Publications by authors named "Chiara D"

57 Publications

Aicardi Syndrome: Key Fetal MRI Features and Prenatal Differential Diagnosis.

Neuropediatrics 2020 08 3;51(4):276-285. Epub 2020 Jul 3.

Department of Pediatric Radiology and Neuroradiology, Children's Hospital V. Buzzi, Milan, Italy.

Objective: This study was aimed to investigate the prenatal findings in Aicardi syndrome (AIC) by intrauterine magnetic resonance imaging (iuMRI) suggesting possible diagnostic criteria and differential diagnosis.

Methods: The iuMRI features of nine AIC confirmed cases were described and then compared with those of postnatal MRI. Furthermore, all iuMRI cases with both corpus callosum (CC) agenesis-dysgenesis and cortical malformation (AIC mimickers) were retrospectively reviewed and compared with iuMRI AIC cases, in order to identify possible neuroradiological predictors of AIC syndrome. For this purpose, Chi-square statistic and binary logistic regression analysis were performed.

Results: In all AIC cases, iuMRI was able to detect CC agenesis-dysgenesis and cortical development anomalies. Postnatal MRI revealed some additional findings mainly including further cystic lesions and in two cases small coloboma. A statistically significant difference between AIC and AIC mimicker were found regarding sex, nodular heterotopias, posterior fossa abnormalities, coloboma, and cortical gyration abnormalities. The most predictive variables in the logistic regression model were cortical gyration abnormalities, coloboma, and sex.

Conclusion: The iuMRI findings may suggest prenatal diagnosis of AIC syndrome with significant impact on parental counseling. Among possible differential diagnoses, tubulinopathies emerged.
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http://dx.doi.org/10.1055/s-0040-1710528DOI Listing
August 2020

Photoaffinity labeling identifies an intersubunit steroid-binding site in heteromeric GABA type A (GABA) receptors.

J Biol Chem 2020 08 15;295(33):11495-11512. Epub 2020 Jun 15.

Department of Neurobiology, Harvard Medical School, Boston, Massachusetts

Allopregnanolone (3α5α-P), pregnanolone, and their synthetic derivatives are potent positive allosteric modulators (PAMs) of GABA receptors (GABARs) with anesthetic, anxiolytic, and anti-convulsant effects. Mutational analysis, photoaffinity labeling, and structural studies have provided evidence for intersubunit and intrasubunit steroid-binding sites in the GABAR transmembrane domain, but revealed only little definition of their binding properties. Here, we identified steroid-binding sites in purified human α1β3 and α1β3γ2 GABARs by photoaffinity labeling with [H]21-[4-(3-(trifluoromethyl)-3H-diazirine-3-yl)benzoxy]allopregnanolone ([H]21-TFDBzox-AP), a potent GABAR PAM. Protein microsequencing established 3α5α-P inhibitable photolabeling of amino acids near the cytoplasmic end of the β subunit M4 (β3Pro-415, β3Leu-417, and β3Thr-418) and M3 (β3Arg-309) helices located at the base of a pocket in the β-α subunit interface that extends to the level of αGln-242, a steroid sensitivity determinant in the αM1 helix. Competition photolabeling established that this site binds with high affinity a structurally diverse group of 3α-OH steroids that act as anesthetics, anti-epileptics, and anti-depressants. The presence of a 3α-OH was crucial: 3-acetylated, 3-deoxy, and 3-oxo analogs of 3α5α-P, as well as 3β-OH analogs that are GABAR antagonists, bound with at least 1000-fold lower affinity than 3α5α-P. Similarly, for GABAR PAMs with the C-20 carbonyl of 3α5α-P or pregnanolone reduced to a hydroxyl, binding affinity is reduced by 1,000-fold, whereas binding is retained after deoxygenation at the C-20 position. These results provide a first insight into the structure-activity relationship at the GABAR β-α subunit interface steroid-binding site and identify several steroid PAMs that act via other sites.
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http://dx.doi.org/10.1074/jbc.RA120.013452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7450100PMC
August 2020

GLOS: GLOve for Speech Recognition.

Annu Int Conf IEEE Eng Med Biol Soc 2019 Jul;2019:3319-3322

Technological advancements in the field of Biomedical Engineering have allowed impaired individuals to use assistive devices in order to improve their quality of life. In the case of deafblind subjects, who experience both visual and auditory loss, the majority of available assistive devices are invasive (e.g. cochlear implants). Non-invasive technological improvements are extremely limited, in part due to the lack of scientific research interest in dual sensory loss [1]. In this paper we aim to present GLOS, a low-cost and non-invasive device that will allow the deafblind individuals to comprehend speeches in real-time. The proposed wearable device uses off-the-shelf components such as a Raspberry Pi 3 board, a simple microphone, and haptic feedback vibrating disks. The recorded speech from the microphone is processed by the board and encoded into 5 haptic vibrating modules attached to a glove. Each haptic vibrating module is placed on a different finger of the right hand. The current available non-invasive solutions do not use speech and therefore they do not allow for live communication (e.g. MyVox [2], Sparsha [3] and Mobile Lorm Glove [4]) or require long procedures to convert the messages (e.g. Finger Braille Teaching System [5]). This new biomedical device aims at overcoming these limitations. The authors tested the device in a preliminary testing and it was shown that GLOS has an average accuracy of 91.67% when tested for the recognition of twenty encoded sentences. The authors were deprived of both visual and hearing inputs and were trained for half hour per day for a period of 30 days.
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http://dx.doi.org/10.1109/EMBC.2019.8857927DOI Listing
July 2019

Identifying Drugs that Bind Selectively to Intersubunit General Anesthetic Sites in the 132 GABAR Transmembrane Domain.

Mol Pharmacol 2019 06 5;95(6):615-628. Epub 2019 Apr 5.

Department of Neurobiology, Harvard Medical School, Boston, Massachusetts (S.S.J., D.C.C., J.B.C.); Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, Boston, Massachusetts (X.Z., K.W.M.); Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, Illinois (P.Y.S., K.S.B.); and the Departamento de Quimica Orgánica, Universidad Autónoma de Madrid, Madrid, Spain (C.J.-B., M.T.)

GABA receptors (GABARs) are targets for important classes of clinical agents (e.g., anxiolytics, anticonvulsants, and general anesthetics) that act as positive allosteric modulators (PAMs). Previously, using photoreactive analogs of etomidate ([H]azietomidate) and mephobarbital [[H]1-methyl-5-allyl-5-(-trifluoromethyl-diazirynylphenyl)barbituric acid ([H]-TFD-MPAB)], we identified two homologous but pharmacologically distinct classes of general anesthetic binding sites in the 132 GABAR transmembrane domain at - ( sites) and -/- ( sites) subunit interfaces. We now use competition photolabeling with [H]azietomidate and [H]TFD-MPAB to identify -substituted propofol analogs and other drugs that bind selectively to intersubunit anesthetic sites. Propofol and 4-chloro-propofol bind with 5-fold selectivity to , while derivatives with bulkier lipophilic substitutions [4-(-butyl)-propofol and 4-(hydroxyl(phenyl)methyl)-propofol] bind with ∼10-fold higher affinity to sites. Similar to TFD-MPAB and propofol, these drugs bind in the presence of GABA with similar affinity to the - and - sites. However, we discovered four compounds that bind with different affinities to the two interface sites. Two of these bind with higher affinity to one of the sites than to the sites. We deduce that 4-benzoyl-propofol binds with >100-fold higher affinity to the - site than to the - or - sites, whereas loreclezole, an anticonvulsant, binds with 5- and 100-fold higher affinity to the - site than to the and - sites. These studies provide a first identification of PAMs that bind selectively to a single intersubunit site in the GABAR transmembrane domain, a property that may facilitate the development of subtype selective GABAR PAMs.
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http://dx.doi.org/10.1124/mol.118.114975DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6505378PMC
June 2019

A photoreactive analog of allopregnanolone enables identification of steroid-binding sites in a nicotinic acetylcholine receptor.

J Biol Chem 2019 05 28;294(19):7892-7903. Epub 2019 Mar 28.

From the Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115 and

Many neuroactive steroids potently and allosterically modulate pentameric ligand-gated ion channels, including GABA receptors (GABAR) and nicotinic acetylcholine receptors (nAChRs). Allopregnanolone and its synthetic analog alphaxalone are GABAR-positive allosteric modulators (PAMs), whereas alphaxalone and most neuroactive steroids are nAChR inhibitors. In this report, we used 11β-(-azidotetrafluorobenzoyloxy)allopregnanolone (FNBzoxy-AP), a general anesthetic and photoreactive allopregnanolone analog that is a potent GABAR PAM, to characterize steroid-binding sites in the αβγδ nAChR in its native membrane environment. We found that FNBzoxy-AP (IC = 31 μm) is 7-fold more potent than alphaxalone in inhibiting binding of the channel blocker [H]tenocyclidine to nAChRs in the desensitized state. At 300 μm, neither steroid inhibited binding of [H]tetracaine, a closed-state selective channel blocker, or of [H]acetylcholine. Photolabeling identified three distinct [H]FNBzoxy-AP-binding sites in the nAChR transmembrane domain: 1) in the ion channel, identified by photolabeling in the M2 helices of βVal-261 and δVal-269 (position M2-13'); 2) at the interface between the αM1 and αM4 helices, identified by photolabeling in αM1 (αCys-222/αLeu-223); and 3) at the lipid-protein interface involving γTrp-453 (M4), a residue photolabeled by small lipophilic probes and promegestone, a steroid nAChR antagonist. Photolabeling in the ion channel and αM1 was higher in the nAChR-desensitized state than in the resting state and inhibitable by promegestone. These results directly indicate a steroid-binding site in the nAChR ion channel and identify additional steroid-binding sites also occupied by other lipophilic nAChR antagonists.
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http://dx.doi.org/10.1074/jbc.RA118.007172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6514614PMC
May 2019

Inhibitable photolabeling by neurosteroid diazirine analog in the β3-Subunit of human hetereopentameric type A GABA receptors.

Eur J Med Chem 2019 Jan 19;162:810-824. Epub 2018 Nov 19.

Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 833 South Wood Street, Chicago, IL, 60612, USA. Electronic address:

Pregnanolone and allopregnanolone-type ligands exert general anesthetic, anticonvulsant and anxiolytic effects due to their positive modulatory interactions with the GABA receptors in the brain. Binding sites for these neurosteroids have been recently identified at subunit interfaces in the transmembrane domain (TMD) of homomeric β3 GABA receptors using photoaffinity labeling techniques, and in homomeric chimeric receptors containing GABA receptor α subunit TMDs by crystallography. Steroid binding sites have yet to be determined in human, heteromeric, functionally reconstituted, full-length, glycosylated GABA receptors. Here, we report on the synthesis and pharmacological characterization of several photoaffinity analogs of pregnanolone and allopregnanolone, of which 21-[4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzoxy]allopregnanolone (21-pTFDBzox-AP) was the most potent ligand. It is a partial positive modulator of the human α1β3 and α1β3γ2L GABA receptors at sub-micromolar concentrations. [H]21-pTFDBzox-AP photoincorporated in a pharmacologically specific manner into the α and β subunits of those receptors, with the β3 subunit photolabeled most efficiently. Importantly, photolabeling by [H]21-pTFDBzox-AP was inhibited by the positive steroid modulators alphaxalone, pregnanolone and allopregnanolone, but not by inhibitory neurosteroid pregnenolone sulfate or by two potent general anesthetics and GABAR positive allosteric modulators, etomidate and an anesthetic barbiturate. The latter two ligands bind to sites at subunit interfaces in the GABAR that are different from those interacting with neurosteroids. 21-pTFDBzox-AP's potency and pharmacological specificity of photolabeling indicate its suitability for characterizing neurosteroid binding sites in native GABA receptors.
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http://dx.doi.org/10.1016/j.ejmech.2018.11.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6912599PMC
January 2019

Photoaffinity Labeling of Pentameric Ligand-Gated Ion Channels: A Proteomic Approach to Identify Allosteric Modulator Binding Sites.

Methods Mol Biol 2017 ;1598:157-197

Department of Pharmaceutical Sciences, College of Pharmacy, Texas A&M Health Sciences Center, Kingsville, TX, USA.

Photoaffinity labeling techniques have been used for decades to identify drug binding sites and to study the structural biology of allosteric transitions in transmembrane proteins including pentameric ligand-gated ion channels (pLGIC). In a typical photoaffinity labeling experiment, to identify drug binding sites, UV light is used to introduce a covalent bond between a photoreactive ligand (which upon irradiation at the appropriate wavelength converts to a reactive intermediate) and amino acid residues that lie within its binding site. Then protein chemistry and peptide microsequencing techniques are used to identify these amino acids within the protein primary sequence. These amino acid residues are located within homology models of the receptor to identify the binding site of the photoreactive probe. Molecular modeling techniques are then used to model the binding of the photoreactive probe within the binding site using docking protocols. Photoaffinity labeling directly identifies amino acids that contribute to drug binding sites regardless of their location within the protein structure and distinguishes them from amino acids that are only involved in the transduction of the conformational changes mediated by the drug, but may not be part of its binding site (such as those identified by mutational studies). Major limitations of photoaffinity labeling include the availability of photoreactive ligands that faithfully mimic the properties of the parent molecule and protein preparations that supply large enough quantities suitable for photoaffinity labeling experiments. When the ligand of interest is not intrinsically photoreactive, chemical modifications to add a photoreactive group to the parent drug, and pharmacological evaluation of these chemical modifications become necessary. With few exceptions, expression and affinity-purification of proteins are required prior to photolabeling. Methods to isolate milligram quantities of highly enriched pLGIC suitable for photoaffinity labeling experiments have been developed. In this chapter, we discuss practical aspects of experimental strategies to identify allosteric modulator binding sites in pLGIC using photoaffinity labeling.
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http://dx.doi.org/10.1007/978-1-4939-6952-4_7DOI Listing
March 2018

Synthesis and pharmacological evaluation of neurosteroid photoaffinity ligands.

Eur J Med Chem 2017 Aug 21;136:334-347. Epub 2017 Apr 21.

Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 833 South Wood Street (M/C 781), Chicago, IL 60612-7231, USA. Electronic address:

Neuroactive steroids are potent positive allosteric modulators of GABA receptors (GABAR), but the locations of their GABAR binding sites remain poorly defined. To discover these sites, we synthesized two photoreactive analogs of alphaxalone, an anesthetic neurosteroid targeting GABAR, 11β-(4-azido-2,3,5,6-tetrafluorobenzoyloxy)allopregnanolone, (F4N3Bzoxy-AP) and 11-aziallopregnanolone (11-AziAP). Both photoprobes acted with equal or higher potency than alphaxalone as general anesthetics and potentiators of GABAR responses, left-shifting the GABA concentration - response curve for human α1β3γ2 GABARs expressed in Xenopus oocytes, and enhancing [H]muscimol binding to α1β3γ2 GABARs expressed in HEK293 cells. With EC of 110 nM, 11-AziAP is one the most potent general anesthetics reported. [H]F4N3Bzoxy-AP and [H]11-AziAP, at anesthetic concentrations, photoincorporated into α- and β-subunits of purified α1β3γ2 GABARs, but labeling at the subunit level was not inhibited by alphaxalone (30 μM). The enhancement of photolabeling by H-azietomidate and H-mTFD-MPAB in the presence of either of the two steroid photoprobes indicates the neurosteroid binding site is different from, but allosterically related to, the etomidate and barbiturate sites. Our observations are consistent with two hypotheses. First, F4N3Bzoxy-AP and 11-aziAP bind to a high affinity site in such a pose that the 11-photoactivatable moiety, that is rigidly attached to the steroid backbone, points away from the protein. Second, F4N3Bzoxy-AP, 11-aziAP and other steroid anesthetics, which are present at very high concentration at the lipid-protein interface due to their high lipophilicity, act via low affinity sites, as proposed by Akk et al. (Psychoneuroendocrinology2009, 34S1, S59-S66).
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http://dx.doi.org/10.1016/j.ejmech.2017.04.043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6071317PMC
August 2017

Unraveling amino acid residues critical for allosteric potentiation of (α4)3(β2)2-type nicotinic acetylcholine receptor responses.

J Biol Chem 2017 06 26;292(24):9988-10001. Epub 2017 Apr 26.

From the Department of Pharmaceutical Sciences, Texas A&M Health Sciences Center, Kingsville, Texas 78363,

Neuronal nicotinic acetylcholine receptors (nAChRs) are promising drug targets to manage several neurological disorders and nicotine addiction. Growing evidence indicates that positive allosteric modulators of nAChRs improve pharmacological specificity by binding to unique sites present only in a subpopulation of nAChRs. Furthermore, nAChR positive allosteric modulators such as NS9283 and CMPI have been shown to potentiate responses of (α4)3(β2)2 but not (α4)2(β2)3 nAChR isoforms. This selective potentiation underlines that the α4:α4 interface, which is present only in the (α4)3(β2)2 nAChR, is an important and promising drug target. In this report we used site-directed mutagenesis to substitute specific amino acid residues and computational analyses to elucidate CMPI's binding mode at the α4:α4 subunit extracellular interface and identified a unique set of amino acid residues that determined its affinity. We found that amino acid residues α4Gly-41, α4Lys-64, and α4Thr-66 were critical for (α4)3(β2)2 nAChR potentiation by CMPI, but not by NS9283, whereas amino acid substitution at α4His-116, a known determinant of NS9283 and of agonist binding at the α4:α4 subunit interface, did not reduce CMPI potentiation. In contrast, substitutions at α4Gln-124 and α4Thr-126 reduced potentiation by CMPI and NS9283, indicating that their binding sites partially overlap. These results delineate the role of amino acid residues contributing to the α4:α4 subunit extracellular interface in nAChR potentiation. These findings also provide structural information that will facilitate the structure-based design of novel therapeutics that target selectively the (α4)3(β2)2 nAChR.
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http://dx.doi.org/10.1074/jbc.M116.771246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5473250PMC
June 2017

General Anesthetic Binding Sites in Human α4β3δ γ-Aminobutyric Acid Type A Receptors (GABAARs).

J Biol Chem 2016 Dec 7;291(51):26529-26539. Epub 2016 Nov 7.

From the Departments of Neurobiology and

Extrasynaptic γ-aminobutyric acid type A receptors (GABARs),which contribute generalized inhibitory tone to the mammalian brain, are major targets for general anesthetics. To identify anesthetic binding sites in an extrasynaptic GABAR, we photolabeled human α4β3δ GABARs purified in detergent with [H]azietomidate and a barbiturate, [H]R-mTFD-MPAB, photoreactive anesthetics that bind with high selectivity to distinct but homologous intersubunit binding sites in the transmembrane domain of synaptic α1β3γ2 GABARs. Based upon H incorporation into receptor subunits resolved by SDS-PAGE, there was etomidate-inhibitable labeling by [H]azietomidate in the α4 and β3 subunits and barbiturate-inhibitable labeling by [H]R-mTFD-MPAB in the β3 subunit. These sites did not bind the anesthetic steroid alphaxalone, which enhanced photolabeling, or DS-2, a δ subunit-selective positive allosteric modulator, which neither enhanced nor inhibited photolabeling. The amino acids labeled by [H]azietomidate or [H]R-mTFD-MPAB were identified by N-terminal sequencing of fragments isolated by HPLC fractionation of enzymatically digested subunits. No evidence was found for a δ subunit contribution to an anesthetic binding site. [H]azietomidate photolabeling of β3Met-286 in βM3 and α4Met-269 in αM1 that was inhibited by etomidate but not by R-mTFD-MPAB established that etomidate binds to a site at the β3-α4 interface equivalent to its site in α1β3γ2 GABARs. [H]Azietomidate and [H]R-mTFD-MPAB photolabeling of β3Met-227 in βM1 established that these anesthetics also bind to a homologous site, most likely at the β3-β3 interface, which suggests a subunit arrangement of β3α4β3δβ3.
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http://dx.doi.org/10.1074/jbc.M116.753335DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5159512PMC
December 2016

Multimodality treatment of brain metastases from renal cell carcinoma in the era of targeted therapy.

Ther Adv Med Oncol 2016 Nov 25;8(6):450-459. Epub 2016 Jul 25.

Universita degli Studi di Roma La Sapienza, Roma, Italy Azienda Ospedaliera Sant'Andrea, Roma, Italy.

In patients with renal cancer, brain metastasis is associated with poor survival and high morbidity. Poor life expectancy is often associated with widespread extracranial metastases. In such patients, a multidisciplinary approach is paramount. Brain metastases-specific therapies may include surgery, radiosurgery, conventional radiation and targeted therapies (TT) or a combination of these treatments. Some factors are important prognostically when choosing the best strategy: performance status, the number, size and location of brain metastases, the extension of systemic metastases and a well-controlled primary tumour. Failure of chemical therapy has always been attributed to an intact blood-brain barrier and acquired drug resistance by renal cancer cells. Recent studies have demonstrated objective responses with TT in a variety of cancer types, including renal cancer. In most cases, these agents have been used in combination and in conjunction with whole-brain radiation therapy and radiosurgery. Local control appears to be better with the combined method if the patient has a good performance status and may improve overall survival. This review summarizes current literature data on multidisciplinary approach in the management of renal brain metastasis with radiation, surgery and TT with an emphasis on potential better outcomes with a combination of current treatment methods.
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http://dx.doi.org/10.1177/1758834016659825DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5066543PMC
November 2016

Multiple Non-Equivalent Interfaces Mediate Direct Activation of GABAA Receptors by Propofol.

Curr Neuropharmacol 2016 ;14(7):772-80

Department of Anesthesiology, Washington University, Campus Box 8054, 660 South Euclid Ave, St. Louis, MO 63110.

Background: Propofol is a sedative agent that at clinical concentrations acts by allosterically activating or potentiating the γ-aminobutyric acid type A (GABAA) receptor. Mutational, modeling, and photolabeling studies with propofol and its analogues have identified potential interaction sites in the transmembrane domain of the receptor. At the "+" of the β subunit, in the β-α interface, meta-azipropofol labels the M286 residue in the third transmembrane domain. Substitution of this residue with tryptophan results in loss of potentiation by propofol. At the "-" side of the β subunit, in the α-β interface (or β-β interface, in the case of homomeric β receptors), ortho-propofol diazirine labels the H267 residue in the second transmembrane domain. Structural modeling indicates that the β(H267) residue lines a cavity that docks propofol with favorable interaction energy.

Method: We used two-electrode voltage clamp to determine the functional effects of mutations to the "+" and "-" sides of the β subunit on activation of the α1β3 GABAA receptor by propofol.

Results: We found that while the individual mutations had a small effect, the combination of the M286W mutation with tryptophan mutations of selected residues at the α-β interface leads to strong reduction in gating efficacy for propofol.

Conclusion: We conclude that α1β3 GABAA receptors can be activated by propofol interactions with the β-β, α-β, and β-α interfaces, where distinct, non-equivalent regions control channel gating. Any interface can mediate activation, hence substitutions at all interfaces are required for loss of activation by propofol.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5050400PMC
http://dx.doi.org/10.2174/1570159x14666160202121319DOI Listing
January 2017

Positive and Negative Allosteric Modulation of an α1β3γ2 γ-Aminobutyric Acid Type A (GABAA) Receptor by Binding to a Site in the Transmembrane Domain at the γ+-β- Interface.

J Biol Chem 2015 Sep 30;290(38):23432-46. Epub 2015 Jul 30.

From the Departments of Neurobiology and

In the process of developing safer general anesthetics, isomers of anesthetic ethers and barbiturates have been discovered that act as convulsants and inhibitors of γ-aminobutyric acid type A receptors (GABAARs) rather than potentiators. It is unknown whether these convulsants act as negative allosteric modulators by binding to the intersubunit anesthetic-binding sites in the GABAAR transmembrane domain (Chiara, D. C., Jayakar, S. S., Zhou, X., Zhang, X., Savechenkov, P. Y., Bruzik, K. S., Miller, K. W., and Cohen, J. B. (2013) J. Biol. Chem. 288, 19343-19357) or to known convulsant sites in the ion channel or extracellular domains. Here, we show that S-1-methyl-5-propyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid (S-mTFD-MPPB), a photoreactive analog of the convulsant barbiturate S-MPPB, inhibits α1β3γ2 but potentiates α1β3 GABAAR responses. In the α1β3γ2 GABAAR, S-mTFD-MPPB binds in the transmembrane domain with high affinity to the γ(+)-β(-) subunit interface site with negative energetic coupling to GABA binding in the extracellular domain at the β(+)-α(-) subunit interfaces. GABA inhibits S-[(3)H]mTFD-MPPB photolabeling of γ2Ser-280 (γM2-15') in this site. In contrast, within the same site GABA enhances photolabeling of β3Met-227 in βM1 by an anesthetic barbiturate, R-[(3)H]methyl-5-allyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (mTFD-MPAB), which differs from S-mTFD-MPPB in structure only by chirality and two hydrogens (propyl versus allyl). S-mTFD-MPPB and R-mTFD-MPAB are predicted to bind in different orientations at the γ(+)-β(-) site, based upon the distance in GABAAR homology models between γ2Ser-280 and β3Met-227. These results provide an explanation for S-mTFD-MPPB inhibition of α1β3γ2 GABAAR function and provide a first demonstration that an intersubunit-binding site in the GABAAR transmembrane domain binds negative and positive allosteric modulators.
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http://dx.doi.org/10.1074/jbc.M115.672006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4645599PMC
September 2015

Anesthetics target interfacial transmembrane sites in nicotinic acetylcholine receptors.

Neuropharmacology 2015 Sep 12;96(Pt B):169-77. Epub 2014 Oct 12.

Dept. of Anesthesia Critical Care & Pain Medicine, Massachusetts General Hospital, Boston, 55 Fruit Street, MA 02114, USA; Dept. of Anaesthesia, Harvard Medical School, 25 Shattuck Street, Boston, MA 02115, USA. Electronic address:

General anesthetics are a heterogeneous group of small amphiphilic ligands that interact weakly at multiple allosteric sites on many pentameric ligand gated ion channels (pLGICs), resulting in either inhibition, potentiation of channel activity, or both. Allosteric principles imply that modulator sites must change configuration and ligand affinity during receptor state transitions. Thus, general anesthetics and related compounds are useful both as state-dependent probes of receptor structure and as potentially selective modulators of pLGIC functions. This review focuses on general anesthetic sites in nicotinic acetylcholine receptors, which were among the first anesthetic-sensitive pLGIC experimental models studied, with particular focus on sites formed by transmembrane domain elements. Structural models place many of these sites at interfaces between two or more pLGIC transmembrane helices both within subunits and between adjacent subunits, and between transmembrane helices and either lipids (the lipid-protein interface) or water (i.e. the ion channel). A single general anesthetic may bind at multiple allosteric sites in pLGICs, producing a net effect of either inhibition (e.g. blocking the ion channel) or enhanced channel gating (e.g. inter-subunit sites). Other general anesthetic sites identified by photolabeling or crystallography are tentatively linked to functional effects, including intra-subunit helix bundle sites and the lipid-protein interface. This article is part of the Special Issue entitled 'The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition'.
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http://dx.doi.org/10.1016/j.neuropharm.2014.10.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4394016PMC
September 2015

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

J Biol Chem 2014 Oct 1;289(40):27456-68. Epub 2014 Aug 1.

From the Departments of Neurobiology and

Propofol acts as a positive allosteric modulator of γ-aminobutyric acid type A receptors (GABAARs), an interaction necessary for its anesthetic potency in vivo as a general anesthetic. Identifying the location of propofol-binding sites is necessary to understand its mechanism of GABAAR modulation. [(3)H]2-(3-Methyl-3H-diaziren-3-yl)ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate (azietomidate) and R-[(3)H]5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (mTFD-MPAB), photoreactive analogs of 2-ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate (etomidate) and mephobarbital, respectively, have identified two homologous but pharmacologically distinct classes of intersubunit-binding sites for general anesthetics in the GABAAR transmembrane domain. Here, we use a photoreactive analog of propofol (2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol ([(3)H]AziPm)) to identify propofol-binding sites in heterologously expressed human α1β3 GABAARs. Propofol, AziPm, etomidate, and R-mTFD-MPAB each inhibited [(3)H]AziPm photoincorporation into GABAAR subunits maximally by ∼ 50%. When the amino acids photolabeled by [(3)H]AziPm were identified by protein microsequencing, we found propofol-inhibitable photolabeling of amino acids in the β3-α1 subunit interface (β3Met-286 in β3M3 and α1Met-236 in α1M1), previously photolabeled by [(3)H]azietomidate, and α1Ile-239, located one helical turn below α1Met-236. There was also propofol-inhibitable [(3)H]AziPm photolabeling of β3Met-227 in βM1, the amino acid in the α1-β3 subunit interface photolabeled by R-[(3)H]mTFD-MPAB. The propofol-inhibitable [(3)H]AziPm photolabeling in the GABAAR β3 subunit in conjunction with the concentration dependence of inhibition of that photolabeling by etomidate or R-mTFD-MPAB also establish that each anesthetic binds to the homologous site at the β3-β3 subunit interface. These results establish that AziPm as well as propofol bind to the homologous intersubunit sites in the GABAAR transmembrane domain that binds etomidate or R-mTFD-MPAB with high affinity.
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http://dx.doi.org/10.1074/jbc.M114.581728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4183786PMC
October 2014

Identifying barbiturate binding sites in a nicotinic acetylcholine receptor with [3H]allyl m-trifluoromethyldiazirine mephobarbital, a photoreactive barbiturate.

Mol Pharmacol 2014 May 21;85(5):735-46. Epub 2014 Feb 21.

Department of Neurobiology, Harvard Medical School, Boston, Massachusetts (A.K.H., D.C.C., J.B.C.); Department of Anesthesia, Critical Care, and Pain Medicine, Massachusetts General Hospital, Boston, Massachusetts (D.S.S.); and Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, Illinois (P.Y.S., K.S.B.).

At concentrations that produce anesthesia, many barbituric acid derivatives act as positive allosteric modulators of inhibitory GABAA receptors (GABAARs) and inhibitors of excitatory nicotinic acetylcholine receptors (nAChRs). Recent research on [(3)H]R-mTFD-MPAB ([(3)H]R-5-allyl-1-methyl-5-(m-trifluoromethyldiazirinylphenyl)barbituric acid), a photoreactive barbiturate that is a potent and stereoselective anesthetic and GABAAR potentiator, has identified a second class of intersubunit binding sites for general anesthetics in the α1β3γ2 GABAAR transmembrane domain. We now characterize mTFD-MPAB interactions with the Torpedo (muscle-type) nAChR. For nAChRs expressed in Xenopus oocytes, S- and R-mTFD-MPAB inhibited ACh-induced currents with IC50 values of 5 and 10 µM, respectively. Racemic mTFD-MPAB enhanced the equilibrium binding of [(3)H]ACh to nAChR-rich membranes (EC50 = 9 µM) and inhibited binding of the ion channel blocker [(3)H]tenocyclidine in the nAChR desensitized and resting states with IC50 values of 2 and 170 µM, respectively. Photoaffinity labeling identified two binding sites for [(3)H]R-mTFD-MPAB in the nAChR transmembrane domain: 1) a site within the ion channel, identified by photolabeling in the nAChR desensitized state of amino acids within the M2 helices of each nAChR subunit; and 2) a site at the γ-α subunit interface, identified by photolabeling of γMet299 within the γM3 helix at similar efficiency in the resting and desensitized states. These results establish that mTFD-MPAB is a potent nAChR inhibitor that binds in the ion channel preferentially in the desensitized state and binds with lower affinity to a site at the γ-α subunit interface where etomidate analogs bind that act as positive and negative nAChR modulators.
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http://dx.doi.org/10.1124/mol.113.090985DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3990015PMC
May 2014

Photoaffinity labeling the propofol binding site in GLIC.

Biochemistry 2014 Jan 30;53(1):135-42. Epub 2013 Dec 30.

Department of Neurobiology, Harvard Medical School , Boston, Massachusetts 02115, United States.

Propofol, an intravenous general anesthetic, produces many of its anesthetic effects in vivo by potentiating the responses of GABA type A receptors (GABAAR), members of the superfamily of pentameric ligand-gated ion channels (pLGICs) that contain anion-selective channels. Propofol also inhibits pLGICs containing cation-selective channels, including nicotinic acetylcholine receptors and GLIC, a prokaryotic proton-gated homologue from Gloeobacter violaceus . In the structure of GLIC cocrystallized with propofol at pH 4 (presumed open/desensitized states), propofol was localized to an intrasubunit pocket at the extracellular end of the transmembrane domain within the bundle of transmembrane α-helices (Nury, H, et al. (2011) Nature 469, 428-431). To identify propofol binding sites in GLIC in solution, we used a recently developed photoreactive propofol analogue (2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol or AziPm) that acts as an anesthetic in vivo and potentiates GABAAR in vitro. For GLIC expressed in Xenopus oocytes, propofol and AziPm inhibited current responses at pH 5.5 (EC20) with IC50 values of 20 and 50 μM, respectively. When [(3)H]AziPm (7 μM) was used to photolabel detergent-solubilized, affinity-purified GLIC at pH 4.4, protein microsequencing identified propofol-inhibitable photolabeling of three residues in the GLIC transmembrane domain: Met-205, Tyr-254, and Asn-307 in the M1, M3, and M4 transmembrane helices, respectively. Thus, for GLIC in solution, propofol and AziPm bind competitively to a site in proximity to these residues, which, in the GLIC crystal structure, are in contact with the propofol bound in the intrasubunit pocket.
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http://dx.doi.org/10.1021/bi401492kDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3935609PMC
January 2014

Photoaffinity labeling of nicotinic receptors: diversity of drug binding sites!

J Mol Neurosci 2014 Jul 26;53(3):480-6. Epub 2013 Oct 26.

Department of Neurobiology, Harvard Medical School, Boston, MA, 02115, USA,

For almost 30 years, photoaffinity labeling and protein microsequencing techniques have been providing novel insights about the structure of nicotinic acetylcholine receptors (nAChR) and the diversity of nAChR drug binding sites. Photoaffinity labeling allows direct identification of amino acid residues contributing to a drug binding site without prior knowledge of the location of the binding site within the nAChR or the orientation of the ligand within the binding site. It also distinguishes amino acids that contribute to allosteric binding sites from those involved in allosteric modulation of gating. While photoaffinity labeling was used initially to identify amino acids contributing to the agonist binding sites and the ion channel, it has been used recently to identify binding sites for allosteric modulators at subunit interfaces in the extracellular and the transmembrane domains, and within a subunit's transmembrane helix bundle. In this article, we review the different types of photoaffinity probes that have been used and the various binding sites that have been identified within the structure of nAChR, with emphasis on our recent studies of allosteric modulator binding sites.
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http://dx.doi.org/10.1007/s12031-013-0150-1DOI Listing
July 2014

Cysteine substitutions define etomidate binding and gating linkages in the α-M1 domain of γ-aminobutyric acid type A (GABAA) receptors.

J Biol Chem 2013 Oct 5;288(42):30373-30386. Epub 2013 Sep 5.

From the Department of Anesthesia Critical Care and Pain Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114,. Electronic address:

Etomidate is a potent general anesthetic that acts as an allosteric co-agonist at GABAA receptors. Photoreactive etomidate derivatives labeled αMet-236 in transmembrane domain M1, which structural models locate in the β+/α- subunit interface. Other nearby residues may also contribute to etomidate binding and/or transduction through rearrangement of the site. In human α1β2γ2L GABAA receptors, we applied the substituted cysteine accessibility method to α1-M1 domain residues extending from α1Gln-229 to α1Gln-242. We used electrophysiology to characterize each mutant's sensitivity to GABA and etomidate. We also measured rates of sulfhydryl modification by p-chloromercuribenzenesulfonate (pCMBS) with and without GABA and tested if etomidate blocks modification of pCMBS-accessible cysteines. Cys substitutions in the outer α1-M1 domain impaired GABA activation and variably affected etomidate sensitivity. In seven of eight residues where pCMBS modification was evident, rates of modification were accelerated by GABA co-application, indicating that channel activation increases water and/or pCMBS access. Etomidate reduced the rate of modification for cysteine substitutions at α1Met-236, α1Leu-232 and α1Thr-237. We infer that these residues, predicted to face β2-M3 or M2 domains, contribute to etomidate binding. Thus, etomidate interacts with a short segment of the outer α1-M1 helix within a subdomain that undergoes significant structural rearrangement during channel gating. Our results are consistent with in silico docking calculations in a homology model that orient the long axis of etomidate approximately orthogonal to the transmembrane axis.
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http://dx.doi.org/10.1074/jbc.M113.494583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3798502PMC
October 2013

Specificity of intersubunit general anesthetic-binding sites in the transmembrane domain of the human α1β3γ2 γ-aminobutyric acid type A (GABAA) receptor.

J Biol Chem 2013 Jul 15;288(27):19343-57. Epub 2013 May 15.

Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, USA.

GABA type A receptors (GABAAR), the brain's major inhibitory neurotransmitter receptors, are the targets for many general anesthetics, including volatile anesthetics, etomidate, propofol, and barbiturates. How such structurally diverse agents can act similarly as positive allosteric modulators of GABAARs remains unclear. Previously, photoreactive etomidate analogs identified two equivalent anesthetic-binding sites in the transmembrane domain at the β(+)-α(-) subunit interfaces, which also contain the GABA-binding sites in the extracellular domain. Here, we used R-[(3)H]5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid (R-mTFD-MPAB), a potent stereospecific barbiturate anesthetic, to photolabel expressed human α1β3γ2 GABAARs. Protein microsequencing revealed that R-[(3)H]mTFD-MPAB did not photolabel the etomidate sites at the β(+)-α(-) subunit interfaces. Instead, it photolabeled sites at the α(+)-β(-) and γ(+)-β(-) subunit interfaces in the transmembrane domain. On the (+)-side, α1M3 was labeled at Ala-291 and Tyr-294 and γ2M3 at Ser-301, and on the (-)-side, β3M1 was labeled at Met-227. These residues, like those in the etomidate site, are located at subunit interfaces near the synaptic side of the transmembrane domain. The selectivity of R-etomidate for the β(+)-α(-) interface relative to the α(+)-β(-)/γ(+)-β(-) interfaces was >100-fold, whereas that of R-mTFD-MPAB for its sites was >50-fold. Each ligand could enhance photoincorporation of the other, demonstrating allosteric interactions between the sites. The structural heterogeneity of barbiturate, etomidate, and propofol derivatives is accommodated by varying selectivities for these two classes of sites. We hypothesize that binding at any of these homologous intersubunit sites is sufficient for anesthetic action and that this explains to some degree the puzzling structural heterogeneity of anesthetics.
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http://dx.doi.org/10.1074/jbc.M113.479725DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3707639PMC
July 2013

Recurrent pericarditis: autoimmune or autoinflammatory?

Autoimmun Rev 2012 Nov 2;12(1):60-5. Epub 2012 Aug 2.

Internal Medicine, Ospedali Riuniti, Bergamo, Italy.

Idiopathic recurrent acute pericarditis (IRAP) represents the most troublesome complication of acute pericarditis and occurs in up to 20-50% of patients. It is generally idiopathic or postcardiac injury. IRAP is a disease of suspected immune-mediated pathogenesis. On the other hand, it has been suggested that some of these patients might have an atypical or subclinical form of an autoinflammatory disease, e.g. genetic disorders characterized by primary dysfunction of the innate immune system and caused by mutations of genes involved in the inflammatory response. We found that IRAP patients were negative for mutations associated with familial Mediterranean fever, but 6% (8/131 patients) carry a mutation in the TNFRSF1A gene, encoding the receptor for tumor necrosis factor-alfa. C-reactive protein (CRP) may be useful to follow the disease activity and guide the appropriate length of therapy, with continuation of the attack doses of the drugs until CRP normalization, at which time tapering may be considered. IRAP often needs a multidrug therapy: NSAIDs or aspirin at high dosages every 6-8h, corticosteroids only rarely, at low dosages and with a very gradual tapering (months) and colchicine at low dosages if tolerated. Anakinra could be a solution for patients who do not tolerate other therapies.
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http://dx.doi.org/10.1016/j.autrev.2012.07.023DOI Listing
November 2012

Allyl m-trifluoromethyldiazirine mephobarbital: an unusually potent enantioselective and photoreactive barbiturate general anesthetic.

J Med Chem 2012 Jul 17;55(14):6554-65. Epub 2012 Jul 17.

Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, 32 Fruit Street, Boston, MA 02114, USA.

We synthesized 5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (14), a trifluoromethyldiazirine-containing derivative of general anesthetic mephobarbital, separated the racemic mixture into enantiomers by chiral chromatography, and determined the configuration of the (+)-enantiomer as S by X-ray crystallography. Additionally, we obtained the (3)H-labeled ligand with high specific radioactivity. R-(-)-14 is an order of magnitude more potent than the most potent clinically used barbiturate, thiopental, and its general anesthetic EC(50) approaches those for propofol and etomidate, whereas S-(+)-14 is 10-fold less potent. Furthermore, at concentrations close to its anesthetic potency, R-(-)-14 both potentiated GABA-induced currents and increased the affinity for the agonist muscimol in human α1β2/3γ2L GABA(A) receptors. Finally, R-(-)-14 was found to be an exceptionally efficient photolabeling reagent, incorporating into both α1 and β3 subunits of human α1β3 GABA(A) receptors. These results indicate R-(-)-14 is a functional general anesthetic that is well-suited for identifying barbiturate binding sites on Cys-loop receptors.
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http://dx.doi.org/10.1021/jm300631eDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3717557PMC
July 2012

Mapping general anesthetic binding site(s) in human α1β3 γ-aminobutyric acid type A receptors with [³H]TDBzl-etomidate, a photoreactive etomidate analogue.

Biochemistry 2012 Jan 23;51(4):836-47. Epub 2012 Jan 23.

Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, United States.

The γ-aminobutyric acid type A receptor (GABA(A)R) is a target for general anesthetics of diverse chemical structures, which act as positive allosteric modulators at clinical doses. Previously, in a heterogeneous mixture of GABA(A)Rs purified from bovine brain, [³H]azietomidate photolabeling of αMet-236 and βMet-286 in the αM1 and βM3 transmembrane helices identified an etomidate binding site in the GABA(A)R transmembrane domain at the interface between the β and α subunits [Li, G. D., et.al. (2006) J. Neurosci. 26, 11599-11605]. To further define GABA(A)R etomidate binding sites, we now use [³H]TDBzl-etomidate, an aryl diazirine with broader amino acid side chain reactivity than azietomidate, to photolabel purified human FLAG-α1β3 GABA(A)Rs and more extensively identify photolabeled GABA(A)R amino acids. [³H]TDBzl-etomidate photolabeled in an etomidate-inhibitable manner β3Val-290, in the β3M3 transmembrane helix, as well as α1Met-236 in α1M1, a residue photolabeled by [³H]azietomidate, while no photolabeling of amino acids in the αM2 and βM2 helices that also border the etomidate binding site was detected. The location of these photolabeled amino acids in GABA(A)R homology models derived from the recently determined structures of prokaryote (GLIC) or invertebrate (GluCl) homologues and the results of computational docking studies predict the orientation of [³H]TDBzl-etomidate bound in that site and the other amino acids contributing to this GABA(A)R intersubunit etomidate binding site. Etomidate-inhibitable photolabeling of β3Met-227 in βM1 by [³H]TDBzl-etomidate and [³H]azietomidate also provides evidence of a homologous etomidate binding site at the β3-β3 subunit interface in the α1β3 GABA(A)R.
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http://dx.doi.org/10.1021/bi201772mDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3274767PMC
January 2012

p-(4-Azipentyl)propofol: a potent photoreactive general anesthetic derivative of propofol.

J Med Chem 2011 Dec 10;54(23):8124-35. Epub 2011 Nov 10.

Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114, United States.

We synthesized 2,6-diisopropyl-4-[3-(3-methyl-3H-diazirin-3-yl)propyl]phenol (p-(4-azipentyl)propofol), or p-4-AziC5-Pro, a novel photoactivable derivative of the general anesthetic propofol. p-4-AziC5-Pro has an anesthetic potency similar to that of propofol. Like propofol, the compound potentiates inhibitory GABA(A) receptor current responses and allosterically modulates binding to both agonist and benzodiazepine sites, assayed on heterologously expressed GABA(A) receptors. p-4-AziC5-Pro inhibits excitatory current responses of nACh receptors expressed in Xenopus oocytes and photoincorporates into native nACh receptor-enriched Torpedo membranes. Thus, p-4-AziC5-Pro is a functional general anesthetic that both modulates and photoincorporates into Cys-loop ligand-gated ion channels, making it an excellent candidate for use in identifying propofol binding sites.
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http://dx.doi.org/10.1021/jm200943fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3580944PMC
December 2011

Numerous classes of general anesthetics inhibit etomidate binding to gamma-aminobutyric acid type A (GABAA) receptors.

J Biol Chem 2010 Mar 18;285(12):8615-20. Epub 2010 Jan 18.

Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, UCLA, Los Angeles, California 90095, USA.

Enhancement of gamma-aminobutyric acid type A receptor (GABA(A)R)-mediated inhibition is a property of most general anesthetics and a candidate for a molecular mechanism of anesthesia. Intravenous anesthetics, including etomidate, propofol, barbiturates, and neuroactive steroids, as well as volatile anesthetics and long-chain alcohols, all enhance GABA(A)R function at anesthetic concentrations. The implied existence of a receptor site for anesthetics on the GABA(A)R protein was supported by identification, using photoaffinity labeling, of a binding site for etomidate within the GABA(A)R transmembrane domain at the beta-alpha subunit interface; the etomidate analog [(3)H]azietomidate photolabeled in a pharmacologically specific manner two amino acids, alpha1Met-236 in the M1 helix and betaMet-286 in the M3 helix (Li, G. D., Chiara, D. C., Sawyer, G. W., Husain, S. S., Olsen, R. W., and Cohen, J. B. (2006) J. Neurosci. 26, 11599-11605). Here, we use [(3)H]azietomidate photolabeling of bovine brain GABA(A)Rs to determine whether other structural classes of anesthetics interact with the etomidate binding site. Photolabeling was inhibited by anesthetic concentrations of propofol, barbiturates, and the volatile agent isoflurane, at low millimolar concentrations, but not by octanol or ethanol. Inhibition by barbiturates, which was pharmacologically specific and stereospecific, and by propofol was only partial, consistent with allosteric interactions, whereas isoflurane inhibition was nearly complete, apparently competitive. Protein sequencing showed that propofol inhibited to the same extent the photolabeling of alpha1Met-236 and betaMet-286. These results indicate that several classes of general anesthetics modulate etomidate binding to the GABA(A)R: isoflurane binds directly to the site with millimolar affinity, whereas propofol and barbiturates inhibit binding but do not bind in a mutually exclusive manner with etomidate.
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http://dx.doi.org/10.1074/jbc.M109.074708DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2838283PMC
March 2010

Conformational changes in the nicotinic acetylcholine receptor during gating and desensitization.

Biochemistry 2010 Jan;49(1):156-65

Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114, USA.

The nicotinic acetylcholine receptor (nAChR) is a member of the important Cys loop ligand-gated ion channel superfamily that modulates neuronal excitability. After they respond to their agonists, their actions are terminated either by removal of ligand or by fast and slow desensitization, processes that play an important role in modulating the duration of conducting states and hence of integrated neuronal behavior. We monitored structural changes occurring during fast and slow desensitization in the transmembrane domain of the Torpedo nAChR using time-resolved photolabeling with the hydrophobic probe 3-(trifluoromethyl)-3-(m-iodophenyl)diazirine (TID). After channel opening, TID photolabels a residue on the delta-subunit's M2-M3 loop and a cluster of four residues on deltaM1 and deltaM2, defining an open state pocket [Arevalo, E., et al. (2005) J. Biol. Chem. 280, 13631-13640]. We now find that photolabeling of this pocket persists during the transition to the fast desensitized state, the extent of photoincorporation decreasing only with the transition to the slow desensitized state. In contrast, the extent of photoincorporation in the channel lumen at the conserved 9'-leucines on the second transmembrane helix (M2-9') decreased successively during the resting to open and open to fast desensitized state transitions, implying that the local conformation is different in each state, a conclusion consistent with the hypothesis that there are separate gates for channel opening and desensitization. Thus, although during fast desensitization there is a conformation change in the channel lumen at the level of M2-9', there is none in the regions of the delta-subunit's M2-M3 loop and the interior of its M1-M4 helix bundle until slow desensitization occurs.
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http://dx.doi.org/10.1021/bi901550pDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2818735PMC
January 2010

[(3)H]chlorpromazine photolabeling of the torpedo nicotinic acetylcholine receptor identifies two state-dependent binding sites in the ion channel.

Biochemistry 2009 Oct;48(42):10066-77

Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, USA.

Chlorpromazine (CPZ), a potent nicotinic acetylcholine receptor (nAChR) noncompetitive antagonist, binds with higher affinity in the ion channel in the desensitized state than in the closed channel state and with low affinity to additional sites in nAChR-rich membranes. For nAChR equilibrated with agonist, we confirm previous reports that [(3)H]CPZ occupies a site near the cytoplasmic end of the M2 ion channel domain, photolabeling positions M2-2, M2-6, and/or M2-9 in each subunit. We find that [(3)H]CPZ also binds at the extracellular end of the channel, photolabeling amino acids at positions M2-16 (alpha,gamma), M2-17 (alpha,beta,delta), and M2-20 (alpha,beta,delta). The photolabeling at the cytoplasmic end of the channel is fully inhibitable by phencyclidine or proadifen, whereas neither drug inhibits [(3)H]CPZ photolabeling at the extracellular end, establishing that positively charged drugs can bind simultaneously at the cytoplasmic and extracellular ends of the ion channel. [(3)H]CPZ photolabeling is not detected in the transmembrane domain outside the ion channel, but it photolabels alphaMet-386 and alphaSer-393 in the cytoplasmic alphaMA helix. In the nAChR equilibrated with alpha-bungarotoxin to stabilize the nAChR in a closed state, [(3)H]CPZ photolabels amino acids at M2-5 (alpha), M2-6 (alpha,beta,delta), and M2-9 (beta,delta), with no labeling at M2-2. These results provide novel information about the modes of drug binding within the nAChR ion channel and indicate that within the nAChR transmembrane domain, the binding of cationic aromatic amine antagonists can be restricted to the ion channel domain, in contrast to the uncharged, allosteric potentiators and inhibitors that also bind within the delta subunit helix bundle and at subunit interfaces.
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http://dx.doi.org/10.1021/bi901271wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2878666PMC
October 2009

Neurosteroids allosterically modulate binding of the anesthetic etomidate to gamma-aminobutyric acid type A receptors.

J Biol Chem 2009 May 12;284(18):11771-5. Epub 2009 Mar 12.

Department of Molecular and Medical Pharmacology, Geffen School of Medicine at UCLA, Los Angeles, California 90095, USA.

Photoaffinity labeling of gamma-aminobutyric acid type A (GABA(A))-receptors (GABA(A)R) with an etomidate analog and mutational analyses of direct activation of GABA(A)R by neurosteroids have each led to the proposal that these structurally distinct general anesthetics bind to sites in GABA(A)Rs in the transmembrane domain at the interface between the beta and alpha subunits. We tested whether the two ligand binding sites might overlap by examining whether neuroactive steroids inhibited etomidate analog photolabeling. We previously identified (Li, G. D., Chiara, D. C., Sawyer, G. W., Husain, S. S., Olsen, R. W., and Cohen, J. B. (2006) J. Neurosci. 26, 11599-11605) azietomidate photolabeling of GABA(A)R alpha1Met-236 and betaMet-286 (in alphaM1 and betaM3). Positioning these two photolabeled amino acids in a single type of binding site at the interface of beta and alpha subunits (two copies per pentamer) is consistent with a GABA(A)R homology model based upon the structure of the nicotinic acetylcholine receptor and with recent alphaM1 to betaM3 cross-linking data. Biologically active neurosteroids enhance rather than inhibit azietomidate photolabeling, as assayed at the level of GABA(A)R subunits on analytical SDS-PAGE, and protein microsequencing establishes that the GABA(A)R-modulating neurosteroids do not inhibit photolabeling of GABA(A)R alpha1Met-236 or betaMet-286 but enhance labeling of alpha1Met-236. Thus modulatory steroids do not bind at the same site as etomidate, and neither of the amino acids identified as neurosteroid activation determinants (Hosie, A. M., Wilkins, M. E., da Silva, H. M., and Smart, T. G. (2006) Nature 444, 486-489) are located at the subunit interface defined by our etomidate site model.
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http://dx.doi.org/10.1074/jbc.C900016200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2673245PMC
May 2009

Time-resolved photolabeling of the nicotinic acetylcholine receptor by [3H]azietomidate, an open-state inhibitor.

Mol Pharmacol 2009 May 13;75(5):1084-95. Epub 2009 Feb 13.

Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA.

Azietomidate is a photoreactive analog of the general anesthetic etomidate that acts as a nicotinic acetylcholine receptor (nAChR) noncompetitive antagonist. We used rapid perfusion electrophysiological techniques to characterize the state dependence and kinetics of azietomidate inhibition of Torpedo californica nAChRs and time-resolved photolabeling to identify the nAChR binding sites occupied after exposure to [(3)H]azietomidate and agonist for 50 ms (open state) or at equilibrium (desensitized state). Azietomidate acted primarily as an open channel inhibitor characterized by a bimolecular association rate constant of k(+) = 5 x 10(5) M(-1) s(-1) and a dissociation rate constant of <3s(-1). Azietomidate at 10 microM, when perfused with acetylcholine (ACh), inhibited the ACh response by approximately 50% after 50 ms; when preincubated for 10 s, it decreased the peak initial response by approximately 15%. Comparison of the kinetics of recovery of ACh responses after exposure to ACh and azietomidate or to ACh alone indicated that at subsecond times, azietomidate inhibited nAChRs without enhancing the kinetics of agonist-induced desensitization. In nAChRs frozen after 50-ms exposure to agonist and [(3)H]azietomidate, amino acids were photolabeled in the ion channel [position M2-20 (alphaGlu-262, betaAsp-268, deltaGln-276)], in deltaM1 (deltaCys-236), and in alphaMA/alphaM4 (alphaGlu-390, alphaCys-412) that were also photolabeled in nAChRs in the equilibrium desensitized state at approximately half the efficiency. These results identify azietomidate binding sites at the extracellular end of the ion channel, in the delta subunit helix bundle, and in the nAChR cytoplasmic domain that seem similar in structure and accessibility in the open and desensitized states of the nAChR.
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http://dx.doi.org/10.1124/mol.108.054353DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2672814PMC
May 2009

Probing the structure of the affinity-purified and lipid-reconstituted torpedo nicotinic acetylcholine receptor.

Biochemistry 2008 Dec;47(48):12787-94

Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, USA.

The Torpedo nicotinic acetylcholine receptor (nAChR) is the only member of the Cys-loop superfamily of ligand-gated ion channels (LGICs) that is available in high abundance in a native membrane preparation. To study the structure of the other LGICs using biochemical and biophysical techniques, detergent solubilization, purification, and lipid reconstitution are usually required. To assess the effects of purification on receptor structure, we used the hydrophobic photoreactive probe 3-trifluoromethyl-3-(m-[(125)I]iodophenyl)diazirine ([(125)I]TID) to compare the state-dependent photolabeling of the Torpedo nAChR before and after purification and reincorporation into lipid. For the purified nAChR, the agonist-sensitive photolabeling within the M2 ion channel domain of positions M2-6, M2-9, and M2-13, the agonist-enhanced labeling of deltaThr274 (deltaM2-18) within the delta subunit helix bundle, and the labeling at the lipid-protein interface (alphaMu4) were the same as for the nAChR in native membranes. However, addition of agonist did not enhance [(125)I]TID photolabeling of deltaIle288 within the deltaM2-M3 loop. These results indicate that after purification and reconstitution of the Torpedo nAChR, the difference in structure between the resting and desensitized states within the M2 ion channel domain was preserved, but not the agonist-dependent change of structure of the deltaM2-M3 loop. To further characterize the pharmacology of [(125)I]TID binding sites in the nAChR in the desensitized state, we examined the effect of phencyclidine (PCP) on [(125)I]TID photolabeling. PCP inhibited [(125)I]TID labeling of amino acids at the cytoplasmic end of the ion channel (M2-2 and M2-6) while potentiating labeling at M2-9 and M2-13 and allosterically modulating the labeling of amino acids within the delta subunit helix bundle.
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http://dx.doi.org/10.1021/bi801476jDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2872774PMC
December 2008