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Contrasting actions of a convulsant barbiturate and its anticonvulsant enantiomer on the α 1 β 3 γ 2L GABA A receptor account for their in vivo effects
Author(s) -
Desai Rooma,
Savechenkov Pavel Y.,
Zolkowska Dorota,
Ge Ri Le,
Rogawski Michael A.,
Bruzik Karol S.,
Forman Stuart A.,
Raines Douglas E.,
Miller Keith W.
Publication year - 2015
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jp270971
Subject(s) - convulsant , enantiomer , chemistry , barbiturate , gabaa receptor , anticonvulsant , stereochemistry , convulsants , receptor , pharmacology , biochemistry , neuroscience , biology , epilepsy
Key Points Most barbiturates are anaesthetics but unexpectedly a few are convulsants whose mechanism of action is poorly understood. We synthesized and characterized a novel pair of chiral barbiturates that are capable of photolabelling their binding sites on GABA A receptors. In mice the S ‐enantiomer is a convulsant, but the R ‐enantiomer is an anticonvulsant. The convulsant S ‐enantiomer binds solely at an inhibitory site. It is both an open state inhibitor and a resting state inhibitor. Its action is pH independent, suggesting the pyrimidine ring plays little part in binding. The inhibitory site is not enantioselective because the R ‐enantiomer inhibits with equal affinity. In contrast, only the anticonvulsant R ‐enantiomer binds to the enhancing site on open channels, causing them to stay open longer. The enhancing site is enantioselective. The in vivo actions of the convulsant S ‐enantiomer are accounted for by its interactions with GABA A receptors.Abstract Most barbiturates are anaesthetics but a few unexpectedly are convulsants. We recently located the anaesthetic sites on GABA A receptors (GABA A Rs) by photolabelling with an anaesthetic barbiturate. To apply the same strategy to locate the convulsant sites requires the creation and mechanistic characterization of a suitable agent. We synthesized enantiomers of a novel, photoactivable barbiturate, 1‐methyl‐5‐propyly‐5‐(m‐trifluoromethyldiazirinyl) phenyl barbituric acid ( m TFD‐MPPB). In mice, S‐m TFD‐MPPB acted as a convulsant, whereas R‐m TFD‐MPPB acted as an anticonvulsant. Using patch clamp electrophysiology and fast solution exchange on recombinant human α 1 β 3 γ 2L GABA A Rs expressed in HEK cells, we found that S‐m TFD‐MPPB inhibited GABA‐induced currents, whereas R‐m TFD‐MPPB enhanced them. S ‐ m TFD‐MPPB caused inhibition by binding to either of two inhibitory sites on open channels with bimolecular kinetics. It also inhibited closed, resting state receptors at similar concentrations, decreasing the channel opening rate and shifting the GABA concentration–response curve to the right. R ‐ m TFD‐MPPB, like most anaesthetics, enhanced receptor gating by rapidly binding to allosteric sites on open channels, initiating a rate‐limiting conformation change to stabilized open channel states. These states had slower closing rates, thus shifting the GABA concentration–response curve to the left. Under conditions when most GABA A Rs were open, an inhibitory action of R ‐ m TFD‐MPPB was revealed that had a similar IC 50 to that of S ‐ m TFD‐MPPB. Thus, the inhibitory sites are not enantioselective, and the convulsant action of S ‐ m TFD‐MPPB results from its negligible affinity for the enhancing, anaesthetic sites. Interactions with these two classes of barbiturate binding sites on GABA A Rs underlie the enantiomers’ different pharmacological activities in mice.