Laura E. Chavez-Noriega

Ric-3 Promotes Functional Expression of the Nicotinic Acetylcholine Receptor α7 Subunit in Mammalian Cells

Expression of functional, recombinant α7 nicotinic acetylcholine receptors in several mammalian cell types, including HEK293 cells, has been problematic. We have isolated the recently described human ric-3 cDNA and co-expressed it in Xenopus oocytes and HEK293 cells with the human nicotinic acetylcholine receptor α7 subunit. In addition to confirming the previously reported effect on α7 receptor expression in Xenopus oocytes we demonstrate that ric-3 promotes the formation of functional α7 receptors in mammalian cells, as determined by whole cell patch clamp recording and surface α-bungarotoxin binding. Upon application of 1 mm nicotine, currents were undetectable in HEK293 cells expressing only the α7 subunit. In contrast, co-expression of α7 and ric-3 cDNAs resulted in currents that averaged 42 pA/pF with kinetics similar to those observed in cells expressing endogenous α7 receptors. Immunoprecipitation studies demonstrate that α7 and ric-3 proteins co-associate. Additionally, cell surface labeling with biotin revealed the presence of α7 protein on the plasma membrane of cells lacking ric-3, but surface α-bungarotoxin staining was only observed in cells co-expressing ric-3. Thus, ric-3 appears to be necessary for proper folding and/or assembly of α7 receptors in HEK293 cells.

Metabotropic Glutamate Receptors: Potential Drug Targets for the Treatment of Schizophrenia

Schizophrenia is a debilitating chronic psychiatric illness affecting 1% of the population. The cardinal features of schizophrenia are positive symptoms (thought disorder, hallucinations, catatonic behavior), negative symptoms (social withdrawal, anhedonia, apathy) and cognitive impairment. Although progress in elucidating the aetiology of schizophrenia has been slow, new insights on the neurochemical and neurophysiological mechanisms underlying the pathophysiology of this illness are beginning to emerge. The glutamate/N-methyl-D-aspartate (NMDA) hypofunction hypothesis of schizophrenia is supported by observations that administration of NMDA glutamate receptor antagonists such as phencyclidine (PCP) or ketamine induces psychosis in humans; moreover, decreased levels of glutamate and changes in several markers of glutamatergic function occur in schizophrenic brain. Administration of PCP or ketamine to rodents elicits an increase in locomotion and stereotypy accompanied by an increase in glutamate efflux in several brain regions. Systemic administration of group II metabotropic glutamate (mGlu) receptor agonists suppresses PCP-induced behavioral effects and the increase in glutamate efflux. Activation of group II mGlu receptors (mGlu2 and mGlu3) decreases glutamate release from presynaptic nerve terminals, suggesting that group II mGlu receptor agonists may be beneficial in the treatment of schizophrenia. In addition, pharmacological manipulations that enhance NMDA function may be efficacious antipsychotics. Selective activation of mGlu5 receptors significantly potentiates NMDA-induced responses, supporting this novel approach for the treatment of schizophrenia. The glutamate hypothesis of schizophrenia predicts that agents that restore the balance in glutamatergic neurotransmission will ameliorate the symptomatology associated with this illness. Development of potent, efficacious, systemically active drugs will help to address the antipsychotic potential of these novel therapeutics. This review will discuss recent progress in elucidating the pharmacology and function of group II mGlu and mGlu5 receptors in the context of current hypotheses on the pathophysiology of schizophrenia and the need for new and better antipsychotics.

Subunit-dependent inhibition of human neuronal nicotinic acetylcholine receptors and other ligand-gated ion channels by dissociative anesthetics ketamine and dizocilpine.

Background: The neuronal mechanisms responsible for dissociative anesthesia remain controversial. N-methyl-D-aspartate (NMDA) receptors are inhibited by ketamine and related drugs at concentrations lower than those required for anesthetic effects. Thus, the authors studied whether ligand-gated ion channels other than NMDA receptors might display a sensitivity to ketamine and dizocilpine that is consistent with concentrations required for anesthesia. Methods: Heteromeric human neuronal nicotinic acetylcholine receptors (hnAChR channels &agr;2&bgr;2, &agr;2&bgr;4, &agr;3&bgr;2, &agr;3&bgr;4, &agr;4&bgr;2 and &agr;4&bgr;4), 5-hydroxytryptamine3 (5-HT3), &agr;1&bgr;2&ggr;2S&ggr;-aminobutyric acid type A (GABAA) and &agr;1 glycine receptors were expressed in Xenopus oocytes, and effects of ketamine and dizocilpine were studied using the two-electrode voltage-clamp technique. Results: Both ketamine and dizocilpine inhibited hnAChRs in a noncompetitive and voltage-dependent manner. Receptors containing &bgr;4 subunits were more sensitive to ketamine and dizocilpine than those containing &bgr;2 subunits. The inhibitor concentration for half-maximal response (IC50) values for ketamine of hnAChRs composed of &bgr;4 subunits were 9.5–29 &mgr;M, whereas those of &bgr;2subunits were 50–92 &mgr;M. Conversely, 5-HT3 receptors were inhibited only by concentrations of ketamine and dizocilpine higher than the anesthetic concentrations. This inhibition was mixed (competitive/noncompetitive). GABAA and glycine receptors were very resistant to dissociative anesthetics. Conclusions: Human nAChRs are inhibited by ketamine and dizocilpine at concentrations possibly achieved in vivo during anesthesia in a subunit-dependent manner, with &bgr; subunits being more critical than &agr; subunits. Conversely, 5-HT3, GABAA, and glycine receptors were relatively insensitive to dissociative anesthetics.

Comparative structure of human neuronal α2–α7 and β2–β4 nicotinic acetylcholine receptor subunits and functional expression of the α2, α3, α4, α7, β2, and β4 subunits

AbstractcDNA clones encoding human neuronal nicotinic acetylcholine receptor α2, α3, α4, α5, α6, α7, β2, β3, and β4 subunits were isolated from brainstem, hippocampus, prefrontal cortex, substantia nigra, thalamus, and IMR32 libraries. Human α2 and α6 and full-length β3 and β4 clones have not been previously reported. Deduced amino acid sequences of the α2, α6, β3, and β4 predicted mature peptides are 503 residues (56.9 kDa), 464 residues (53.7 kDa), 440 residues (50.8 kDa), and 477 residues (54.1 kDa), respectively. These sequences show 84 (α2), 87 (α6), 89 (β3), and 84% (β4) identity to the corresponding rat sequences. The amino termini of the human α2 and β3 mature peptides contain 23 and six additional residues, respectively, compared to those of rat α2 and β3. Recombinant receptors were expressed inXenopus laevis oocytes injected with in vitro transcripts encoding either α7 alone or α2, α3, or α4 in pairwise combination with β2 or β4. Inward currents were elicited by the application of acetylcholine (1–100 µM) and other agonists; these responses were blocked 65–97% by application of 10 µM d-tubocurare, confirming functional expression of human nicotinic receptors.

Characterization of the recombinant human neuronal nicotinic acetylcholine receptors α3β2 and α4β2 stably expressed in HEK293 cells

Abstract HEK293 cells were stably transfected with the cDNAs encoding full-length human neuronal nicotinic acetylcholine receptor (nAChR) subunit combinations α3β2 or α4β2. [ 3 H]-(±)Epibatidine ([ 3 H]-(±)EPI) bound to membranes from A3B2 (α3β2) and A4B2.2 (α4β2) cells with K d values of 7.5 and 33.4 pM and B max values of 497 and 1564 fmol/mg protein, respectively. Concentration-dependent increases in intracellular free Ca 2+ concentration were elicited by nAChR agonists with a rank order of potency of EPI>1,1-dimethyl-4-phenylpiperazinium (DMPP)>nicotine (NIC)=suberyldicholine (SUB)>cytisine (CYT)=acetylcholine (ACh) for A3B2 cells and EPI>CYT=SUB=NIC=DMPP>ACh for A4B2.2 cells. Antagonists of nAChRs blocked NIC-induced responses with a rank order of potency of d-tubocurarine (d-Tubo)=mecamylamine (MEC)>dihydro-β-erythroidine (DHβE) in A3B2 cells and MEC=DHβE>d-Tubo in A4B2.2 cells. Whole-cell patch clamp recordings indicate that the decay rate of macroscopic ACh-induced currents is faster in A3B2 than in A4B2.2 cells and that A3B2 cells are less sensitive to ACh than A4B2.2 cells. ACh currents elicited in α3β2 and α4β2 human nAChRs are maximally potentiated at 20 and 2 mM external Ca 2+ , respectively. Our results indicate that stably expressed α3β2 and α4β2 human nAChRs are pharmacologically and functionally distinct.

Human Neuronal Nicotinic Acetylcholine Receptors Expressed in Xenopus Oocytes Predict Efficacy of Halogenated Compounds That Disobey the Meyer-Overton Rule

BACKGROUND According to the Meyer-Overton rule, anesthetic potency of a substance can be predicted by its lipid solubility, but a group of halogenated volatile compounds predicted to induce anesthesia does not obey this rule. Thus, these compounds are useful tools for studies of molecular targets of anesthetics. Human neuronal nicotinic acetylcholine receptor (hnAChR) subunits have been recently cloned, which allowed the authors to assess whether these receptors could differentiate among volatile anesthetic and nonimmobilizer compounds. This study provides the first data regarding anesthetic sensitivity of hnAChRs. METHODS alpha2beta4, alpha3beta4, and alphaabeta2 hnAChRs were expressed in Xenopus oocytes, and effects of volatile anesthetics isoflurane and F3 (1-chioro-1,2,2-triflurocyclobutane, 1A) and nonimmobilizers F6 (1,2-dichlorohexafluorocyclobutane, 2N) and F8 (2,3-dichlorooctafluorobutane) on the peak acetylcholine-gated currents were studied using the two-electrode voltage-clamp technique. RESULTS Isoflurane and F3 inhibited all the hnAChRs tested in a concentration-dependent manner. Isoflurane at a concentration corresponding to 1 minimum alveolar concentration (MAC) inhibited 83, 69, and 71% of ACh-induced currents in alpha2beta4, alpha3beta4, and alpha4beta2 hnAChRs, respectively, and 1 MAC of F3 inhibited 64, 44, and 61% of currents gated in those receptors. F6 (8-34 microM) did not cause any changes in currents gated by any of the receptors tested. F8 (4-18 microM) did not alter the currents gated in either alpha3beta4 or alpha4beta2 receptors, but caused a small potentiation of alpha2beta4 hnAChRs without a concentration-response relation. CONCLUSION The in vivo potency and effectiveness of volatile anesthetic and nonimmobilizer compounds were consistent with their actions on hnAChRs expressed in a recombinant expression system, suggesting a potential participation of these receptors in the mechanisms of anesthesia.

Conformationally restricted analogues of nicotine and anabasine

A series of conformationally restricted analogues of nicotine has been synthesized and evaluated as agonists of neuronal acetylcholine receptors. Compound 2 (SIB-1663), which selectively activated human recombinant alpha 2 beta 4 and alpha 4 beta 4 nAChRs, was shown to be active in animal models of Parkinsons disease and pain.

Novel Potential Therapeutics for Schizophrenia: Focus on the Modulation of Metabotropic Glutamate Receptor Function

Schizophrenia is the most disabling psychiatric disorder and one of the worlds top ten causes of long-term disability, affecting 1% of the population worldwide. The major symptoms of schizophrenia , psychosis (positive symptoms), apathy, social withdrawal (negative symptoms) and cognitive impairment, become manifest in late adolescence/early adulthood and persist thereafter, resulting in chronic disability. The pharmacotherapy of schizophrenia has evolved from typical antipsychotics (dopamine D2 receptor antagonists) to atypical antipsychotics (mixed D2 and serotonin 5-HT2A antagonists with activity at various other receptors) with improved efficacy and side effect profile. More recently, the glutamate/ N-methyl-D- aspartate glutamate receptor (NMDAR) hypothesis of schizophrenia has been formulated. This hypothesis is supported by the observation that administration of NMDAR blockers to human volunteers is psychotomimetic and administration to schizophrenic patients exacerbates pre-existing symptoms. This has generated an interest to develop novel antipsychotics focused on the identification of novel molecular targets and susceptibility genes that result in deregulation of glutamatergic , GABAergic and dopaminergic neurotransmission. In particular, metabotropic glutamate (mGlu) receptors mGlu2/3 and mGlu5 are prominently expressed in relevant forebrain regions and their activation modulates glutamatergic transmission and NMDAR function in the mammalian brain. The activity of mGlu2/3 and mGlu5 receptor agonists and more recently, the activity shown by selective mGlu2 and mGlu5 receptor allosteric potentiators in preclinical models of psychosis are promising. Further evaluation of the efficacy and side effect profile of potent, selective and brain-penetrant mGlu receptor activators may provide novel therapeutic avenues for the treatment of schizophrenia.

Recombinant human receptors and functional assays in the discovery of altinicline (SIB-1508Y), a novel acetylcholine-gated ion channel (nAChR) agonist

Neuronal nicotinic acetylcholine receptors (nAChRs) are a class of ion channels with significant potential as molecular targets for the design of drugs to treat a variety of CNS disorders. The discovery that neuronal nAChRs are further subdivided into multiple subtypes suggests that drugs which act selectively at specific nAChR subtypes might effectively treat Parkinsons disease (PD), Alzheimers disease (AD), schizophrenia, ADHD, depression, anxiety or pain without the accompanying adverse side effects associated with non-selective agents such as nicotine (1) and epibatidine. Altinicline (SIB-1508Y) is a novel, small molecule designed to selectively activate neuronal nAChRs and is undergoing clinical evaluation for the treatment of PD. It was selected from a series of compounds primarily on the basis of results from functional assays, including (a) measurement of Ca2+ flux in stable cell lines expressing specific recombinant human neuronal nAChR subtypes; (b) determination of in vitro and in vivo neurotransmitter release; (c) in vivo models of PD. Biological data on both altinicline and the series of compounds from which it was selected are reported.