Neurosteroids and their potential as a safer class of general anesthetics

Neurosteroids (NS) are a class of steroids that are synthesized within the central nervous system (CNS). Various NS can either enhance or inhibit CNS excitability and they play important biological roles in brain development, brain function and as mediators of mood. One class of NS, 3α-hydroxy-pregnane steroids such as allopregnanolone (AlloP) or pregnanolone (Preg), inhibits neuronal excitability; these endogenous NS and their analogues have been therapeutically applied as anti-depressants, anti-epileptics and general anesthetics. While NS have many favorable properties as anesthetics (e.g. rapid onset, rapid recovery, minimal cardiorespiratory depression, neuroprotection), they are not currently in clinical use, largely due to problems with formulation. Recent advances in understanding NS mechanisms of action and improved formulations have rekindled interest in development of NS as sedatives and anesthetics. In this review, the synthesis of NS, and their mechanism of action will be reviewed with specific emphasis on their binding sites and actions on γ-aminobutyric acid type A (GAB

Development of connectivity in a motoneuronal network in Drosophila larvae.

BACKGROUND: Much of our understanding of how neural networks develop is based on studies of sensory systems, revealing often highly stereotyped patterns of connections, particularly as these diverge from the presynaptic terminals of sensory neurons. We know considerably less about the wiring strategies of motor networks, where connections converge onto the dendrites of motoneurons. Here, we investigated patterns of synaptic connections between identified motoneurons with sensory neurons and interneurons in the motor network of the Drosophila larva and how these change as it develops. RESULTS: We find that as animals grow, motoneurons increase the number of synapses with existing presynaptic partners. Different motoneurons form characteristic cell-type-specific patterns of connections. At the same time, there is considerable variability in the number of synapses formed on motoneuron dendrites, which contrasts with the stereotypy reported for presynaptic terminals of sensory neurons. Where two motoneurons of the same cell type contact a common interneuron partner, each postsynaptic cell can arrive at a different connectivity outcome. Experimentally changing the positioning of motoneuron dendrites shows that the geography of dendritic arbors in relation to presynaptic partner terminals is an important determinant in shaping patterns of connectivity. CONCLUSIONS: In the Drosophila larval motor network, the sets of connections that form between identified neurons manifest an unexpected level of variability. Synapse number and the likelihood of forming connections appear to be regulated on a cell-by-cell basis, determined primarily by the postsynaptic dendrites of motoneuron terminals.L.C. was supported by a Fyssen Foundation post-doctoral fellowship. This work was supported by a Biotechnology and Biological Sciences Research Council (UK) grant (BB/I022414/1) to M.L., a Wellcome Trust Programme Grant (WT075934) to Michael Bate and M.L., a Grass Foundation fellowship to A.S.M., and a Sir Isaac Newton Trust grant to A.S.M. and M.L. The work benefited from facilities supported by a Wellcome Trust Equipment Grant (WT079204) and contributions by the Sir Isaac Newton Trust in Cambridge.This paper was originally published in Current Biology (Couton L, Mauss AS, Yunusov T, Diegelmann S, Evers JF, Landgraf M, Current Biology 2015, 25, 568–576, doi:10.1016/j.cub.2014.12.056

Tuning the nuclei-induced spin relaxation of localized electrons by the quantum Zeno and anti-Zeno effects

Quantum measurement back action is fundamentally unavoidable when manipulating electron spins. Here we demonstrate that this back action can be efficiently exploited to tune the spin relaxation of localized electrons induced by the hyperfine interaction. In optical pump-probe experiments, powerful probe pulses suppress the spin relaxation of electrons on Si donors in an InGaAs epilayer due to the quantum Zeno effect. By contrast, an increase of the probe power leads to a speed-up of the spin relaxation for electrons in InGaAs quantum dots due to the quantum anti-Zeno effect. The microscopic description shows that the transition between the two regimes occurs when the spin dephasing time is comparable to the probe pulse repetition period

Photoaffinity labeling with cholesterol analogues precisely maps a cholesterol-binding site in voltage-dependent anion channel-1

Voltage-dependent anion channel-1 (VDAC1) is a highly regulated β-barrel membrane protein that mediates transport of ions and metabolites between the mitochondria and cytosol of the cell. VDAC1 co-purifies with cholesterol and is functionally regulated by cholesterol, among other endogenous lipids. Molecular modeling studies based on NMR observations have suggested five cholesterol-binding sites in VDAC1, but direct experimental evidence for these sites is lacking. Here, to determine the sites of cholesterol binding, we photolabeled purified mouse VDAC1 (mVDAC1) with photoactivatable cholesterol analogues and analyzed the photolabeled sites with both top-down mass spectrometry (MS), and bottom-up MS paired with a clickable, stable isotope-labeled tag, FLI-tag. Using cholesterol analogues with a diazirine in either the 7 position of the steroid ring (LKM38) or the aliphatic tail (KK174), we mapped a binding pocket in mVDAC1 localized to Thr83 and Glu73, respectively. When Glu73 was mutated to a glutamine, KK174 no longer photolabeled this residue, but instead labeled the nearby Tyr62 within this same binding pocket. The combination of analytical strategies employed in this work permits detailed molecular mapping of a cholesterol-binding site in a protein, including an orientation of the sterol within the site. Our work raises the interesting possibility that cholesterol-mediated regulation of VDAC1 may be facilitated through a specific binding site at the functionally important Glu73 residue

Kinetic and Structural Determinants for GABA-A Receptor Potentiation by Neuroactive Steroids

Endogenous neurosteroids and synthetic neuroactive steroid analogs are among the most potent and efficacious potentiators of the mammalian GABA-A receptor. The compounds interact with one or more sites on the receptor leading to an increase in the channel open probability through a set of changes in the open and closed time distributions. The endogenous neurosteroid allopregnanolone potentiates the α1β2γ2L GABA-A receptor by enhancing the mean duration and prevalence of the longest-lived open time component and by reducing the prevalence of the longest-lived intracluster closed time component. Thus the channel mean open time is increased and the mean closed time duration is decreased, resulting in potentiation of channel function. Some of the other previously characterized neurosteroids and steroid analogs act through similar mechanisms while others affect a subset of these parameters. The steroids modulate the GABA-A receptor through interactions with the membrane-spanning region of the receptor. However, the number of binding sites that mediate the actions of steroids is unclear. We discuss data supporting the notions of a single site vs. multiple sites mediating the potentiating actions of steroids

Multiple functional neurosteroid binding sites on GABAA receptors

Neurosteroids are endogenous modulators of neuronal excitability and nervous system development and are being developed as anesthetic agents and treatments for psychiatric diseases. While gamma amino-butyric acid Type A (GABAA) receptors are the primary molecular targets of neurosteroid action, the structural details of neurosteroid binding to these proteins remain ill defined. We synthesized neurosteroid analogue photolabeling reagents in which the photolabeling groups were placed at three positions around the neurosteroid ring structure, enabling identification of binding sites and mapping of neurosteroid orientation within these sites. Using middle-down mass spectrometry (MS), we identified three clusters of photolabeled residues representing three distinct neurosteroid binding sites in the human α1β3 GABAA receptor. Novel intrasubunit binding sites were identified within the transmembrane helical bundles of both the α1 (labeled residues α1-N408, Y415) and β3 (labeled residue β3-Y442) subunits, adjacent to the extracellular domains (ECDs). An intersubunit site (labeled residues β3-L294 and G308) in the interface between the β3(+) and α1(-) subunits of the GABAA receptor pentamer was also identified. Computational docking studies of neurosteroid to the three sites predicted critical residues contributing to neurosteroid interaction with the GABAA receptors. Electrophysiological studies of receptors with mutations based on these predictions (α1-V227W, N408A/Y411F, and Q242L) indicate that both the α1 intrasubunit and β3-α1 intersubunit sites are critical for neurosteroid action