The Role of Calcium-activated Potassium Channel in Mitochondria-Associated ER Membrane and Its Functional Link to Cell Survival and Death

The process of apoptosis is not only regulated by molecular gens but it is also regulated by cellular ionic homeostasis especially K+ homeostasis in the cell. In the past decade, molecular mechanisms of ionic regulation of apoptosis have been extensively investigated. The ionic mechanism of apoptosis are involves Ca2+ influx and accumulation of intracellular Ca2+ is convincing evidence to excessive K+ efflux resulting in early steps in apoptosis. The BK channels play a critical role in mediating the K+ efflux linked with apoptotic cell shrinkage. Mitochondria-associated ER membranes (MAMs) control Ca2+ influx between ER and mitochondria. The BKα subunits are localized in the inner mitochondrial and ER membrane and directly interact with other BK channel associated proteins (BKAPs) like, IP3R-1, calreticulin at the ER face of the MAMs, and the molecular chaperone grp78, which bridges the IP3R-1 with voltage-dependent anion channel (VDAC-1) of the outer mitochondrial membrane (OMM). The present chapter clearly depicts that how BK channels are associated with BKAPs and how they are involved in apoptosis through regulation of K+ efflux

Preemptive analgesic effect of intrathecal applications of neuroactive steroids in a rodent model of post-surgical pain: Evidence for the role of T-type calcium channels

Preemptive management of post-incisional pain remains challenging. Here, we examined the role of preemptive use of neuroactive steroids with activity on low-voltage activated T-type C

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

Further evidence that inhibition of neuronal voltage-gated calcium channels contributes to the hypnotic effect of neurosteroid analogue, 3β-OH

We recently reported that a neurosteroid analogue with T-channel-blocking properties (3β,5β,17β)-3-hydroxyandrostane-17-carbonitrile (3β-OH), induced hypnosis in rat pups without triggering neuronal apoptosis. Furthermore, we found that the inhibition of the C

Differential effects of the novel neurosteroid hypnotic (3β,5β,17β)-3-hydroxyandrostane-17-carbonitrile on electroencephalogram activity in male and female rats

BACKGROUND: We recently showed that a neurosteroid analogue, (3β,5β,17β)-3-hydroxyandrostane-17-carbonitrile (3β-OH), induced hypnosis in rats. The aim of the present study was to evaluate the hypnotic and anaesthetic potential of 3β-OH further using electroencephalography. METHODS: We used behavioural assessment and cortical electroencephalogram (EEG) spectral power analysis to examine hypnotic and anaesthetic effects of 3β-OH (30 and 60 mg kg RESULTS: We found dose-dependent sex differences in 3β-OH-induced hypnosis and EEG changes. Both male and female rats responded similarly to i.p. 3β-OH 30 mg kg CONCLUSIONS: Based on its behavioural effects and EEG signature, 3β-OH is a potent hypnotic in rats, with female rats being more sensitive than male rats

The enantiomer of allopregnanolone prevents pressure-mediated retinal degeneration via autophagy

In a

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

Comparison of Steroid Modulation of Spontaneous Inhibitory Postsynaptic Currents in Cultured Hippocampal Neurons and Steady-State Single-Channel Currents from Heterologously Expressed a1b2g2L GABA A Receptors

ABSTRACT Neuroactive steroids are efficacious modulators of g-aminobutyric acid type A receptor (GABA A ) receptor function. The effects of steroids on the GABA A receptor are typically determined by comparing steady-state single-channel open probability or macroscopic peak responses elicited by GABA in the absence and presence of a steroid. Due to differences in activation conditions (exposure duration, concentration of agonist), it is not obvious whether modulation measured using typical experimental protocols can be used to accurately predict the effect of a modulator on native receptors under physiologic conditions. In the present study, we examined the effects of 14 neuroactive steroids and analogs on the properties of spontaneous inhibitory postsynaptic currents (sIPSCs) in cultured rat hippocampal neurons. The goal was to determine whether the magnitude of modulation of the decay time course of sIPSCs correlates with the extent of modulation and kinetic properties of potentiation as determined in previous singlechannel studies. The steroids were selected to cover a wide range of efficacy on heterologously expressed rat a1b2g2L GABA A receptors, ranging from essentially inert to highly efficacious (strong potentiators of single-channel and macroscopic peak responses). The data indicate a strong correlation between prolongation of the decay time course of sIPSCs and potentiation of single-channel open probability. Furthermore, changes in intracluster closed time distributions were the single best predictor of prolongation of sIPSCs. We infer that the information obtained in steady-state single-channel recordings can be used to forecast modulation of synaptic currents