Identifying Drugs that Bind Selectively to Intersubunit General Anesthetic Sites in the α1β3γ2 GABAAR Transmembrane Domain

Jayakar, Selwyn S.; Zhou, Xiaojuan; Chiara, David C.; Jarava‐Barrera, Carlos; Savechenkov, Pavel Y.; Bruzik, Karol S.; Tortosa, Mariola; Miller, Keith W.; Cohen, Jonathan B.

doi:10.1124/mol.118.114975

Cited by 23 publications

(26 citation statements)

References 54 publications

(91 reference statements)

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“…[ 3 H]muscimol binding in α 1 β 3 GABA A Rs and acts through a binding site distinct from NS(53,54). The mutations targeting NS binding sites resulted in modest decreases in [ 3 H]muscimol binding enhancement by etomidate; however, the α 1 β 3 (M286W) mutation which abolishes etomidate potentiation and activation of GABA A Rs(55,56), also abolished[ 3 H]muscimol binding enhancement (Figure 6E).…”

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confidence: 96%

Site-specific effects of neurosteroids on GABAA receptor activation and desensitization

Sugasawa

Cheng

Bracamontes

et al. 2020

eLife

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This study examines how site-specific binding to three identified neurosteroid binding sites in the α1β3 GABAA receptor (GABAAR) contributes to neurosteroid allosteric modulation. We found that the potentiating neurosteroid, allopregnanolone, but not its inhibitory 3β-epimer epi-allopregnanolone, binds to the canonical β3(+)–α1(-) intersubunit site that mediates receptor activation by neurosteroids. In contrast, both allopregnanolone and epi-allopregnanolone bind to intrasubunit sites in the β3 subunit, promoting receptor desensitization and the α1 subunit promoting effects that vary between neurosteroids. Two neurosteroid analogues with diazirine moieties replacing the 3-hydroxyl (KK148 and KK150) bind to all three sites, but do not potentiate GABAAR currents. KK148 is a desensitizing agent, whereas KK150 is devoid of allosteric activity. These compounds provide potential chemical scaffolds for neurosteroid antagonists. Collectively, these data show that differential occupancy and efficacy at three discrete neurosteroid binding sites determine whether a neurosteroid has potentiating, inhibitory, or competitive antagonist activity on GABAARs.

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confidence: 96%

Site-specific effects of neurosteroids on GABAA receptor activation and desensitization

Sugasawa

Cheng

Bracamontes

et al. 2020

eLife

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“…This could affect the overall predicted rank order of the four higher-affinity intersubunit propofol sites, and introduce the modest quantitative discrepancies with the results of Ref. (34). We would not expect these changes to affect the observed relationship between solvation and affinity.…”

Section: Resultsmentioning

confidence: 80%

“…Overall, our results are consistent with results collected via mutagenesis and photolabeling involving GABA A receptors over the past two decades, particularly those reported in Refs. (26,29,30,34,40,58,59,(67)(68)(69)(70). These studies have focused exclusively on protein-mediated mechanisms of anesthetic binding.…”

Section: Resultsmentioning

confidence: 99%

“…In all high affinity (K D < 1µM) intersubunit propofol sites, more than five water molecules are displaced on average upon ligand binding. Very recent photolabeling protection studies (34) have indicated that propofol has a five-fold higher affinity for the two + sites ( ↵ + -↵and + -↵ -) than for the two sites (↵ + -and + --). As shown in Table 2, we have measured a range of values (0.2 -20 µ M) across three calculations involving ↵ + / sites.…”

Section: Methionine Residues Determine Lipid Accessibility To Intersumentioning

confidence: 99%

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Competitive dewetting underlies site-specific binding of general anesthetics to GABA(A) receptors

Murlidaran

Hénin

Brannigan

2019

Preprint

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GABA(A) receptors are pentameric ligand-gated ion channels playing a critical role in the modulation of neuronal excitability. These inhibitory receptors, gated by -aminobutyric acid (GABA), can be potentiated and even directly activated by intravenous and inhalational anesthetics. Intersubunit cavities in the transmembrane domain have been consistently identified as putative binding sites by numerous experiment and simulation results. Synaptic GABA(A) receptors are predominantly found in a 2↵:2 :1 stoichiometry, with four unique inter-subunit interfaces. Experimental and computational results have suggested a perplexing specificity, given that cavity-lining residues are highly conserved, and the functional effects of general anesthetics are only weakly sensitive to most mutations of cavity residues. Here we use Molecular Dynamics simulations and thermodynamically rigorous alchemical free energy perturbation (AFEP) techniques to calculate affinities of the intravenous anesthetic propofol and the inhaled anesthetic sevoflurane to all intersubunit sites in a heteromeric GABA(A) receptor. We find that the best predictor of general anesthetic affinity for the intersubunit cavity sites is water displacement: combinations of anesthetic and binding site that displace more water molecules have higher affinities than those that displace fewer. The amount of water displacement is, in turn, a function of size of the general anesthetic, successful competition of the general anesthetic with water for the few hydrogen bonding partners in the site, and inaccessibility of the site to lipid acyl chains. The latter explains the surprisingly low affinity of GAs for the ↵ intersubunit site, which is missing a bulky methionine residue at the cavity entrance and can be occupied by acyl chains in the unbound state. Simulations also identify sevoflurane binding sites in the subunit centers and in the pore, but predict that these are lower affinity than the intersubunit sites. SignificanceAfter over a century of research, it is established that general anesthetics interact directly with hydrophobic cavities in proteins. We still do not know why not all small hydrophobic molecules can act as general anesthetics, or why not all hydrophobic cavities bind these molecules. General anesthetics can even select among homologous sites on one critical target, the GABA(A) heteropentamer, although the origins of selectivity are unknown. Here we used rigorous free energy calculations to find that binding affinity correlates with the number of released water molecules, which in turn depends upon the lipid content of the cavity without bound anesthetic. Results suggest a mechanism that reconciles lipid-centered and protein-centered theories, and which can directly inform design of new anesthetics.

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“…In contrast, mutations in the α1 and β3 intrasubunit sites, but not Table 1). To confirm that the effect of these mutations on NS effect are steroid-specific, we also tested their effect on etomidate, which enhances [ 3 H]muscimol binding in α1β3 GABAARs and acts through 23 a binding site distinct from NS (48,49). The mutations targeting NS binding sites resulted in modest 24 decreases in [ 3 H]muscimol binding enhancement by etomidate; however, the α1β3(M286W) mutation which 12 enhancement ( Figure 6E).…”

mentioning

confidence: 91%

Site-specific effects of neurosteroids on GABA_A receptor activation and desensitization

Sugasawa

Cheng

Bracamontes

et al. 2020

Preprint

View full text Add to dashboard Cite

1This study examines how site-specific binding to the three identified neurosteroid binding sites in the 2 α1β3 GABAA receptor (GABAAR) contributes to neurosteroid allosteric modulation. We found that the 3 potentiating neurosteroid, allopregnanolone, but not its inhibitory 3β-epimer epi-allopregnanolone, binds 4 to the canonical β3(+)-α1(-) intersubunit site that mediates receptor activation by neurosteroids. In contrast, 5 both allopregnanolone and epi-allopregnanolone bind to intrasubunit sites in the β3 subunit, promoting 6 receptor desensitization and the α1 subunit promoting ligand-specific effects. Two neurosteroid analogues 7 with diazirine moieties replacing the 3-hydroxyl (KK148 and KK150) bind to all three sites, but do not 8 potentiate GABAAR currents. KK148 is a desensitizing agent, whereas KK150 is devoid of allosteric 9 activity. These compounds provide potential chemical scaffolds for site-specific and general neurosteroid 10 antagonists. Collectively, these data show that differential occupancy and efficacy at three discrete 11 neurosteroid binding sites determine whether a neurosteroid has potentiating, inhibitory, or competitive 12 antagonist activity on GABAARs. 13 14 15 16

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Identifying Drugs that Bind Selectively to Intersubunit General Anesthetic Sites in the α1β3γ2 GABA_AR Transmembrane Domain

Cited by 23 publications

References 54 publications

Site-specific effects of neurosteroids on GABAA receptor activation and desensitization

Site-specific effects of neurosteroids on GABAA receptor activation and desensitization

Competitive dewetting underlies site-specific binding of general anesthetics to GABA(A) receptors

Site-specific effects of neurosteroids on GABA_A receptor activation and desensitization

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