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.

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.