmitter release. We measured the effects of isoflurane, a prototypical halogenated ether volatile anesthetic, on the prokaryotic voltage-gated Na(+) channel from Bacillus halodurans (NaChBac). Using whole-cell patch-clamp recording, human embryonic kidney 293 cells transfected with NaChBac displayed large inward currents (I(Na)) that activated at potentials of -60 mV or higher with a peak voltage of activation of 0 mV (from a holding potential of -80 mV) or -10 mV (from a holding potential of -100 mV). Isoflurane inhibited I(Na) in a concentration-dependent manner over a clinically relevant concentration range; inhibition was significantly more potent from a holding potential of -80 mV (IC(50) = 0.35 mM) than from -100 mV (IC(50) = 0.48 mM). Isoflurane positively shifted the voltage dependence of peak activation, and it negatively shifted the voltage dependence of end steady-state activation. The voltage dependence of inactivation was negatively shifted with no change in slope factor. Enhanced inactivation of I(Na) was 8-fold more sensitive to isoflurane than reduction of channel opening. In addition to tonic block of closed and/or open channels, isoflurane enhanced use-dependent block by delaying recovery from inactivation. These results indicate that a prokaryotic voltage-gated Na(+) channel, like mammalian voltage-gated Na(+) channels, is inhibited by clinical concentrations of isoflurane involving multiple state-dependent mechanisms. NaChBac should provide a useful model for structure-function studies of volatile anesthetic actions on voltage-gated ion channels.

Distance measurements reveal a common topology of prokaryotic voltage-gated ion channels in the lipid bilayer.

Voltage-dependent ion channels are fundamental to the physiology of excitable cells because they underlie the generation and propagation of the action potential and excitation-contraction coupling. To understand how ion channels work, it is important to determine their structures in different conformations in a membrane environment. The validity of the crystal structure for the prokaryotic K(+) channel, K(V)AP, has been questioned based on discrepancies with biophysical data from functional eukaryotic channels, underlining the need for independent structural data under native conditions. We investigated the structural organization of two prokaryotic voltage-gated channels, NaChBac and K(V)AP, in liposomes by using luminescence resonance energy transfer. We describe here a transmembrane packing representation of the voltage sensor and pore domains of the prokaryotic Na channel, NaChBac. We find that NaChBac and K(V)AP share a common arrangement in which the structures of the Na and K selective pores and voltage-sensor domains are conserved. The packing arrangement of the voltage-sensing region as determined by luminescence resonance energy transfer differs significantly from that of the K(V)AP crystal structure, but resembles that of the eukaryotic K(V)1.2 crystal structure. However, the voltage-sensor domain in prokaryotic channels is closer to the pore domain than in the K(V)1.2 structure. Our results indicate that prokaryotic and eukaryotic channels that share similar functional properties have similar helix arrangements, with differences arising likely from the later introduction of additional structural elements.