Validation of the Nasal Obstruction Symptom Evaluation Scale in Mexican Adults.

BACKGROUND: Nasal obstruction is a feeling of impaired airflow through the nose, caused by diverse factors with high prevalence. There are numerous methods to study it, being the most important subjective instrument the Nasal Obstruction Symptom Evaluation score, NOSE, with good structural validity and internal consistency. It has been translated and validated in several languages, including European Spanish, but hasn't been validated in Mexico. AIM: Adapt and validate the NOSE scale to the Spanish language for a Mexican adult population. METHODS: This study was conducted from May-August 2015. The scale was translated from English to Spanish and assessed by two experts to confirm its acceptability, being retranslated later. Adult subjects with native Mexican Spanish were recruited from the outpatient clinic, with chronic rhinopathy and without evidence of respiratory pathology. Both groups filled the scale, and anthropometric data were collected. A subgroup completed the instrument again after one month. STATA 15.1 was used for analysis. RESULTS: The sample were 261 subjects. Cronbach alpha was 0.89, intraclass correlation coefficient 0.84, agreement limits -9.62 to 16.29. There was a statistically significant difference in score between groups. The area under the ROC curve was 0.855, with 83% sensibility and 76% specificity for a 20 total score. Moderate to strong item-total association was found with Spearman. The response model showed moderate item discrimination. In the factorial analysis, one factor was found, with an Eigenvalue of 3.21. CONCLUSION: The adaptation of the NOSE scale is reliable and valid for its application in Mexican adult population.

Structural and Functional Characterization of a Nav1.5-Mitochondrial Couplon.

RATIONALE: The cardiac sodium channel NaV1.5 has a fundamental role in excitability and conduction. Previous studies have shown that sodium channels cluster together in specific cellular subdomains. Their association with intracellular organelles in defined regions of the myocytes, and the functional consequences of that association, remain to be defined. OBJECTIVE: To characterize a subcellular domain formed by sodium channel clusters in the crest region of the myocytes and the subjacent subsarcolemmal mitochondria. METHODS AND RESULTS: Through a combination of imaging approaches including super-resolution microscopy and electron microscopy we identified, in adult cardiac myocytes, a NaV1.5 subpopulation in close proximity to subjacent subsarcolemmal mitochondria; we further found that subjacent subsarcolemmal mitochondria preferentially host the mitochondrial NCLX (Na+/Ca2+ exchanger). This anatomic proximity led us to investigate functional changes in mitochondria resulting from sodium channel activity. Upon TTX (tetrodotoxin) exposure, mitochondria near NaV1.5 channels accumulated more Ca2+ and showed increased reactive oxygen species production when compared with interfibrillar mitochondria. Finally, crosstalk between NaV1.5 channels and mitochondria was analyzed at a transcriptional level. We found that SCN5A (encoding NaV1.5) and SLC8B1 (which encode NaV1.5 and NCLX, respectively) are negatively correlated both in a human transcriptome data set (Genotype-Tissue Expression) and in human-induced pluripotent stem cell-derived cardiac myocytes deficient in SCN5A. CONCLUSIONS: We describe an anatomic hub (a couplon) formed by sodium channel clusters and subjacent subsarcolemmal mitochondria. Preferential localization of NCLX to this domain allows for functional coupling where the extrusion of Ca2+ from the mitochondria is powered, at least in part, by the entry of sodium through NaV1.5 channels. These results provide a novel entry-point into a mechanistic understanding of the intersection between electrical and structural functions of the heart.

COVID-19 y sus imaginarios socioculturales en Latinoamérica: una herramienta para la salud pública / COVID-19 and its socio-cultural imagery in Latin America: a tool for public health

RESUMEN Objetivo Identificar el contenido y organización de las representaciones sociales que tiene la población urbana de tres ciudades de América Latina sobre el COVID-19. Materiales y Métodos Estudio cualitativo hecho desde la antropología cognitiva a través del análisis estructural de las representaciones sociales. Se utilizaron técnicas de listados libres y comparación de pares de febrero a abril de 2020 en Pereira (Colombia), en la zona metropolitana de Guadalajara (México) y en León (también en México). Participaron 97 personas de ambos sexos (97 en la primera fase y 71 en la segunda), mayores de edad y que su ocupación o profesión no tuvieran relación con el área de salud; se realizó muestreo no probabilístico-propositivo. Resultados El imaginario sociocultural está asociado con términos como contagio y pandemia, con énfasis en los términos relacionados con la prevención de la enfermedad. Conclusiones Los saberes de la población sobre el COVID-19 privilegian aspectos clínicos y epidemiológicos, así como las medidas de contención y mitigación.

ABSTRACT Objective Identify the content and organization of social representations (SR) against COVID-19 in urban population of three cities in Latin America, 2020. Materials and Methods Qualitative study from cognitive anthropology through the structural analysis of SR. Free listing and pair comparison techniques were used from February-April 2020, in Pereira (Colombia), Guadalajara Metropolitan Area (ZMG)-(Mexico), León (Mexico). 97 participants were included in the first phase and 71 in the second phase, of both sexes, of legal age, whose occupation and profession had no relation to the health area; non-probability-purposeful sampling was performed. Results The socio-cultural imaginary is associated with terms such as contagion, pandemic, but highlighting the terms related to disease prevention. Conclusions The knowledge of the population about COVID-19 privileges clinical and epidemiological aspects, as well as containment and mitigation measures.

Nanoarchitecture and dynamics of the mouse enteric glycocalyx examined by freeze-etching electron tomography and intravital microscopy.

The glycocalyx is a highly hydrated, glycoprotein-rich coat shrouding many eukaryotic and prokaryotic cells. The intestinal epithelial glycocalyx, comprising glycosylated transmembrane mucins, is part of the primary host-microbe interface and is essential for nutrient absorption. Its disruption has been implicated in numerous gastrointestinal diseases. Yet, due to challenges in preserving and visualizing its native organization, glycocalyx structure-function relationships remain unclear. Here, we characterize the nanoarchitecture of the murine enteric glycocalyx using freeze-etching and electron tomography. Micrometer-long mucin filaments emerge from microvillar-tips and, through zigzagged lateral interactions form a three-dimensional columnar network with a 30 nm mesh. Filament-termini converge into globular structures ~30 nm apart that are liquid-crystalline packed within a single plane. Finally, we assess glycocalyx deformability and porosity using intravital microscopy. We argue that the columnar network architecture and the liquid-crystalline packing of the filament termini allow the glycocalyx to function as a deformable size-exclusion filter of luminal contents.

[COVID-19 and its socio-cultural imagery in Latin America: a tool for public health]. / COVID-19 y sus imaginarios socioculturales en Latinoamérica: una herramienta para la salud pública.

OBJECTIVE: Identify the content and organization of social representations (SR) against COVID-19 in urban population of three cities in Latin America, 2020. MATERIALS AND METHODS: Qualitative study from cognitive anthropology through the structural analysis of SR. Free listing and pair comparison techniques were used from February-April 2020, in Pereira (Colombia), Guadalajara Metropolitan Area (ZMG)-(Mexico), León (Mexico). 97 participants were included in the first phase and 71 in the second phase, of both sexes, of legal age, whose occupation and profession had no relation to the health area; non-probability-purposeful sampling was performed. RESULTS: The socio-cultural imaginary is associated with terms such as contagion, pandemic, but highlighting the terms related to disease prevention. CONCLUSIONS: The knowledge of the population about COVID-19 privileges clinical and epidemiological aspects, as well as containment and mitigation measures.

OBJETIVO: Identificar el contenido y organización de las representaciones sociales que tiene la población urbana de tres ciudades de América Latina sobre el COVID-19. MATERIALES Y MÉTODOS: Estudio cualitativo hecho desde la antropología cognitiva a través del análisis estructural de las representaciones sociales. Se utilizaron técnicas de listados libres y comparación de pares de febrero a abril de 2020 en Pereira (Colombia), en la zona metropolitana de Guadalajara (México) y en León (también en México). Participaron 97 personas de ambos sexos (97 en la primera fase y 71 en la segunda), mayores de edad y que su ocupación o profesión no tuvieran relación con el área de salud; se realizó muestreo no probabilístico-propositivo. RESULTADOS: El imaginario sociocultural está asociado con términos como contagio y pandemia, con énfasis en los términos relacionados con la prevención de la enfermedad. CONCLUSIONES: Los saberes de la población sobre el COVID-19 privilegian aspectos clínicos y epidemiológicos, así como las medidas de contención y mitigación.

Embarazos adolescentes y representaciones sociales (León, Guanajuato, México, 2016-2017) / Adolescent pregnancies and social representations (León, Guanajuato, Mexico, 2016-2017) / Gravidez na adolescência e representações sociais (León, Guanajuato, México, 2016-2017)

Resumen (analítico): La intencionalidad del embarazo se ha tratado de explicar desde diversos marcos teóricos, pero no siempre se considera el contexto cultural; por ello persisten complicaciones conceptuales y metodológicas. A lo largo de este estudio se pretendió identificar las representaciones sociales que las adolescentes de León (Guanajuato, México) tienen del embarazo no deseado y no planeado, estudio basado en el enfoque teórico de las representaciones sociales, con el uso de técnicas asociativas derivadas de la antropología cognitiva, específicamente listados libres y comparación de pares. Participaron 72 mujeres de 15 a 19 años. Las representaciones sociales del embarazo no planeado se centraron en aspectos económicos y emocionales negativos; el embarazo no deseado se asoció con aspectos emocionales negativos (sentimientos de enojo y culpa por la situación), además de que se consideró el aborto como solución.

Abstract (analytical): A number of authors have tried to explain the intentionality of pregnancy from different theoretical frameworks, however the cultural context is not always considered, and as a result conceptual and methodological complications persist. This study aims to identify the social representations that adolescents in León, Guanajuato construct in relation to their unwanted and unplanned pregnancies. This study is based on the theoretical approach of social representations and uses associative techniques derived from cognitive anthropology, specifically free listings and peer comparison. 72 women aged 15 to 19 participated. The social representations of unplanned pregnancy focused on negative economic and emotional aspects. Social representations of unwanted pregnancies were associated with negative emotional aspects (feelings of anger and guilt towards the situation), and abortion was considered as a solution.

Resumo (analítico): A intencionalidade da gravidez tem sido tratada para explicar a partir de vários referenciais teóricos, no entanto, o contexto cultural nem sempre é considerado, por isso persistem complicações conceituais e metodológicas. Este estudo tem como objetivo identificar as representações sociais que adolescentes em León, Guanajuato, têm de gravidez indesejada e não planejada. Estudo baseado na abordagem teórica das representações sociais, com o uso de técnicas associativas derivadas da antropologia cognitiva, especificamente listagens livres e comparação de pares. Participaram 72 mulheres com idades entre 15 e 19 anos. As representações sociais da gravidez não planejada focaram aspectos econômicos e emocionais negativos; a gravidez indesejada foi associada a aspectos emocionais negativos (sentimentos de raiva e culpa pela situação) e o aborto foi considerado uma solução.

Localized Myosin II Activity Regulates Assembly and Plasticity of the Axon Initial Segment.

The axon initial segment (AIS) is the site of action potential generation and a locus of activity-dependent homeostatic plasticity. A multimeric complex of sodium channels, linked via a cytoskeletal scaffold of ankyrin G and beta IV spectrin to submembranous actin rings, mediates these functions. The mechanisms that specify the AIS complex to the proximal axon and underlie its plasticity remain poorly understood. Here we show phosphorylated myosin light chain (pMLC), an activator of contractile myosin II, is highly enriched in the assembling and mature AIS, where it associates with actin rings. MLC phosphorylation and myosin II contractile activity are required for AIS assembly, and they regulate the distribution of AIS components along the axon. pMLC is rapidly lost during depolarization, destabilizing actin and thereby providing a mechanism for activity-dependent structural plasticity of the AIS. Together, these results identify pMLC/myosin II activity as a common link between AIS assembly and plasticity.

Sodium Channel Remodeling in Subcellular Microdomains of Murine Failing Cardiomyocytes.

BACKGROUND: Cardiac sodium channel (NaV1.5) dysfunction contributes to arrhythmogenesis during pathophysiological conditions. Nav1.5 localizes to distinct subcellular microdomains within the cardiomyocyte, where it associates with region-specific proteins, yielding complexes whose function is location specific. We herein investigated sodium channel remodeling within distinct cardiomyocyte microdomains during heart failure. METHODS AND RESULTS: Mice were subjected to 6 weeks of transverse aortic constriction (TAC; n=32) to induce heart failure. Sham-operated on mice were used as controls (n=20). TAC led to reduced left ventricular ejection fraction, QRS prolongation, increased heart mass, and upregulation of prohypertrophic genes. Whole-cell sodium current (INa) density was decreased by 30% in TAC versus sham-operated on cardiomyocytes. On macropatch analysis, INa in TAC cardiomyocytes was reduced by 50% at the lateral membrane (LM) and by 40% at the intercalated disc. Electron microscopy and scanning ion conductance microscopy revealed remodeling of the intercalated disc (replacement of [inter-]plicate regions by large foldings) and LM (less identifiable T tubules and reduced Z-groove ratios). Using scanning ion conductance microscopy, cell-attached recordings in LM subdomains revealed decreased INa and increased late openings specifically at the crest of TAC cardiomyocytes, but not in groove/T tubules. Failing cardiomyocytes displayed a denser, but more stable, microtubule network (demonstrated by increased α-tubulin and Glu-tubulin expression). Superresolution microscopy showed reduced average NaV1.5 cluster size at the LM of TAC cells, in line with reduced INa. CONCLUSIONS: Heart failure induces structural remodeling of the intercalated disc, LM, and microtubule network in cardiomyocytes. These adaptations are accompanied by alterations in NaV1.5 clustering and INa within distinct subcellular microdomains of failing cardiomyocytes.

Image-based model of the spectrin cytoskeleton for red blood cell simulation.

We simulate deformable red blood cells in the microcirculation using the immersed boundary method with a cytoskeletal model that incorporates structural details revealed by tomographic images. The elasticity of red blood cells is known to be supplied by both their lipid bilayer membranes, which resist bending and local changes in area, and their cytoskeletons, which resist in-plane shear. The cytoskeleton consists of spectrin tetramers that are tethered to the lipid bilayer by ankyrin and by actin-based junctional complexes. We model the cytoskeleton as a random geometric graph, with nodes corresponding to junctional complexes and with edges corresponding to spectrin tetramers such that the edge lengths are given by the end-to-end distances between nodes. The statistical properties of this graph are based on distributions gathered from three-dimensional tomographic images of the cytoskeleton by a segmentation algorithm. We show that the elastic response of our model cytoskeleton, in which the spectrin polymers are treated as entropic springs, is in good agreement with the experimentally measured shear modulus. By simulating red blood cells in flow with the immersed boundary method, we compare this discrete cytoskeletal model to an existing continuum model and predict the extent to which dynamic spectrin network connectivity can protect against failure in the case of a red cell subjected to an applied strain. The methods presented here could form the basis of disease- and patient-specific computational studies of hereditary diseases affecting the red cell cytoskeleton.

Plakophilin-2 is required for transcription of genes that control calcium cycling and cardiac rhythm.

Plakophilin-2 (PKP2) is a component of the desmosome and known for its role in cell-cell adhesion. Mutations in human PKP2 associate with a life-threatening arrhythmogenic cardiomyopathy, often of right ventricular predominance. Here, we use a range of state-of-the-art methods and a cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mouse to demonstrate that in addition to its role in cell adhesion, PKP2 is necessary to maintain transcription of genes that control intracellular calcium cycling. Lack of PKP2 reduces expression of Ryr2 (coding for Ryanodine Receptor 2), Ank2 (coding for Ankyrin-B), Cacna1c (coding for CaV1.2) and Trdn (coding for triadin), and protein levels of calsequestrin-2 (Casq2). These factors combined lead to disruption of intracellular calcium homeostasis and isoproterenol-induced arrhythmias that are prevented by flecainide treatment. We propose a previously unrecognized arrhythmogenic mechanism related to PKP2 expression and suggest that mutations in PKP2 in humans may cause life-threatening arrhythmias even in the absence of structural disease.It is believed that mutations in desmosomal adhesion complex protein plakophilin 2 (PKP2) cause arrhythmia due to loss of cell-cell communication. Here the authors show that PKP2 controls the expression of proteins involved in calcium cycling in adult mouse hearts, and that lack of PKP2 can cause arrhythmia in a structurally normal heart.

Multilevel analyses of SCN5A mutations in arrhythmogenic right ventricular dysplasia/cardiomyopathy suggest non-canonical mechanisms for disease pathogenesis.

AIMS: Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy (ARVD/C) is often associated with desmosomal mutations. Recent studies suggest an interaction between the desmosome and sodium channel protein Nav1.5. We aimed to determine the prevalence and biophysical properties of mutations in SCN5A (the gene encoding Nav1.5) in ARVD/C. METHODS AND RESULTS: We performed whole-exome sequencing in six ARVD/C patients (33% male, 38.2 ± 12.1 years) without a desmosomal mutation. We found a rare missense variant (p.Arg1898His; R1898H) in SCN5A in one patient. We generated induced pluripotent stem cell-derived cardiomyocytes (hIPSC-CMs) from the patient's peripheral blood mononuclear cells. The variant was then corrected (R1898R) using Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 technology, allowing us to study the impact of the R1898H substitution in the same cellular background. Whole-cell patch clamping revealed a 36% reduction in peak sodium current (P = 0.002); super-resolution fluorescence microscopy showed reduced abundance of NaV1.5 (P = 0.005) and N-Cadherin (P = 0.026) clusters at the intercalated disc. Subsequently, we sequenced SCN5A in an additional 281 ARVD/C patients (60% male, 34.8 ± 13.7 years, 52% desmosomal mutation-carriers). Five (1.8%) subjects harboured a putatively pathogenic SCN5A variant (p.Tyr416Cys, p.Leu729del, p.Arg1623Ter, p.Ser1787Asn, and p.Val2016Met). SCN5A variants were associated with prolonged QRS duration (119 ± 15 vs. 94 ± 14 ms, P < 0.01) and all SCN5A variant carriers had major structural abnormalities on cardiac imaging. CONCLUSIONS: Almost 2% of ARVD/C patients harbour rare SCN5A variants. For one of these variants, we demonstrated reduced sodium current, Nav1.5 and N-Cadherin clusters at junctional sites. This suggests that Nav1.5 is in a functional complex with adhesion molecules, and reveals potential non-canonical mechanisms by which Nav1.5 dysfunction causes cardiomyopathy.

Nanoscale visualization of functional adhesion/excitability nodes at the intercalated disc.

Intercellular adhesion and electrical excitability are considered separate cellular properties. Studies of myelinated fibres, however, show that voltage-gated sodium channels (VGSCs) aggregate with cell adhesion molecules at discrete subcellular locations, such as the nodes of Ranvier. Demonstration of similar macromolecular organization in cardiac muscle is missing. Here we combine nanoscale-imaging (single-molecule localization microscopy; electron microscopy; and 'angle view' scanning patch clamp) with mathematical simulations to demonstrate distinct hubs at the cardiac intercalated disc, populated by clusters of the adhesion molecule N-cadherin and the VGSC NaV1.5. We show that the N-cadherin-NaV1.5 association is not random, that NaV1.5 molecules in these clusters are major contributors to cardiac sodium current, and that loss of NaV1.5 expression reduces intercellular adhesion strength. We speculate that adhesion/excitability nodes are key sites for crosstalk of the contractile and electrical molecular apparatus and may represent the structural substrate of cardiomyopathies in patients with mutations in molecules of the VGSC complex.

The cardiac connexome: Non-canonical functions of connexin43 and their role in cardiac arrhythmias.

Connexin43 is the major component of gap junctions, an anatomical structure present in the cardiac intercalated disc that provides a low-resistance pathway for direct cell-to-cell passage of electrical charge. Recent studies have shown that in addition to its well-established function as an integral membrane protein that oligomerizes to form gap junctions, Cx43 plays other roles that are independent of channel (or perhaps even hemi-channel) formation. This article discusses non-canonical functions of Cx43. In particular, we focus on the role of Cx43 as a part of a protein interacting network, a connexome, where molecules classically defined as belonging to the mechanical junctions, the gap junctions and the sodium channel complex, multitask and work together to bring about excitability, electrical and mechanical coupling between cardiac cells. Overall, viewing Cx43 as a multi-functional protein, beyond gap junctions, opens a window to better understand the function of the intercalated disc and the pathological consequences that may result from changes in the abundance or localization of Cx43 in the intercalated disc subdomain.

Ultrastructure of the intercellular space in adult murine ventricle revealed by quantitative tomographic electron microscopy.

AIMS: Progress in tissue preservation (high-pressure freezing), data acquisition (tomographic electron microscopy, TEM), and analysis (image segmentation and quantification) have greatly improved the level of information extracted from ultrastructural images. Here, we combined these methods and developed analytical tools to provide an in-depth morphometric description of the intercalated disc (ID) in adult murine ventricle. As a point of comparison, we characterized the ultrastructure of the ID in mice heterozygous-null for the desmosomal gene plakophilin-2 (PKP2; mice dubbed PKP2-Hz). METHODS AND RESULTS: Tomographic EM images of thin sections of adult mouse ventricular tissue were processed by image segmentation analysis. Novel morphometric routines allowed us to generate the first quantitative description of the ID intercellular space based on three-dimensional data. We show that complex invaginations of the cell membrane significantly increased the total ID surface area. In addition, PKP2-Hz samples showed increased average intercellular spacing, ID surface area, and membrane tortuosity, as well as reduced number and length of mechanical junctions compared with control. Finally, we observed membranous structures reminiscent of junctional sarcoplasmic reticulum at the ID, which were significantly more abundant in PKP2-Hz hearts. CONCLUSION: We have developed a systematic method to characterize the ultrastructure of the intercellular space in the adult murine ventricle and have provided a quantitative description of the structure of the intercellular membranes and of the intercellular space. We further show that PKP2 deficiency associates with ultrastructural defects. The possible importance of the intercellular space in cardiac behaviour is discussed.

Organization and dynamics of the nonhomologous end-joining machinery during DNA double-strand break repair.

Nonhomologous end-joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs), involving synapsis and ligation of the broken strands. We describe the use of in vivo and in vitro single-molecule methods to define the organization and interaction of NHEJ repair proteins at DSB ends. Super-resolution fluorescence microscopy allowed the precise visualization of XRCC4, XLF, and DNA ligase IV filaments adjacent to DSBs, which bridge the broken chromosome and direct rejoining. We show, by single-molecule FRET analysis of the Ku/XRCC4/XLF/DNA ligase IV NHEJ ligation complex, that end-to-end synapsis involves a dynamic positioning of the two ends relative to one another. Our observations form the basis of a new model for NHEJ that describes the mechanism whereby filament-forming proteins bridge DNA DSBs in vivo. In this scheme, the filaments at either end of the DSB interact dynamically to achieve optimal configuration and end-to-end positioning and ligation.

Super-resolution imaging reveals that loss of the C-terminus of connexin43 limits microtubule plus-end capture and NaV1.5 localization at the intercalated disc.

AIMS: It is well known that connexin43 (Cx43) forms gap junctions. We recently showed that Cx43 is also part of a protein-interacting network that regulates excitability. Cardiac-specific truncation of Cx43 C-terminus (mutant 'Cx43D378stop') led to lethal arrhythmias. Cx43D378stop localized to the intercalated disc (ID); cell-cell coupling was normal, but there was significant sodium current (INa) loss. We proposed that the microtubule plus-end is at the crux of the Cx43-INa relation. Yet, specific localization of relevant molecular players was prevented due to the resolution limit of fluorescence microscopy. Here, we use nanoscale imaging to establish: (i) the morphology of clusters formed by the microtubule plus-end tracking protein 'end-binding 1' (EB1), (ii) their position, and that of sodium channel alpha-subunit NaV1.5, relative to N-cadherin-rich sites, and (iii) the role of Cx43 C-terminus on the above-mentioned parameters and on the location-specific function of INa. METHODS AND RESULTS: Super-resolution fluorescence localization microscopy in murine adult cardiomyocytes revealed EB1 and NaV1.5 as distinct clusters preferentially localized to N-cadherin-rich sites. Extent of co-localization decreased in Cx43D378stop cells. Macropatch and scanning patch clamp showed reduced INa exclusively at cell end, without changes in unitary conductance. Experiments in Cx43-modified HL1 cells confirmed the relation between Cx43, INa, and microtubules. CONCLUSIONS: NaV1.5 and EB1 localization at the cell end is Cx43-dependent. Cx43 is part of a molecular complex that determines capture of the microtubule plus-end at the ID, facilitating cargo delivery. These observations link excitability and electrical coupling through a common molecular mechanism.

Molecular mechanism of fascin function in filopodial formation.

Filopodia are cell surface protrusions that are essential for cell migration. This finger-like structure is supported by rigid tightly bundled actin filaments. The protein responsible for actin bundling in filopodia is fascin. However, the mechanism by which fascin functions in filopodial formation is not clear. Here we provide biochemical, cryo-electron tomographic, and x-ray crystal structural data demonstrating the unique structural characteristics of fascin. Systematic mutagenesis studies on 100 mutants of fascin indicate that there are two major actin-binding sites on fascin. Crystal structures of four fascin mutants reveal concerted conformational changes in fascin from inactive to active states in the process of actin bundling. Mutations in any one of the actin-binding sites impair the cellular function of fascin in filopodial formation. Altogether, our data reveal the molecular mechanism of fascin function in filopodial formation.

An aspartate residue in the external vestibule of GLYT2 (glycine transporter 2) controls cation access and transport coupling.

Synaptic glycine levels are controlled by GLYTs (glycine transporters). GLYT1 is the main regulator of synaptic glycine concentrations and catalyses Na+-Cl--glycine co-transport with a 2:1:1 stoichiometry. In contrast, neuronal GLYT2 supplies glycine to the presynaptic terminal with a 3:1:1 stoichiometry. We subjected homology models of GLYT1 and GLYT2 to molecular dynamics simulations in the presence of Na+. Using molecular interaction potential maps and in silico mutagenesis, we identified a conserved region in the GLYT2 external vestibule likely to be involved in Na+ interactions. Replacement of Asp471 in this region reduced Na+ affinity and Na+ co-operativity of transport, an effect not produced in the homologous position (Asp295) in GLYT1. Unlike the GLYT1-Asp295 mutation, this Asp471 mutant increased sodium leakage and non-stoichiometric uncoupled ion movements through GLYT2, as determined by simultaneously measuring current and [3H]glycine accumulation. The homologous Asp471 and Asp295 positions exhibited distinct cation-sensitive external accessibility, and they were involved in Na+ and Li+-induced conformational changes. Although these two cations had opposite effects on GLYT1, they had comparable effects on accessibility in GLYT2, explaining the inhibitory and stimulatory responses to lithium exhibited by the two transporters. On the basis of these findings, we propose a role for Asp471 in controlling cation access to GLYT2 Na+ sites, ion coupling during transport and the subsequent conformational changes.

Molecular basis of the differential interaction with lithium of glycine transporters GLYT1 and GLYT2.

Glycine synaptic levels are controlled by glycine transporters (GLYTs) catalyzing Na(+)/Cl(-)/glycine cotransport. GLYT1 displays a 2:1 :1 stoichiometry and is the main regulator of extracellular glycine concentrations. The neuronal GLYT2, with higher sodium coupling (3:1 :1), supplies glycine to the pre-synaptic terminal to refill synaptic vesicles. In this work, using structural homology modelling and molecular dynamics simulations of GLYTs, we predict the conservation of the two sodium sites present in the template (leucine transporter from Aquifex aeolicus), and confirm its use by mutagenesis and functional analysis. GLYTs Na1 and Na2 sites show differential cation selectivity, as inferred from the action of lithium, a non-transport-supporting ion, on Na(+)-site mutants. GLYTs lithium responses were unchanged in Na1-site mutants, but abolished or inverted in mutants of Na2 site, which binds lithium in the presence of low sodium concentrations and therefore, controls lithium responses. Here, we report, for the first time, that lithium exerts opposite actions on GLYTs isoforms. Glycine transport by GLYT1 is inhibited by lithium whereas GLYT2 transport is stimulated, and this effect is more evident at increased glycine concentrations. In contrast to GLYT1, high and low affinity lithium-binding processes were detected in GLYT2.