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 ␣1␤2␥2 GABA A R function in a barbiturate-like manner. At millimolar concentrations, inhibitory effects were apparent. Similar allosteric effects were not observed for homomeric 1 GABA or glycine ␣1 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.

The central nervous system convulsant picrotoxin (PTX) inhibits GABA A and glutamate-gated Cl ؊ 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 ␣1 receptors from non-use-facilitated to usefacilitated. In wild type ␣1 receptors, PTX inhibited glycine-gated Cl ؊ 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 (␣1(S15Q) receptors) converted the mechanism of PTX blockade from competitive to non-competitive. However, more notable was the fact that in ␣1(S15Q) receptors, PTX had insignificant effects on peak current amplitude and dramatically enhanced current decay kinetics. Similar results were found in ␣1(S15N) receptors. The reciprocal mutation in the ␤2 subunit of ␣1␤2 GABA A receptors (␣1␤2(N15S) 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.Glycine receptors belong to a superfamily of ligand-gated chloride channels that include GABA A 1 receptors, GABA C receptors, and glutamate-gated chloride channels (1). In native tissue, glycine receptors exist as either ␣ homomers or ␣␤ heteromers (1). They comprise five subunits (usually three ␣ subunits and two ␤ subunits) arranged asymmetrically around the ion pore. Each subunit is made up of a large extracellular N-terminal region, four transmembrane domains (TM), and a large cytoplasmic domain; TMII forms the channel lumen (2). Glycine receptors are targets of therapeutics such as anesthetics as well as toxins like the central nervous system convulsant picrotoxin (1).Picrotoxin inhibits all known anionic ligand-gated Cl Ϫ channels (3-5). The mechanism of action and the exact location of picrotoxin binding are still unknown (6 -12). However, several studies have indicated that TMII is the probable site for picrotoxin action (6, 13-22) (Fig. 1). For example, the TMII of the glycine ␤ subunit was found to be responsible for conferring resistance to picrotoxin in heteromeric glycine ␣ n ␤ receptors (n ϭ 1-3) (6). Subsequent work has defined the existence of a phenylalanine residue at the 6Ј position of the TMII glycine ␤ subunit in conferring insensitivity to picrotoxin (16). In addition, other TMII residues (2Ј and 19Ј) have also been implicated directly or indirectly in the mechanism by which picrotoxin inhibits these channels (13, 15,16). The mutations at positions 2Ј and 19Ј have been shown to affect the type of the inhibition (competitive versus non-competitive) by picrotoxin ...

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.

ABSTRACT:We previously reported on the synthesis of substituted phenyl-4-hydroxy-1-piperidyl indole analogues with nanomolar affinity at D2 dopamine receptors, ranging from 10-to 100-fold selective for D2 compared to the D3 dopamine receptor subtype. More recently, we evaluated a panel of aripiprazole analogues, identifying several analogues that also exhibit D2 vs D3 dopamine receptor binding selectivity. These studies further characterize the intrinsic efficacy of the compound with the greatest binding selectivity from each chemical class, 1-((5-methoxy-1H-indol-3-yl)methyl)-4-(4-(methylthio)phenyl)piperidin-4-ol (SV 293) and 7-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butoxy)-3,4-dihydroquinolin-2(1H)-one (SV-III-130s), using an adenylyl cyclase inhibition assay, a G-protein-coupled inward-rectifying potassium (GIRK) channel activation assay, and a cell based phospho-MAPK (pERK1/2) assay. SV 293 was found to be a neutral antagonist at D2 dopamine receptors using all three assays. SV-III-130s is a partial agonist using an adenylyl cyclase inhibition assay but an antagonist in the GIRK and phospho ERK1/2 assays. To define the molecular basis for the binding selectivity, the affinity of these two compounds was evaluated using (a) wild type human D2 and D3 receptors and (b) a panel of chimeric D2/D3 dopamine receptors. Computer-assisted modeling techniques were used to dock these compounds to the human D2 and D3 dopamine receptor subtypes. It is hoped that these studies on D2 receptor selective ligands will be useful in the future design of (a) receptor selective ligands used to define the function of D2-like receptor subtypes, (b) novel pharmacotherapeutic agents, and/or (c) in vitro and in vivo imaging agents. KEYWORDS: Dopamine receptors, binding selectivity, functional selectivity, GPCR structure, D2-like dopamine receptors, GPCR model building, ligand−receptor docking T here are three dopaminergic pathways in the brain: the nigrostriatal pathway, the mesocorticolimbic pathway, and the tuberoinfundibular pathway. These pathways are involved in movement coordination, cognition, emotion, memory, reward, and regulation of prolactin secretion. Alterations in the dopaminergic pathways are thought to be involved in the pathogenesis of neurological, neuropsychiatric, and hormonal disorders. 1−6 Modulation of the dopaminergic pathways is also thought to occur as a consequence of acute or chronic abuse of pyschostimulants. 7,8 Previous studies have defined two types of dopamine receptors, the D1-like (D1 and D5 subtypes) and D2-like (D2, D3, and D4 subtypes) receptors. D1-like receptors are linked to the activation of adenylyl cyclase via coupling to the Gs/Golf class of G proteins. 9 Stimulation of the D2-like receptors results in coupling with the Gi/Go class of G proteins, leading to the inhibition of adenylyl cyclase activity. 10,11 Agonist activation of D2-like receptors can also lead to (a) activation of G-protein-coupled inward rectifying potassium (GIRK) channels, (b) stimulation of mitogenesis, (c) an increase in...

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