SUR2A-mediated regulation of KATP channels is affected by residues belonging to the C terminus of the first nucleotide binding domain (NBD1). We studied the C-terminal region of NBD1 by comparing experiments using NBD1 S615-D914 and NBD1 S615-K972 constructs to studies of NBD1 S615-L933 also performed in our laboratory. Our NMR data suggests that the C-terminal region of NBD1 from residues Q915 to L933 is disordered and transiently contacts the NBD1 core, which may affect NBD1 phosphorylation. Tryptophan quenching fluorescence experiments corroborate that the Q915-L933 C-terminal tail contacts the NBD1 core. Fluorescence thermal denaturation experiments suggest that NBD1 S615-D914 has a higher affinity for MgATP compared with NBD1 S615-L933, implying that the C-terminal tail varies MgATP binding. Additional experiments were performed to identify soluble constructs of hSUR1 NBD1 and rSUR2A NBD2 that would allow detailed biophysical studies of these domains. Some of the constructs studied showed improved solubility and stability.

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Les ataxies épisodiques (EA) d’origine génétique sont un groupe de maladies possédant un phénotype et génotype hétérogènes, mais ont en commun la caractéristique d’un dysfonctionnement cérébelleux intermittent. Les EA de type 1 et 2 sont les plus largement reconnues des ataxies épisodiques autosomiques dominantes et sont causées par un dysfonctionnement des canaux ioniques voltage-dépendants dans les neurones. La présente étude se concentrera sur les mutations causant l'EA-1, retrouvées dans le senseur de voltage (VSD) de Kv1.1, un canal très proche de la famille des canaux Shaker. Nous avons caractérisé les propriétés électrophysiologiques de six mutations différentes à la position F244 et partiellement celles des mutations T284 A/M, R297 K/Q/A/H, I320T, L375F, L399I et S412 C/I dans la séquence du Shaker grâce à la technique du ‘’cut open voltage clamp’’ (COVC). Les mutations de la position F244 situées sur le S1 du canal Shaker sont caractérisées par un décalement des courbes QV et GV vers des potentiels dépolarisants et modifient le couplage fonctionnel entre le domaine VSD et le pore. Un courant de fuite est observé durant la phase d'activation des courants transitoires et peut être éliminé par l'application du 4-AP (4-aminopyridine) ou la réinsertion de l'inactivation de type N mais pas par le TEA (tétraéthylamonium). Dans le but de mieux comprendre les mécanismes moléculaires responsables de la stabilisation d’un état intermédiaire, nous avons étudié séparément la neutralisation des trois premières charges positives du S4 (R1Q, R2Q et R3Q). Il en est ressorti l’existence d’une interaction entre R2 et F244. Une seconde interface entre S1 et le pore proche de la surface extracellulaire agissant comme un second point d'ancrage et responsable des courants de fuite a été mis en lumière. Les résultats suggèrent une anomalie du fonctionnement du VSD empêchant la repolarisation normale de la membrane des cellules nerveuses affectées à la suite d'un potentiel d'action. / The genetic episodic ataxias form a group of disorders with heterogeneous phenotype and genotype, but share the common feature of intermittent cerebellar dysfunction. Episodic ataxia (EA) types 1 and 2 are most widely recognised amongst the autosomal dominant episodic ataxias and are caused by dysfunction of neuronal voltage-gated ion channels. The present study focuses on mutations causing EA-1 located in the voltage sensor domains (VSDs) of Kv1.1. A member of the Shaker channel family. Here, we have characterised the electrophysiological properties of six different mutations at the position of F244 and we also reported the partiality effects of these following mutations T284A/M, R297K/Q/A/H, I320T, L375F, L399I S412C/I on Shaker sequence using the cut open voltage clamp technique (COVC). We have shown that mutations of F244 in the S1 of the Shaker Kv channel positively shift the voltage dependence of the VSD movement and alter functional coupling between VSD and pore domain. The mutations causing immobilization of the VSD movement during activation and deactivation and responsible for creating a leak current during activation, are removed by the application of 4-AP (4-aminopyridine) or by reinsertion of N-type inactivation but not by TEA (tetraethylamonium). Insights into the molecular mechanisms responsible for the stabilization of the intermediate state have been investigated by separately neutralizing the first three charges (R1Q, R2Q and R3Q) in the S4 segment. The result suggests an interaction between R2 and F244 mutants. It was established that a second co-evolved interface exists between S1 and the pore helix near the extracellular surface and it acts as a second anchor point. It is also responsible for generation of leak currents. The results suggest a dysfunction of the VSD in which the affected nerve cells cannot efficiently repolarize following an action potential because of altered delayed rectifier function

SUR2A-mediated regulation of KATP channels is affected by residues belonging to the C terminus of the first nucleotide binding domain (NBD1). We studied the C-terminal region of NBD1 by comparing experiments using NBD1 S615-D914 and NBD1 S615-K972 constructs to studies of NBD1 S615-L933 also performed in our laboratory. Our NMR data suggests that the C-terminal region of NBD1 from residues Q915 to L933 is disordered and transiently contacts the NBD1 core, which may affect NBD1 phosphorylation. Tryptophan quenching fluorescence experiments corroborate that the Q915-L933 C-terminal tail contacts the NBD1 core. Fluorescence thermal denaturation experiments suggest that NBD1 S615-D914 has a higher affinity for MgATP compared with NBD1 S615-L933, implying that the C-terminal tail varies MgATP binding. Additional experiments were performed to identify soluble constructs of hSUR1 NBD1 and rSUR2A NBD2 that would allow detailed biophysical studies of these domains. Some of the constructs studied showed improved solubility and stability.

Gout is one form of inflammatory arthritis, which is caused by the precipitation of crystals of monosodium urate (MSU) in the joints. Acute gout is associated with sudden and painful inflammatory episodes characterized by high neutrophil infiltration. In spite of years of study gout treatment remains a challenge due to its relative ineficcacy. Thus, search for new and efficient therapies is necessary. The objective of this study was to investigate the involvement of glibenclamide in a model of acute gout in rats induced by MSU. MSU crystals produced nociception and edema when injected into the ankle joint of rats. Treatment with glibenclamide (3 mg/kg, s.c.) or dexamethasone (8 mg/kg, s.c., used as a positive control) decreased spontaneous nociception (67% ± 11 and 70 ± 7% inhibition, respectively) and edema (28 ± 7% and 77 ± 7% inhibition, respectively) induced 6 hours after MSU injection. The number of leukocyte infiltrates in the synovial fluid as well as the release of interleukin 1β (IL-1β) and prostaglandin E2 (PGE2) significantly increased at 6 hours after injection of MSU joint, but these effects were not reversed by treatment with glibenclamide (3 mg/kg, s.c.). In contrast, dexamethasone reduced the leukocyte infiltration and release of IL-1β and PGE2. To confirm if the dose of glibenclamide was able to block the KATP channels, we determined the levels of glucose in the blood of animals. Glibenclamide decreased (23 ± 2%) and dexamethasone increased the blood glucose of the rats compared to vehicle-treated animals / MSU. Therefoe, the effects of glibenclamide on nociception and edema induced MSU, suggests that this sulfonylurea may be an interesting option as an adjunct therapy in pain observed in acute attacks of gout. / A gota é uma forma de artrite inflamatória, causada pela precipitação de cristais de urato monossódico (MSU) nas articulações. A forma aguda de gota está associada a episódios inflamatórios súbitos e dolorosos caracterizados por uma grande infiltração de neutrófilos. Apesar dos anos de estudo sobre a gota, o seu tratamento ainda é um desafio pela relativa ineficácia dos fármacos disponíveis no mercado. Assim, a busca por novos agentes terapêuticos mais efetivos e seguros se faz necessário. Desta forma, o objetivo deste estudo foi investigar o possível potencial farmacológico da glibenclamida em um modelo de gota aguda induzida por MSU em ratos. Os cristais de MSU produziram nocicepção e edema quando injetados na articulação do tornozelo de ratos. O tratamento com glibenclamida (3 mg/kg, s.c.) ou dexametasona (8 mg/kg, s.c., usada como controle positivo) reduziu a nocicepção espontânea (67 ± 11% e 70 ± 7% de inibição, respectivamente) e o edema (28 ± 7% e 77 ± 7% de inibição, respectivamente) induzidos pelo MSU, 6 horas após a injeção do cristal. O número de leucócitos infiltrados no líquido sinovial, assim como a liberação de interleucina 1β (IL-1β) e de prostaglandina E2 (PGE2) foram consideravelmente aumentados, 6 horas após a injeção de MSU na articulação, porém esses efeitos não foram revertidos pelo tratamento com glibenclamida (3 mg/kg, s.c.). Em contrapartida, dexametasona reduziu a infiltração de leucócitos e a liberação de IL-1β e de PGE2. Para confirmar se a dose utilizada de glibenclamida foi capaz de bloquear os canais de KATP, foi avaliado os níveis de glicose no sangue dos animais. A glibenclamida reduziu (23 ± 2%) e a dexametasona aumentou a glicemia dos ratos quando comparado aos animais tratados com veículo /MSU. Assim, frente aos efeitos desempenhados pela glibenclamida sobre a nocicepção e edema induzidos pelo MSU, sugere-se que esta sulfonilureia possa ser uma opção interessante como um tratamento adjuvante na dor observada em ataques agudos de gota.

Chemical reactions involving small number of reacting molecules are noisy processes. They are simulated using stochastic simulation algorithms like the Gillespie SSA, which are valid when the reaction environment is well-mixed. This is not the case in reactions occuring on biological media like cell membranes, where alternative simulation methods have to be used to account for the crowded nature of the reacting environment. Ion channels, which are membrane proteins controlling the flow of ions into and out of the cell, offer excellent single molecule conditions to test stochastic simulation schemes in crowded biological media. Single molecule reactions are of great importance in determining the functions of biological molecules. Access to their experimental data have increased the scope of com-putational modeling of biological processes. Recently, single molecule experiments have revealed the non-Markovian nature of chemical reactions, due to a phenomenon called `dynamic disorder', which makes the rate constants a deterministic function of time or a random process. This happens when there are additional slow scale conformational transitions, giving the molecule a memory of its previous states. In a previous work, the hTREK1 two pore domain potassium channel was revealed to have long term memory in its kinetics, prompting alternate non-Markovian schemes to analyze its gating. Traditionally, ion channel gating is modeled as Markovian transitions between fixed states. In this work, we have used single channel data from hTREK1 ion channel and have provided a simple diffusion model for its gating. The main assumption of this model is that the ion channel diffuses through a continuum of states on its potential energy landscape, which is derived from the steady state probability distribution of ionic current recorded from patch clamp experiments. A stochastic differential equation (SDE) driven by Gaussian white noise is proposed to model this motion in an asymmetric double well potential. The method is computationally very simple and efficient and reproduces the amplitude histogram very well. For the case when ligands are added, leading to incorporation of long term memory in the kinetics, the SDE is modified to run on coloured noise. This has been done by introducing an auxiliary variable into the equation. It has been shown that increasing the noise correlation with ligand concentration improves the fits to the experimental data. This has been validated for several datasets. These methods are more advantageous for simulation than the Markovian models as they are true to the physical picture of gating and also computationally very efficient. Reproducing the whole raw data trace takes no more than a few seconds with our scheme, with the only input being the amplitude histogram and four parameters. Finally a quantitative model based on a modified version of the Chemical Langevin equation is given, which works on random rate parameters. This model is computationally simple to implement and reproduces the catalytic activity of the channel as a function of time. From the computational analysis undertaken in this work, we can infer that ion channel activity can be modeled using the framework of non-Markovian processes, lending credence to the recent understanding that single molecule reactions are basically processes with long-term memory. Since the ion channel is basically a protein, we can also hypothesize that the some of the properties that make proteins so vital to living organ-isms could be attributed to long-term memory in their folding kinetics, giving them the ability to sample specific regions of their conformation space, which are of interest to biological functions.

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