RESUMEN
Expression of functional, recombinant alpha7 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 alpha7 subunit. In addition to confirming the previously reported effect on alpha7 receptor expression in Xenopus oocytes we demonstrate that ric-3 promotes the formation of functional alpha7 receptors in mammalian cells, as determined by whole cell patch clamp recording and surface alpha-bungarotoxin binding. Upon application of 1 mm nicotine, currents were undetectable in HEK293 cells expressing only the alpha7 subunit. In contrast, co-expression of alpha7 and ric-3 cDNAs resulted in currents that averaged 42 pA/pF with kinetics similar to those observed in cells expressing endogenous alpha7 receptors. Immunoprecipitation studies demonstrate that alpha7 and ric-3 proteins co-associate. Additionally, cell surface labeling with biotin revealed the presence of alpha7 protein on the plasma membrane of cells lacking ric-3, but surface alpha-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 alpha7 receptors in HEK293 cells.
Asunto(s)
Proteínas/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Animales , Western Blotting , Bungarotoxinas/metabolismo , Línea Celular , Conductividad Eléctrica , Regulación de la Expresión Génica , Humanos , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular , Neuronas/metabolismo , Oocitos/metabolismo , Unión Proteica , Subunidades de Proteína/análisis , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Receptores Nicotínicos/análisis , Xenopus , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
In the present study, we describe the characterization of a positive allosteric modulator at metabotropic glutamate subtype 2 receptors (mGluR2). N-(4-(2-Methoxyphenoxy)-phenyl-N-(2,2,2-trifluoroethylsulfonyl)-pyrid-3-ylmethylamine (LY487379) is a selective positive allosteric modulator at human mGluR2 and is without activity at human mGluR3. Furthermore, LY487379 has no intrinsic agonist or antagonist activity at hmGluR2, as determined by functional guanosine 5'(gamma-[35S]thio)triphosphate ([35S]GTPgammaS) binding, single-cell Ca2+ imaging, and electrophysiological studies. However, LY487379 markedly potentiated glutamate-stimulated [35S]GTPgammaS binding in a concentration-dependent manner at hmGluR2, shifting the glutamate dose-response curve leftward by 3-fold and increasing the maximum levels of [35S]GTPgammaS stimulation. This effect of LY487479 was also observed to a greater extent on the concentration-response curves to selective hmGluR2/3 agonists. In radioligand binding studies to rat cortical membranes, LY487379 increased the affinity of the radiolabeled agonist, [3H]DCG-IV, without affecting the binding affinity of the radiolabeled antagonist, [3H]LY341495. In rat hippocampal slices, coapplication of LY487379 potentiated synaptically evoked mGluR2 responses. Finally, to elucidate the site of action, we systematically exchanged segments and single amino acids between hmGluR2 and hmGluR3. Substitution of Ser688 and/or Gly689 in transmembrane IV along with Asn735 located in transmembrane segment V, with the homologous amino acids of hmGluR3, completely eliminated LY487379 allosteric modulation of hmGluR2. We propose that this allosteric binding site defines a pocket that is different from the orthosteric site located in the amino terminal domain.
Asunto(s)
Glicina/análogos & derivados , Piridinas/farmacología , Receptores de Glutamato Metabotrópico/metabolismo , Sulfonamidas/farmacología , Regulación Alostérica , Secuencia de Aminoácidos , Aminoácidos/farmacología , Animales , Sitios de Unión , Células CHO , Calcio/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Corteza Cerebral/citología , Cricetinae , Ciclopropanos/farmacología , Giro Dentado/efectos de los fármacos , Giro Dentado/metabolismo , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Glicina/farmacología , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Humanos , Datos de Secuencia Molecular , Vía Perforante/efectos de los fármacos , Vía Perforante/metabolismo , Estructura Terciaria de Proteína , Ratas , Homología de Secuencia de Aminoácido , Tritio , Xantenos/farmacologíaRESUMEN
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.