Comparison of intranasal naloxone and intranasal nalmefene in a translational model assessing the impact of synthetic opioid overdose on respiratory depression and cardiac arrest.

Introduction: Using a validated translational model that quantitatively predicts opioid-induced respiratory depression and cardiac arrest, we compared cardiac arrest events caused by synthetic opioids (fentanyl, carfentanil) following rescue by intranasal (IN) administration of the μ-opioid receptor antagonists naloxone and nalmefene.

Methods: This translational model was originally developed by Mann et al. (Clin Pharmacol Ther 2022) to evaluate the effectiveness of intramuscular (IM) naloxone.

Fluorometric Estimation of Glutathione in Cultured Microglial Cell Lysate.

Glutathione is one of the major antioxidant defense components present in cells. It is predominantly present as reduced glutathione (GSH) and converted into oxidized glutathione (GSSG) while reducing the free radicals like hydroxyl ions (OH-). For the measurement of GSH and GSSG, o-phthalaldehyde (OPT) has been used as a fluorescent reagent.

Modal affinities of endplate acetylcholine receptors caused by loop C mutations.

The time course of the endplate current is determined by the rate and equilibrium constants for acetylcholine receptor (AChR) activation. We measured these constants in single-channel currents from AChRs with mutations at the neurotransmitter-binding sites, in loop C. The main findings are: (a) Almost all perturbations of loop C generate heterogeneity in the channel open probability ("modes").

Function of the M1 π-helix in endplate receptor activation and desensitization.

Key Points: A conserved proline in M1 causes a kink between α and π helical segments. The kink is under greater tension in the resting versus active conformation. The kink and the agonist do not interact directly.

Catch-and-hold activation of muscle acetylcholine receptors having transmitter binding site mutations.

Agonists turn on receptors because their target sites have a higher affinity in the active versus resting conformation of the protein. We used single-channel electrophysiology to measure the lower-affinity (LA) and higher-affinity (HA) equilibrium dissociation constants for acetylcholine in adult-type muscle mouse nicotinic receptors (AChRs) having mutations of agonist binding site amino acids. For a series of agonists and for all mutations of αY93, αG147, αW149, αY190, αY198, εW55, and δW57, the change in LA binding energy was approximately half that in HA binding energy.

Functional anatomy of an allosteric protein.

Synaptic receptors are allosteric proteins that switch on and off to regulate cell signalling. Here, we use single-channel electrophysiology to measure and map energy changes in the gating conformational change of a nicotinic acetylcholine receptor. Two separated regions in the α-subunits--the transmitter-binding sites and αM2-αM3 linkers in the membrane domain--have the highest ϕ-values (change conformation the earliest), followed by the extracellular domain, most of the membrane domain and the gate.

Function of interfacial prolines at the transmitter-binding sites of the neuromuscular acetylcholine receptor.

The neuromuscular acetylcholine (ACh) receptor has two conserved prolines in loop D of the complementary subunit at each of its two transmitter-binding sites (α-ε and α-δ). We used single-channel electrophysiology to estimate the energy changes caused by mutations of these prolines with regard to unliganded gating (ΔG0) and the affinity change for ACh that increases the open channel probability (ΔGB). The effects of mutations of ProD2 (εPro-121/δPro-123) were greater than those of its neighbor (εPro-120/δPro-122) and were greater at α-ε versus α-δ.

Loop C and the mechanism of acetylcholine receptor-channel gating.

Agonist molecules at the two neuromuscular acetylcholine (ACh) receptor (AChR) transmitter-binding sites increase the probability of channel opening. In one hypothesis for AChR activation ("priming"), the capping of loop C at each binding site transfers energy independently to the distant gate over a discrete structural pathway. We used single-channel analyses to examine the experimental support for this proposal with regard to brief unliganded openings, the effects of loop-C modifications, the effects of mutations to residues either on or off the putative pathway, and state models for describing currents at low [ACh].

Action of nicotine and analogs on acetylcholine receptors having mutations of transmitter-binding site residue αG153.

A primary target for nicotine is the acetylcholine receptor channel (AChR). Some of the ability of nicotine to activate differentially AChR subtypes has been traced to a transmitter-binding site amino acid that is glycine in lower affinity and lysine in higher affinity AChRs. We studied the effects of mutations of this residue (αG153) in neuromuscular AChRs activated by nicotine and eight other agonists including nornicotine and anabasine.

Sources of energy for gating by neurotransmitters in acetylcholine receptor channels.

Nicotinic acetylcholine receptors (AChRs) mediate signaling in the central and peripheral nervous systems. The AChR gating conformational change is powered by a low- to high-affinity change for neurotransmitters at two transmitter binding sites. We estimated (from single-channel currents) the components of energy for gating arising from binding site aromatic residues in the α-subunit.

The intrinsic energy of the gating isomerization of a neuromuscular acetylcholine receptor channel.

Nicotinic acetylcholine receptor (AChR) channels at neuromuscular synapses rarely open in the absence of agonists, but many different mutations increase the unliganded gating equilibrium constant (E0) to generate AChRs that are active constitutively. We measured E0 for two different sets of mutant combinations and by extrapolation estimated E0 for wild-type AChRs. The estimates were 7.

Design and control of acetylcholine receptor conformational change.

Allosteric proteins use energy derived from ligand binding to promote a global change in conformation. The "gating" equilibrium constant of acetylcholine receptor-channels (AChRs) is influenced by ligands, mutations, and membrane voltage. We engineered AChRs to have specific values of this constant by combining these perturbations, and then calculated the corresponding values for a reference condition.

Glycine hinges with opposing actions at the acetylcholine receptor-channel transmitter binding site.

The extent to which agonists activate synaptic receptor-channels depends on both the intrinsic tendency of the unliganded receptor to open and the amount of agonist binding energy realized in the channel-opening process. We examined mutations of the nicotinic acetylcholine receptor transmitter binding site (α subunit loop B) with regard to both of these parameters. αGly147 is an "activation" hinge where backbone flexibility maintains high values for intrinsic gating, the affinity of the resting conformation for agonists and net ligand binding energy.

Energetics of gating at the apo-acetylcholine receptor transmitter binding site.

Acetylcholine receptor channels switch between conformations that have a low versus high affinity for the transmitter and conductance for ions (R<-->R*; gating). The forward isomerization, which begins at the transmitter binding sites and propagates approximately 50 A to the narrow region of the pore, occurs by approximately the same sequence of molecular events with or without agonists present at the binding sites. To pinpoint the forces that govern the R versus R* agonist affinity ratio, we measured single-channel activation parameters for apo-receptors having combinations of mutations of 10 transmitter binding site residues in the alpha (Y93, G147, W149, G153, Y190, C192, and Y198), epsilon (W55 and P121), or delta (W57) subunit.

Energy and structure of the M2 helix in acetylcholine receptor-channel gating.

We studied single-channel currents from neuromuscular acetylcholine receptor-channels with mutations in the pore-lining, M2 helix of the epsilon-subunit. Three parameters were quantified: 1), the diliganded gating equilibrium constant (E(2)), which reflects the energy difference between C(losed) and O(pen) conformations; 2), the correlation between the opening rate constant and E(2) on a log-log scale (Phi), which illuminates the energy character of the residue (C- versus O-like) within the C<-->O isomerization process; and 3), the open-channel current amplitude (i(0)), which reports whether a mutation alters the energetics of ion permeation. The largest E(2) changes were observed in the cytoplasmic half of epsilonM2 (5', 9', 12', 13', and 16'), with smaller changes apparent for residues > or =17'.

Unliganded gating of acetylcholine receptor channels.

We estimated the unliganded opening and closing rate constants of neuromuscular acetylcholine receptor-channels (AChRs) having mutations that increased the gating equilibrium constant. For some mutant combinations, spontaneous openings occurred in clusters. For 25 different constructs, the unliganded gating equilibrium constant (E(0)) was correlated with the product of the predicted fold-increase in the diliganded gating equilibrium constant caused by each mutation alone.

Gating at the mouth of the acetylcholine receptor channel: energetic consequences of mutations in the alphaM2-cap.

Gating of nicotinic acetylcholine receptors from a C(losed) to an O(pen) conformation is the initial event in the postsynaptic signaling cascade at the vertebrate nerve-muscle junction. Studies of receptor structure and function show that many residues in this large, five-subunit membrane protein contribute to the energy difference between C and O. Of special interest are amino acids located at the two transmitter binding sites and in the narrow region of the channel, where C<-->O gating motions generate a low<-->high change in the affinity for agonists and in the ionic conductance, respectively.

Acetylcholine receptor gating: movement in the alpha-subunit extracellular domain.

Acetylcholine receptor channel gating is a brownian conformational cascade in which nanometer-sized domains ("Phi blocks") move in staggering sequence to link an affinity change at the transmitter binding sites with a conductance change in the pore. In the alpha-subunit, the first Phi-block to move during channel opening is comprised of residues near the transmitter binding site and the second is comprised of residues near the base of the extracellular domain. We used the rate constants estimated from single-channel currents to infer the gating dynamics of Y127 and K145, in the inner and outer sheet of the beta-core of the alpha-subunit.

Acetylcholine receptor gating at extracellular transmembrane domain interface: the "pre-M1" linker.

Charged residues in the beta10-M1 linker region ("pre-M1") are important in the expression and function of neuromuscular acetylcholine receptors (AChRs). The perturbation of a salt bridge between pre-M1 residue R209 and loop 2 residue E45 has been proposed as being a principle event in the AChR gating conformational "wave." We examined the effects of mutations to all five residues in pre-M1 (positions M207-P211) plus E45 in loop 2 in the mouse alpha(1)-subunit.

Acetylcholine receptor gating at extracellular transmembrane domain interface: the cys-loop and M2-M3 linker.

Acetylcholine receptor channel gating is a propagated conformational cascade that links changes in structure and function at the transmitter binding sites in the extracellular domain (ECD) with those at a "gate" in the transmembrane domain (TMD). We used Phi-value analysis to probe the relative timing of the gating motions of alpha-subunit residues located near the ECD-TMD interface. Mutation of four of the seven amino acids in the M2-M3 linker (which connects the pore-lining M2 helix with the M3 helix), including three of the four residues in the core of the linker, changed the diliganded gating equilibrium constant (K(eq)) by up to 10,000-fold (P272 > I274 > A270 > G275).

A stepwise mechanism for acetylcholine receptor channel gating.

Muscle contraction is triggered by the opening of acetylcholine receptors at the vertebrate nerve-muscle synapse. The M2 helix of this allosteric membrane protein lines the channel, and contains a 'gate' that regulates the flow of ions through the pore. We used single-molecule kinetic analysis to probe the transition state of the gating conformational change and estimate the relative timing of M2 motions in the alpha-subunit of the murine acetylcholine receptor.

Synthesis, beta-adrenergic blocking activity and beta-receptor binding affinities of 1-substituted-3-(2-isopropyl-5-methyl-phenoxy)-propan-2-ol oxalates.

The compounds 1-isopropylamino-3-(2-isopropyl-5-methyl-phenoxy)-propan-2-ol oxalate (5) and 1-tert-butylamino-3-(2-isopropyl-5-methyl-phenoxy)-propan-2-ol oxalate (6) were synthesized from thymol (1), a naturally occurring agent in Thymus vulgaris L. Pharmacological evaluation of 5 and 6 were carried out using mouse ECG and isolated rat uterus models. Pretreatment of 5 (100 microg/kg, i.