In Vivo Photoadduction of Anesthetic Ligands in Mouse Brain Markedly Extends Sedation and Hypnosis.

Photoaffinity ligands are best known as tools used to identify the specific binding sites of drugs to their molecular targets. However, photoaffinity ligands have the potential to further define critical neuroanatomic targets of drug action. In the brains of WT male mice, we demonstrate the feasibility of using photoaffinity ligands in vivo to prolong anesthesia via targeted yet spatially restricted photoadduction of azi-m-propofol (aziPm), a photoreactive analog of the general anesthetic propofol. Systemic administration of aziPm with bilateral near-ultraviolet photoadduction in the rostral pons, at the border of the parabrachial nucleus and locus coeruleus, produced a 20-fold increase in the duration of sedative and hypnotic effects compared with control mice without UV illumination. Photoadduction that missed the parabrachial-coerulean complex also failed to extend the sedative or hypnotic actions of aziPm and was indistinguishable from nonadducted controls. Paralleling the prolonged behavioral and EEG consequences of on target in vivo photoadduction, we conducted electrophysiologic recordings in rostral pontine brain slices. Using neurons within the locus coeruleus to further highlight the cellular consequences of irreversible aziPm binding, we demonstrate transient slowing of spontaneous action potentials with a brief bath application of aziPm that becomes irreversible on photoadduction. Together, these findings suggest that photochemistry-based strategies are a viable new approach for probing CNS physiology and pathophysiology.SIGNIFICANCE STATEMENT Photoaffinity ligands are drugs capable of light-induced irreversible binding, which have unexploited potential to identify the neuroanatomic sites of drug action. We systemically administer a centrally acting anesthetic photoaffinity ligand in mice, conduct localized photoillumination within the brain to covalently adduct the drug at its in vivo sites of action, and successfully enrich irreversible drug binding within a restricted 250 µm radius. When photoadduction encompassed the pontine parabrachial-coerulean complex, anesthetic sedation and hypnosis was prolonged 20-fold, thus illustrating the power of in vivo photochemistry to help unravel neuronal mechanisms of drug action.

Propofol directly binds to and inhibits TLR7.

Sedatives/anesthetics are important medical tools to facilitate medical care and increase patients' comfort. Increasingly, there is recognition that sedatives/anesthetics can modulate immune functions. Toll-like receptors (TLRs) are major pattern recognition receptors involved in the recognition of microbial components. TLR7 recognizes single-strand RNA virus such as influenza and SARS-CoV2 viruses and initiates interferon (IFN) responses. IFN production triggered by TLR7 stimulation is a critical anti-viral response. For example, patients with TLR7 variants including loss-of- function variants were associated with severe COVID-19. Taken together, it is important to determine if sedatives/anesthetics mitigate TLR7 function. We have previously showed that TLR7-mediated activation was not affected by volatile anesthetics. However, we found that propofol attenuated TLR7 activation among intravenous sedatives in the reporter assay. TLR7 agonist R837 stimulation increased TNF-α, IL-1ß, IL-6, IL-10, and IFN-ß mRNA levels in bone marrow-derived dendritic cells, while these levels were attenuated by propofol. Our murine lung slice experiments showed that propofol attenuated IFN production. R837 increased IFN-ß expression in the lungs, and propofol attenuated IFN-ß expression in an in vivo model of R837 intranasal instillation. We also found that propofol directly bound to and hindered its association of TLR7 with MyD88. Our analysis using fropofol, propofol derivative showed that the hydroxyl group in propofol was important for propofol-TLR7 interaction.

Preclinical and translational models for delirium: Recommendations for future research from the NIDUS delirium network.

Delirium is a common, morbid, and costly syndrome that is closely linked to Alzheimer's disease (AD) and AD-related dementias (ADRD) as a risk factor and outcome. Human studies of delirium have advanced our knowledge of delirium incidence and prevalence, risk factors, biomarkers, outcomes, prevention, and management. However, understanding of delirium neurobiology remains limited. Preclinical and translational models for delirium, while challenging to develop, could advance our knowledge of delirium neurobiology and inform the development of new prevention and treatment approaches. We discuss the use of preclinical and translational animal models in delirium, focusing on (1) a review of current animal models, (2) challenges and strategies for replicating elements of human delirium in animals, and (3) the utility of biofluid, neurophysiology, and neuroimaging translational markers in animals. We conclude with recommendations for the development and validation of preclinical and translational models for delirium, with the goal of advancing awareness in this important field.

The effect of anesthetics on toll like receptor 9.

Toll like receptors (TLRs) are critical receptors to respond to danger signals, and their functions are relevant in the perioperative period. We previously reported that volatile anesthetics directly bound to TLR2 and TLR4 and attenuated their functions. Given that TLR9 can respond to mitochondrial DNA, a danger signal that is released upon tissue injury, we examined the role of anesthetics on TLR9 function. Our reporter assay showed that volatile anesthetics isoflurane and sevoflurane increased the activation of TLR9, while propofol attenuated it. TLR9 activation occurs via its dimerization. The dimerization is facilitated by unmethylated cytosine-phosphate-guanine (CpG) DNA as well as DNA containing cytosine at the second position from 5'-end (5'-xCx DNA). Our structural analysis using photoactivable anesthetics and rigid docking simulation showed that isoflurane and sevoflurane bound to both TLR9 dimer interface and 5'-xCx DNA binding site. Propofol bound to the TLR9 antagonist binding site. This is the first illustration that anesthetics can affect the binding of nucleic acids to their receptor. This study sets the foundation for the effect of anesthetics on TLR9 and will pave the way for future studies to determine the significance of such interactions in the clinical setting.

Improving perioperative brain health: an expert consensus review of key actions for the perioperative care team.

Delirium and postoperative neurocognitive disorder are the commonest perioperative complications in patients more than 65 yr of age. However, data suggest that we often fail to screen patients for preoperative cognitive impairment, to warn patients and families of risk, and to take preventive measures to reduce the incidence of perioperative neurocognitive disorders. As part of the American Society of Anesthesiologists (ASA) Perioperative Brain Health Initiative, an international group of experts was invited to review published best practice statements and guidelines. The expert group aimed to achieve consensus on a small number of practical recommendations that could be implemented by anaesthetists and their partners to reduce the incidence of perioperative neurocognitive disorders. Six statements were selected based not only on the strength of the evidence, but also on the potential for impact and the feasibility of widespread implementation. The actions focus on education, cognitive and delirium screening, non-pharmacologic interventions, pain control, and avoidance of antipsychotics. Strategies for effective implementation are discussed. Anaesthetists should be key members of multidisciplinary perioperative care teams to implement these recommendations.

Anesthetic Effects on the Progression of Parkinson Disease in the Rat DJ-1 Model.

BACKGROUND: Parkinson disease is a chronic and progressive movement disorder characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). The causes of Parkinson disease are not clear but may involve genetic susceptibilities and environmental factors. As in other neurodegenerative disorders, individuals predisposed to Parkinson disease may have an accelerated onset of symptoms following perioperative stress such as anesthesia, surgery, pain, and inflammation. We hypothesized that anesthesia alone accelerates the onset of Parkinson disease-like pathology and symptoms. METHODS: A presymptomatic Parkinson rat model (the protein, DJ-1, encoded by the Park7 gene [DJ-1], PARK7 knockout) was exposed to a surgical plane of isoflurane or 20% oxygen balanced with nitrogen for 2 hours on 3 occasions between 6 and 7 months of age. Acute and long-term motor and neuropathological effects were examined from 7 to 12 months of age in male DJ-1 rats, using the ladder rung, rotarod, and novel object recognition assays, as well as the immunohistochemical localization of tyrosine hydroxylase in dopaminergic neurons in the substantia nigra and ionized calcium-binding adaptor protein-1 (Iba-1) microglial activation in the substantia nigra and hippocampus. RESULTS: In the acute group, after the third anesthetic exposure at 7 months of age, the isoflurane group had a significant reduction in the density of dopaminergic neurons in the SNpc compared to controls. However, this reduction was not associated with increased microglial activation in the hippocampus or substantia nigra. With the ladder rung motor skills test, there was no effect of anesthetic exposure on the total number of foot faults or the ladder rung pattern in the acute group. The rotarod test also detected no differences before and after the third exposure in controls. For the long-term group, immunohistochemical analyses detected no differences in the density of dopaminergic neurons or microglial cells compared to unexposed DJ-1 rats from 8 to 12 months of age. The ladder rung test in the long-term group showed no differences in the total number of foot faults with time and exposure or between ladder rung patterns. The rotarod test detected no significant effect of exposure with time or between groups at any time point. The novel object recognition task in the long-term group revealed no differences in short- or long-term memory or in the number of rearings as a function of exposure. CONCLUSIONS: Multiple isoflurane exposures in this rat model of Parkinson disease transiently enhanced dopaminergic neurodegeneration in the SNpc that resolved over time and had no effects on progression in this Parkinson disease-like phenotype.

The role of propofol hydroxyl group in 5-lipoxygenase recognition.

Propofol is a clinically important intravenous anesthetic. We previously reported that it directly inhibited 5-lipoxygenase (5-LOX), a key enzyme for leukotriene biosynthesis. Because the hydroxyl group in propofol (propofol 1-hydroxyl) is critical for its anesthetic effect, we examined if its presence would be inevitable for 5-lipoxygenase recognition. Fropofol is developed by substituting the hydroxy group in propofol with fluorine. We found that propofol 1-hydroxyl was important for 5-lipoxygenase recognition, but it was not absolutely necessary. Azi-fropofol bound to 5-LOX at one of the two propofol binding sites of 5-LOX (pocket around Phe-187), suggesting that propofol 1-hydroxyl is important for 5-LOX inhibition at the other propofol binding site (pocket around Val-431). Interestingly, 5-hydroperoxyeicosatetraenoic acid (5-HpETE) production was significantly increased by stimulation with calcium ionophore A23187 in HEK293 cells expressing 5-LOX, suggesting that the fropofol binding site is important for the conversion from 5-HpETE to leukotriene A4. We also indicated that propofol 1-hydroxyl might have contributed to interaction with wider targets among our body.

Volatile anesthetics isoflurane and sevoflurane directly target and attenuate Toll-like receptor 4 system.

General anesthesia has been the requisite component of surgical procedures for over 150 yr. Although immunomodulatory effects of volatile anesthetics have been growingly appreciated, the molecular mechanism has not been understood. In septic mice, the commonly used volatile anesthetic isoflurane attenuated the production of 5-lipoxygenase products and IL-10 and reduced CD11b and intercellular adhesion molecule-1 expression on neutrophils, suggesting the attenuation of TLR4 signaling. We confirmed the attenuation of TLR4 signaling in vitro and their direct binding to TLR4-myeloid differentiation-2 (MD-2) complex by photolabeling experiments. The binding sites of volatile anesthetics isoflurane and sevoflurane were located near critical residues for TLR4-MD-2 complex formation and TLR4-MD-2-LPS dimerization. Additionally, TLR4 activation was not attenuated by intravenous anesthetics, except for a high concentration of propofol. Considering the important role of TLR4 system in the perioperative settings, these findings suggest the possibility that anesthetic choice may modulate the outcome in patients or surgical cases in which TLR4 activation is expected.-Okuno, T., Koutsogiannaki, S., Hou, L., Bu, W., Ohto, U., Eckenhoff, R. G., Yokomizo, T., Yuki, K. Volatile anesthetics isoflurane and sevoflurane directly target and attenuate Toll-like receptor 4 system.

Perioperative Neurocognitive Disorder: State of the Preclinical Science.

The purpose of this article is to provide a succinct summary of the different experimental approaches that have been used in preclinical postoperative cognitive dysfunction research, and an overview of the knowledge that has accrued. This is not intended to be a comprehensive review, but rather is intended to highlight how the many different approaches have contributed to our understanding of postoperative cognitive dysfunction, and to identify knowledge gaps to be filled by further research. The authors have organized this report by the level of experimental and systems complexity, starting with molecular and cellular approaches, then moving to intact invertebrates and vertebrate animal models. In addition, the authors' goal is to improve the quality and consistency of postoperative cognitive dysfunction and perioperative neurocognitive disorder research by promoting optimal study design, enhanced transparency, and "best practices" in experimental design and reporting to increase the likelihood of corroborating results. Thus, the authors conclude with general guidelines for designing, conducting and reporting perioperative neurocognitive disorder rodent research.

Common general anesthetic propofol impairs kinesin processivity.

Propofol is the most widely used i.v. general anesthetic to induce and maintain anesthesia. It is now recognized that this small molecule influences ligand-gated channels, including the GABAA receptor and others. Specific propofol binding sites have been mapped using photoaffinity ligands and mutagenesis; however, their precise target interaction profiles fail to provide complete mechanistic underpinnings for the anesthetic state. These results suggest that propofol and other common anesthetics, such as etomidate and ketamine, may target additional protein networks of the CNS to contribute to the desired and undesired anesthesia end points. Some evidence for anesthetic interactions with the cytoskeleton exists, but the molecular motors have received no attention as anesthetic targets. We have recently discovered that propofol inhibits conventional kinesin-1 KIF5B and kinesin-2 KIF3AB and KIF3AC, causing a significant reduction in the distances that these processive kinesins can travel. These microtubule-based motors are highly expressed in the CNS and the major anterograde transporters of cargos, such as mitochondria, synaptic vesicle precursors, neurotransmitter receptors, cell signaling and adhesion molecules, and ciliary intraflagellar transport particles. The single-molecule results presented show that the kinesin processive stepping distance decreases 40-60% with EC50 values <100 nM propofol without an effect on velocity. The lack of a velocity effect suggests that propofol is not binding at the ATP site or allosteric sites that modulate microtubule-activated ATP turnover. Rather, we propose that a transient propofol allosteric site forms when the motor head binds to the microtubule during stepping.

Using Medicare claims in identifying Alzheimer's disease and related dementias.

INTRODUCTION: This study develops a measure of Alzheimer's disease and related dementias (ADRD) using Medicare claims. METHODS: Validation resembles the approach of the American Psychological Association, including (1) content validity, (2) construct validity, and (3) predictive validity. RESULTS: We found that four items-a Medicare claim recording ADRD 1 year ago, 2 years ago, 3 years ago, and a total stay of 6 months in a nursing home-exhibit a pattern of association consistent with a single underlying ADRD construct, and presence of any two of these four items predict a direct measure of cognitive function and also future claims for ADRD. DISCUSSION: Our four items are internally consistent with the measurement of a single quantity. The presence of any two items do a better job than a single claim when predicting both a direct measure of cognitive function and future ADRD claims.

An allosteric propofol-binding site in kinesin disrupts kinesin-mediated processive movement on microtubules.

Microtubule-based molecular motors mediate transport of intracellular cargo to subdomains in neurons. Previous evidence has suggested that the anesthetic propofol decreases the average run-length potential of the major anterograde transporters kinesin-1 and kinesin-2 without altering their velocity. This effect on kinesin has not been observed with other inhibitors, stimulating considerable interest in the underlying mechanism. Here, we used a photoactive derivative of propofol, meta-azipropofol (AziPm), to search for potential propofol-binding sites in kinesin. Single-molecule motility assays confirmed that AziPm and propofol similarly inhibit kinesin-1 and kinesin-2. We then applied AziPm in semiquantitative radiolabeling and MS microsequencing assays to identify propofol-binding sites within microtubule-kinesin complexes. The radiolabeling experiments suggested preferential AziPm binding to the ATP-bound microtubule-kinesin complex. The photolabeled residues were contained within the kinesin motor domain rather than at the motor domain-ß-tubulin interface. No residues within the P-loop of kinesin were photolabeled, indicating an inhibitory mechanism that does not directly affect ATPase activity and has an effect on run length without changing velocity. Our results also indicated that when the kinesin motor interacts with the microtubule during its processive run, a site forms in kinesin to which propofol can then bind and allosterically disrupt the kinesin-microtubule interaction, resulting in kinesin detachment and run termination. The discovery of the propofol-binding allosteric site in kinesin may improve our understanding of the strict coordination of the motor heads during the processive run. We hypothesize that propofol's potent effect on intracellular transport contributes to various components of its anesthetic action.

Identification of binding sites contributing to volatile anesthetic effects on GABA type A receptors.

Most general anesthetics enhance GABA type A (GABAA) receptor activity at clinically relevant concentrations. Sites of action of volatile anesthetics on the GABAA receptor remain unknown, whereas sites of action of many intravenous anesthetics have been identified in GABAA receptors by using photolabeling. Here, we used photoactivatable analogs of isoflurane (AziISO) and sevoflurane (AziSEVO) to locate their sites on α1ß3γ2L and α1ß3 GABAA receptors. As with isoflurane and sevoflurane, AziISO and AziSEVO enhanced the currents elicited by GABA. AziISO and AziSEVO each labeled 10 residues in α1ß3 receptors and 9 and 8 residues, respectively, in α1ß3γ2L receptors. Photolabeled residues were concentrated in transmembrane domains and located in either subunit interfaces or in the interface between the extracellular domain and the transmembrane domain. The majority of these transmembrane residues were protected from photolabeling with the addition of excess parent anesthetic, which indicated specificity. Binding sites were primarily located within α+/ß- and ß+/α- subunit interfaces, but residues in the α+/γ- interface were also identified, which provided a basis for differential receptor subtype sensitivity. Isoflurane and sevoflurane did not always share binding sites, which suggests an unexpected degree of selectivity.-Woll, K. A., Zhou, X., Bhanu, N. V., Garcia, B. A., Covarrubias, M., Miller, K. W., Eckenhoff, R. G. Identification of binding sites contributing to volatile anesthetic effects on GABA type A receptors.

Prevalence and Multivariable Factors Associated With Preoperative Cognitive Impairment in Outpatient Surgery in the United States.

Preoperative cognitive impairment increases the risk of adverse events after surgery but its prevalence in outpatient surgery has not been defined. We aimed to determine the prevalence and multivariable factors associated with cognitive impairment in individuals who present for outpatient surgery. We used data from the Health and Retirement Study, a longitudinal panel survey of older Americans. Of 1836 participants who reported having outpatient surgery, we found that 16.1% had evidence of cognitive impairment. Significant multivariable factors associated with preoperative cognitive impairment included non-Hispanic African American race, prior stroke, preoperative functional dependence, and lower socioeconomic status and education level.

Fluorine-19 NMR and computational quantification of isoflurane binding to the voltage-gated sodium channel NaChBac.

Voltage-gated sodium channels (NaV) play an important role in general anesthesia. Electrophysiology measurements suggest that volatile anesthetics such as isoflurane inhibit NaV by stabilizing the inactivated state or altering the inactivation kinetics. Recent computational studies suggested the existence of multiple isoflurane binding sites in NaV, but experimental binding data are lacking. Here we use site-directed placement of 19F probes in NMR experiments to quantify isoflurane binding to the bacterial voltage-gated sodium channel NaChBac. 19F probes were introduced individually to S129 and L150 near the S4-S5 linker, L179 and S208 at the extracellular surface, T189 in the ion selectivity filter, and all phenylalanine residues. Quantitative analyses of 19F NMR saturation transfer difference (STD) spectroscopy showed a strong interaction of isoflurane with S129, T189, and S208; relatively weakly with L150; and almost undetectable with L179 and phenylalanine residues. An orientation preference was observed for isoflurane bound to T189 and S208, but not to S129 and L150. We conclude that isoflurane inhibits NaChBac by two distinct mechanisms: (i) as a channel blocker at the base of the selectivity filter, and (ii) as a modulator to restrict the pivot motion at the S4-S5 linker and at a critical hinge that controls the gating and inactivation motion of S6.

Intravenous anesthetic propofol binds to 5-lipoxygenase and attenuates leukotriene B4 production.

Propofol is an intravenous anesthetic that produces its anesthetic effect, largely via the GABAA receptor in the CNS, and also reduces the N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced neutrophil respiratory burst. Because fMLP-stimulated neutrophils produce leukotriene (LT)B4, we examined the effect of propofol on LTB4 production in vivo and in vitro Cecal ligation and puncture surgery was performed in mice, with or without exposure to propofol. Propofol attenuated the production of 5-lipoxygenase (5-LOX)-related arachidonic acid (AA) derivatives in the peritoneal fluid. Also, in the in vitro experiments on fMLP-stimulated neutrophils and 5-LOX-transfected human embryonic kidney cells, propofol attenuated the production of 5-LOX-related AA derivatives. Based on these results, we hypothesized that propofol would directly affect 5-LOX function. Using meta-azi-propofol (AziPm), we photolabeled stable 5-LOX protein, which had been used to solve the X-ray crystallographic structure of 5-LOX, and examined the binding site(s) of propofol on 5-LOX. Two propofol binding pockets were identified near the active site of 5-LOX. Alanine scanning mutagenesis was performed for the residues of 5-LOX in the vicinity of propofol, and we evaluated the functional role of these pockets in LTB4 production. We demonstrated that these pockets were functionally important for 5-LOX activity. These two pockets can be used to explore a novel 5-LOX inhibitor in the future.-Okuno, T., Koutsogiannaki, S., Ohba, M., Chamberlain, M., Bu, W., Lin, F.-Y., Eckenhoff, R. G., Yokomizo T., Yuki, K. Intravenous anesthetic propofol binds to 5-lipoxygenase and attenuates leukotriene B4 production.

Best Practices for Postoperative Brain Health: Recommendations From the Fifth International Perioperative Neurotoxicity Working Group.

As part of the American Society of Anesthesiology Brain Health Initiative goal of improving perioperative brain health for older patients, over 30 experts met at the fifth International Perioperative Neurotoxicity Workshop in San Francisco, CA, in May 2016, to discuss best practices for optimizing perioperative brain health in older adults (ie, >65 years of age). The objective of this workshop was to discuss and develop consensus solutions to improve patient management and outcomes and to discuss what older adults should be told (and by whom) about postoperative brain health risks. Thus, the workshop was provider and patient oriented as well as solution focused rather than etiology focused. For those areas in which we determined that there were limited evidence-based recommendations, we identified knowledge gaps and the types of scientific knowledge and investigations needed to direct future best practice. Because concerns about perioperative neurocognitive injury in pediatric patients are already being addressed by the SmartTots initiative, our workshop discussion (and thus this article) focuses specifically on perioperative cognition in older adults. The 2 main perioperative cognitive disorders that have been studied to date are postoperative delirium and cognitive dysfunction. Postoperative delirium is a syndrome of fluctuating changes in attention and level of consciousness that occurs in 20%-40% of patients >60 years of age after major surgery and inpatient hospitalization. Many older surgical patients also develop postoperative cognitive deficits that typically last for weeks to months, thus referred to as postoperative cognitive dysfunction. Because of the heterogeneity of different tools and thresholds used to assess and define these disorders at varying points in time after anesthesia and surgery, a recent article has proposed a new recommended nomenclature for these perioperative neurocognitive disorders. Our discussion about this topic was organized around 4 key issues: preprocedure consent, preoperative cognitive assessment, intraoperative management, and postoperative follow-up. These 4 issues also form the structure of this document. Multiple viewpoints were presented by participants and discussed at this in-person meeting, and the overall group consensus from these discussions was then drafted by a smaller writing group (the 6 primary authors of this article) into this manuscript. Of course, further studies have appeared since the workshop, which the writing group has incorporated where appropriate. All participants from this in-person meeting then had the opportunity to review, edit, and approve this final manuscript; 1 participant did not approve the final manuscript and asked for his/her name to be removed.

Sites Contributing to TRPA1 Activation by the Anesthetic Propofol Identified by Photoaffinity Labeling.

In addition to inducing anesthesia, propofol activates a key component of the pain pathway, the transient receptor potential ankyrin 1 ion channel (TRPA1). Recent mutagenesis studies suggested a potential activation site within the transmembrane domain, near the A-967079 cavity. However, mutagenesis cannot distinguish between protein-based and ligand-based mechanisms, nor can this site explain the complex modulation by propofol. Thus more direct approaches are required to reveal potentially druggable binding sites. Here we apply photoaffinity labeling using a propofol derivative, meta-azipropofol, for direct identification of binding sites in mouse TRPA1. We confirm that meta-azipropofol activates TRPA1 like the parent anesthetic, and identify two photolabeled residues (V954 and E969) in the S6 helix. In combination with docking to closed and open state models of TRPA1, photoaffinity labeling suggested that the A-967079 cavity is a positive modulatory site for propofol. Further, the photoaffinity labeling of E969 indicated pore block as a likely mechanism for propofol inhibition at high concentrations. The direct identification of drug-binding sites clarifies the molecular mechanisms of important TRPA1 agonists, and will facilitate drug design efforts to modulate TRPA1.

A Novel Bifunctional Alkylphenol Anesthetic Allows Characterization of γ-Aminobutyric Acid, Type A (GABAA), Receptor Subunit Binding Selectivity in Synaptosomes.

Propofol, an intravenous anesthetic, is a positive modulator of the GABAA receptor, but the mechanistic details, including the relevant binding sites and alternative targets, remain disputed. Here we undertook an in-depth study of alkylphenol-based anesthetic binding to synaptic membranes. We designed, synthesized, and characterized a chemically active alkylphenol anesthetic (2-((prop-2-yn-1-yloxy)methyl)-5-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenol, AziPm-click (1)), for affinity-based protein profiling (ABPP) of propofol-binding proteins in their native state within mouse synaptosomes. The ABPP strategy captured ∼4% of the synaptosomal proteome, including the unbiased capture of five α or ß GABAA receptor subunits. Lack of γ2 subunit capture was not due to low abundance. Consistent with this, independent molecular dynamics simulations with alchemical free energy perturbation calculations predicted selective propofol binding to interfacial sites, with higher affinities for α/ß than γ-containing interfaces. The simulations indicated hydrogen bonding is a key component leading to propofol-selective binding within GABAA receptor subunit interfaces, with stable hydrogen bonds observed between propofol and α/ß cavity residues but not γ cavity residues. We confirmed this by introducing a hydrogen bond-null propofol analogue as a protecting ligand for targeted-ABPP and observed a lack of GABAA receptor subunit protection. This investigation demonstrates striking interfacial GABAA receptor subunit selectivity in the native milieu, suggesting that asymmetric occupancy of heteropentameric ion channels by alkylphenol-based anesthetics is sufficient to induce modulation of activity.

Molecular mechanism of anesthetic-induced depression of myocardial contraction.

Isoflurane and propofol are known to depress cardiac contraction, but the molecular mechanisms involved are not known. In this study, we determined whether decreasing myofilament Ca(2+) responsiveness underlies anesthesia-induced depression of contraction and uncovered the molecular targets of isoflurane and propofol. Force and intracellular Ca(2+) ([Ca(2+)]i) were measured in rat trabeculae superfused with Krebs-Henseleit solution, with or without propofol or isoflurane. Photoaffinity labeling of myofilament proteins with meta-Azi-propofol (AziPm) and Azi-isoflurane (Azi-iso) and molecular docking were also used. Both propofol and isoflurane dose dependently depressed force from low doses (propofol, 27 ± 6 µM; isoflurane, 1.0 ± 0.1%) to moderate doses (propofol, 87 ± 4 µM; isoflurane, 3.0 ± 0.25%), without significant alteration [Ca(2+)]i During steady-state activations in both intact and skinned preparations, propofol and isoflurane depressed maximum Ca(2+)-activated force and increased the [Ca(2+)]i required for 50% of activation. Myofibrils photolabeled with AziPm and Azi-iso identified myosin, actin, and myosin light chain as targets of the anesthetics. Several adducted residues in those proteins were located in conformationally sensitive regions that underlie contractile function. Thus, propofol and isoflurane decrease force development by directly depressing myofilament Ca(2+) responsiveness and have binding sites in key regions for contraction in both actin and myosin.-Meng, T., Bu, W., Ren, X., Chen, X., Yu, J., Eckenhoff, R. G., Gao, W. D. Molecular mechanism of anesthetic-induced depression of myocardial contraction.