Propofol directly binds and inhibits skeletal muscle ryanodine receptor 1 (RyR1).

As the primary Ca 2+ release channel in skeletal muscle sarcoplasmic reticulum (SR), mutations in the type 1 ryanodine receptor (RyR1) or its binding partners underlie a constellation of muscle disorders, including malignant hyperthermia (MH). In patients with MH mutations, exposure to triggering drugs such as the halogenated volatile anesthetics biases RyR1 to an open state, resulting in uncontrolled Ca 2+ release, sarcomere tension and heat production. Restoration of Ca 2+ into the SR also consumes ATP, generating a further untenable metabolic load. When anesthetizing patients with known MH mutations, the non-triggering intravenous general anesthetic propofol is commonly substituted for triggering anesthetics. Evidence of direct binding of anesthetic agents to RyR1 or its binding partners is scant, and the atomic-level interactions of propofol with RyR1 are entirely unknown. Here, we show that propofol decreases RyR1 opening in heavy SR vesicles and planar lipid bilayers, and that it inhibits activator-induced Ca 2+ release from SR in human skeletal muscle. In addition to confirming direct binding, photoaffinity labeling using m- azipropofol (AziP m ) revealed several putative propofol binding sites on RyR1. Prediction of binding affinity by molecular dynamics simulation suggests that propofol binds at least one of these sites at clinical concentrations. These findings invite the hypothesis that in addition to propofol not triggering MH, it may also be protective against MH by inhibiting induced Ca 2+ flux through RyR1.

Cryo-EM analysis of scorpion toxin binding to Ryanodine Receptors reveals subconductance that is abolished by PKA phosphorylation.

Calcins are peptides from scorpion venom with the unique ability to cross cell membranes, gaining access to intracellular targets. Ryanodine Receptors (RyR) are intracellular ion channels that control release of Ca2+ from the endoplasmic and sarcoplasmic reticulum. Calcins target RyRs and induce long-lived subconductance states, whereby single-channel currents are decreased. We used cryo-electron microscopy to reveal the binding and structural effects of imperacalcin, showing that it opens the channel pore and causes large asymmetry throughout the cytosolic assembly of the tetrameric RyR. This also creates multiple extended ion conduction pathways beyond the transmembrane region, resulting in subconductance. Phosphorylation of imperacalcin by protein kinase A prevents its binding to RyR through direct steric hindrance, showing how posttranslational modifications made by the host organism can determine the fate of a natural toxin. The structure provides a direct template for developing calcin analogs that result in full channel block, with potential to treat RyR-related disorders.

Calcium-release channels: structure and function of IP3 receptors and ryanodine receptors.

Ca2+-release channels are giant membrane proteins that control the release of Ca2+ from the endoplasmic and sarcoplasmic reticulum. The two members, ryanodine receptors (RyRs) and inositol-1,4,5-trisphosphate receptors (IP3Rs), are evolutionarily related and are both activated by cytosolic Ca2+. They share a common architecture, but RyRs have evolved additional modules in the cytosolic region. Their massive size allows for the regulation by tens of proteins and small molecules, which can affect the opening and closing of the channels. In addition to Ca2+, other major triggers include IP3 for the IP3Rs and depolarization of the plasma membrane for a particular RyR subtype expressed in skeletal muscle. Their size has made them popular targets for study via electron microscopic methods, with current structures culminating near 3 Å. The available structures have provided many new mechanistic insights into the binding of auxiliary proteins and small molecules, how these can regulate channel opening, and the mechanisms of disease-associated mutations. They also help scrutinize previously proposed binding sites, as some of these are now incompatible with the structures. Many questions remain around the structural effects of posttranslational modifications, additional binding partners, and the higher order complexes these channels can make in situ. This review summarizes our current knowledge about the structures of Ca2+-release channels and how this informs on their function.

Binding Sites and the Mechanism of Action of Propofol and a Photoreactive Analogue in Prokaryotic Voltage-Gated Sodium Channels.

Propofol, one of the most commonly used intravenous general anesthetics, modulates neuronal function by interacting with ion channels. The mechanisms that link propofol binding to the modulation of distinct ion channel states, however, are not understood. To tackle this problem, we investigated the prokaryotic ancestors of eukaryotic voltage-gated Na+ channels (Navs) using unbiased photoaffinity labeling (PAL) with a diazirine derivative of propofol (AziPm), electrophysiological methods, and mutagenesis. AziPm inhibits Nav function in a manner that is indistinguishable from that of the parent compound by promoting activation-coupled inactivation. In several replicates (8/9) involving NaChBac and NavMs, we found adducts at residues located at the C-terminal end of the S4 voltage sensor, the S4-S5 linker, and the N-terminal end of the S5 segment. However, the non-inactivating mutant NaChBac-T220A yielded adducts that were different from those found in the wild-type counterpart, which suggested state-dependent changes at the binding site. Then, using molecular dynamics simulations to further elucidate the structural basis of Nav modulation by propofol, we show that the S4 voltage sensors and the S4-S5 linkers shape two distinct propofol binding sites in a conformation-dependent manner. Supporting the PAL and MD simulation results, we also found that Ala mutations of a subset of adducted residues have distinct effects on gating modulation of NaChBac and NavMs by propofol. The results of this study provide direct insights into the structural basis of the mechanism through which propofol binding promotes activation-coupled inactivation to inhibit Nav channel function.

Pathological conformations of disease mutant Ryanodine Receptors revealed by cryo-EM.

Ryanodine Receptors (RyRs) are massive channels that release Ca2+ from the endoplasmic and sarcoplasmic reticulum. Hundreds of mutations are linked to malignant hyperthermia (MH), myopathies, and arrhythmias. Here, we explore the first MH mutation identified in humans by providing cryo-EM snapshots of the pig homolog, R615C, showing that it affects an interface between three solenoid regions. We also show the impact of apo-calmodulin (apoCaM) and how it can induce opening by bending of the bridging solenoid, mediated by its N-terminal lobe. For R615C RyR1, apoCaM binding abolishes a pathological 'intermediate' conformation, distributing the population to a mixture of open and closed channels, both different from the structure without apoCaM. Comparisons show that the mutation primarily affects the closed state, inducing partial movements linked to channel activation. This shows that disease mutations can cause distinct pathological conformations of the RyR and facilitate channel opening by disrupting interactions between different solenoid regions.

Mechanistic insights into volatile anesthetic modulation of K2P channels.

K2P potassium channels are known to be modulated by volatile anesthetic (VA) drugs and play important roles in clinically relevant effects that accompany general anesthesia. Here, we utilize a photoaffinity analog of the VA isoflurane to identify a VA-binding site in the TREK1 K2P channel. The functional importance of the identified site was validated by mutagenesis and biochemical modification. Molecular dynamics simulations of TREK1 in the presence of VA found multiple neighboring residues on TREK1 TM2, TM3, and TM4 that contribute to anesthetic binding. The identified VA-binding region contains residues that play roles in the mechanisms by which heat, mechanical stretch, and pharmacological modulators alter TREK1 channel activity and overlaps with positions found to modulate TASK K2P channel VA sensitivity. Our findings define molecular contacts that mediate VA binding to TREK1 channels and suggest a mechanistic basis to explain how K2P channels are modulated by VAs.

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.

Alkylphenol inverse agonists of HCN1 gating: H-bond propensity, ring saturation and adduct geometry differentially determine efficacy and potency.

BACKGROUND AND PURPOSE: In models of neuropathic pain, inhibition of HCN1 is anti-hyperalgesic. 2,6-di-iso-propyl phenol (propofol) and its non-anesthetic congener, 2,6-di-tert-butyl phenol, inhibit HCN1 channels by stabilizing closed state(s). EXPERIMENTAL APPROACH: Using in vitro electrophysiology and kinetic modeling, we systematically explore the contribution of ligand architecture to alkylphenol-channel coupling. KEY RESULTS: When corrected for changes in hydrophobicity (and propensity for intra-membrane partitioning), the decrease in potency upon 1-position substitution (NCO∼OH >> SH >>> F) mirrors the ligands' H-bond acceptor (NCO > OH > SH >>> F) but not donor profile (OH > SH >>> NCO∼F). H-bond elimination (OH to F) corresponds to a ΔΔG of ∼4.5 kCal mol-1 loss of potency with little or no disruption of efficacy. Substitution of compact alkyl groups (iso-propyl, tert-butyl) with shorter (ethyl, methyl) or more extended (sec-butyl) adducts disrupts both potency and efficacy. Ring saturation (with the obligate loss of both planarity and π electrons) primarily disrupts efficacy. CONCLUSIONS AND IMPLICATIONS: A hydrophobicity-independent decrement in potency at higher volumes suggests the alkylbenzene site has a volume of ≥800 Å3. Within this, a relatively static (with respect to ligand) H-bond donor contributes to initial binding with little involvement in generation of coupling energy. The influence of π electrons/ring planarity and alkyl adducts on efficacy reveals these aspects of the ligand present towards a face of the channel that undergoes structural changes during opening. The site's characteristics suggest it is "druggable"; introduction of other adducts on the ring may generate higher potency inverse agonists.

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 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.

High-Throughput Screening to Identify Anesthetic Ligands Using Xenopus laevis Tadpoles.

General anesthetics are considered among the most significant advances in modern medicine; however, they are also some of the most dangerous commonly administered drugs. Despite this, the discovery of novel anesthetics has been slow, with few clinically used agents regardless of their nearly 200-year history. Xenopus laevis frogs have a long history as a model organism and provide a vital bridge between in vitro and preclinical mammalian assays. The provided protocols are efficient and cost-effective and therefore readily amendable for high-throughput evaluation of novel anesthetic ligands. By using the X. laevis bioassay, a researcher is capable of determining relative general anesthetic tolerance and/or cross-tolerance for candidate nonvolatile and/or volatile ligands.

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.

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.

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.

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.