Anecdotal reports have surfaced concerning misuse of the HIV antiretroviral medication efavirenz ((4S)-6-chloro-4-(2-cyclopropylethynyl)-4-(trifluoromethyl)-2,4-dihydro-1H-3,1-benzoxazin-2-one) by HIV patients and non-infected teens who crush the pills and smoke the powder for its psychoactive effects. Molecular profiling of the receptor pharmacology of efavirenz pinpointed interactions with multiple established sites of action for other known drugs of abuse including catecholamine and indolamine transporters, and GABA A and 5-HT 2A receptors. In rodents, interaction with the 5-HT 2A receptor, a primary site of action of lysergic acid diethylamine (LSD), appears to dominate efavirenz's behavioral profile. Both LSD and efavirenz reduce ambulation in a novel open-field environment. Efavirenz occasions drug-lever responding in rats discriminating LSD from saline, and this effect is abolished by selective blockade of the 5-HT 2A receptor. Similar to LSD, efavirenz induces head-twitch responses in wild-type, but not in 5-HT 2A -knockout, mice. Despite having GABA A -potentiating effects (like benzodiazepines and barbiturates), and interactions with dopamine transporter, serotonin transporter, and vesicular monoamine transporter 2 (like cocaine and methamphetamine), efavirenz fails to maintain responding in rats that selfadminister cocaine, and it fails to produce a conditioned place preference. Although its molecular pharmacology is multifarious, efavirenz's prevailing behavioral effect in rodents is consistent with LSD-like activity mediated via the 5-HT 2A receptor. This finding correlates, in part, with the subjective experiences in humans who abuse efavirenz and with specific dose-dependent adverse neuropsychiatric events, such as hallucinations and night terrors, reported by HIV patients taking it as a medication.

Receptor endocytosis is an important mechanism for regulating the synaptic efficacy of neurotransmitters. There is strong evidence that GABA A receptor endocytosis is clathrin-dependent; however, this process is not well understood. Here we demonstrate that in HEK 293 cells, endocytosis of GABA A receptors composed of either ␣ 1 ␤ 2 ␥ 2 L or ␣ 1 ␤ 2 subunits is blocked by the dominant negative dynamin construct K44A. Furthermore, we identify a dileucine AP2 adaptin-binding motif within the receptor ␤ 2 subunit that is critical for endocytosis. Internalization of GABA A receptors lacking this motif is dramatically inhibited, and the receptors appear to accumulate on the cell surface. Patch clamp analysis of receptors lacking the dileucine motif show that there is an increase in the peak amplitude of GABA-gated chloride currents compared with wild-type receptors. Additionally, GABA-gated chloride currents in HEK 293 cells expressing wild-type receptors are increased by introduction of a peptide corresponding to the dileucine motif region of the receptor ␤ 2 subunit but not by a control peptide containing alanine substitutions for the dileucine motif. In mouse brain cerebral cortical neurons, the dileucine motif peptide increases GABA-gated chloride currents of native GABA A receptors. This is the first report to our knowledge that an AP2 adaptin dileucine recognition motif is critical for the endocytosis of ligand-gated ion channels belonging to this superfamily.The GABA A receptor is a ligand-gated chloride channel that, upon activation by GABA 1 (␥-aminobutyric acid), mediates increases in chloride conductance resulting in membrane hyperpolarization and neuronal inhibition (1). The role of these receptors in hyperexcitability states, such as epilepsy and anxiety, is widely recognized. Importantly, GABA A receptors mediate the effects of benzodiazepines and barbiturates, two frequently prescribed classes of therapeutic agents. The GABA A receptor is a pentameric receptor composed of multiple subunits, each containing four membrane-spanning regions (M1-M4) with a large intracellular loop between M3 and M4. A number of subunits exist (␣ 1Ϫ6 , ␤ 1Ϫ3 , ␥ 1Ϫ3 , ␦, , ⑀, ), and receptors composed of ␣ 1 ␤ 2 ␥ 2 L subunits are believed to represent the predominant GABA A receptor subtype in the brain (1).Receptor endocytosis is known to regulate the cell surface expression of neurotransmitter receptors, and such regulation is an important mechanism for controlling the synaptic efficacy of neurotransmitters (2). Although GABA A receptors undergo endocytosis, the mechanism is not well understood. Several lines of evidence indicate that GABA A receptor endocytosis may be clathrin/dynamin-dependent. These include the presence of GABA A receptors in clathrin-coated vesicles isolated from brain (3), the colocalization of the receptor with transferrin receptors (4), and the colocalization and co-immunoprecipitation of hippocampal GABA A receptors with the clathrin adaptor complex AP2 adaptin (5). Additionally, peptides that dis...

L-type voltage-gated Ca 2؉ channels (VGCC) play an important role in dendritic development, neuronal survival, and synaptic plasticity. Recent studies have demonstrated that the gonadal steroid estrogen rapidly induces Ca 2؉ influx in hippocampal neurons, which is required for neuroprotection and potentiation of LTP. The mechanism by which estrogen rapidly induces this Ca 2؉ influx is not clearly understood. We show by electrophysiological studies that extremely low concentrations of estrogens acutely potentiate VGCC in hippocampal neurons, hippocampal slices, and HEK-293 cells transfected with neuronal L-type VGCC, in a manner that was estrogen receptor (ER)-independent. Equilibrium, competitive, and whole-cell binding assays indicate that estrogen directly interacts with the VGCC. Furthermore, a L-type VGCC antagonist to the dihydropyridine site displaced estrogen binding to neuronal membranes, and the effects of estrogen were markedly attenuated in a mutant, dihydropyridineinsensitive L-type VGCC, demonstrating a direct interaction of estrogens with L-type VGCC. Thus, estrogen-induced potentiation of calcium influx via L-type VGCC may link electrical events with rapid intracellular signaling seen with estrogen exposure leading to modulation of synaptic plasticity, neuroprotection, and memory formation.estrogen receptors ͉ signaling ͉ estradiol ͉ memory A large body of evidence shows that estrogens exert multiple rapid effects on the structure and function of neurons in a variety of brain regions, including the hippocampus (1). For example, estrogens rapidly potentiate kainite-induced currents in hippocampal neurons from wild-type (2) as well as from estrogenreceptor (ER)-␣ knockout (3) mice and induce rapid spine synapse formation in the CA1 hippocampus of ovariectomized (OVX) rats (4). Furthermore, acute application of estrogens to hippocampal slices increases NMDA and AMPA receptor transmission (5), induces long-term potentiation (LTP) and long-term depression (LTD) (6), and rapidly modulates neuronal excitability in rat medial amygdala (7) and hippocampus(8).It is well known that estrogens interact with cell membrane components and initiate signaling events leading to a rise in intracellular Ca 2ϩ , and activation of Src kinase, G protein-coupled receptor (GPCR), MAPK, PI3K/AKT, PKA, and adenylyl cyclase (9). The mechanism(s) by which estrogens induce these rapid and diverse effects remains largely unknown. Ca 2ϩ is a second messenger that can trigger the modification of synaptic efficacy. A plasticity-induction protocol like repetitive low-frequency synaptic stimulation (10) induces the elevation of postsynaptic intracellular Ca 2ϩ . The level of intracellular Ca 2ϩ concentration can activate numerous kinases like CAMK, PKA, PKC, MAPK, PI3K, or phosphatases (11-15), which, respectively, phosphorylate or dephosphorylate ion channels, transcription factors, and other proteins that are involved in synaptic plasticity and memory formation. Because voltage-gated Ca 2ϩ channels (VGCC)-mediated extracellular Ca ...

These results suggest that sigma-1 receptor activation can regulate calcium homeostasis and signaling in RGCs, likely by directly influencing the activity of L-type voltage-gated calcium channels. Regulation of calcium influx in RGCs by sigma-1 receptor ligands may represent in part the neuroprotective effect of sigma-1 receptors.

Despite the presence of the multiple subunits (␣, ␤, ␥, and ␦) and their isoforms for ␥-aminobutyric acid, type A (GABA A ) receptors in mammalian brains, the ␣x␤2␥2 subtypes appear to be the prototype GABA A receptors sharing many properties with native neuronal receptors. In order to gain insight into their subunit stoichiometry and orientation, we prepared a tandem construct of the ␣6 and ␤2 subunit cDNAs where the carboxyl-terminal of ␣6 is linked to the amino-terminal of ␤2 via a linker encoding 10 glutamine residues. Transfection of human embryonic kidney 293 cells with the tandem construct alone failed to induce GABA-dependent Cl ؊ currents, but its cotransfection with the cDNA for ␣6 or ␥2, but not ␤2, led to the appearance of GABA currents which were picrotoxin-sensitive and, in the case of ␥2 containing receptors, responded to a benzodiazepine agonist, U-92330. The high affinity GABA site, however, was detected with [ 3 H]muscimol binding in all combinations of the receptor subunits, including the tandem construct alone or with the ␤2. No appreciable differences were found in their K d (2.5 nM) and B max values (1.4 pmol/mg of protein). These data are consistent with the view that the polypeptides arising from the tandem construct were expressed with the high affinity GABA site, but unable to form GABA channels. The requirement of a specific monomeric subunit (␣6 or ␥2) for the tandem construct to express Cl ؊ currents supports a pentameric structure of GABA A receptors consisting of two ␣6, two ␤2, and one ␥2 for the ␣6␤2␥2 and three ␣6 and two ␤2 for the ␣6␤2 subtype.GABA A 1 receptors, responsible for inhibitory neurotransmission in mammalian brains, are ligand-gated Cl Ϫ channels made of various subunits (␣, ␤, ␥, and ␦) (1-3). Each subunit consists of several isoforms and contains four transmembranespanning segments (M1 to M4) (1-5). Despite the existence of the multiple subunits and their isoforms, combinations of ␣x, ␤2, and ␥2 subunits produced Cl Ϫ channels sharing many functional characteristics with native neuronal receptors and displaying the ability to respond to all the GABA A receptor ligands known up-to-date (1, 3, 6, 7). Such cloned GABA A receptors have been proposed to be of pentameric structure with M2 lining the pore in analogy with another member of the four transmembrane ligand-gated channel family, acetylcholine receptors (1-3). Recent studies, including immunoprecipitation with subunit specific antibodies, have shown the presence of two ␣ subunits per GABA A receptor (8 -11). Further experimental evidence is needed, however, about the stoichiometry of the recombinant GABA A receptors of ␣x␤2␥2 and their modes of association. One way to gain insight into this structural issue is to predetermine the alignment of subunits via gene fusion and to study such fused gene products. Similar approaches have been successful with potassium channels made of their subunits in concatameric or tandem linkages (12,13). In this study we prepared a tandem construct of ␣6 and ␤2 subunit cDNAs of th...