Assessment of direct gating and allosteric modulatory effects of meprobamate in recombinant GABAA receptors

Abstract

Meprobamate is a schedule II anxiolytic and the primary metabolite of the muscle relaxant carisoprodol. Meprobamate modulates GABAA (γ-aminobutyric acid type A) receptors, and has barbiturate-like activity. To gain insight into its actions, we have conducted a series of studies using recombinant GABAA receptors. In αxβzγ GABAA receptors (where x = 1–6 and z = 1–3), the ability to enhance GABA-mediated current was evident for all α subunit isoforms, with the largest effect observed in α5-expressing receptors. Direct gating was present with all α subunits, although attenuated in α3-expressing receptors. Allosteric and direct effects were comparable in α1β1γ2 and α1β2γ2 receptors, whereas allosteric effects were enhanced in α1β2 compared to α1β2γ2 receptors. In “extrasynaptic” (α1β3δ and α4β3δ) receptors, meprobamate enhanced EC20 and saturating GABA currents, and directly activated these receptors. The barbiturate antagonist bemegride attenuated direct effects of meprobamate. Whereas pentobarbital directly gated homomeric β3 receptors, meprobamate did not, and instead blocked the spontaneously open current present in these receptors. In wild type homomeric ρ1 receptors, pentobarbital and meprobamate were ineffective in direct gating; a mutation known to confer sensitivity to pentobarbital did not confer sensitivity to meprobamate. Our results provide insight into the actions of meprobamate and parent therapeutic agents such as carisoprodol. Whereas in general actions of meprobamate were comparable to those of carisoprodol, differential effects of meprobamate at some receptor subtypes suggest potential advantages of meprobamate may be exploited. A re-assessment of previously synthesized meprobamate-related carbamate molecules for myorelaxant and other therapeutic indications is warranted.

Amino acid substitutions in the human homomeric β3 GABAA receptor that enable activation by GABA

“…The major subtype in the central nervous system is the ␣ 1 ␤ 2 ␥ 2 GABA A R. The ␤ 1 , ␤ 3 , and subunits can form homomers when recombinantly expressed in vitro. Although the homomeric ␤ 3 has not been identified in vivo, it is of considerable interest as the first GABA A R to yield to high-resolution structural analysis (12) and for functional studies because histaminergic ligands and propofol activate the receptor (13)(14)(15)(16). Recently, four heteromeric GABA A R structures were published, including the major subtype (17)(18)(19).…”

The dual modulatory effects of efavirenz on GABA A receptors are mediated via two distinct sites

“…Dual effects of efavirenz present in α1, α2 and α4-containing GABA A receptors are mediated by two distinct sites. In fact, similar dual modulation has also been demonstrated in other GABA A receptor modulators such as pentobarbital (Akk and Steinbach, 2000; Akk et al, 2004), lactones (Rho et al, 1997), carisoprodol (Kumar et al, 2015) and meprobamate (Kumar and Dillon, 2016). Dual modulatory effects of efavirenz on GABA A receptor is also dependent on [GABA].…”

A Single Amino Acid Residue at Transmembrane Domain 4 of the α Subunit Influences Carisoprodol Direct Gating Efficacy at GABAA Receptors

“…These results are consistent with our contention that carisoprodol mediates these two actions through distinct sites on the GABA A receptor. In addition, as noted recently (Kumar and Dillon, 2016), an array of meprobamate-related dicarbamate molecules were generated years ago, when both meprobamate and carisoprodol were being widely prescribed (Ludwig et al, 1969). Many of these molecules showed promise as muscle relaxants in preclinical studies; however, to our knowledge, none advanced to market.…”

“…Carisoprodol's less potent metabolite, meprobamate, also displays reduced direct gating effects in a3-expressing receptors (Kumar and Dillon, 2016) compared with all other a subunits. We thus evaluated the ability of these mutations to impact direct gating by meprobamate.…”