Contrasting actions of a convulsant barbiturate and its anticonvulsant enantiomer on the α₁β₃γ_2L GABA_A receptor account for their in vivo effects

Abstract

Key Pointsr Most barbiturates are anaesthetics but unexpectedly a few are convulsants whose mechanism of action is poorly understood.r 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. 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 (mTFD-MPPB). In mice, S-mTFD-MPPB acted as a convulsant, whereas R-mTFD-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-mTFD-MPPB inhibited GABA-induced currents, whereas R-mTFD-MPPB enhanced them. S-mTFD-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-mTFD-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-mTFD-MPPB was revealed that had a similar IC 50 to that of S-mTFD-MPPB. Thus, the inhibitory sites are not enantioselective, and the convulsant action of S-mTFD-MPPB results from its negligible affinity for the enhancing, anaesthetic sites.

Correction for Inhibition Leads to an Allosteric Co-Agonist Model for Pentobarbital Modulation and Activation of α1β3γ2L GABAA Receptors

“…Recently, a convulsant barbiturate photolabel, S-mTFD-MPPB, was reported to photolabel residues in the γ + –β – transmembrane interface of α1β3γ2 receptors [ 25 ]. Moreover, S-mTFD-MPPB inhibits α1β3γ2, but not α1β3 GABA A receptors, indicating that the γ + –β – site is crucial for inhibition [ 26 ]. These data suggest that occlusion of the open chloride channel may not underlie inhibition by PB, and suggest an alternative mechanism whereby inhibitory barbiturates bind at an allosteric site, possibly overlapping with the γ + –β – allosteric agonist site.…”

“…These data suggest that occlusion of the open chloride channel may not underlie inhibition by PB, and suggest an alternative mechanism whereby inhibitory barbiturates bind at an allosteric site, possibly overlapping with the γ + –β – allosteric agonist site. Rightward shifts in GABA-response curves also suggest that inhibitory barbiturates act as inverse agonists [ 26 ]. It is notable that photolabeling identifies only one inhibitory barbiturate site, while our data and that of most other studies of PB are consistent with multiple inhibitory sites, while occupation of one site inhibits channel conductance.…”

Enantiomeric barbiturates bind distinct inter- and intrasubunit binding sites in a nicotinic acetylcholine receptor (nAChR)

“…Torpedo nAChR-rich membranes, isolated from T. californica electric organs (Aquatic Research Consultants, San Pedro, CA) as described (52), contained ϳ1 nmol of [ 3 H]ACh binding sites/mg of protein, as determined by an ultracentifugation assay. R-and S-mTFD-MPPB, R-mTFD-MPAB, and [ 3 H]S-mTFD-MPPB (49.9 Ci/mmol) were synthesized previously (22,24,25). Diisopropylphosphofluoridate, tetracaine, Carb, PCP, propofol, proadifen, and CNBr were from Sigma-Aldrich.…”

“…Discussion S-mTFD-MPPB, a convulsant in vivo, acts as an inhibitor of ␣␤␥ GABA A Rs, whereas R-mTFD-MPAB, which differs only by chirality and the presence of a 5-allyl rather than 5-propyl substituent, acts as an anesthetic in vivo and as an ␣␤␥ GABA A R PAM (22,24). In ␣1␤3␥2 GABA A Rs, photoaffinity labeling studies established that S-mTFD-MPPB and R-mTFD-MPAB bind to the same binding site in the TMD at the ␥ ϩ -␤ Ϫ subunit interface, but with the opposite state dependence and in different orientations (23,25).…”

“…R-and S-mTFD-MPAB act as GABA A R PAMs (22). S-mTFD-MPPB, differing from R-mTFD-MPAB only in terms of chirality and a 5-propyl versus 5-allyl substituent, acts in vivo as a convulsant and as an ␣1␤3␥2 GABA A R inhibitor, whereas R-mTFD-MPPB acts as a PAM (24). Direct GABA A R photolabeling with [ 3 H]S-mTFD-MPPB established that it binds to the same sites as R-mTFD-MPAB, but with opposite state dependence; R-mTFD-MPAB binds with highest affinity in the presence of GABA, whereas S-mTFD-MPPB binds preferentially in the presence of the inverse agonist bicuculline (25).…”