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.

Balloon Kyphoplasty vs Vertebroplasty: A Systematic Review of Height Restoration in Osteoporotic Vertebral Compression Fractures.

Purpose of Review: This systematic review comprehensively compared balloon kyphoplasty and vertebroplasty with respect to height restoration and pain relief. Recent Findings: PRISMA guidelines were utilized to compare balloon kyphoplasty and vertebroplasty, focusing on the primary outcome of height restoration and the secondary outcomes of pain relief and functionality. A total of 33 randomized controlled trials were included; 20 reviewed balloon kyphoplasty, 7 reviewed vertebroplasty, and 6 compared vertebroplasty to balloon kyphoplasty. Both treatments restored some vertebral body height and showed benefits in pain reduction and improved patient-reported functionality. Summary: Balloon kyphoplasty and vertebroplasty are effective treatments for vertebral compression fractures and this review suggests that balloon kyphoplasty may be favored for vertebral height restoration. Further studies are needed to conclude whether balloon kyphoplasty or vertebroplasty is superior for alleviating pain.

National Institutes of Health Career Development (K) Awards to Young Surgeons: An Academic Milestone or One-hit Wonder?

OBJECTIVE: To assess contemporary trends in the National Institutes of Health (NIH) Career Development (K) Awards within the Departments of Surgery and its impact on the likelihood of achieving independent R01 grants. BACKGROUND: The NIH provides K-type Career Development Awards to nurture young clinicians toward a productive academic career, thereby maintaining a pipeline of physician-scientists. However, the impact of K awards on career trajectory of surgeons remains unclear. METHODS: The NIH grant data was queried for all new K08/K23 grants awarded to Departments of Surgery (1999-2019). Principal Investigators' data and grant-related information was obtained. RESULTS: The NIH awarded 298 K08/23 surgical grants ($41,893,170) over the last 2 decades. Median budget increased from $116,370 to $167,508 (P<0.001). Of grantees, 83.2% were MDs, 15.1% MD/PhD, and 1.7% PhDs, with 25.2% being women. Principal Investigators' were mostly practicing surgeons (91.1%) with fellowship training (82.4%) and young in their careers {4 [interquartile ranges (IQR) 4] years of experience}. Vascular surgery (15.9%), Complex General Surgical Oncology (15.1%), and Trauma/Critical Care (14.6%) were the most frequent specialties. Awards were associated with 3,336 publications [median 8/project (IQR 13)]. The majority of K grantees (77.2%) currently hold an academic faculty position. Only 32.2% of awardees received independent R01 grant funding, at a median of 5.5 years (IQR 5) after their K awards. Sex (P = 0.71), previous fellowship training (P = 0.63), type of surgical specialty (P = 0.72), or MD/PhD degree (P = 0.75) were not associated with increased likelihood of achieving a subsequent R01 award. CONCLUSION: Although the majority of K awardees maintain an academic career, only a limited number of grantees progress to obtain NIH R01 funding. Increased mentorship, financial support, and infrastructure are needed to facilitate career development awardees opportunities to enhance their ability to achieve independent funding.

Regulation and drug modulation of a voltage-gated sodium channel: Pivotal role of the S4-S5 linker in activation and slow inactivation.

Voltage-gated sodium (NaV) channels control excitable cell functions. While structural investigations have revealed conformation details of different functional states, the mechanisms of both activation and slow inactivation remain unclear. Here, we identify residue T140 in the S4-S5 linker of the bacterial voltage-gated sodium channel NaChBac as critical for channel activation and drug effects on inactivation. Mutations at T140 either attenuate activation or render the channel nonfunctional. Propofol, a clinical anesthetic known to inhibit NaChBac by promoting slow inactivation, binds to a pocket between the S4-S5 linker and S6 helix in a conformation-dependent manner. Using 19F-NMR to quantify site-specific binding by saturation transfer differences (STDs), we found strong STDs in inactivated, but not activated, NaChBac. Molecular dynamics simulations show a highly dynamic pocket in the activated conformation, limiting STD buildup. In contrast, drug binding to this pocket promotes and stabilizes the inactivated states. Our results provide direct experimental evidence showing distinctly different associations between the S4-S5 linker and S6 helix in activated and inactivated states. Specifically, an exchange occurs between interaction partners T140 and N234 of the same subunit in activation, and T140 and N225 of the domain-swapped subunit in slow inactivation. The drug action on slow inactivation of prokaryotic NaV channels seems to have a mechanism similar to the recently proposed "door-wedge" action of the isoleucine-phenylalanine-methionine (IFM) motif on the fast inactivation of eukaryotic NaV channels. Elucidating this gating mechanism points to a possible direction for conformation-dependent drug development.

National Institutes of Health Research Funding to Academic Surgical Oncologists: Who Are We and Where Do We Stand?

BACKGROUND: The National Institutes of Health (NIH) is the primary public funding source for surgical research in the United States. Surgical oncology is a highly academic career, but NIH funding for surgical oncologists (SOs) is not well characterized. METHODS: The NIH RePORTER (Research Portfolio Online Reporting Tools Expenditures and Results) was queried to identify R01-and-equivalents grants awarded to departments of surgery (DoS) between 2008 and 2018. Surgical oncologists were considered to be those who completed a Society of Surgical Oncology (SSO)-accredited fellowship (breast or complex surgical oncology). RESULTS: Of 1101 projects, 510 (46.3%) were led by practicing surgeons. Among these, general surgeons accounted for most grants (31%), followed by SOs (20.8%). Women represented 211 (24.1%) of the grantees. However, SOs had a higher proportion of female investigators than other surgeons (30.0% vs. 16.1%; P = 0.001). The SO grantees had fewer years of experience (YoE) (12 years; interquartile range [IQR], 8.75 vs. 13 years; IQR, 13 years; P = 0.003), lower senior status (≥ 24 YoE), fewer investigators (4.0% vs. 18.9%; P < 0.001), and fewer PhD holders (30.8% vs. 65.5%; P < 0.001) than the overall cohort. Projects led by SOs accounted for 1121 publications (14.1%), with a higher proportion of high-impact articles (26.3% vs. 9.7%; P < 0.001), and were more likely to hold a registered patent (odds ratio [OR], 3.30; 95% confidence interval [CI], 1.24-8.74; P = 0.016). CONCLUSION: Among surgical subspecialties, SSO-accredited surgeons accounted for the largest share of the NIH grants. The SO grantees were younger in their career and had higher-impact scholarly productivity. A smaller proportion of female SOs received NIH grants than males, but this gender disparity was less significant among SOs than among other surgical specialties. Fellowship programs should continue to stimulate groundbreaking research by integrating grant-writing training and mentorship.

Synthesis and Characterization of a Diazirine-Based Photolabel of the Nonanesthetic Fropofol.

The mechanisms of general anesthetics have been debated in the literature for many years and continue to be of great interest. As anesthetic molecules are notoriously difficult to study due to their low binding affinities and multitude of binding partners, it is advantageous to have additional tools to study these interactions. Fropofol is a hydroxyl to fluorine-substituted propofol analogue that is able to antagonize the actions of propofol. Understanding fropofol's ability to antagonize propofol would facilitate further characterization of the binding interactions of propofol that may contribute to its anesthetic actions. However, the study of fropofol's molecular interactions has many of the same difficulties as its parent compound. Here, we present the synthesis and characterization of ortho-azi-fropofol (AziFo) as a suitable photoaffinity label (PAL) of fropofol that can be used to covalently label proteins of interest to characterize fropofol's binding interactions and their contribution to general anesthetic antagonism.

Patterns of National Institutes of Health Grant Funding to Surgical Research and Scholarly Productivity in the United States.

OBJECTIVE: The aim of this study was to assess the contemporary trends in National Institutes of Health (NIH) grants awarded to surgical investigators, including potential disparities. BACKGROUND: The NIH remains the primary public funding source for surgical research in the United States; however, the patterns for grants and grantees are poorly understood. METHODS: NIH RePORTER was queried for new grants (R01, -03, -21) awarded to Departments of Surgery (DoS). Principal investigators' (PIs) data were extracted from publicly available information from their institutions' websites and/or professional social media accounts. RESULTS: The NIH awarded 1101 new grants (total: $389,006,782; median: $313,030) between 2008 and 2018. Funding to DoS has doubled in the last 10 years ($22,983,500-2008 to $49,446,076-2018). Midwest/Southeast institutions and surgical oncologists accounted for majority of the grants (31.9% and 24.5%, respectively). Only 24.7% of the projects were led by female PIs, who were predominantly nonphysician PhD scientists (52% vs 37.7% PhD-only male PIs; P = 0.002). During this time, there was a significant increase from 12.4% to 31.7% in grants awarded to PIs with >15 years of experience. These grants were associated with 8215 publications; however, only 13.2% were published in high-impact journals (impact factor ≥10). 4.4% of the grants resulted in patents, and these were associated with higher award amounts ($345,801 vs $311,350; P = 0.030). On multivariate analysis, combined MD/PhD degree [odds ratio (OR) 5.98; 95% confidence interval (CI) 2.18-16.39; P < 0.001] was associated with improved odds of patent creation; conversely, practicing surgeon PIs affected patent creation negatively (OR 0.31; 95% CI 0.11-0.85; P = 0.024). CONCLUSION: In the last decade, a greater proportion of NIH grants in DoS were awarded to more experienced investigators. Disparities exist among grantees, and female investigators are underrepresented, especially among practicing surgeons.

Fropofol prevents disease progression in mice with hypertrophic cardiomyopathy.

AIMS: Increased myofilament contractility is recognized as a crucial factor in the pathogenesis of hypertrophic cardiomyopathy (HCM). Direct myofilament desensitization might be beneficial in preventing HCM disease progression. Here, we tested whether the small molecule fropofol prevents HCM phenotype expression and disease progression by directly depressing myofilament force development. METHODS AND RESULTS: Force, intracellular Ca2+, and steady-state activation were determined in isolated trabecular muscles from wild-type (WT) and transgenic HCM mice with heterozygous human α-myosin heavy chain R403Q mutation (αMHC 403/+). αMHC 403/+ HCM mice were treated continuously with fropofol by intraperitoneal infusion for 12 weeks. Heart tissue was analysed with histology and real-time PCR of prohypertrophic and profibrotic genes. Fropofol decreased force in a concentration-dependent manner without significantly altering [Ca2+]i in isolated muscles from both WT and αMHC 403/+ HCM mouse hearts. Fropofol also depressed maximal Ca2+-activated force and increased the [Ca2+]i required for 50% activation during steady-state activation. In whole-animal studies, chronic intra-abdominal administration of fropofol prevented hypertrophy development and diastolic dysfunction. Chronic fropofol treatment also led to attenuation of prohypertrophic and profibrotic gene expression, reductions in cell size, and decreases in tissue fibrosis. CONCLUSIONS: Direct inhibition of myofilament contraction by fropofol prevents HCM disease phenotypic expression and progression, suggesting that increased myofilament contractile force is the primary trigger for hypertrophy development and HCM disease progression.

Azi-medetomidine: Synthesis and Characterization of a Novel α2 Adrenergic Photoaffinity Ligand.

Agonists at the α2 adrenergic receptor produce sedation, increase focus, provide analgesia, and induce centrally mediated hypotension and bradycardia, yet neither their dynamic interactions with adrenergic receptors nor their modulation of neuronal circuit activity is completely understood. Photoaffinity ligands of α2 adrenergic agonists have the potential both to capture discrete moments of ligand-receptor interactions and to prolong naturalistic drug effects in discrete regions of tissue in vivo. We present here the synthesis and characterization of a novel α2 adrenergic agonist photolabel based on the imidazole medetomidine called azi-medetomidine. Azi-medetomidine shares protein association characteristics with its parent compound in experimental model systems and by molecular dynamics simulation of interactions with the α2A adrenergic receptor. Azi-medetomidine acts as an agonist at α2A adrenergic receptors, and produces hypnosis in Xenopus laevis tadpoles. Azi-medetomidine competes with the α2 agonist clonidine at α2A adrenergic receptors, which is potentiated by photolabeling, and azi-medetomidine labels moieties on the α2A adrenergic receptor as determined by mass spectrometry in a manner consistent with a simulated model. This novel α2 adrenergic agonist photolabel can serve as a powerful tool for in vitro and in vivo investigations of adrenergic signaling.

Propofol inhibits the voltage-gated sodium channel NaChBac at multiple sites.

Voltage-gated sodium (NaV) channels are important targets of general anesthetics, including the intravenous anesthetic propofol. Electrophysiology studies on the prokaryotic NaV channel NaChBac have demonstrated that propofol promotes channel activation and accelerates activation-coupled inactivation, but the molecular mechanisms of these effects are unclear. Here, guided by computational docking and molecular dynamics simulations, we predict several propofol-binding sites in NaChBac. We then strategically place small fluorinated probes at these putative binding sites and experimentally quantify the interaction strengths with a fluorinated propofol analogue, 4-fluoropropofol. In vitro and in vivo measurements show that 4-fluoropropofol and propofol have similar effects on NaChBac function and nearly identical anesthetizing effects on tadpole mobility. Using quantitative analysis by 19F-NMR saturation transfer difference spectroscopy, we reveal strong intermolecular cross-relaxation rate constants between 4-fluoropropofol and four different regions of NaChBac, including the activation gate and selectivity filter in the pore, the voltage sensing domain, and the S4-S5 linker. Unlike volatile anesthetics, 4-fluoropropofol does not bind to the extracellular interface of the pore domain. Collectively, our results show that propofol inhibits NaChBac at multiple sites, likely with distinct modes of action. This study provides a molecular basis for understanding the net inhibitory action of propofol on NaV channels.

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 General Anesthetic Target Protein-Binding Sites by Photoaffinity Labeling and Mass Spectrometry.

General anesthetics are unique in that they represent a diverse range of chemical structures. Therefore, it is not surprising that the desired and undesired molecular targets, and binding sites therein, are as equally diverse and unique. Photoaffinity labeling has proven to be a valuable strategy for the identification of anesthetic molecular targets, as well as binding sites within those targets. In combination with the advances in mass spectrometry-based proteomics, along with the ability to comprehensively map posttranslational modifications, the method is likely to undergo continued improvement. Here, we provide the fundamentals for the design and development of an anesthetic photolabel. We also outline a protocol for the identification of photolabeled residues by mass spectrometry. The major steps include the photolabeling experiment, sample preparation, high-resolution mass spectrometry, and data analysis. The protocol can be used as a foundation for further optimization for the specific protein of interest and conditions of an experiment. The use of photoaffinity labeling adds an advantageous alternative and/or complementary approach to increase understanding of anesthetic molecular mechanisms.

Photoaffinity Ligand for the Inhalational Anesthetic Sevoflurane Allows Mechanistic Insight into Potassium Channel Modulation.

Sevoflurane is a commonly used inhaled general anesthetic. Despite this, its mechanism of action remains largely elusive. Compared to other anesthetics, sevoflurane exhibits distinct functional activity. In particular, sevoflurane is a positive modulator of voltage-gated Shaker-related potassium channels (Kv1.x), which are key regulators of action potentials. Here, we report the synthesis and validation of azisevoflurane, a photoaffinity ligand for the direct identification of sevoflurane binding sites in the Kv1.2 channel. Azisevoflurane retains major sevoflurane protein binding interactions and pharmacological properties within in vivo models. Photoactivation of azisevoflurane induces adduction to amino acid residues that accurately reported sevoflurane protein binding sites in model proteins. Pharmacologically relevant concentrations of azisevoflurane analogously potentiated wild-type Kv1.2 and the established mutant Kv1.2 G329T. In wild-type Kv1.2 channels, azisevoflurane photolabeled Leu317 within the internal S4-S5 linker, a vital helix that couples the voltage sensor to the pore region. A residue lining the same binding cavity was photolabeled by azisevoflurane and protected by sevoflurane in the Kv1.2 G329T. Mutagenesis of Leu317 in WT Kv1.2 abolished sevoflurane voltage-dependent positive modulation. Azisevoflurane additionally photolabeled a second distinct site at Thr384 near the external selectivity filter in the Kv1.2 G329T mutant. The identified sevoflurane binding sites are located in critical regions involved in gating of Kv channels and related ion channels. Azisevoflurane has thus emerged as a new tool to discover inhaled anesthetic targets and binding sites and investigate contributions of these targets to general anesthesia.

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.

Shedding Light on Anesthetic Mechanisms: Application of Photoaffinity Ligands.

Anesthetic photoaffinity ligands have had an increasing presence within anesthesiology research. These ligands mimic parent general anesthetics and allow investigators to study anesthetic interactions with receptors and enzymes; identify novel targets; and determine distribution within biological systems. To date, nearly all general anesthetics used in medicine have a corresponding photoaffinity ligand represented in the literature. In this review, we examine all aspects of the current methodologies, including ligand design, characterization, and deployment. Finally we offer points of consideration and highlight the future outlook as more photoaffinity ligands emerge within the field.

Macroscopic and macromolecular specificity of alkylphenol anesthetics for neuronal substrates.

We used a photoactive general anesthetic called meta-azi-propofol (AziPm) to test the selectivity and specificity of alkylphenol anesthetic binding in mammalian brain. Photolabeling of rat brain sections with [(3)H]AziPm revealed widespread but heterogeneous ligand distribution, with [(3)H]AziPm preferentially binding to synapse-dense areas compared to areas composed largely of cell bodies or myelin. With [(3)H]AziPm and propofol, we determined that alkylphenol general anesthetics bind selectively and specifically to multiple synaptic protein targets. In contrast, the alkylphenol anesthetics do not bind to specific sites on abundant phospholipids or cholesterol, although [(3)H]AziPm shows selectivity for photolabeling phosphatidylethanolamines. Together, our experiments suggest that alkylphenol anesthetic substrates are widespread in number and distribution, similar to those of volatile general anesthetics, and that multi-target mechanisms likely underlie their pharmacology.

Role for the propofol hydroxyl in anesthetic protein target molecular recognition.

Propofol is a widely used intravenous general anesthetic. We synthesized 2-fluoro-1,3-diisopropylbenzene, a compound that we call "fropofol", to directly assess the significance of the propofol 1-hydroxyl for pharmacologically relevant molecular recognition in vitro and for anesthetic efficacy in vivo. Compared to propofol, fropofol had a similar molecular volume and only a small increase in hydrophobicity. Isothermal titration calorimetry and competition assays revealed that fropofol had higher affinity for a protein site governed largely by van der Waals interactions. Within another protein model containing hydrogen bond interactions, propofol demonstrated higher affinity. In vivo, fropofol demonstrated no anesthetic efficacy, but at high concentrations produced excitatory activity in tadpoles and mice; fropofol also antagonized propofol-induced hypnosis. In a propofol protein target that contributes to hypnosis, α1ß2γ2L GABAA receptors, fropofol demonstrated no significant effect alone or on propofol positive allosteric modulation of the ion channel, suggesting an additional requirement for the 1-hydroxyl within synaptic GABAA receptor site(s). However, fropofol caused similar adverse cardiovascular effects as propofol by a dose-dependent depression of myocardial contractility. Our results directly implicate the propofol 1-hydroxyl as contributing to molecular recognition within protein targets leading to hypnosis, but not necessarily within protein targets leading to side effects of the drug.

Multiple propofol-binding sites in a γ-aminobutyric acid type A receptor (GABAAR) identified using a photoreactive propofol analog.

Propofol acts as a positive allosteric modulator of γ-aminobutyric acid type A receptors (GABAARs), an interaction necessary for its anesthetic potency in vivo as a general anesthetic. Identifying the location of propofol-binding sites is necessary to understand its mechanism of GABAAR modulation. [(3)H]2-(3-Methyl-3H-diaziren-3-yl)ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate (azietomidate) and R-[(3)H]5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (mTFD-MPAB), photoreactive analogs of 2-ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate (etomidate) and mephobarbital, respectively, have identified two homologous but pharmacologically distinct classes of intersubunit-binding sites for general anesthetics in the GABAAR transmembrane domain. Here, we use a photoreactive analog of propofol (2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol ([(3)H]AziPm)) to identify propofol-binding sites in heterologously expressed human α1ß3 GABAARs. Propofol, AziPm, etomidate, and R-mTFD-MPAB each inhibited [(3)H]AziPm photoincorporation into GABAAR subunits maximally by ∼ 50%. When the amino acids photolabeled by [(3)H]AziPm were identified by protein microsequencing, we found propofol-inhibitable photolabeling of amino acids in the ß3-α1 subunit interface (ß3Met-286 in ß3M3 and α1Met-236 in α1M1), previously photolabeled by [(3)H]azietomidate, and α1Ile-239, located one helical turn below α1Met-236. There was also propofol-inhibitable [(3)H]AziPm photolabeling of ß3Met-227 in ßM1, the amino acid in the α1-ß3 subunit interface photolabeled by R-[(3)H]mTFD-MPAB. The propofol-inhibitable [(3)H]AziPm photolabeling in the GABAAR ß3 subunit in conjunction with the concentration dependence of inhibition of that photolabeling by etomidate or R-mTFD-MPAB also establish that each anesthetic binds to the homologous site at the ß3-ß3 subunit interface. These results establish that AziPm as well as propofol bind to the homologous intersubunit sites in the GABAAR transmembrane domain that binds etomidate or R-mTFD-MPAB with high affinity.

Mechanisms revealed through general anesthetic photolabeling.

General anesthetic photolabels are used to reveal molecular targets and molecular binding sites of anesthetic ligands. After identification, the relevance of anesthetic substrates or binding sites can be tested in biological systems. Halothane and photoactive analogs of isoflurane, propofol, etomidate, neurosteroids, anthracene, and long chain alcohols have been used in anesthetic photolabeling experiments. Interrogated protein targets include the nicotinic acetylcholine receptor, GABAA receptor, tubulin, leukocyte function-associated antigen-1, and protein kinase C. In this review, we summarize insights revealed by photolabeling these targets, as well as general features of anesthetics, such as their propensity to partition to mitochondria and bind voltage-dependent anion channels. The theory of anesthetic photolabel design and the experimental application of photoactive ligands are also discussed.

Photoaffinity labeling the propofol binding site in GLIC.

Propofol, an intravenous general anesthetic, produces many of its anesthetic effects in vivo by potentiating the responses of GABA type A receptors (GABAAR), members of the superfamily of pentameric ligand-gated ion channels (pLGICs) that contain anion-selective channels. Propofol also inhibits pLGICs containing cation-selective channels, including nicotinic acetylcholine receptors and GLIC, a prokaryotic proton-gated homologue from Gloeobacter violaceus . In the structure of GLIC cocrystallized with propofol at pH 4 (presumed open/desensitized states), propofol was localized to an intrasubunit pocket at the extracellular end of the transmembrane domain within the bundle of transmembrane α-helices (Nury, H, et al. (2011) Nature 469, 428-431). To identify propofol binding sites in GLIC in solution, we used a recently developed photoreactive propofol analogue (2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol or AziPm) that acts as an anesthetic in vivo and potentiates GABAAR in vitro. For GLIC expressed in Xenopus oocytes, propofol and AziPm inhibited current responses at pH 5.5 (EC20) with IC50 values of 20 and 50 µM, respectively. When [(3)H]AziPm (7 µM) was used to photolabel detergent-solubilized, affinity-purified GLIC at pH 4.4, protein microsequencing identified propofol-inhibitable photolabeling of three residues in the GLIC transmembrane domain: Met-205, Tyr-254, and Asn-307 in the M1, M3, and M4 transmembrane helices, respectively. Thus, for GLIC in solution, propofol and AziPm bind competitively to a site in proximity to these residues, which, in the GLIC crystal structure, are in contact with the propofol bound in the intrasubunit pocket.