Propofol Binding Research Articles

Three classes of propofol binding sites on GABAA receptors

Propofol is a widely used anesthetic and sedative that acts as a positive allosteric modulator (PAM) of gamma-aminobutyric acid type A (GABAA) receptors. Several potential propofol binding sites that may mediate this effect have been identified using propofol-analogue photoaffinity labeling. o-PD labels β-H267, a pore-lining residue, whereas AziPm labels residues β-M286, β-M227 and α-I239 in the two membrane-facing interfaces (β(+)/α(-) and α(+)/β(-)) between α and β subunits. This study used photoaffinity labeling of α1β3 GABAA receptors to reconcile the apparently conflicting results obtained with AziPm and o-PD labeling, focusing on whether β3-H267 identifies specific propofol binding site(s). The results show that propofol, but not AziPm protects β3-H267 from labeling by o-PD, whereas both propofol and o-PD protect against AziPm labeling of β3-M286, β3-M227 and α1I239. These data indicate that there are three distinct classes of propofol binding sites, with AziPm binding to two of the classes and o-PD to all three. Analysis of binding stoichiometry using native mass spectrometry in β3 homomeric receptors, demonstrated a minimum of five AziPm labeled residues and three o-PD labeled residues per pentamer, suggesting that there are two distinct propofol binding sites per β-subunit. The native MS data, coupled with photolabeling performed in the presence of zinc, indicate that the binding site(s) identified by o-PD are adjacent to, but not within the channel pore, since the pore at the 17’ H267 residue can accommodate only one propofol molecule. These data validate the existence of three classes of specific propofol binding sites on α1β3 GABAA receptors.

Propofol binding sites on ryanodine receptor 1

Propofol binding sites on ryanodine receptor 1

Anesthesia, Coming of Age in the World of Modern In Silico Drug Design.

Anesthesia, Coming of Age in the World of Modern In Silico Drug Design.

Direct visualization of general anesthetic propofol on neurons by stimulated Raman scattering microscopy

SummaryThe consensus for the precise mechanism of action of general anesthetics is through allosteric interactions with GABA receptors in neurons. However, it has been speculated that these anesthetics may also interact with the plasma membrane on some level. Owing to the small size of anesthetics, direct visualization of these interactions is difficult to achieve. We demonstrate the ability to directly visualize a deuterated analog of propofol in living cells using stimulated Raman scattering (SRS) microscopy. Our findings support the theory that propofol is highly concentrated and interacts primarily through non-specific binding to the plasma membrane of neurons. Additionally, we show that SRS microscopy can be used to monitor the dynamics of propofol binding using real-time, live-cell imaging. The strategy used to visualize propofol can be applied to other small molecule drugs that have been previously invisible to traditional imaging techniques

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.

Modulation of α1β3γ2 GABAA receptors expressed in X. laevis oocytes using a propofol photoswitch tethered to the transmembrane helix

Tethered photoswitches are molecules with two photo-dependent isomeric forms, each with different actions on their biological targets. They include reactive chemical groups capable of covalently binding to their target. Our aim was to develop a β-subunit-tethered propofol photoswitch (MAP20), as a tool to better study the mechanism of anesthesia through the GABAA α1β3γ2 receptor. We used short spacers between the tether (methanethiosulfonate), the photosensitive moiety (azobenzene), and the ligand (propofol), to allow a precise tethering adjacent to the putative propofol binding site at the β+α- interface of the receptor transmembrane helices (TMs). First, we used molecular modeling to identify possible tethering sites in β3TM3 and α1TM1, and then introduced cysteines in the candidate positions. Two mutant subunits [β3(M283C) and α1(V227C)] showed photomodulation of GABA responses after incubation with MAP20 and illumination with lights at specific wavelengths. The α1β3(M283C)γ2 receptor showed the greatest photomodulation, which decreased as GABA concentration increased. The location of the mutations that produced photomodulation confirmed that the propofol binding site is located in the β+α- interface close to the extracellular side of the transmembrane helices. Tethering the photoswitch to cysteines introduced in the positions homologous to β3M283 in two other subunits (α1W288 and γ2L298) also produced photomodulation, which was not entirely reversible, probably reflecting the different nature of each interface. The results are in agreement with a binding site in the β+α- interface for the anesthetic propofol.

Mechanistic basis of propofol-induced disruption of kinesin processivity

Propofol is a widely used general anesthetic to induce and maintain anesthesia, and its effects are thought to occur through impact on the ligand-gated channels including the GABAA receptor. Propofol also interacts with a large number of proteins including molecular motors and inhibits kinesin processivity, resulting in significant decrease in the run length for conventional kinesin-1 and kinesin-2. However, the molecular mechanism by which propofol achieves this outcome is not known. The structural transition in the kinesin neck-linker region is crucial for its processivity. In this study, we analyzed the effect of propofol and its fluorine derivative (fropofol) on the transition in the neck-linker region of kinesin. Propofol binds at two crucial surfaces in the leading head: one at the microtubule-binding interface and the other in the neck-linker region. We observed in both the cases the order-disorder transition of the neck-linker was disrupted and kinesin lost its signal for forward movement. In contrast, there was not an effect on the neck-linker transition with propofol binding at the trailing head. Free-energy calculations show that propofol at the microtubule-binding surface significantly reduces the microtubule-binding affinity of the kinesin head. While propofol makes pi-pi stacking and H-bond interactions with the propofol binding cavity, fropofol is unable to make a suitable interaction at this binding surface. Therefore, the binding affinity of fropofol is much lower compared to propofol. Hence, this study provides a mechanism by which propofol disrupts kinesin processivity and identifies transitions in the ATPase stepping cycle likely affected.

Propofol sedation-induced alterations in brain connectivity reflect parvalbumin interneurone distribution in human cerebral cortex

BackgroundPropofol, a commonly used intravenous anaesthetic, binds to type A gamma aminobutyric acid (GABA) receptors in mammalian brain. Previous work on its anaesthetic action has characterised either the biochemistry underlying propofol binding or the associated changes in brain network dynamics during sedation. Despite these advances, no study has focused on understanding how propofol action at the cellular level results in changes in brain network connectivity. MethodsWe used human whole-brain microarray data to generate distribution maps for genes that mark the primary GABAergic cortical interneurone subtypes (somatostatin, parvalbumin [PV], and 5-hydroxytryptamine 3A. Next, 25 healthy participants underwent propofol-induced sedation during resting state functional MRI scanning. We used partial least squares analysis to identify the brain regions in which connectivity patterns were most impacted by propofol sedation. We then correlated these multimodal cortical patterns to determine if a specific interneurone subtype was disproportionately expressed in brain regions in which connectivity patterns were altered during sedation. ResultsBrain networks that were significantly altered by propofol sedation had a high density of PV-expressing GABAergic interneurones. Brain networks that anticorrelated during normal wakefulness, namely the default mode network and attentional and frontoparietal control networks, increased in correlation during sedation. ConclusionsPV-expressing interneurones are highly expressed in brain regions with altered connectivity profiles during propofol-induced sedation. This study also demonstrates the utility of leveraging multiple datasets to address multiscale neurobiological problems.

Novel Approach for Characterizing Propofol Binding Affinities to Serum Albumins from Different Species.

The interaction between the main carrier (serum albumin, SA) of endogenous and exogenous compounds in the bloodstream of different species (human, bovine, canine, rat, rabbit, and sheep) and a general anesthetic agent (propofol, PR) was investigated using an experimental technique (high-performance liquid chromatography) and computational methods (molecular docking, molecular dynamics, sequence, and phylogenetic analyses). The obtained results revealed the differences in the PR binding affinity to various homologous forms of this protein with reliable statistics (R2 = 0.9 and p-value < 0.005), correlating with the evolutionary relationships among SAs from different species. Additionally, the protein conformational changes (root-mean-square deviation ≈ 1.0 Å) and amino acid conservation of binding sites in protein domains were detected, contributing to the SA–PR binding modes. Overall, the outcomes from this study might provide a novel methodology to assess protein–ligand interactions and to gain some interesting insights into drug pharmacokinetics and pharmacodynamics to explain its variations among different species.

A Comprehensive In Silico Study Towards Understanding the Inhibitory Mechanism of Lactoperoxidase by Dapsone and Propofol.

Lactoperoxidase (LPO) is a member of the mammalian heme peroxidase family and is an enzyme of the innate immune system. It possesses a covalently linked heme prosthetic group (a derivative of protoporphyrin IX) in its active site. LPO catalyzes the oxidation of halides and pseudohalides in the presence of hydrogen peroxide (H2O2) and shows a broad range of the antimicrobial activity. In this study, we have used two pharmaceutically important drug molecules, namely dapsone and propofol, which were earlier reported as potent inhibitors of LPO. At the same time, the stereochemistry and mode of binding of dapsone and propofol to LPO are still not known because of the lack of the crystal structures of LPO with these two drugs. In order to fill this gap, we utilized molecular docking and molecular dynamics (MD) simulation studies of LPO in its native and complex forms with dapsone and propofol. From the docking results, the estimated binding free energies (ΔG) of -9.25 kcal/mol (Ki = 0.16 μM) and -7.05 kcal/mol (Ki = 6.79 μM) were observed for dapsone, and propofol, respectively. The standard error of the Auto Dock program is 2.5 kcal/mol; therefore, molecular docking results alone were inconclusive. To further validate the docking results, we performed MD simulation on unbound, and two drugs bounded LPO structures. Interestingly, MD simulations results explained that the structural stability of LPO-Propofol complex was higher than LPO-Dapsone complex. The results obtained from this study establish the mode of binding and interaction pattern of the dapsone and propofol to LPO as inhibitors.

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.

Misdiagnosis of CTX due to propofol: The interference of total intravenous propofol anaesthesia with bile acid profiling.

BackgroundCerebrotendinous xanthomatosis (CTX) is a rare genetic disorder, characterised by chronic diarrhoea, xanthomas, cataracts, and neurological deterioration. CTX is caused by CYP27A1 deficiency, which leads to abnormal cholesterol and bile acid metabolism. Urinary bile acid profiling (increased m/z 627: glucuronide‐5β‐cholestane‐pentol) serves as diagnostic screening for CTX. However, this led to a false positive CTX diagnosis in two patients, who had received total intravenous anaesthesia (TIVA) with propofol.MethodsTo determine the influence of propofol on bile acid profiling, 10 urinary samples and 2 blood samples were collected after TIVA with propofol Fresenius 7 to 10 mg/kg/h from 12 subjects undergoing scoliosis correction. Urinary bile acids were analysed using flow injection negative electrospray mass spectrometry. Propofol binding to recombinant CYP27A1, the effects of propofol on recombinant CYP27A1 activity, and CYP27A1 expression in liver organoids were investigated using spectral binding, enzyme activity assays, and qPCR, respectively. Accurate masses were determined with high‐resolution mass spectrometry.ResultsAbnormal urinary profiles were identified in all subjects after TIVA, with a trend correlating propofol dose per kilogramme and m/z 627 peak intensity. Propofol only induced a weak CYP27A1 response in the spectral binding assay, minimally affected CYP27A1 activity and did not affect CYP27A1 expression. The accurate mass of m/z 627 induced by propofol differed >10 PPM from m/z 627 observed in CTX.ConclusionsTIVA with propofol invariably led to a urinary profile misleadingly suggestive of CTX, but not through CYP27A1 inhibition. To avoid further misdiagnoses, propofol administration should be considered when interpreting urinary bile acid profiles.

Propofol inhibits prokaryotic voltage-gated Na+ channels by promoting activation-coupled inactivation.

Propofol is widely used in the clinic for the induction and maintenance of general anesthesia. As with most general anesthetics, however, our understanding of its mechanism of action remains incomplete. Local and general anesthetics largely inhibit voltage-gated Na+ channels (Navs) by inducing an apparent stabilization of the inactivated state, associated in some instances with pore block. To determine the biophysical and molecular basis of propofol action in Navs, we investigated NaChBac and NavMs, two prokaryotic Navs with distinct voltage dependencies and gating kinetics, by whole-cell patch clamp electrophysiology in the absence and presence of propofol at clinically relevant concentrations (2-10 µM). In both Navs, propofol induced a hyperpolarizing shift of the pre-pulse inactivation curve without any significant effects on recovery from inactivation at strongly hyperpolarized voltages, demonstrating that propofol does not stabilize the inactivated state. Moreover, there was no evidence of fast or slow pore block by propofol in a non-inactivating NaChBac mutant (T220A). Propofol also induced hyperpolarizing shifts of the conductance-voltage relationships with negligible effects on the time constants of deactivation at hyperpolarized voltages, indicating that propofol does not stabilize the open state. Instead, propofol decreases the time constants of macroscopic activation and inactivation. Adopting a kinetic scheme of Nav gating that assumes preferential closed-state recovery from inactivation, a 1.7-fold acceleration of the rate constant of activation and a 1.4-fold acceleration of the rate constant of inactivation were sufficient to reproduce experimental observations with computer simulations. In addition, molecular dynamics simulations and molecular docking suggest that propofol binding involves interactions with gating machinery in the S4-S5 linker and external pore regions. Our findings show that propofol is primarily a positive gating modulator of prokaryotic Navs, which ultimately inhibits the channels by promoting activation-coupled inactivation.

Expansion of a Transmembrane Cavity Facilitates Anesthetic Potentiation of a Pentameric Ligand Gated Ion Channel

Ligand-gated ion channels are important molecular targets of general anesthetics, some of which have been in use since the mid 19th century. However, the molecular mechanism of these drug-receptor interactions is poorly understood, creating barriers to the development of new anesthetics. Biochemical and electrophysiological studies have shown that general anesthetics bind to small pockets in Cys-loop receptors, also known as pentameric ligand-gated ion channels. In addition, recent X-ray structures of the Gloeobacter violaceus ligand-gated ion channel show differential binding of the common anesthetic propofol in the presence of sensitizing mutations. In this work, we used molecular dynamics simulations to investigate how anesthetic binding relates to receptor structure. We found a correlation between anesthetic occupancy and binding site expansion. This correlation was removed by a mutation that increased functional sensitivity to anesthetic potentiation. Contraction of the apo binding site from the expanded conformation was associated with a pinching motion of two transmembrane helices, this motion being less pronounced in the mutant than in the wild-type channel. The sensitizing mutation also influenced flexibility of the mutated amino acid side chain and the bound anesthetic. These novel dynamic observations enabled us to refine an allosteric mechanism for bimodal anesthetic modulation in the Cys-loop receptor family, and provided testable hypotheses for further structure-function experiments.

Flexibility of a Transmembrane Helix Underlies Dramatic Reversal of Net Anesthetic Effects in a Pentameric Ligand-Gated Ion Channel

Mechanisms of action for general anesthetics have evolved substantially from purely lipid-based theories to models involving specific protein interfaces, with the capacity to allosterically inhibit as well as potentiate human Cys-loop receptors. However, we still lack a structurally detailed mechanism for these bimodal allosteric effects. Recently, we mapped an allosteric model for anesthetic modulation involving multiple state-dependent binding sites, based on new co-crystal structures of the bacterial homolog GLIC and the general anesthetic propofol. Notably, a point mutation (M205W) in the first transmembrane helix facilitated propofol binding in a membrane-accessible intrasubunit site, and conferred primarily potentiating rather than inhibitory net anesthetic effects. Molecular dynamics simulations correlated the potentiated phenotype with changes in backbone flexibility of the first transmembrane helix. Here, we used mutagenesis and two-electrode voltage-clamp electrophysiology to reveal that helix-perturbing residues at the same transmembrane position lead to potent potentiation even by subclinical concentrations of propofol. Interestingly, these variants were so sensitive to propofol that they exhibited persistent potentiation for up to 40 minutes after exposure to moderate concentrations of propofol, in contrast to an almost instantaneous washout in wild-type channels. The extent of this effect depended on propofol exposure time, suggesting involvement of the membrane as a reservoir for a lipid-accessible binding site. Persistent potentiation also appeared to be independent of an inhibitory effect observed at high acute doses, consistent with the presence of a discrete, water-accessible site for inhibition. The resulting mechanism for general anesthetic modulation could bridge the lipid-based Meyer-Overton theory, postulated more than a century ago, with more complex receptor-based models, informing both our understanding of allostery and the development of new anesthetics.

Propofol Is an Allosteric Agonist with Multiple Binding Sites on Concatemeric Ternary GABAA Receptors.

GABAA receptors can be directly activated and potentiated by the intravenous anesthetic propofol. Previous photolabeling, modeling, and functional data have identified two binding domains through which propofol acts on the GABAA receptor. These domains are defined by the β(M286) residue at the β"+"-α"-" interface in the transmembrane region and the β(Y143) residue near the β"-" surface in the junction between the extracellular and transmembrane domains. In the ternary receptor, there are predicted to be two copies of each class of sites, for a total of four sites per receptor. We used β2α1γ2L and β2α1 concatemeric constructs to determine the functional effects of the β(Y143W) and β(M286W) mutations to gain insight into the number of functional binding sites for propofol and the energetic contributions stemming from propofol binding to the individual sites. A mutation of each of the four sites affected the response to propofol, indicating that each of the four sites is functional in the wild-type receptor. The mutations mainly impaired stabilization of the open state by propofol, i.e., reduced gating efficacy. The effects were similar for mutations at either site and were largely additive and independent of the presence of other Y143W or M286W mutations in the receptor. The two classes of sites appeared to differ in affinity for propofol, with the site affected by M286W having about a 2-fold higher affinity. Our analysis indicates there may be one or two additional functionally equivalent binding sites for propofol, other than those modified by substitutions at β(Y143) and β(M286).

In silico investigation of propofol binding sites in human serum albumin using explicit and implicit solvation models

All-atom molecular dynamics (MD) simulations are presented on general anesthetic propofol bound to human serum albumin (HSA) due to the drug pharmacokinetics and pharmacodynamics in the circulatory system. We implemented the explicit and implicit solvation models to compare the binding affinity of propofol at the different binding sites (PR1 and PR2) in the HSA protein. Only the implicit solvation models provided the evidence in accordance with the experimental data indicating that the HSA-ligand interactions are dominanted by hydrophobic forces due to the higher drug affinity at the PR1 position with a ΔGMM-PB/SA value of −23.44kcalmol−1. Overall, this study provides important information on the accuracy of explicit and implicit solvation models to characterize the propofol interaction with different HSA binding sites.

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.

Multiple Mechanisms of Propofol Inhibition of the Voltage-Gated Sodium Channel Nachbac: A 19F NMR Investigation

Voltage-gated sodium channels are important molecular targets for anesthetics. The intravenous general anesthetic propofol was found to inhibit NaChBac, a prokaryotic sodium channel from Bacillus halodurans, but little is known about where propofol binds and how propofol binding inhibits the function of this channel. Here, we used 19F-NMR saturation transfer difference (STD) spectroscopy to quantify propofol interactions with NaChBac and to understand the inhibitory mechanisms by propofol. 19F probes were introduced, one at a time, by tagging 1,1,1-trifluoro-3-bromo acetone to site-directed single cysteine mutations that had been computationally predicted to be involved in propofol binding. Quantitative 19F-NMR STD between the 19F probes and 4-fluoropropofol, a fluorinated propofol analog show that propofol binds with different affinities to the sites in different regions of NaChBac. The order of fluoropropofol binding strength, reflected in the cross relaxation rate constant (σ), from the strongest to the weakest, is as follows: the channel activation gate at the cytoplasmic end of the pore, the voltage sensing domain near the gating charge-carrying residues, the ion selectivity filter at the end of the P1 loop, the S4-S5 linker, and the central cavity of the pore. No STD was detectable in the extracellular interface of channel. The finding of multiple binding sites is consistent with the previous observations with the volatile anesthetics isoflurane and sevoflurane. Our results suggest that propofol inhibits NaChBac through multiple sites with distinct mechanisms. As a channel blocker, propofol interferes with the activation gate and selectivity filter and obstructs conductance by binding to the inner pore. As an inhibitory modulator, propofol binding interrupts charge movement in the voltage-sensing domain and restricts the pivot motion of the S4-S5 linker. The study provides a molecular basis for the understanding of propofol inhibitory action on voltage-gated sodium channels. This research was supported by grants from the NIH.

Structural and Functional Evidence for Multi-Site Allostery Mediated by General Anesthetics in a Model Ligand-Gated Ion Channel

General anesthetics act as either positive or negative allosteric modulators of several pentameric ligand-gated ion channels, including physiologically important receptors for γ-aminobutyric acid and acetylcholine. Although functional studies have implicated conserved sites of modulation in this channel family, the limited scope and resolution of structural data for human Cys-loop receptors have hampered mechanistic studies of anesthetic action. We previously showed the prokaryotic homolog GLIC to be a useful model system that recapitulates functional modulation of human ion channels, and enables structure determination both in apparent open and nonconducting states. Specifically, anesthetic inhibition of GLIC can be removed, reversed, or rendered bimodal by site-directed mutations in the transmembrane domain (TMD). In this work, we provide crystallographic and electrophysiological evidence for a multi-site mechanism of bimodal modulation by anesthetizing agents, including the common surgical medication propofol. With the pore in an apparent nonconducting state, propofol bound at the intracellular (lower) end of the channel pore, similar to other inhibitors. Consistent with this result, single hydrophobic substitutions at the lower-pore site enhanced functional inhibition. Conversely, in the apparent open state, anesthetics bound to one or more contiguous sites in the extracellular-facing (upper) end of the TMD, particularly for variants in which anesthetic inhibition was reduced or reversed. In one novel variant exhibiting anesthetic potentiation, propofol binding in the upper-TMD converted the channel from an apparent nonconducting- to open-pore conformation under otherwise identical conditions, providing direct evidence for potentiation via specific sites in the upper TMD. Based on these findings, we propose a structural model for allostery in which anesthetic binding to spatially distinct TMD cavities differentially stabilizes opposing functional states of pentameric ligand-gated ion channels, providing valuable insights into channel modulation and drug development.