A single serine residue confers tetrodotoxin insensitivity on the rat sensory-neuron-specific sodium channel SNS

AbstractSensory neurons express a sodium channel (SNS) that is highly resistant to block by tetrodotoxin (IC50=60 μM). SNS is 65% homologous to the cardiac sodium channel, in which a single hydrophilic residue in the SS2 segment is critical for tetrodotoxin resistance. By site-directed mutagenesis, we have substituted phenylalanine for serine at the equivalent position in SNS: this mutated (S356F) SNS channel is functionally similar to wild-type SNS when expressed in Xenopus oocytes, but is potently blocked by tetrodotoxin and saxitoxin with IC50s of 2.8 nM and 8.2 nM, respectively. These data provide clues to the rational design of selective blockers of SNS with potential as analgesic drugs

Stem starch reserves studied by on-solid reactions coupled with reflectance detections in water stressed grapevines

Wine grape is usually grown under water deficit conditions that could negatively impact plant reserves, including the organic carbon storage in perennial organs such as woody stems. Assessing the carbohydrate status in woody organs is therefore of interest as it can influence vegetative growth in the successive season. In this study, we aimed to apply an on-solid colour reaction (using Lugol's iodine solution) coupled with reflectance spectroscopy detection to assess the grapevine canes' starch accumulation in response to short drought periods. We used two Vitis vinifera cultivars ('Cabernet Sauvignon' and 'Syrah') that were subjected to three different water conditions (well-watered; early water stress; late water stress) during the growing season as case study. We sampled woody stem tissue during winter rest. The results showed that water stress reduced the starch storage in 'Syrah', especially when imposed late and recovery time was not enough for carbon restoration, while 'Cabernet Sauvignon' was not affected. The results showed that the sensitivity of the method used here is adequate to assess starch accumulation differences due to drought treatments in grapevine canes. Moreover, the analytical approach appears fast, low cost, and promising for future physiological and agronomical research applications

Mechanism of activation of the prokaryotic channel ELIC by propylamine: A single-channel study.

Prokaryotic channels, such as Erwinia chrysanthemi ligand-gated ion channel (ELIC) and Gloeobacter violaceus ligand-gated ion channel, give key structural information for the pentameric ligand-gated ion channel family, which includes nicotinic acetylcholine receptors. ELIC, a cationic channel from E. chrysanthemi, is particularly suitable for single-channel recording because of its high conductance. Here, we report on the kinetic properties of ELIC channels expressed in human embryonic kidney 293 cells. Single-channel currents elicited by the full agonist propylamine (0.5-50 mM) in outside-out patches at -60 mV were analyzed by direct maximum likelihood fitting of kinetic schemes to the idealized data. Several mechanisms were tested, and their adequacy was judged by comparing the predictions of the best fit obtained with the observable features of the experimental data. These included open-/shut-time distributions and the time course of macroscopic propylamine-activated currents elicited by fast theta-tube applications (50-600 ms, 1-50 mM, -100 mV). Related eukaryotic channels, such as glycine and nicotinic receptors, when fully liganded open with high efficacy to a single open state, reached via a preopening intermediate. The simplest adequate description of their activation, the "Flip" model, assumes a concerted transition to a single intermediate state at high agonist concentration. In contrast, ELIC open-time distributions at saturating propylamine showed multiple components. Thus, more than one open state must be accessible to the fully liganded channel. The "Primed" model allows opening from multiple fully liganded intermediates. The best fits of this type of model showed that ELIC maximum open probability (99%) is reached when at least two and probably three molecules of agonist have bound to the channel. The overall efficacy with which the fully liganded channel opens was ∼102 (∼20 for α1β glycine channels). The microscopic affinity for the agonist increased as the channel activated, from 7 mM for the resting state to 0.15 mM for the partially activated intermediate state

Heterogeneity of cell membrane structure studied by single molecule tracking

Heterogeneity in cell membrane structure, typified by microdomains with different biophysical and biochemical properties, is thought to impact on a variety of cell functions. Integral membrane proteins act as nanometre-sized probes of the lipid environment and their thermally-driven movements can be used to report local variations in membrane properties. In the current study, we have used total internal reflection fluorescence microscopy (TIRFM) combined with super-resolution tracking of multiple individual molecules, in order to create high-resolution maps of local membrane viscosity. We used a quadrat sampling method and show how statistical tests for membrane heterogeneity can be conducted by analysing the paths of many molecules that pass through the same unit area of membrane. We describe experiments performed on cultured primary cells, stable cell lines and ex vivo tissue slices using a variety of membrane proteins, under different imaging conditions. In some cell types, we find no evidence for heterogeneity in mobility across the plasma membrane, but in others we find statistically significant differences with some regions of membrane showing significantly higher viscosity than others

Mechanism of gating and partial agonist action in the glycine receptor

Ligand-gated ion channels mediate signal transduction at chemical synapses and transition between resting, open, and desensitized states in response to neurotransmitter binding. Neurotransmitters that produce maximum open channel probabilities (Po) are full agonists, whereas those that yield lower than maximum Po are partial agonists. Cys-loop receptors are an important class of neurotransmitter receptors, yet a structure-based understanding of the mechanism of partial agonist action has proven elusive. Here, we study the glycine receptor with the full agonist glycine and the partial agonists taurine and γ-amino butyric acid (GABA). We use electrophysiology to show how partial agonists populate agonist-bound, closed channel states and cryo-EM reconstructions to illuminate the structures of intermediate, pre-open states, providing insights into previously unseen conformational states along the receptor reaction pathway. We further correlate agonist-induced conformational changes to Po across members of the receptor family, providing a hypothetical mechanism for partial and full agonist action at Cys-loop receptors

Starting, building and sustaining a program of research in emergency medicine in Canada

Objective: To develop pragmatic recommendations for starting, building and sustaining a program of research in emergency medicine (EM) in Canada at sites with limited infrastructure and/or prior research experience. Methods: At the direction of the Canadian Association of Emergency Physicians (CAEP) academic section, we assembled an expert panel of 10 EM researchers with experience building programs of research. Using a modified Delphi approach, our panel developed initial recommendations for (1) starting, (2) building, and (3) sustaining a program of research in EM. These recommendations were peer-reviewed by emergency physicians and researchers from each of the panelist’s home institutions and tested for face and construct validity, as well as ease of comprehension. The recommendations were then iteratively revised based on feedback and suggestions from peer review and amended again after being presented at the 2020 CAEP academic symposium. Results: Our panel created 15 pragmatic recommendations for those intending to start (formal research training, find mentors, local support, develop a niche, start small), build (funding, build a team, collaborate, publish, expect failure) and sustain (become a mentor, obtain leadership roles, lead national studies, gain influence, prioritize wellness) a program of EM research in centers without an established research culture. Additionally, we suggest four recommendations for department leads aiming to foster a program of research within their departments. Conclusion: These recommendations serve as guidance for centres wanting to establish a program of research in EM

Multiplying the serum aminotransferase by the acetaminophen concentration to predict toxicity following overdose The APAP × AT multiplication product

Context. The first available predictors of hepatic injury following acetaminophen (APAP) overdose are the serum APAP and aminotransferases [AT, i.e., aspartate (AST) aminotransferase or alanine (ALT) aminotransferase]. Objective. We describe the initial value, rate of change, and interrelationship between these biomarkers in patients who develop hepatotoxicity despite treatment following acute overdose. A new parameter, the APAP × AT multiplication product, is proposed for early risk stratification. Methods. We conducted a descriptive study of individuals selected from a multicenter retrospective cohort of patients hospitalized for APAP poisoning. We selected those acute APAP overdose patients who subsequently developed AT >1,000 IU/L. Rising serum AT values were compared to simultaneously measured (or estimated) falling serum APAP. The APAP × AT was expressed relative to initiation of acetylcysteine therapy and grouped by time to meeting hepatotoxicity criteria. Results. In the 94 cases studied, serum APAP concentrations were still appreciable Because serum AT rose rapidly (doubling time 9.5 h ) and APAP fell slowly (half-life 4.8 h), the multiplication product remained elevated during the first 12-24 h of antidotal therapy, especially among patients who developed earlier hepatotoxicity (AT > 1,000 IU/L). Discussion and conclusions. The APAP × AT multiplication product, calculated at the time of presentation and after several h of antidotal therapy, holds promise as a new risk predictor following APAP overdose. It requires neither graphical interpretation nor accurate time of ingestion, two limitations to current risk stratification

Galanin-immunoreactivity identifies a distinct population of inhibitory interneurons in laminae I-III of the rat spinal cord

Background: Inhibitory interneurons constitute 30-40% of neurons in laminae I-III and have an important anti-nociceptive role. However, because of the difficulty in classifying them we know little about their organisation. Previous studies have identified 3 non-overlapping groups of inhibitory interneuron, which contain neuropeptide Y (NPY), neuronal nitric oxide synthase (nNOS) or parvalbumin, and have shown that these differ in postsynaptic targets. Some inhibitory interneurons contain galanin and the first aim of this study was to determine whether these form a different population from those containing NPY, nNOS or parvalbumin. We also estimated the proportion of neurons and GABAergic axons that contain galanin in laminae I-III. Results: Galanin cells were concentrated in laminae I-IIo, with few in laminae IIi-III. Galanin showed minimal co-localisation with NPY, nNOS or parvalbumin in laminae I-II, but most galanin-containing cells in lamina III were nNOS-positive. Galanin cells constituted similar to 7%, 3% and 2% of all neurons in laminae I, II and III, and we estimate that this corresponds to 26%, 10% and 5% of the GABAergic neurons in these laminae. However, galanin was only found in similar to 6% of GABAergic boutons in laminae I-IIo, and similar to 1% of those in laminae IIi-III. Conclusions: These results show that galanin, NPY, nNOS and parvalbumin can be used to define four distinct neurochemical populations of inhibitory interneurons. Together with results of a recent study, they suggest that the galanin and NPY populations account for around half of the inhibitory interneurons in lamina I and a quarter of those in lamina I

High risk clinical characteristics for subarachnoid haemorrhage in patients with acute headache: prospective cohort study

Objective To identify high risk clinical characteristics for subarachnoid haemorrhage in neurologically intact patients with headache