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Astrocytes are greatly impacted by oxidative stress, which can also be related to neurodegenerative diseases. Therefore, preventing the production of reactive oxygen species (ROS) is crucial for maintaining healthy cells. Large conductance Ca2+-activated big potassium (BK) channel openers are effective in eliminating the effects of oxidative stress. The present study aims to determine if NS11021, a BK channel opener, protects the astrocytcs from harmful effects of hydrogen peroxide (H2O2), which is an oxidative stress inducer. For this purpose, primary astrocyte cultures were incubated with H2O2, NS11021, and Iberiotoxin both separately and together. H2O2 decreased cell viability by approximately 50% and increased the number of ROS-positive astrocytes. However, NS11021, but not Iberiotoxin, reversed the deleterious effects of H2O2 on cell viability and decreased ROS production. Moreover, dysregulations in Cyclin D1/CDK6/p21 gene expressions under conditions of oxidative stress were regulated again by the opener. To the best of our knowledge, this study has been the first to reveal that NS11021 reversed the deleterious effects of H2O2 on cell viability by regulating ROS production in astrocytcs. Its effect may also be related to the regulation of cell cycle at the transcriptional level. NS11021 may also be used as an agent for the treatment of oxidative-stress related dysfunction of astrocytes.

Epilepsy is one of the most common neurological diseases and it causes profound morbidity and mortality. We identified the first de novo variant in KCNMA1 (c. 2984 A > G (p.(N995S)))-encoding the BK channel-that causes epilepsy, but not paroxysmal dyskinesia, in two independent families. The c. 2984 A > G (p.(N995S)) variant markedly increased the macroscopic potassium current by increasing both the channel open probability and channel open dwell time. The c. 2984 A > G (p.(N995S)) variant did not affect the calcium sensitivity of the channel. We also identified three other variants of unknown significance (c. 1554 G > T (p.(K518N)), c. 1967A > C (p.(E656A)), and c. 3476 A > G (p.(N1159S))) in three separate patients with divergent epileptic phenotypes. However, these variants did not affect the BK potassium current, and are therefore unlikely to be disease-causing. These results demonstrate that BK channel variants can cause epilepsy without paroxysmal dyskinesia. The underlying molecular mechanism can be increased activation of the BK channel by increased sensitivity to the voltage-dependent activation without affecting the sensitivity to the calcium-dependent activation. Our data suggest that the BK channel may represent a drug target for the treatment of epilepsy. Our data highlight the importance of functional electrophysiological studies of BK channel variants in distinguishing whether a genomic variant of unknown significance is a disease-causing variant or a benign variant.

Photochemical processes provide versatile triggers of chemical reactions. Here, we use a photoactivated lipid switch to modulate the folding and assembly of a protein channel within a model biological membrane. In contrast to the information rich field of water-soluble protein folding, there is only a limited understanding of the assembly of proteins that are integral to biological membranes. It is however possible to exploit the foreboding hydrophobic lipid environment and control membrane protein folding via lipid bilayer mechanics. Mechanical properties such as lipid chain lateral pressure influence the insertion and folding of proteins in membranes, with different stages of folding having contrasting sensitivities to the bilayer properties. Studies to date have relied on altering bilayer properties through lipid compositional changes made at equilibrium, and thus can only be made before or after folding. We show that light-activation of photoisomerisable di-(5-[[4-(4-butylphenyl)azo]phenoxy]pentyl)phosphate (4-Azo-5P) lipids influences the folding and assembly of the pentameric bacterial mechanosensitive channel MscL. The use of a photochemical reaction enables the bilayer properties to be altered during folding, which is unprecedented. This mechanical manipulation during folding, allows for optimisation of different stages of the component insertion, folding and assembly steps within the same lipid system. The photochemical approach offers the potential to control channel assembly when generating synthetic devices that exploit the mechanosensitive protein as a nanovalve.

The perception of complex acoustic stimuli begins with the deconstruction of sound into its frequency components. This spectral processing occurs first and foremost in the inner ear. In vertebrates, two very different strategies of frequency analysis have evolved. In nonmammalian vertebrates, the sensory hair cells of the inner ear are intrinsically electrically tuned to a narrow band of acoustic frequencies. This electrical tuning relies on the interplay between BK channels and voltage-gated calcium channels. Systematic variations in BK channel density and kinetics establish a gradient in electrical resonance that enables the coding of a broad range of acoustic frequencies. In contrast, mammalian hair cells are extrinsically tuned by mechanical properties of the cochlear duct. Even so, mammalian hair cells also express BK channels. These BK channels play critical roles in various aspects of mammalian auditory signaling, from developmental maturation to protection against acoustic trauma. This review summarizes the anatomical localization, biophysical properties, and functional contributions of BK channels in vertebrate inner ears. Areas of future research, based on an updated understanding of the biology of both BK channels and the inner ear, are also highlighted. Investigation of BK channels in the inner ear continues to provide fertile research grounds for examining both BK channel biophysics and the molecular mechanisms underlying signal processing in the auditory periphery.

The tension-driven gating process of MscL from Mycobacterium tuberculosis, Tb-MscL, has been addressed at near-atomic detail using coarse-grained molecular dynamics simulations. To perform the simulations, a novel coarse-grained peptide model based on a thermodynamic parameterization of the amino-acid side chains has been applied. Both the wild-type Tb-MscL and its gain-of-function mutant V21 D embedded in a solvated lipid bilayer have been studied. To mimic hypoosmotic shock conditions, simulations were performed at increasing levels of membrane tension approaching the rupture threshold of the lipid bilayer. Both the wild-type and the mutant channel are found to undergo significant conformational changes in accordance with an irislike expansion mechanism, reaching a conducting state on a microsecond timescale. The most pronounced expansion of the pore has been observed for the V21 D mutant, which is consistent with the experimentally shown gain-of-function phenotype of the V21 D mutant.

Toward the realization of nanoscale device control, we report a molecular valve embedded in a membrane that can be opened by illumination with long-wavelength ultraviolet (366 nanometers) light and then resealed by visible irradiation. The valve consists of a channel protein, the mechanosensitive channel of large conductance (MscL) from Escherichia coli , modified by attachment of synthetic compounds that undergo light-induced charge separation to reversibly open and close a 3-nanometer pore. The system is compatible with a classical encapsulation system, the liposome, and external photochemical control over transport through the channel is achieved.

GUVs have been widely used for studies on lipid mobility, membrane dynamics and lipid domain ( raft) formation, using single molecule techniques like fluorescence correlation spectroscopy. Reports on membrane protein dynamics in these types of model membranes are by far less advanced due to the difficulty of incorporating proteins into GUVs in a functional state. We have used sucrose to prevent four distinct membrane protein(s) ( complexes) from inactivating during the dehydration step of the GUV-formation process. The amount of sucrose was optimized such that the proteins retained 100% biological activity, and many proteo-GUVs were obtained. Although GUVs could be formed by hydration of lipid mixtures composed of neutral and anionic lipids, an alternate current electric field was required for GUV formation from neutral lipids. Distribution, lateral mobility, and function of an ATP-binding cassette transport system, an ion-linked transporter, and a mechanosensitive channel in GUVs were determined by confocal imaging, fluorescence correlation spectroscopy, patch-clamp measurements, and biochemical techniques. In addition, we show that sucrose slows down the lateral mobility of fluorescent lipid analogs, possibly due to hydrogen-bonding with the lipid headgroups, leading to larger complexes with reduced mobility.

The functions of the mechanosensitive channels from Lactococcus lactis were determined by biochemical, physiological, and electrophysiological methods. Patch-clamp studies showed that the genes yncB and mscL encode MscS and MscL-like channels, respectively, when expressed in Escherichia coli or if the gene products were purified and reconstituted in proteoliposomes. However, unless yncB was expressed in trans, wild type membranes of L. lactis displayed only MscL activity. Membranes prepared from an mscL disruption mutant did not show any mechanosensitive channel activity, irrespective of whether the cells had been grown on low or high osmolarity medium. In osmotic downshift assays, wild type cells survived and retained 20% of the glycine betaine internalized under external high salt conditions. On the other hand, the mscL disruption mutant retained 40% of internalized glycine betaine and was significantly compromised in its survival upon osmotic downshifts. The data strongly suggest that L. lactis uses MscL as the main mechanosensitive solute release system to protect the cells under conditions of osmotic downshift.

The mechanosensitive channel protein of large conductance, MscL, from Escherichia coli has been implicated in protein efflux, but the passage of proteins through the channel has never been demonstrated. We used dual-color fluorescence-burst analysis to evaluate the efflux of fluorescent labeled compounds through MscL. The method correlates the fluctuations in intensity of fluorescent labeled membranes and encapsulated (macro)molecules (labeled with second fluorophore) for each liposome diffusing through the observation volume. The analysis provides quantitative information on the concentration of macromolecules inside the liposomes and the fraction of functional channel proteins. For MscL, reconstituted in large unilamellar vesicles, we show that insulin, bovine pancreas trypsin inhibitor, and other compounds smaller than 6.5kDa can pass through MscL, whereas larger macromolecules cannot.

International audience; Objective: Autism is a complex, largely genetic psychiatric disorder. In the majority of cases, the cause of autism is not known, but there is strong evidence for a genetic etiology. To identify candidate genes, the physical mapping of balanced chromosomal aberrations is a powerful strategy, since several genes have been characterized in numerous disorders. In this study, the authors analyzed a balanced reciprocal translocation arising de novo in a subject with autism and mental retardation. Method: The authors performed the physical mapping of the balanced 9q23/ 10q22 translocation by fluorescent in situ hybridization experiments using bacterial artificial chromosome clones covering the areas of interest. Results: Findings revealed that the KCNMA1 gene, which encodes the alpha- subunit of the large conductance Ca2+-activated K+ ( BKCa) channel, a synaptic regulator of neuronal excitability, is physically disrupted. Further molecular and functional analyses showed the haploinsufficiency of this gene as well as decreased activity of the coded BKCa channel. This activity can be enhanced in vitro by addition of a BKCa channel opener (BMS-204352). Further mutational analyses on 116 autistic subjects led to the identification of an amino acid substitution located in a highly conserved domain of the protein not found in comparison subjects. Conclusions: These results suggest a possible association between a functional defect of the BKCa channel and autistic disorder and raise the hypothesis that deficits in synaptic transmission may contribute to the physiopathology of autism and mental deficiency.