Carisoprodol-mediated modulation of GABAA receptors: in vitro and in vivo studies.

Carisoprodol is a frequently prescribed muscle relaxant. In recent years, this drug has been increasingly abused. The effects of carisoprodol have been attributed to its metabolite, meprobamate, a controlled substance that produces sedation via GABA(A) receptors (GABA(A)Rs). Given the structural similarities between carisoprodol and meprobamate, we used electrophysiological and behavioral approaches to investigate whether carisoprodol directly affects GABA(A)R function. In whole-cell patch-clamp studies, carisoprodol allosterically modulated and directly activated human alpha1beta2gamma2 GABA(A)R function in a barbiturate-like manner. At millimolar concentrations, inhibitory effects were apparent. Similar allosteric effects were not observed for homomeric rho1 GABA or glycine alpha1 receptors. In the absence of GABA, carisoprodol produced picrotoxin-sensitive, inward currents that were significantly larger than those produced by meprobamate, suggesting carisoprodol may directly produce GABAergic effects in vivo. When administered to mice via intraperitoneal or oral routes, carisoprodol elicited locomotor depression within 8 to 12 min after injection. Intraperitoneal administration of meprobamate depressed locomotor activity in the same time frame. In drug discrimination studies with carisoprodol-trained rats, the GABAergic ligands pentobarbital, chlordiazepoxide, and meprobamate each substituted for carisoprodol in a dose-dependent manner. In accordance with findings in vitro, the discriminative stimulus effects of carisoprodol were antagonized by a barbiturate antagonist, bemegride, but not by the benzodiazepine site antagonist, flumazenil. The results of our studies in vivo and in vitro collectively suggest the barbiturate-like effects of carisoprodol may not be due solely to its metabolite, meprobamate. Furthermore, the functional traits we have identified probably contribute to the abuse potential of carisoprodol.

Stoichiometric analysis of the TM2 6' phenylalanine mutation on desensitization in alpha1beta2 and alpha1beta2gamma2 GABA A receptors.

The presence of phenylalanine (F) at the 6' position of transmembrane domain 2 (TM2) in the alpha4 subunit of alpha4beta2 nicotinic receptors enhances desensitization. As the GABA A receptor affords the ability to study the influence of as few as one and as many as five Fs at this position, we have used it to investigate potential subunit- and stoichiometry-dependent effects of the TM2 6'F mutation on desensitization. Whereas the presence of one F at this position decreased extent of desensitization, desensitization was increased in all configurations that included two or more Fs at the TM2 6' position; desensitization was particularly rapid with 3 or 4 F residues present. Our results demonstrate the ability of F residues at the TM2 6' position to modulate desensitization is likely conserved in the cys-loop family of ligand-gated ion channels. Moreover, our findings demonstrate both stoichiometric- and subunit-dependent effects of the ability of this mutation to regulate desensitization in GABA A receptors.

ERK/MAPK pathway regulates GABAA receptors.

The GABAA receptor is a ligand-gated ion channel whose function and activity can be regulated by ligand binding or alternatively may be influenced indirectly through the phosphorylation of specific subunits that comprise the GABAA receptor pentamer. With respect to phosphorylation, most studies have focused on either beta or gamma subunits, whereas the role of the alpha subunit as a relevant target of signaling kinases is largely unknown. Interestingly, we found a putative phosphorylation site for extracellular-signal regulated kinase (ERK), a key effector of the MAPK pathway, in almost all known alpha subunits of the GABAA receptor, including the ubiquitously expressed alpha1 subunit. To determine whether this putative ERK phosphorylation site was functionally relevant, we evaluated if ERK inhibition (through pharmacological inhibition of its upstream kinase, MEK) altered GABA-gated currents. Using HEK293 cells stably transfected with the alpha1beta2gamma2 form of the GABAA receptor, we found that UO126 reduced basal ERK phosphorylation and resulted in an enhancement of GABA-induced peak current amplitudes. Further, the enhancement of GABA-gated currents required an intact intracellular environment as it was robust in perforated patch recordings (which preserves the intracellular milieu), but absent in conventional whole-cell recordings (which dialyzes the cytosolic contents), supporting the involvement of an intracellular signaling pathway. Finally, mutation of the ERK phosphorylation site (T375-->A) prevented the UO126-induced enhancement of GABA-gated currents. Collectively, our results implicate the MAPK pathway as a negative modulator of GABAA receptor function, whose influence on GABA-gated currents may be mediated by phosphorylation of the alpha subunit.

Enantioselectivity of alpha-benzyl-alpha-methyl-gamma-butyrolactone-mediated modulation of anticonvulsant activity and GABA(A) receptor function.

Alkyl-substituted butyrolactones have both inhibitory and stimulatory effects on GABA(A) receptors. Lactones with small alkyl substitutions at the alpha-position positively modulate the channel, whereas beta-substituted lactones tend to inhibit the GABA(A) receptor. These compounds mediate inhibition through the picrotoxin site of the receptor. A distinct binding site that mediates the stimulatory actions of lactones is presumed to exist, although no definitive evidence to support this claim exists. In the present study, we used in vivo and in vitro assays to evaluate the effects of the enantiomers of a novel lactone, alpha-benzyl-alpha-methyl-gamma-butyrolactone (alpha-BnMeGBL), on the GABA(A) receptor. R-(-)-alpha-BnMeGBL was 2-fold more potent than the S-(+)-alpha-BnMeGBL in blocking pentylenetetrazol-induced seizures in CF-1 mice. The (+)-enantiomer inhibited binding of t-butylbicyclophosporothionate with a higher affinity than the (-)-enantiomer (IC(50) of 0.68 and 1.1 mM, respectively). Whole cell patch-clamp recordings from recombinant alpha1beta2gamma2 receptors stably expressed in HEK293 cells demonstrated that both compounds stimulated GABA-activated current. The maximal stimulation was approximately 2-fold greater with (+)-alpha-BnMeGBL than that seen with (-)-alpha-BnMeGBL. Both enantiomers of alpha-BnMeGBL directly gated the GABA(A) receptor at mM concentrations, in a nonstereoselective manner. Our data demonstrate the stimulatory actions of alpha-BnMeGBL on GABA(A) receptor function display enantioselectivity and provide strong evidence for the existence of a true "lactone site" on the receptor.

Identification of a novel residue within the second transmembrane domain that confers use-facilitated block by picrotoxin in glycine alpha 1 receptors.

The central nervous system convulsant picrotoxin (PTX) inhibits GABA(A) and glutamate-gated Cl(minus sign) channels in a use-facilitated fashion, whereas PTX inhibition of glycine and GABA(C) receptors displays little or no use-facilitated block. We have identified a residue in the extracellular aspect of the second transmembrane domain that converted picrotoxin inhibition of glycine alpha1 receptors from non-use-facilitated to use-facilitated. In wild type alpha1 receptors, PTX inhibited glycine-gated Cl(minus sign) current in a competitive manner and had equivalent effects on peak and steady-state currents, confirming a lack of use-facilitated block. Mutation of the second transmembrane domain 15'-serine to glutamine (alpha1(S15'Q) receptors) converted the mechanism of PTX blockade from competitive to non-competitive. However, more notable was the fact that in alpha1(S15'Q) receptors, PTX had insignificant effects on peak current amplitude and dramatically enhanced current decay kinetics. Similar results were found in alpha1(S15'N) receptors. The reciprocal mutation in the beta2 subunit of alpha1beta2 GABA(A) receptors (alpha1beta2(N15'S) receptors) decreased the magnitude of use-facilitated PTX inhibition. Our results implicate a specific amino acid at the extracellular aspect of the ion channel in determining use-facilitated characteristics of picrotoxin blockade. Moreover, the data are consistent with the suggestion that picrotoxin may interact with two domains in ligand-gated anion channels.