Background γ-Aminobutyric acid type A (GABAA) receptors mediate important effects of intravenous general anesthetics. Photolabel derivatives of etomidate, propofol, barbiturates, and a neurosteroid get incorporated in GABAA receptor transmembrane helices M1 and M3 adjacent to intersubunit pockets. However, photolabels have not been consistently targeted at heteromeric αβγ receptors and do not form adducts with all contact residues. Complementary approaches may further define anesthetic sites in typical GABAA receptors. Methods Two mutation-based strategies, substituted tryptophan sensitivity and substituted cysteine modification–protection, combined with voltage-clamp electrophysiology in Xenopus oocytes, were used to evaluate interactions between four intravenous anesthetics and six amino acids in M1 helices of α1, β3, and γ2L GABAA receptor subunits: two photolabeled residues, α1M236 and β3M227, and their homologs. Results Tryptophan substitutions at α1M236 and positional homologs β3L231 and γ2L246 all caused spontaneous channel gating and reduced γ-aminobutyric acid EC50. Substituted cysteine modification experiments indicated etomidate protection at α1L232C and α1M236C, R-5-allyl-1-methyl-5-(m-trifluoromethyl-diazirinylphenyl) barbituric acid protection at β3M227C and β3L231C, and propofol protection at α1M236C and β3M227C. No alphaxalone protection was evident at the residues the authors explored, and none of the tested anesthetics protected γ2I242C or γ2L246C. Conclusions All five intersubunit transmembrane pockets of GABAA receptors display similar allosteric linkage to ion channel gating. Substituted cysteine modification and protection results were fully concordant with anesthetic photolabeling at α1M236 and β3M227 and revealed overlapping noncongruent sites for etomidate and propofol in β+–α– interfaces and R-5-allyl-1-methyl-5-(m-trifluoromethyl-diazirinylphenyl) barbituric acid and propofol in α+–β– and γ+–β– interfaces. The authors’ results identify the α+–γ– transmembrane interface as a potentially unique orphan modulator site.

Propofol, etomidate, and barbiturate anesthetics are allosteric coagonists at pentameric a1b3g2 GABA A receptors, modulating channel activation via four biochemically established intersubunit transmembrane pockets. Etomidate selectively occupies the two b 1 /a 2 pockets, the barbiturate photolabel R-5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid (R-mTFD-MPAB) occupies homologous a 1 /b 2 and g 1 /b 2 pockets, and propofol occupies all four. Functional studies of mutations at M2-159 or M3-369 loci abutting these pockets provide conflicting results regarding their relative contributions to propofol modulation. We electrophysiologically measured GABA-dependent channel activation in a1b3g2L or receptors with single M2-159 (a1S270I, b3N265M, and g2S280W) or M3-369 (a1A291W, b3M286W, and g2S301W) mutations, in the absence and presence of equipotent clinical range concentrations of etomidate, R-mTFD-MPAB, and propofol. Estimated open probabilities were calculated and analyzed using global two-state Monod-Wyman-Changeux models to derive log(d) parameters proportional to anesthetic-induced channel modulating energies (where d is the allosteric anesthetic shift factor). All mutations reduced the log(d) values for anesthetics occupying both abutting and nonabutting pockets. The Dlog(d) values [log(d, mutant) 2 log(d, wild type)] for M2-159 mutations abutting an anesthetic's biochemically established binding sites were consistently larger than the Dlog(d) values for nonabutting mutations, although this was not true for the M3-369 mutant Dlog(d) values. The sums of the anesthetic-associated Dlog(d) values for sets of M2-159 or M3-369 mutations were all much larger than the wild-type log(d) values. Mutant Dlog(d) values qualitatively reflect anesthetic site occupancy patterns. However, the lack of Dlog(d) additivity undermines quantitative comparisons of distinct site contributions to anesthetic modulation because the mutations impaired both abutting anesthetic binding effects and positive cooperativity between anesthetic binding sites.

Background Neurosteroids like alphaxalone are potent anxiolytics, anticonvulsants, amnestics, and sedative-hypnotics, with effects linked to enhancement of γ-aminobutyric acid type A (GABAA) receptor gating in the central nervous system. Data locating neurosteroid binding sites on synaptic αβγ GABAA receptors are sparse and inconsistent. Some evidence points to outer transmembrane β+–α− interfacial pockets, near sites that bind the anesthetics etomidate and propofol. Other evidence suggests that steroids bind more intracellularly in β+–α− interfaces. Methods The authors created 12 single-residue β3 cysteine mutations: β3T262C and β3T266C in β3-M2; and β3M283C, β3Y284C, β3M286C, β3G287C, β3F289C, β3V290C, β3F293C, β3L297C, β3E298C, and β3F301C in β3-M3 helices. The authors coexpressed α1 and γ2L with each mutant β3 subunit in Xenopus oocytes and electrophysiologically tested each mutant for covalent sulfhydryl modification by the water-soluble reagent para-chloromercuribenzenesulfonate. Then, the authors assessed whether receptor-bound alphaxalone, etomidate, or propofol blocked cysteine modification, implying steric hindrance. Results Eleven mutant β3 subunits, when coexpressed with α1 and γ2L, formed functional channels that displayed varied sensitivities to the three anesthetics. Exposure to para-chloromercuribenzenesulfonate produced irreversible functional changes in ten mutant receptors. Protection by alphaxalone was observed in receptors with β3V290C, β3F293C, β3L297C, or β3F301C mutations. Both etomidate and propofol protected receptors with β3M286C or β3V290C mutations. Etomidate also protected β3F289C. In α1β3γ2L structural homology models, all these protected residues are located in transmembrane β+–α− interfaces. Conclusions Alphaxalone binds in transmembrane β+–α− pockets of synaptic GABAA receptors that are adjacent and intracellular to sites for the potent anesthetics etomidate and propofol.

Neuroactive steroids are potent positive allosteric modulators of GABAA receptors (GABAAR), but the locations of their GABAAR binding sites remain poorly defined. To discover these sites, we synthesized two photoreactive analogs of alphaxalone, an anesthetic neurosteroid targeting GABAAR, 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 GABAAR responses, left-shifting the GABA concentration – response curve for human α1β3γ2 GABAARs expressed in Xenopus oocytes, and enhancing [3H]muscimol binding to α1β3γ2 GABAARs expressed in HEK293 cells. With EC50 of 110 nM, 11-AziAP is one the most potent general anesthetics reported. [3H]F4N3Bzoxy-AP and [3H]11-AziAP, at anesthetic concentrations, photoincorporated into α- and β-subunits of purified α1β3γ2 GABAARs, but labeling at the subunit level was not inhibited by alphaxalone (30 μM). The enhancement of photolabeling by 3H-azietomidate and 3H-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. (Psychoneuroendocrinology 2009, 34S1, S59–S66).

Pregnanolone and allopregnanolone-type ligands exert general anesthetic, anticonvulsant and anxiolytic effects due to their positive modulatory interactions with the GABA A 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 A receptors using photoaffinity labeling techniques, and in homomeric chimeric receptors containing GABA A receptor α subunit TMDs by crystallography. Steroid binding sites have yet to be determined in human, heteromeric, functionally reconstituted, full-length, glycosylated GABA A 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-3yl)benzoxy]allopregnanolone (21-pTFDBzox-AP) was the most potent ligand. It is a partial positive modulator of the human α1β3 and α1β3γ2L GABA A receptors at sub-micromolar concentrations. [ 3 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 [ 3 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 GABA A R positive allosteric modulators, etomidate and an anesthetic barbiturate. The latter two ligands bind to sites at subunit

Background Clusters of COVID‐19 cases amplify the pandemic and are critical targets for intervention, but comprehensive cluster‐level data are not collected systematically by federal or most state public health entities. This analysis characterizes COVID‐19 clusters among vulnerable populations housed in congregate living settings across an entire community and describes early mitigation efforts. Methods The Cuyahoga County Board of Health identified and interviewed COVID‐19 cases and exposed contacts, assessing possible connections to congregate living facilities within its jurisdiction from March 7, 2020, to May 15, 2020, during the first phase of the pandemic, while state of Ohio stay‐at‐home orders were in effect. A multi‐disciplinary team‐based response network was mobilized to support active case finding and develop facility‐focused containment strategies. Results We identified a cascade of 45 COVID‐19 clusters across community facilities (corrections, nursing, assisted living, intermediate care, extended treatment, shelters, group homes). Attack rates were highest within small facilities ( P < .01) and large facilities requiring extensive support to implement effective containment measures. For 25 clusters, we identified an index case who frequently (88%) was a healthcare worker. Engagement of clinical, community, and government partners through public health coordination efforts created opportunities to rapidly develop and coordinate effective response strategies to support the facilities facing the dawning impact of the pandemic. Conclusions Active cluster investigations can uncover the dynamics of community transmission affecting both residents of congregate settings and their caregivers and help to target efforts toward populations with ongoing challenges in access to detection and control resources.