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Propofol binds and inhibits skeletal muscle ryanodine receptor 1. BACKGROUND: As the primary Ca2+ release channel in skeletal muscle sarcoplasmic reticulum (SR), mutations in type 1 ryanodine receptor (RyR1) or its binding partners underlie a constellation of muscle disorders, including malignant hyperthermia (MH). In patients with MH mutations, triggering agents including halogenated volatile anaesthetics bias RyR1 to an open state resulting in uncontrolled Ca2+ release, increased sarcomere tension, and heat production. Propofol does not trigger MH and is commonly used for patients at risk of MH. The atomic-level interactions of any anaesthetic with RyR1 are unknown. METHODS: RyR1 opening was measured by [3H]ryanodine binding in heavy SR vesicles (wild type) and single-channel recordings of MH mutant R615C RyR1 in planar lipid bilayers, each exposed to propofol or the photoaffinity ligand analogue m-azipropofol (AziPm). Activator-mediated wild-type RyR1 opening as a function of propofol concentration was measured by Fura-2 Ca2+ imaging of human skeletal myotubes. AziPm binding sites, reflecting propofol binding, were identified on RyR1 using photoaffinity labelling. Propofol binding affinity to a photoadducted site was predicted using molecular dynamics (MD) simulation. RESULTS: Both propofol and AziPm decreased RyR1 opening in planar lipid bilayers (P<0.01) and heavy SR vesicles, and inhibited activator-induced Ca2+ release from human skeletal myotube SR. Several putative propofol binding sites on RyR1 were photoadducted by AziPm. MD simulation predicted propofol KD values of 55.8 μM and 1.4 μM in the V4828 pocket in open and closed RyR1, respectively. CONCLUSIONS: Propofol demonstrated direct binding and inhibition of RyR1 at clinically plausible concentrations, consistent with the hypothesis that propofol partially mitigates malignant hyperthermia by inhibition of induced Ca2+ flux through RyR1.

Detailed methods are available in the Supplementary material. This was conducted as described.8 Heavy SR (HSR) vesicles were provided by Francisco Alvarado (Cardiovascular Research Center, School of Medicine and Public Health, University of Wisconsin, Madison, WI, USA). Propofol (1–100 μM) or AziPm (1–48 μM) was added to HSR protein in 200 mM KCl, 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.2), 5 nM [3H]ryanodine (56 Ci mmol−1), 1 mM EGTA, and enough CaCl2 to set free [Ca2+] at 100 nM (pCa 7) or 10 μM (pCa 5). Inclusion of 20 μM unlabelled ryanodine in some samples allowed for nonspecific binding estimations. After incubation for 2 h at 37°C, filters were washed and [3H]ryanodine determined with liquid scintillation counting. Experiments were done in triplicate.

gh CaCl2 to set free [Ca2+] at 100 nM (pCa 7) or 10 μM (pCa 5). Inclusion of 20 μM unlabelled ryanodine in some samples allowed for nonspecific binding estimations. After incubation for 2 h at 37°C, filters were washed and [3H]ryanodine determined with liquid scintillation counting. Experiments were done in triplicate. Frozen rabbit or pig skeletal muscle (∼200 g) was blended, centrifuged, filtered, and centrifuged again at higher speed at 4°C. Pellets were solubilised in calcium-free buffer containing 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS), and 0.2% soybean phosphatidylcholine with 100 μl of protease inhibitor cocktail. His-GST-FKBP12.6 (∼5 mg, made in-house) was added, followed by ultracentrifugation. The supernatant was incubated at 4°C with pre-equilibrated GS4B resin (Cytiva, Marlborough, MA, USA). RyR1 was eluted from the resin using TEV protease (made in-house). The eluents were further concentrated and purified with gel filtration using Superose 6 10/300 GL (Cytiva, Marlborough, MA, USA). Fractions containing RyR1 complexes measured by absorbance at 280 nm and were concentrated to ∼2 mg ml-1. RyR1 was reconstituted into proteoliposomes as described.9 Briefly, a 5:3 mixture of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (Avanti Polar Lipids, Alabaster, AL, USA) were dried into a thin film and solubilised with 400 μl of rabbit RyR1 (0.7 mg ml−1). Following dialysis, the samples were aliquoted, flash-frozen in liquid nitrogen, and stored at −80°C.

n-glycero-3-phosphoethanolamine (DOPE) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (Avanti Polar Lipids, Alabaster, AL, USA) were dried into a thin film and solubilised with 400 μl of rabbit RyR1 (0.7 mg ml−1). Following dialysis, the samples were aliquoted, flash-frozen in liquid nitrogen, and stored at −80°C. Using the Orbit mini setup and EDR2 software (Nanion Technologies, Livingston, NJ, USA), recordings were obtained in parallel with multielectrode-cavity-array chips (Ionera Technologies, Freiburg, Germany). The cis and trans chambers contained symmetrical solutions of 250 mM HEPES, 150 mM KCl, 1 mM EGTA (pH 7.3), 0.2 mM CaCl2 ([Ca2+] free = 0.1 μM). To promote fusion to prepared suspended bilayers, 5% glycerol was incorporated into the proteoliposomes and 1–2 μl RyR1 proteoliposomes were added to the cis chamber. To further promote fusion, voltage was maintained at +40 mV. Recordings were started at the point of successful RyR1 insertion. Propofol was introduced to the cis chamber and concentrations determined by absorbance at 270 nm.10 All RyR1 measurements were conducted at 22°C and constant voltage of –60 mV. Recordings were filtered at final bandwidth of 10 kHz. Clampfit software (10.6, Molecular Devices, San Jose, CA, USA) was used to analyse current traces and only channels with a conductance > 700 pS were included in the analysis.11

t 270 nm.10 All RyR1 measurements were conducted at 22°C and constant voltage of –60 mV. Recordings were filtered at final bandwidth of 10 kHz. Clampfit software (10.6, Molecular Devices, San Jose, CA, USA) was used to analyse current traces and only channels with a conductance > 700 pS were included in the analysis.11 Human skeletal muscle cells were maintained in growth medium in a 5% CO2 atmosphere at 37°C. After passage and to induce differentiation, plated cells were incubated overnight in growth medium, then in differentiation medium, the latter changed every other day. Multinuclear myotubes typically formed within 5–6 days. Differentiated myotubes were loaded with 1.5 μM Fura-2 AM marker in 20% bovine serum albumin (BSA) and incubated for 15 min to allow de-esterification. Fura-2 binds to intracellular Ca2+, with the ratio of emission at 340 nm and 380 nm directly related to the concentration of Ca2+.12, 13, 14 Emission ratio was measured using a fluorescent microscope with a cooled high-speed digital video camera and MetaFluor software (version 7.10.4.407, MetaMorph 2020, Molecular Devices, LLC, San Jose, CA, USA). Changes in Fura-2 fluorescence were measured for each drug concentration (n=40–50 cells): ryanodine (2–1000 nM) or propofol (2–300 μM). Data were normalised to the maximal response of cells, and IC50 calculated by fitting to Hill curves using PRISM 10 software (GraphPad Software, San Diego, CA, USA).

an Jose, CA, USA). Changes in Fura-2 fluorescence were measured for each drug concentration (n=40–50 cells): ryanodine (2–1000 nM) or propofol (2–300 μM). Data were normalised to the maximal response of cells, and IC50 calculated by fitting to Hill curves using PRISM 10 software (GraphPad Software, San Diego, CA, USA). AziPm (5 μM) was added (with and without 200 μM propofol to determine specificity) to purified RyR1-FKBP12.6 at a final protein concentration of 1 μg μl−1. Samples were equilibrated on ice in the dark for 5 min then irradiated for 30 min at 350 nm with an RPR-3000 Rayonet lamp in 1-mm path length quartz cuvettes through a 295-nm glass filter (Newport Corporation, Franklin, MA, USA). After UV exposure, proteins were precipitated in acetone, pelleted, washed, and air-dried before resuspension in 50 mM Tris–HCl, pH 8.0, 1% Triton X-100, and 0.5% SDS. Insoluble debris was pelleted and resuspended in NH4HCO3. Samples were treated with dithiothreitol (DTT) and iodoacetamide (IAA) before sequencing-grade modified trypsin was added at a 1:20 protease/protein ratio (w/w) with additional of 0.2% (w/v%) ProteaseMAX (Promega, Madison, WI, USA) surfactant. Proteins were digested and then diluted with NH4HCO3 and 0.02% ProteaseMAX surfactant before the addition of sequencing-grade chymotrypsin at 1:20 protease/protein ratio (w/w). Proteins were digested and acidified before centrifugation to remove insoluble debris. Finally, the sample was desalted using C18 stage tips, dried under vacuum and resuspended in 0.1% formic acid before mass spectrometry.

actant before the addition of sequencing-grade chymotrypsin at 1:20 protease/protein ratio (w/w). Proteins were digested and acidified before centrifugation to remove insoluble debris. Finally, the sample was desalted using C18 stage tips, dried under vacuum and resuspended in 0.1% formic acid before mass spectrometry. Photolabelled proteins were separated by SDS-PAGE; the rRyR1 band was excised, destained, dehydrated, and dried before proteins were reduced by 5 mM DTT and 50 mM NH4HCO3. Samples were then alkylated with 55 mM IAA in 50 mM NH4HCO3, dehydrated, and dried before resuspension in 0.2% ProteaseMAX surfactant and 50 mM NH4HCO3. After this point the digestion, suspension, and extraction were essentially identical to the above in-solution scheme.

eins were reduced by 5 mM DTT and 50 mM NH4HCO3. Samples were then alkylated with 55 mM IAA in 50 mM NH4HCO3, dehydrated, and dried before resuspension in 0.2% ProteaseMAX surfactant and 50 mM NH4HCO3. After this point the digestion, suspension, and extraction were essentially identical to the above in-solution scheme. Mass spectrometry was performed as reported.15 Briefly, desalted peptides were injected into a Thermo LTQ Orbitrap XL Mass Spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) or an Orbitrap Elite Hybrid Ion Trap mass spectrometer. Peptides were eluted with 100 min with linear gradients of acetonitrile in 0.1% formic acid. Spectral analysis was conducted using MaxQuant16 to search b and y ions against the rRyR1 sequence. All analyses included dynamic oxidation of methionine (+15.9949 m/z) as well as static alkylation of cysteine (+57.0215 m/z; iodoacetamide alkylation). Photolabelled peptides were searched for the additional dynamic AziPm modifications. Both in-solution and in-gel sequential trypsin/chymotrypsin digests were searched without enzyme specification with a false discovery rate of 0.01. Samples were analysed in triplicate and samples containing no photoaffinity ligand (controls) were run to identify false positives.

additional dynamic AziPm modifications. Both in-solution and in-gel sequential trypsin/chymotrypsin digests were searched without enzyme specification with a false discovery rate of 0.01. Samples were analysed in triplicate and samples containing no photoaffinity ligand (controls) were run to identify false positives. We used cryo-electron microscopy models (PDB: 6X34, open state; and 6X36, closed-state) of pig R615C RyR1.9 Only the central pore domain of RyR1, itself a functional channel,17 was simulated, as the entire protein is computationally impractical for demanding free energy perturbation (FEP) molecular dynamics (MD) simulations. Systems were constructed using CHARMM-GUI.18 All simulations were conducted using NAMD 2.14 or 319 with CHARMM36 force field, existing propofol parameters20, 21, 22 and TIP3P water. Production simulations were conducted in the isothermic–isobaric ensemble with Langevin thermostat. The lipid bilayers consisted of 70% 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 30% cholesterol.

d using NAMD 2.14 or 319 with CHARMM36 force field, existing propofol parameters20, 21, 22 and TIP3P water. Production simulations were conducted in the isothermic–isobaric ensemble with Langevin thermostat. The lipid bilayers consisted of 70% 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 30% cholesterol. Streamlined Alchemical Free Energy Perturbation (SAFEP) methodology23,24 was used to determine the absolute binding free energy of propofol (ΔGbind∘). SAFEP uses a limited set of restraints on the ligand to maintain its bound conformation during alchemical transformations and improve sampling of states that most contribute to ΔGbind∘. The restraints are then corrected to yield an accurate absolute ΔGbind∘. The overall expression is:ΔGbind∘=–ΔGsite∗+ΔGDBC−ΔGV∘+ΔGbulk∗where ΔGbulk∗ is the energy of decoupling the unbound ligand from solvated to gas phase, ΔGV∘ and ΔGDBC energies of volumetric and distance-from-bound-conformation (DBC) restraints, respectively, and –ΔGsite∗ is the energy of coupling the ligand from gas phase to the protein-bound state. ΔGbulk∗ and ΔGsite∗ were calculated using FEP MD, ΔGDBC using thermodynamic integration, and ΔGV∘ parametrically. The Bennett acceptance ratio method was used to calculate free energy differences in FEP calculations.

This was conducted as described.8 Heavy SR (HSR) vesicles were provided by Francisco Alvarado (Cardiovascular Research Center, School of Medicine and Public Health, University of Wisconsin, Madison, WI, USA). Propofol (1–100 μM) or AziPm (1–48 μM) was added to HSR protein in 200 mM KCl, 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.2), 5 nM [3H]ryanodine (56 Ci mmol−1), 1 mM EGTA, and enough CaCl2 to set free [Ca2+] at 100 nM (pCa 7) or 10 μM (pCa 5). Inclusion of 20 μM unlabelled ryanodine in some samples allowed for nonspecific binding estimations. After incubation for 2 h at 37°C, filters were washed and [3H]ryanodine determined with liquid scintillation counting. Experiments were done in triplicate.

Frozen rabbit or pig skeletal muscle (∼200 g) was blended, centrifuged, filtered, and centrifuged again at higher speed at 4°C. Pellets were solubilised in calcium-free buffer containing 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS), and 0.2% soybean phosphatidylcholine with 100 μl of protease inhibitor cocktail. His-GST-FKBP12.6 (∼5 mg, made in-house) was added, followed by ultracentrifugation. The supernatant was incubated at 4°C with pre-equilibrated GS4B resin (Cytiva, Marlborough, MA, USA). RyR1 was eluted from the resin using TEV protease (made in-house). The eluents were further concentrated and purified with gel filtration using Superose 6 10/300 GL (Cytiva, Marlborough, MA, USA). Fractions containing RyR1 complexes measured by absorbance at 280 nm and were concentrated to ∼2 mg ml-1.

RyR1 was reconstituted into proteoliposomes as described.9 Briefly, a 5:3 mixture of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (Avanti Polar Lipids, Alabaster, AL, USA) were dried into a thin film and solubilised with 400 μl of rabbit RyR1 (0.7 mg ml−1). Following dialysis, the samples were aliquoted, flash-frozen in liquid nitrogen, and stored at −80°C.

Using the Orbit mini setup and EDR2 software (Nanion Technologies, Livingston, NJ, USA), recordings were obtained in parallel with multielectrode-cavity-array chips (Ionera Technologies, Freiburg, Germany). The cis and trans chambers contained symmetrical solutions of 250 mM HEPES, 150 mM KCl, 1 mM EGTA (pH 7.3), 0.2 mM CaCl2 ([Ca2+] free = 0.1 μM). To promote fusion to prepared suspended bilayers, 5% glycerol was incorporated into the proteoliposomes and 1–2 μl RyR1 proteoliposomes were added to the cis chamber. To further promote fusion, voltage was maintained at +40 mV. Recordings were started at the point of successful RyR1 insertion. Propofol was introduced to the cis chamber and concentrations determined by absorbance at 270 nm.10 All RyR1 measurements were conducted at 22°C and constant voltage of –60 mV. Recordings were filtered at final bandwidth of 10 kHz. Clampfit software (10.6, Molecular Devices, San Jose, CA, USA) was used to analyse current traces and only channels with a conductance > 700 pS were included in the analysis.11

Human skeletal muscle cells were maintained in growth medium in a 5% CO2 atmosphere at 37°C. After passage and to induce differentiation, plated cells were incubated overnight in growth medium, then in differentiation medium, the latter changed every other day. Multinuclear myotubes typically formed within 5–6 days. Differentiated myotubes were loaded with 1.5 μM Fura-2 AM marker in 20% bovine serum albumin (BSA) and incubated for 15 min to allow de-esterification. Fura-2 binds to intracellular Ca2+, with the ratio of emission at 340 nm and 380 nm directly related to the concentration of Ca2+.12, 13, 14 Emission ratio was measured using a fluorescent microscope with a cooled high-speed digital video camera and MetaFluor software (version 7.10.4.407, MetaMorph 2020, Molecular Devices, LLC, San Jose, CA, USA). Changes in Fura-2 fluorescence were measured for each drug concentration (n=40–50 cells): ryanodine (2–1000 nM) or propofol (2–300 μM). Data were normalised to the maximal response of cells, and IC50 calculated by fitting to Hill curves using PRISM 10 software (GraphPad Software, San Diego, CA, USA).

AziPm (5 μM) was added (with and without 200 μM propofol to determine specificity) to purified RyR1-FKBP12.6 at a final protein concentration of 1 μg μl−1. Samples were equilibrated on ice in the dark for 5 min then irradiated for 30 min at 350 nm with an RPR-3000 Rayonet lamp in 1-mm path length quartz cuvettes through a 295-nm glass filter (Newport Corporation, Franklin, MA, USA).

After UV exposure, proteins were precipitated in acetone, pelleted, washed, and air-dried before resuspension in 50 mM Tris–HCl, pH 8.0, 1% Triton X-100, and 0.5% SDS. Insoluble debris was pelleted and resuspended in NH4HCO3. Samples were treated with dithiothreitol (DTT) and iodoacetamide (IAA) before sequencing-grade modified trypsin was added at a 1:20 protease/protein ratio (w/w) with additional of 0.2% (w/v%) ProteaseMAX (Promega, Madison, WI, USA) surfactant. Proteins were digested and then diluted with NH4HCO3 and 0.02% ProteaseMAX surfactant before the addition of sequencing-grade chymotrypsin at 1:20 protease/protein ratio (w/w). Proteins were digested and acidified before centrifugation to remove insoluble debris. Finally, the sample was desalted using C18 stage tips, dried under vacuum and resuspended in 0.1% formic acid before mass spectrometry.

Photolabelled proteins were separated by SDS-PAGE; the rRyR1 band was excised, destained, dehydrated, and dried before proteins were reduced by 5 mM DTT and 50 mM NH4HCO3. Samples were then alkylated with 55 mM IAA in 50 mM NH4HCO3, dehydrated, and dried before resuspension in 0.2% ProteaseMAX surfactant and 50 mM NH4HCO3. After this point the digestion, suspension, and extraction were essentially identical to the above in-solution scheme.

Mass spectrometry was performed as reported.15 Briefly, desalted peptides were injected into a Thermo LTQ Orbitrap XL Mass Spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) or an Orbitrap Elite Hybrid Ion Trap mass spectrometer. Peptides were eluted with 100 min with linear gradients of acetonitrile in 0.1% formic acid. Spectral analysis was conducted using MaxQuant16 to search b and y ions against the rRyR1 sequence. All analyses included dynamic oxidation of methionine (+15.9949 m/z) as well as static alkylation of cysteine (+57.0215 m/z; iodoacetamide alkylation). Photolabelled peptides were searched for the additional dynamic AziPm modifications. Both in-solution and in-gel sequential trypsin/chymotrypsin digests were searched without enzyme specification with a false discovery rate of 0.01. Samples were analysed in triplicate and samples containing no photoaffinity ligand (controls) were run to identify false positives.

We used cryo-electron microscopy models (PDB: 6X34, open state; and 6X36, closed-state) of pig R615C RyR1.9 Only the central pore domain of RyR1, itself a functional channel,17 was simulated, as the entire protein is computationally impractical for demanding free energy perturbation (FEP) molecular dynamics (MD) simulations. Systems were constructed using CHARMM-GUI.18 All simulations were conducted using NAMD 2.14 or 319 with CHARMM36 force field, existing propofol parameters20, 21, 22 and TIP3P water. Production simulations were conducted in the isothermic–isobaric ensemble with Langevin thermostat. The lipid bilayers consisted of 70% 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 30% cholesterol.

We measured [3H]ryanodine binding, reflecting the proportion of open RyR1 in HSR vesicles, as a function of m-azipropofol (AziPm) and propofol concentrations.8 At low [Ca2+] (pCa 7, 100 nM), AziPm and propofol each reduced the proportion of open channels, with IC50 values of 6.9 μM and 5.8 μM, respectively. At activating [Ca2+] (pCa 5, 10 μM), a similar trend was observed, with IC50 values of 4.8 μM and 6.7 μM, respectively (Fig. 1). With AziPm, pCa 7 was a sufficient activating concentration to observe its inhibitory effects.Fig 1[3H]Ryanodine binding to skeletal muscle heavy sarcoplasmic reticulum vesicles as a function of propofol (left) or AziPm concentration (right). Values expressed as percentage of control (0 μM propofol or AziPm).Fig 1 [3H]Ryanodine binding to skeletal muscle heavy sarcoplasmic reticulum vesicles as a function of propofol (left) or AziPm concentration (right). Values expressed as percentage of control (0 μM propofol or AziPm).

We measured [3H]ryanodine binding, reflecting the proportion of open RyR1 in HSR vesicles, as a function of m-azipropofol (AziPm) and propofol concentrations.8 At low [Ca2+] (pCa 7, 100 nM), AziPm and propofol each reduced the proportion of open channels, with IC50 values of 6.9 μM and 5.8 μM, respectively. At activating [Ca2+] (pCa 5, 10 μM), a similar trend was observed, with IC50 values of 4.8 μM and 6.7 μM, respectively (Fig. 1). With AziPm, pCa 7 was a sufficient activating concentration to observe its inhibitory effects.Fig 1[3H]Ryanodine binding to skeletal muscle heavy sarcoplasmic reticulum vesicles as a function of propofol (left) or AziPm concentration (right). Values expressed as percentage of control (0 μM propofol or AziPm).Fig 1 [3H]Ryanodine binding to skeletal muscle heavy sarcoplasmic reticulum vesicles as a function of propofol (left) or AziPm concentration (right). Values expressed as percentage of control (0 μM propofol or AziPm). The R615C mutation predisposes pigs to MH-like porcine stress syndrome,25 serving as a model of human MH; the analogous human mutation is R614C. We measured the channel open probability of homozygous pig R615C RyR1 reconstituted in planar lipid bilayers (PLBs) as a function of AziPm and propofol concentrations with an activating concentration of Ca2+ (40 μM). Without drug, channel open probability was 0.11. This decreased, respectively, to 0.03, 0.07, and 0.04 with 10 μM AziPm, 10 μM propofol, and 30 μM propofol (Fig. 2).Fig 2Ryanodine receptor 1 channel opening probability as a function of propofol and AziPm concentration in phospholipid bilayers. ∗∗P<0.01 vs control, Mann-Whitney test (n=5).Fig 2

n probability was 0.11. This decreased, respectively, to 0.03, 0.07, and 0.04 with 10 μM AziPm, 10 μM propofol, and 30 μM propofol (Fig. 2).Fig 2Ryanodine receptor 1 channel opening probability as a function of propofol and AziPm concentration in phospholipid bilayers. ∗∗P<0.01 vs control, Mann-Whitney test (n=5).Fig 2 Ryanodine receptor 1 channel opening probability as a function of propofol and AziPm concentration in phospholipid bilayers. ∗∗P<0.01 vs control, Mann-Whitney test (n=5). We studied the effect of propofol on intracellular Ca2+ concentration in wild-type human skeletal muscle myotubes (HSM) in the presence of the RyR1 activator ryanodine. Higher 340:380 nm ratios measured using Fura-2 Ca2+ imaging indicate increased Ca2+ release attributable to RyR1 opening. Without propofol, the 340:380 nm ratio saturated at ∼ 1 μM ryanodine (Fig. 3), indicating that ryanodine at 1 μM is maximally potent. When propofol was added to HSM Fura-2 preparations containing 1 μM ryanodine, the 340:380 nm ratio decreased as propofol concentration increased, suggesting propofol inhibits RyR1 opening (mean IC50 = 29.5 μM [se=0.1 μM]).Fig 3A 340:380 nm ratio with activators and ryanodine as a function of propofol concentration. Greater open probability with higher ryanodine concentration is implied (left). At a constant concentration of ryanodine, increasing propofol concentration results in lower open probability.Fig 3

n IC50 = 29.5 μM [se=0.1 μM]).Fig 3A 340:380 nm ratio with activators and ryanodine as a function of propofol concentration. Greater open probability with higher ryanodine concentration is implied (left). At a constant concentration of ryanodine, increasing propofol concentration results in lower open probability.Fig 3 A 340:380 nm ratio with activators and ryanodine as a function of propofol concentration. Greater open probability with higher ryanodine concentration is implied (left). At a constant concentration of ryanodine, increasing propofol concentration results in lower open probability.

n IC50 = 29.5 μM [se=0.1 μM]).Fig 3A 340:380 nm ratio with activators and ryanodine as a function of propofol concentration. Greater open probability with higher ryanodine concentration is implied (left). At a constant concentration of ryanodine, increasing propofol concentration results in lower open probability.Fig 3 A 340:380 nm ratio with activators and ryanodine as a function of propofol concentration. Greater open probability with higher ryanodine concentration is implied (left). At a constant concentration of ryanodine, increasing propofol concentration results in lower open probability. AziPm is chemically and functionally similar to propofol,26 so photoadducted residues likely indicate propofol binding sites. Photoaffinity labelling was conducted with RyR1 purified from skeletal muscle of wild-type rabbit (Oryctolagus cuniculus), wild-type pig (Sus scrofa), and R615C pig. Rabbit and pig RyR1 have 97% sequence identity with human RyR1; most nonidentical residues reside outside the transmembrane domain. A preparation of RyR1 and FKBP12.6 (calstabin-2, which stabilises the closed state likely favoured by propofol and AziPm) was incubated with AziPm and irradiated.26 Photoadducted RyR1 residues were identified using mass spectrometry. Sequence coverage was 83%, 87.5%, and 80.2% across rabbit, pig WT, and pig R615C proteins, respectively. Coverage maps and spectra are in Supplementary Figures S2–S7. No photolabelled peptides were identified in nonphotolabelled control samples. Pretreatment with 200 μM propofol prevented adduction by AziPm in most sites (Supplementary Tables S1 - S3) implying AziPm and propofol binding sites are similar. Taken together with the PLB data, these data suggest that AziPm and propofol bind and act in the same locations on RyR1. The identified sites are listed in Table 1 and depicted in Figure 4. Unless otherwise noted, we refer to all photoadducted sites by their sequence locations in the rabbit RyR1 unless present only in pig RyR1. Many of the sites are adjacent to functionally significant RyR1 regions.27Table 1AziPm photoadducted sites in ryanodine receptor 1. WT, wild type.Table 1DomainRabbitPig WTPig R615CNotesCytoplasmicV1689V1689Junctional solenoid (JSol)T2069L2068R2072JSolI2183I2183Bridging solenoid (BSol)M2440BSolC2555BSolCore solenoidM3638M3634M3634BSolL3798L3793L3793Central solenoid (CSol)I4058I4053I4053CSolD4220I4213I4213Thumb-and-forefinger (TaF), near ATP binding siteTransmembraneY4554L4553L4553Pseudo voltage sensing domain (pVSD)F4568A4572A4572S1Y4715Y4713Y4713S2–S3 linker; near caffeine siteI4737I4735I4735S2–S3 linker; caffeine siteL4827V4828I4824S4–S5 linkerV4828L4850L4848L4848S5L4909L4909PoreFig 4(a) Ryanodine receptor 1 (RyR1) residues photoadducted by AziPm, shown as black spheres.

embraneY4554L4553L4553Pseudo voltage sensing domain (pVSD)F4568A4572A4572S1Y4715Y4713Y4713S2–S3 linker; near caffeine siteI4737I4735I4735S2–S3 linker; caffeine siteL4827V4828I4824S4–S5 linkerV4828L4850L4848L4848S5L4909L4909PoreFig 4(a) Ryanodine receptor 1 (RyR1) residues photoadducted by AziPm, shown as black spheres. For visual clarity only two of the four monomers are shown. (b) V4828 site in open-state RyR1 occupied by propofol. (c) V4828 site in closed-state RyR1 occupied by propofol. Representative poses taken from equilibrium molecular dynamics simulations.Fig 4 AziPm photoadducted sites in ryanodine receptor 1. WT, wild type. (a) Ryanodine receptor 1 (RyR1) residues photoadducted by AziPm, shown as black spheres. For visual clarity only two of the four monomers are shown. (b) V4828 site in open-state RyR1 occupied by propofol. (c) V4828 site in closed-state RyR1 occupied by propofol. Representative poses taken from equilibrium molecular dynamics simulations.

ptor 1 (RyR1) residues photoadducted by AziPm, shown as black spheres. For visual clarity only two of the four monomers are shown. (b) V4828 site in open-state RyR1 occupied by propofol. (c) V4828 site in closed-state RyR1 occupied by propofol. Representative poses taken from equilibrium molecular dynamics simulations. The photoadducted V4828 residue in the S4–S5 linker forms part of a binding pocket surrounded by lipophilic α-helices, and is adjacent to the pore lumen (Fig. 1). Mutations in this region (T4825I, H4832Y in rabbit) increase Ca2+ affinities for activation and decrease it for deactivation in vitro: channel open probabilities are increased at both low and high [Ca2+], but not at intermediate concentrations,27 with predicted affinity similar to clinical concentration. The specific photoadducted atom (e.g. side chain or carbonyl) is by definition not identified. As neither the bound pose of the nonphotolysed parent ligand nor its affinity are determined experimentally, we used MD simulation to predict these. Because it would be computationally prohibitive to simulate the entire RyR1, we included only the pore-containing transmembrane domain (TMD, residues 4546–5029), itself a functional channel,17 embedded in a lipid bilayer surrounded by 0.15 M KCl. We initialised each system by manually placing a single propofol molecule in one V4828 pocket followed by minimisation and equilibration MD simulation. This approach imposes no energetic penalty while the ligand is in the pocket, allowing it to locate a local energetic minimum.28

lipid bilayer surrounded by 0.15 M KCl. We initialised each system by manually placing a single propofol molecule in one V4828 pocket followed by minimisation and equilibration MD simulation. This approach imposes no energetic penalty while the ligand is in the pocket, allowing it to locate a local energetic minimum.28 In 50 ns production MD simulations, propofol remained in a stable orientation (Fig. 4). In both open- and closed-state RyR1, its hydroxyl group remained oriented away from V4828, although with different orientations. We used SAFEP MD23 to predict its binding affinity, with aqueous propofol as the unbound reference point. The predicted Gibbs free energy of propofol binding to open-state RyR1 was ΔGbind∘ = –5.9 [se 0.1 kcal mol−1], corresponding to KD = 55.8 μM (95% confidence interval [CI]: 40.3–77.3 μM). This includes aqueous phase decoupling energy ΔGbulk∗ = 1.0 kcal mol−1, restraint correction ΔGDBC = 0.9 [sd 0.1 kcal mol−1], and volumetric correction ΔGV∘ = 0.4 kcal mol−1. For closed-state RyR1, ΔGbind∘ = –8.4 kcal mol−1 [se 0.1 kcal mol−1], corresponding to KD = 1.4 μM (95% CI: 1.0–2.0 μM), with ΔGDBC = 1.6 kcal mol−1 [sd 0.1 kcal mol−1] and the same ΔGbulk∗ and ΔGV∘. Convergence was excellent (Fig. S1 in the supplementary material). As KD depends logarithmically on ΔG, small changes in ΔG lead to large changes in KD.

The R615C mutation predisposes pigs to MH-like porcine stress syndrome,25 serving as a model of human MH; the analogous human mutation is R614C. We measured the channel open probability of homozygous pig R615C RyR1 reconstituted in planar lipid bilayers (PLBs) as a function of AziPm and propofol concentrations with an activating concentration of Ca2+ (40 μM). Without drug, channel open probability was 0.11. This decreased, respectively, to 0.03, 0.07, and 0.04 with 10 μM AziPm, 10 μM propofol, and 30 μM propofol (Fig. 2).Fig 2Ryanodine receptor 1 channel opening probability as a function of propofol and AziPm concentration in phospholipid bilayers. ∗∗P<0.01 vs control, Mann-Whitney test (n=5).Fig 2 Ryanodine receptor 1 channel opening probability as a function of propofol and AziPm concentration in phospholipid bilayers. ∗∗P<0.01 vs control, Mann-Whitney test (n=5).

We studied the effect of propofol on intracellular Ca2+ concentration in wild-type human skeletal muscle myotubes (HSM) in the presence of the RyR1 activator ryanodine. Higher 340:380 nm ratios measured using Fura-2 Ca2+ imaging indicate increased Ca2+ release attributable to RyR1 opening. Without propofol, the 340:380 nm ratio saturated at ∼ 1 μM ryanodine (Fig. 3), indicating that ryanodine at 1 μM is maximally potent. When propofol was added to HSM Fura-2 preparations containing 1 μM ryanodine, the 340:380 nm ratio decreased as propofol concentration increased, suggesting propofol inhibits RyR1 opening (mean IC50 = 29.5 μM [se=0.1 μM]).Fig 3A 340:380 nm ratio with activators and ryanodine as a function of propofol concentration. Greater open probability with higher ryanodine concentration is implied (left). At a constant concentration of ryanodine, increasing propofol concentration results in lower open probability.Fig 3 A 340:380 nm ratio with activators and ryanodine as a function of propofol concentration. Greater open probability with higher ryanodine concentration is implied (left). At a constant concentration of ryanodine, increasing propofol concentration results in lower open probability.

AziPm is chemically and functionally similar to propofol,26 so photoadducted residues likely indicate propofol binding sites. Photoaffinity labelling was conducted with RyR1 purified from skeletal muscle of wild-type rabbit (Oryctolagus cuniculus), wild-type pig (Sus scrofa), and R615C pig. Rabbit and pig RyR1 have 97% sequence identity with human RyR1; most nonidentical residues reside outside the transmembrane domain. A preparation of RyR1 and FKBP12.6 (calstabin-2, which stabilises the closed state likely favoured by propofol and AziPm) was incubated with AziPm and irradiated.26 Photoadducted RyR1 residues were identified using mass spectrometry. Sequence coverage was 83%, 87.5%, and 80.2% across rabbit, pig WT, and pig R615C proteins, respectively. Coverage maps and spectra are in Supplementary Figures S2–S7. No photolabelled peptides were identified in nonphotolabelled control samples. Pretreatment with 200 μM propofol prevented adduction by AziPm in most sites (Supplementary Tables S1 - S3) implying AziPm and propofol binding sites are similar. Taken together with the PLB data, these data suggest that AziPm and propofol bind and act in the same locations on RyR1. The identified sites are listed in Table 1 and depicted in Figure 4. Unless otherwise noted, we refer to all photoadducted sites by their sequence locations in the rabbit RyR1 unless present only in pig RyR1. Many of the sites are adjacent to functionally significant RyR1 regions.27Table 1AziPm photoadducted sites in ryanodine receptor 1. WT, wild type.Table 1DomainRabbitPig WTPig R615CNotesCytoplasmicV1689V1689Junctional solenoid (JSol)T2069L2068R2072JSolI2183I2183Bridging solenoid (BSol)M2440BSolC2555BSolCore solenoidM3638M3634M3634BSolL3798L3793L3793Central solenoid (CSol)I4058I4053I4053CSolD4220I4213I4213Thumb-and-forefinger (TaF), near ATP binding siteTransmembraneY4554L4553L4553Pseudo voltage sensing domain (pVSD)F4568A4572A4572S1Y4715Y4713Y4713S2–S3 linker; near caffeine siteI4737I4735I4735S2–S3 linker; caffeine siteL4827V4828I4824S4–S5 linkerV4828L4850L4848L4848S5L4909L4909PoreFig 4(a) Ryanodine receptor 1 (RyR1) residues photoadducted by AziPm, shown as black spheres.

The photoadducted V4828 residue in the S4–S5 linker forms part of a binding pocket surrounded by lipophilic α-helices, and is adjacent to the pore lumen (Fig. 1). Mutations in this region (T4825I, H4832Y in rabbit) increase Ca2+ affinities for activation and decrease it for deactivation in vitro: channel open probabilities are increased at both low and high [Ca2+], but not at intermediate concentrations,27 with predicted affinity similar to clinical concentration. The specific photoadducted atom (e.g. side chain or carbonyl) is by definition not identified. As neither the bound pose of the nonphotolysed parent ligand nor its affinity are determined experimentally, we used MD simulation to predict these. Because it would be computationally prohibitive to simulate the entire RyR1, we included only the pore-containing transmembrane domain (TMD, residues 4546–5029), itself a functional channel,17 embedded in a lipid bilayer surrounded by 0.15 M KCl. We initialised each system by manually placing a single propofol molecule in one V4828 pocket followed by minimisation and equilibration MD simulation. This approach imposes no energetic penalty while the ligand is in the pocket, allowing it to locate a local energetic minimum.28

Propofol decreases RyR1 open probability in PLBs and HSM and inhibits [3H]ryanodine binding to SR vesicles. Photoaffinity labelling identified several propofol binding sites on RyR1. Overall, these data show that propofol both binds RyR1 in specific sites and inhibits pore opening. The plasma concentration of propofol at human loss of consciousness was estimated29 to be ∼10 μM, within an order of magnitude of our IC50 for both propofol inhibition of RyR1 in HSM and predicted binding affinity in the V4828 pocket. Prior studies showed RyR1 inhibition only at high propofol concentrations, which we believe is a result of incomplete solubilisation and lack of free propofol concentration measurements, allowing the possibility of differential binding between sites and unknown allosteric interactions among them.30, 31, 32 All our different approaches agree with respect to concentration dependence. As propofol avidly binds serum proteins,33 the plasma concentration required to elicit RyR1 inhibition would be higher than in an otherwise protein-free environment as with our HSM experiments.

own allosteric interactions among them.30, 31, 32 All our different approaches agree with respect to concentration dependence. As propofol avidly binds serum proteins,33 the plasma concentration required to elicit RyR1 inhibition would be higher than in an otherwise protein-free environment as with our HSM experiments. In all four subunits, the RyR1 TMD has at least six transmembrane helices of which four encode a (pseudo-)voltage-sensing domain, similar to the inositol-3,4,5-triphosphate (IP3) receptor and voltage-gated ion channels in the Nav, Kv, and Cav families. Propofol inhibition of channels with TMDs similar to that of the RyR1 is not unique.34, 35, 36 Moreover, binding in the conserved S4–5 linker domain appears to be a canonical feature in ion channels such as NaChBac, NavMs, and Kv1.2.34,37 Because propofol binding sites are distributed across this enormous protein, we hypothesise that an allosteric mechanism at least partly underlies its effects. For example, disruption of subunit cooperativity may be important for the function of RyR2.38

al feature in ion channels such as NaChBac, NavMs, and Kv1.2.34,37 Because propofol binding sites are distributed across this enormous protein, we hypothesise that an allosteric mechanism at least partly underlies its effects. For example, disruption of subunit cooperativity may be important for the function of RyR2.38 This study has some limitations. As we did not evaluate a functional model of MH using intact muscle, our prediction that propofol inhibits the clinical presentation of MH is hypothetical and may be trigger-dependent. For example, propofol was unable to reverse heat generation in MH pigs exposed to a high concentration of halothane, a strong trigger.39 Further, AziPm might adduct sites that the parent ligand propofol does not bind, or vice versa, as the diazirine group and halogens render it chemically distinct from the parent ligand, and because the RyR1 sequence was incompletely covered. However, at least in apoferritin, photolabelling, crystallography and fluorescence competition placed AziPm and propofol in the same site.26 It is reassuring that identical or highly analogous residues were adducted in RyR1 purified from three different sources, and no apparent photochemical selectivity for specific residues was observed.

apoferritin, photolabelling, crystallography and fluorescence competition placed AziPm and propofol in the same site.26 It is reassuring that identical or highly analogous residues were adducted in RyR1 purified from three different sources, and no apparent photochemical selectivity for specific residues was observed. We compared photoadduction in wild-type and the only readily available mutant protein, pig R615C RyR1. Despite substantial global conformational changes induced by the mutation,9 the photoadducted residues were the same with one exception (L2068 in WT vs R2072 in R615C). This suggests that the presence of the R615C mutation decreases the energy barrier for RyR1 activation, rather than altering anaesthetic binding. With the caveat that true in vivo RyR1 effect-site concentrations resulting from clinically relevant propofol doses are unknown and therefore not directly comparable to IC50 and KD values reported here, our results invite the hypothesis that clinical manifestations of MH would be actively inhibited by propofol boluses or infusions, subject to its pharmacokinetics. Skeletal muscle weakness from RyR1 inhibition could conceivably result from propofol administration in both wild-type and mutant RyR1, which might partly explain clinically observed muscle relaxation. However, results from human testing are mixed.40,41

by propofol boluses or infusions, subject to its pharmacokinetics. Skeletal muscle weakness from RyR1 inhibition could conceivably result from propofol administration in both wild-type and mutant RyR1, which might partly explain clinically observed muscle relaxation. However, results from human testing are mixed.40,41 Our findings potentially transfer to other disease states arising from increased RyR1 activation. For example, calcium dysregulation could be an upstream mechanism in neurodegenerative disorders. As all three RyR1 subtypes: RyR1, RyR2, and RyR3, are present in neuronal endoplasmic reticulum, RyR channel dysfunction is correlated to disease progression.42, 43, 44 Thus, in addition to potential salutary effects in MH, this invites the hypothesis that a propofol-based anaesthetic is less likely to aggravate these disorders than known triggering anaesthetics.

National Institute of General Medical Sciences, US 10.13039/100000002National Institutes of Health (R01GM135633 to RGE, FVP, TTJ; K08GM139031 to TTJ). TTJ was supported by the 10.13039/100005831Foundation for Anesthesia Education and Research (MRTG-BS-Joseph).