Cross Pharmacological, Biochemical and Computational Studies of a Human Kv3.1b Inhibitor from Androctonus australis Venom.

The voltage-gated K+ channels Kv3.1 display fast activation and deactivation kinetics and are known to have a crucial contribution to the fast-spiking phenotype of certain neurons. AahG50, as a natural product extracted from Androctonus australis hector venom, inhibits selectively Kv3.1 channels. In the present study, we focused on the biochemical and pharmacological characterization of the component in AahG50 scorpion venom that potently and selectively blocks the Kv3.1 channels. We used a combined optimization through advanced biochemical purification and patch-clamp screening steps to characterize the peptide in AahG50 active on Kv3.1 channels. We described the inhibitory effect of a toxin on Kv3.1 unitary current in black lipid bilayers. In silico, docking experiments are used to study the molecular details of the binding. We identified the first scorpion venom peptide inhibiting Kv3.1 current at 170 nM. This toxin is the alpha-KTx 15.1, which occludes the Kv3.1 channel pore by means of the lysine 27 lateral chain. This study highlights, for the first time, the modulation of the Kv3.1 by alpha-KTx 15.1, which could be an interesting starting compound for developing therapeutic biomolecules against Kv3.1-associated diseases.

First Evidence of Kv3.1b Potassium Channel Subtype Expression during Neuronal Serotonergic 1C11 Cell Line Development.

Kv3.1 channel is abundantly expressed in neurons and its dysfunction causes sleep loss, neurodegenerative diseases and depression. Fluoxetine, a serotonin selective reuptake inhibitor commonly used to treat depression, acts also on Kv3.1. To define the relationship between Kv3.1 and serotonin receptors (SR) pharmacological modulation, we showed that 1C11, a serotonergic cell line, expresses different voltage gated potassium (VGK) channels subtypes in the presence (differentiated cells (1C11D)) or absence (not differentiated cells (1C11ND)) of induction. Only Kv1.2 and Kv3.1 transcripts increase even if the level of Kv3.1b transcripts is highest in 1C11D and, after fluoxetine, in 1C11ND but decreases in 1C11D. The Kv3.1 channel protein is expressed in 1C11ND and 1C11D but is enhanced by fluoxetine only in 1C11D. Whole cell measurements confirm that 1C11 cells express (VGK) currents, increasing sequentially as a function of cell development. Moreover, SR 5HT1b is highly expressed in 1C11D but fluoxetine increases the level of transcript in 1C11ND and significantly decreases it in 1C11D. Serotonin dosage shows that fluoxetine at 10 nM blocks serotonin reuptake in 1C11ND but slows down its release when cells are differentiated through a decrease of 5HT1b receptors density. We provide the first experimental evidence that 1C11 expresses Kv3.1b, which confirms its major role during differentiation. Cells respond to the fluoxetine effect by upregulating Kv3.1b expression. On the other hand, the possible relationship between the fluoxetine effect on the kinetics of 5HT1b differentiation and Kv3.1bexpression, would suggest the Kv3.1b channel as a target of an antidepressant drug as well as it was suggested for 5HT1b.

[«â€ Group medical practices†¼ project†: collaboration between primary care medicine and institutional public psychiatry]. / Projet «â€ cabinets de groupe†¼â€ : collaboration entre médecine de premier recours et psychiatrie.

Collaboration between primary care medicine and psychiatry is a well-known challenge. In order to improve access to psychological care for patients undergoing primary care, the «â€…group medical practices ¼ project proposes a collaborative care model in which a psychiatrist employed by a public psychiatric institution integrates group medical practices in order to provide assistance to primary care physicians. It is thus able to evaluate patients directly in the practices and to offer supervision and consilium spaces to primary care physicians.

La collaboration entre la médecine de premier recours et la psychiatrie représente une difficulté bien connue. Pour améliorer l'accès aux soins psychiques des patients suivis en médecine de premier recours, le projet «â€…cabinets de groupe ¼ propose un modèle de soins collaboratifs dans lequel un psychiatre employé d'une institution psychiatrique publique intègre des cabinets de groupe afin d'apporter son aide aux médecins de premier recours (MPR). Il est ainsi en mesure, d'une part, d'évaluer les patients directement au sein des cabinets et, d'autre part, d'offrir des espaces de supervision et de consilium aux MPR.

Xenopus Oocyte's Conductance for Bioactive Compounds Screening and Characterization.

BACKGROUND: Astaxanthin (ATX) is a lipophilic compound found in many marine organisms. Studies have shown that ATX has many strong biological properties, including antioxidant, antiviral, anticancer, cardiovascular, anti-inflammatory, neuro-protective and anti-diabetic activities. However, no research has elucidated the effect of ATX on ionic channels. ATX can be extracted from shrimp by-products. Our work aims to characterize ATX cell targets to lend value to marine by-products. METHODS: We used the Xenopus oocytes cell model to characterize the pharmacological target of ATX among endogenous Xenopus oocytes' ionic channels and to analyze the effects of all carotenoid-extract samples prepared from shrimp by-products using a supercritical fluid extraction (SFE) method. RESULTS: ATX inhibits amiloride-sensitive sodium conductance, xINa, in a dose-dependent manner with an IC50 of 0.14 µg, a maximum inhibition of 75% and a Hill coefficient of 0.68. It does not affect the potential of half activation, but significantly changes the kinetics, according to the slope factor values. The marine extract prepared from shrimp waste at 10 µg inhibits xINa in the same way as ATX 0.1 µg does. When ATX was added to the entire extract at 10 µg, inhibition reached that induced with ATX 1 µg. CONCLUSIONS: ATX and the shrimp Extract inhibit amiloride-sensitive sodium channels in Xenopus oocytes and the TEVC method makes it possible to measure the ATX inhibitory effect in bioactive SFE-Extract samples.

A novel depolarizing activity of scorpion venom fraction M1 due to activation of skeletal muscle nicotinic receptors.

A depolarizing activity following interaction with nicotinic acetylcholine receptors (nAchRs) in skeletal muscle cells, was observed for the first time in the non-toxic venom fraction (M1) of the yellow scorpion Buthus occitanus tunetanus (Bot). The effects of M1 fraction were tested on cultured rat myotubes by recording changes in [Ca2+]i. When applied, M1 (10 microg/mL) induced a transient increase of [Ca2+]i which could be blocked by a prior application of alpha-Bungarotoxin (alpha-Bg-Tx).

Depression of cardiac L-type calcium current by a scorpion venom fraction M1 following muscarinic receptors interaction involving adenylate cyclase pathway.

The effects of a non-toxic fraction, called M1, from Buthus occitanus tunetanus (Bot) scorpion were studied on rat cardiac contraction and calcium transient and current. A decrease in both rate and tension on isolated intact hearts as well as in calcium transient induced by depolarizing 100 K(+) solution on isolated ventricular cardiomyocytes was firstly observed. Studies with the whole cell patch clamp method showed that M1 decreased the L-type calcium current (ICa(L)) in a dose-dependent manner with an IC50 of 0.36 microg/mL and a Hill coefficient of 0.95. This effect was blocked and reversed by the specific muscarinic receptors antagonist atropine, 1 microM, and was completely prevented when cardiomyocytes were pretreated with Pertussis toxin, 1 microg/mL, to block the alpha subunit of the PTX-sensitive G proteins. These results show that M1 fraction of Bot inhibits basal calcium current by interacting with muscarinic receptors and suggest that this inhibition could be attributed to inhibition of adenylate cyclase activity by a mechanism involving PTX-sensitive G proteins.

A new type of scorpion Na+-channel-toxin-like polypeptide active on K+ channels.

We have purified and characterized two peptides, named KAaH1 and KAaH2 (AaH polypeptides 1 and 2 active on K+ channels, where AaH stands for Androctonus australis Hector), from the venom of A. australis Hector scorpions. Their sequences contain 58 amino acids including six half-cysteines and differ only at positions 26 (Phe/Ser) and 29 (Lys/Gln). Although KAaH1 and KAaH2 show important sequence similarity with anti-mammal beta toxins specific for voltage-gated Na+ channels, only weak beta-like effects were observed when KAaH1 or KAaH2 (1 microM) were tested on brain Nav1.2 channels. In contrast, KAaH1 blocks Kv1.1 and Kv1.3 channels expressed in Xenopus oocytes with IC50 values of 5 and 50 nM respectively, whereas KAaH2 blocks only 20% of the current on Kv1.1 and is not active on Kv1.3 channels at a 100 nM concentration. KAaH1 is thus the first member of a new subfamily of long-chain toxins mainly active on voltage-gated K+ channels. NMR spectra of KAaH1 and KAaH2 show good dispersion of signals but broad lines and poor quality. Self-diffusion NMR experiments indicate that lines are broadened due to a conformational exchange on the millisecond time scale. NMR and CD indicate that both polypeptides adopt a similar fold with alpha-helical and b-sheet structures. Homology-based molecular models generated for KAaH1 and KAaH2 are in accordance with CD and NMR data. In the model of KAaH1, the functionally important residues Phe26 and Lys29 are close to each other and are located in the alpha-helix. These residues may constitute the so-called functional dyad observed for short alpha-KTx scorpion toxins in the beta-sheet.

Inhibition of human Kv3.1 current expressed in Xenopus oocytes by the toxic venom fraction of Androctonus australis hector.

AahG50, the toxic fraction of Androctonus australis hector venom, was studied on human Kv3.1 channels activation, stably expressed in Xenopus oocytes using the two-electrode voltage clamp technique. AahG50 reduced Kv3.1 currents in a reversible concentration-dependent manner, with an IC50 value and a Hill coefficient of 40.4 ± 0.2 µg/ml and 1.3 ± 0.05, respectively. AahG50 inhibited IKv3.1 without modifying the current activation kinetics. The AahG50-induced inhibition of Kv3.1 channels was voltage-dependent, with a gradual increase at lower concentrations and over the voltage range of channels opening. However, at higher concentrations, the inhibition exhibited voltage dependence only in the first range of channels opening from -20 to +10 mV, but demonstrates a low degree of voltage-dependence when channels are fully activated. In the literature, toxins have previously been isolated from AahG50, KAaH1 and KAaH2 and were reported not to have any effect on IKv3.1. The present article's findings suggest that AahG50 may contain a peptidic component active on Kv3.1 channels, which inhibits IKv3.1 in a selective manner.