RESUMEN
Although considerable evidence suggests that the chemical synapse is a lynchpin underlying affective disorders, how molecular insults differentially affect specific synaptic connections remains poorly understood. For instance, Neurexin 1a and 2 (NRXN1 and NRXN2) and CNTNAP2 (also known as CASPR2), all members of the neurexin superfamily of transmembrane molecules, have been implicated in neuropsychiatric disorders. However, their loss leads to deficits that have been best characterized with regard to their effect on excitatory cells. Notably, other disease-associated genes such as BDNF and ERBB4 implicate specific interneuron synapses in psychiatric disorders. Consistent with this, cortical interneuron dysfunction has been linked to epilepsy, schizophrenia and autism. Using a microarray screen that focused upon synapse-associated molecules, we identified Cntnap4 (contactin associated protein-like 4, also known as Caspr4) as highly enriched in developing murine interneurons. In this study we show that Cntnap4 is localized presynaptically and its loss leads to a reduction in the output of cortical parvalbumin (PV)-positive GABAergic (γ-aminobutyric acid producing) basket cells. Paradoxically, the loss of Cntnap4 augments midbrain dopaminergic release in the nucleus accumbens. In Cntnap4 mutant mice, synaptic defects in these disease-relevant neuronal populations are mirrored by sensory-motor gating and grooming endophenotypes; these symptoms could be pharmacologically reversed, providing promise for therapeutic intervention in psychiatric disorders.
Asunto(s)
Dopamina/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Transducción de Señal , Transmisión Sináptica/genética , Ácido gamma-Aminobutírico/metabolismo , Animales , Antipsicóticos/farmacología , Conducta Animal/efectos de los fármacos , Conducta Animal/fisiología , Sinapsis Eléctricas/genética , Sinapsis Eléctricas/ultraestructura , Femenino , Genotipo , Humanos , Masculino , Ratones , Polimorfismo de Nucleótido SimpleRESUMEN
Previous findings suggest that neuroadaptations downstream of D-1 dopamine (DA) receptor stimulation in nucleus accumbens (NAc) are involved in the enhancement of drug reward by chronic food restriction (FR). Given the high co-expression of D-1 and GluR1 AMPA receptors in NAc, and the regulation of GluR1 channel conductance and trafficking by D-1-linked intracellular signaling cascades, the present study examined effects of the D-1 agonist, SKF-82958, on NAc GluR1 phosphorylation, intracranial electrical self-stimulation reward (ICSS), and reversibility of reward effects by a polyamine GluR1 antagonist, 1-NA-spermine, in ad libitum fed (AL) and FR rats. Systemically administered SKF-82958, or brief ingestion of a 10% sucrose solution, increased NAc GluR1 phosphorylation on Ser845, but not Ser831, with a greater effect in FR than AL rats. Microinjection of SKF-82958 in NAc shell produced a reward-potentiating effect that was greater in FR than AL rats, and was reversed by co-injection of 1-NA-spermine. GluR1 abundance in whole cell and synaptosomal fractions of NAc did not differ between feeding groups, and microinjection of AMPA, while affecting ICSS, did not exert greater effects in FR than AL rats. These results suggest a role of NAc GluR1 in the reward-potentiating effect of D-1 DA receptor stimulation and its enhancement by FR. Moreover, GluR1 involvement appears to occur downstream of D-1 DA receptor stimulation rather than reflecting a basal increase in GluR1 expression or function. Based on evidence that phosphorylation of GluR1 on Ser845 primes synaptic strengthening, the present results may reflect a mechanism via which FR normally facilitates reward-related learning to re-align instrumental behavior with environmental contingencies under the pressure of negative energy balance.
Asunto(s)
Privación de Alimentos/fisiología , Núcleo Accumbens/efectos de los fármacos , Núcleo Accumbens/fisiología , Receptores AMPA/metabolismo , Receptores de Dopamina D1/metabolismo , Recompensa , Animales , Benzazepinas/farmacología , Sacarosa en la Dieta , Agonistas de Dopamina/farmacología , Ingestión de Alimentos/fisiología , Estimulación Eléctrica , Antagonistas de Aminoácidos Excitadores/farmacología , Masculino , Neuronas/efectos de los fármacos , Neuronas/fisiología , Fosforilación/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores AMPA/antagonistas & inhibidores , Receptores de Dopamina D1/agonistas , Autoadministración , Espermina/farmacología , Sinaptosomas/efectos de los fármacos , Sinaptosomas/fisiología , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/metabolismoRESUMEN
Voltage gated Ca(2+) channel (VGCC) auxiliary beta subunits increase membrane expression of the main pore-forming alpha(1) subunits and finely tune channel activation and inactivation properties. In expression studies, co-expression of beta subunits also reduced neuronal Ca(2+) channel regulation by heterotrimeric G protein. Biochemical studies suggest that VGCC beta subunits and G protein betagamma can compete for overlapping interaction sites on VGCC alpha(1) subunits, suggesting a dynamic association of these subunits with alpha(1). In this work we have analyzed the stability of the alpha(1)/beta association under physiological conditions. Regulation of the alpha(1A) Ca(2+) channel inactivation properties by beta(1b) and beta(2a) subunits had two major effects: a shift in voltage-dependent inactivation (E(in)), and an increase of the non-inactivating current (R(in)). Unexpectedly, large variations in magnitude of the effects were recorded on E(in), when beta(1b) was expressed, and R(in), when beta(2a) was expressed. These variations were not proportional to the current amplitude, and occurred at similar levels of beta subunit expression. beta(2a)-induced variations of R(in) were, however, inversely proportional to the magnitude of G protein block. These data underline the two different mechanisms used by beta(1b) and beta(2a) to regulate channel inactivation, and suggest that the VGCC beta subunit can unbind the alpha1 subunit in physiological situations.
Asunto(s)
Canales de Calcio/metabolismo , Animales , Western Blotting , Canales de Calcio/química , Canales de Calcio/genética , Electrofisiología , Encefalina Ala(2)-MeFe(4)-Gli(5)/metabolismo , Mutación , Oocitos/metabolismo , Unión Proteica , Subunidades de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Xenopus laevisRESUMEN
Stargazin or [gamma]2, the product of the gene mutated in the stargazer mouse, is a homologue of the [gamma]1 protein, an accessory subunit of the skeletal muscle L-type Ca2+ channel. [gamma]2 is selectively expressed in the brain, and considered to be a putative neuronal Ca2+ channel subunit based mainly on homology to [gamma]1. Two new members of the [gamma] family expressed in the brain have recently been identified: [gamma]3 and [gamma]4. We have co-expressed, in Xenopus oocytes, the human [gamma]2, [gamma]3 and [gamma]4 subunits with the P/Q-type (Ca(V)2.1) Ca2+ channel and different regulatory subunits ([alpha]2-[delta]; [beta]1, [beta]2, [beta]3 or [beta]4). Subcellular distribution of the [gamma] subunits confirmed their membrane localization. Ba2+ currents, recorded using two-electrode voltage clamp, showed that the effects of the [gamma] subunits on the electrophysiological properties of the channel are, most of the time, minor. However, a fraction of the oocytes expressing [beta] subunits displayed an unusual slow-inactivating Ba2+ current. Expression of both [beta] and [gamma] subunits increased the appearance of the slow-inactivating current. Our data support a role for the [gamma] subunit as a brain Ca2+ channel modulatory subunit and suggest that [beta] and [gamma] subunits are involved in a switch between two regulatory modes of the P/Q-type channel inactivation.
Asunto(s)
Canales de Calcio Tipo N , Activación del Canal Iónico/fisiología , Animales , Bario/farmacocinética , Calcio/metabolismo , Canales de Calcio Tipo N/química , Canales de Calcio Tipo N/genética , Canales de Calcio Tipo N/metabolismo , Línea Celular , Femenino , Expresión Génica/fisiología , Humanos , Riñón/citología , Cinética , Oocitos/fisiología , Técnicas de Placa-Clamp , Estructura Terciaria de Proteína , Transfección , Xenopus laevisRESUMEN
Spinocerebellar ataxia type 6 (SCA6) is a dominantly inherited degenerative disorder of the cerebellum characterized by nearly selective and progressive death of Purkinje cells. The underlying mutation in SCA6 consists of an expansion of a trinucleotide CAG repeat in the 3' region of the gene, CACNA1A, encoding the alpha(1A) subunit of the neuronal P/Q-type voltage-gated calcium channel. Although it is known that this mutation results in an expanded tract of glutamine residues in some alpha(1A) splice forms, the distribution of these splice forms and the role of this mutation in the highly selective Purkinje cell degeneration seen in SCA6 have yet to be elucidated. Using specific antisera we demonstrate that the pathological expansion in SCA6 can potentially be expressed in multiple isoforms of the alpha(1A) subunit, and that these isoforms are abundantly expressed in the cerebellum, particularly in the Purkinje cell bodies and dendrites. Using alpha(1A) subunit chimeras expressing SCA6 mutations, we show that the SCA6 polyglutamine expansion shifts the voltage dependence of channel activation and rate of inactivation only when expressed with beta(4) subunits and impairs normal G-protein regulation of P/Q channels. These findings suggest the possibility that SCA6 is a channelopathy, and that the underlying mutation in SCA6 causes Purkinje cell degeneration through excessive entry of calcium ions.
Asunto(s)
Canales de Calcio Tipo P/fisiología , Canales de Calcio Tipo Q/fisiología , Canales de Calcio/genética , Neuronas/fisiología , Péptidos/genética , Ataxias Espinocerebelosas/genética , Expansión de Repetición de Trinucleótido , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Anticuerpos , Canales de Calcio/química , Canales de Calcio/fisiología , Canales de Calcio Tipo P/genética , Canales de Calcio Tipo Q/genética , Exones , Femenino , Humanos , Masculino , Potenciales de la Membrana , Datos de Secuencia Molecular , Oocitos/fisiología , Fragmentos de Péptidos/inmunología , Conejos , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/biosíntesis , Xenopus laevisRESUMEN
The effects of 2,5-di(tert-butyl)-1,4-benzohydroquinone (tBHQ), a synthetic phenolic antioxidant and a blocker of the sarco-endoplasmic ATPase, were evaluated on low and high voltage-activated Ca(2+) currents (ICas) with rodent dorsal root ganglion, hippocampal, and motor neurons. In all cell types tested, tBHQ (IC(50) = 35 microM) blocked ICa at concentrations used to inhibit sarco-endoplasmic ATPase. This effect was specific to tBHQ because the other sarco-endoplasmic reticulum calcium ATPase pump inhibitors (thapsigargin and cyclopiazonic acid) had no effect. Selective blockade of the N-type current with omega-conotoxin GVIA and of P- (motoneuron) or Q-type currents (hippocampal neuron) with omega-agatoxin IVA indicated that tBHQ inhibited N, P, and Q types of ICa. tBHQ had no effect on nitrendipine-sensitive (L-type) and residual drug-resistant (R-type) ICa, nor on the low voltage-activated T-type ICa. Contrary to neuronal cells, the L-type ICa was inhibited by tBHQ in a differentiated mouse neuroblastoma and rat glioma hybrid cell line. Injection of cDNAs encoding the alpha1A, alpha1B, alpha1C, and alpha1E subunits into oocytes showed that tBHQ blocked ICas at the level of the pore-forming protein. This effect of tBHQ on ICa should be considered when interpreting results obtained with tBHQ used on neuronal preparations. It also may be useful for developing new strategies for the generation of more potent intracellular calcium transient inhibitors.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/metabolismo , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Hidroquinonas/farmacología , Neuronas Motoras/efectos de los fármacos , Animales , Calcio/metabolismo , Canales de Calcio/efectos de los fármacos , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo N/efectos de los fármacos , Canales de Calcio Tipo N/metabolismo , Canales de Calcio Tipo P/efectos de los fármacos , Canales de Calcio Tipo P/metabolismo , Canales de Calcio Tipo Q/efectos de los fármacos , Canales de Calcio Tipo Q/metabolismo , Canales de Calcio Tipo R/metabolismo , Canales de Calcio Tipo T/metabolismo , Células Cultivadas , ADN Complementario/genética , ADN Complementario/metabolismo , Retículo Endoplásmico/enzimología , Inhibidores Enzimáticos/farmacología , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Glioma/patología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Células Híbridas/efectos de los fármacos , Células Híbridas/metabolismo , Ratones , Neuronas Motoras/metabolismo , Neuroblastoma/patología , Ratas , Ratas Sprague-Dawley , Tapsigargina/farmacologíaRESUMEN
Ca(2+) channel inactivation is a key element in controlling the level of Ca(2+) entry through voltage-gated Ca(2+) channels. Interaction between the pore-forming alpha(1) subunit and the auxiliary beta subunit is known to be a strong modulator of voltage-dependent inactivation. Here, we demonstrate that an N-terminal membrane anchoring site (MAS) of the beta(2a) subunit strongly reduces alpha(1A) (Ca(V)2.1) Ca(2+) channel inactivation. This effect can be mimicked by the addition of a transmembrane segment to the N terminus of the beta(2a) subunit. Inhibition of inactivation by beta(2a) also requires a link between MAS and another important molecular determinant, the beta interaction domain (BID). Our data suggest that mobility of the Ca(2+) channel I-II loop is necessary for channel inactivation. Interaction of this loop with other identified intracellular channel domains may constitute the basis of voltage-dependent inactivation. We thus propose a conceptually novel mechanism for slowing of inactivation by the beta(2a) subunit, in which the immobilization of the channel inactivation gate occurs by means of MAS and BID.
Asunto(s)
Canales de Calcio/metabolismo , Activación del Canal Iónico/fisiología , Subunidades de Proteína , Animales , Bario/farmacología , Sitios de Unión/genética , Canales de Calcio/efectos de los fármacos , Canales de Calcio/genética , Canales de Calcio Tipo P/efectos de los fármacos , Canales de Calcio Tipo P/genética , Canales de Calcio Tipo P/metabolismo , Canales de Calcio Tipo Q/efectos de los fármacos , Canales de Calcio Tipo Q/genética , Canales de Calcio Tipo Q/metabolismo , Línea Celular , Quelantes/farmacología , Electrofisiología , Técnica del Anticuerpo Fluorescente , Glutatión Transferasa/genética , Humanos , Activación del Canal Iónico/efectos de los fármacos , Mutagénesis Sitio-Dirigida , Oocitos/citología , Oocitos/metabolismo , Técnicas de Placa-Clamp , Estructura Terciaria de Proteína/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Relación Estructura-Actividad , Transfección , XenopusRESUMEN
Ca2+ channel auxiliary beta subunits have been shown to modulate voltage-dependent inactivation of various types of Ca2+ channels. The beta1 and beta2 subunits, that are differentially expressed with the L-type alpha1 Ca2+ channel subunit in heart, muscle and brain, can specifically modulate the Ca2+-dependent inactivation kinetics. Their expression in Xenopus oocytes with the alpha1C subunit leads, in both cases, to biphasic Ca2+ current decays, the second phase being markedly slowed by expression of the beta2 subunit. Using a series of beta subunit deletion mutants and chimeric constructs of beta1 and beta2 subunits, we show that the inhibitory site located on the amino-terminal region of the beta2a subunit is the major element of this regulation. These results thus suggest that different splice variants of the beta2 subunit can modulate, in a specific way, the Ca2+ entry through L-type Ca2+ channels in different brain or heart regions.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/farmacología , Empalme Alternativo , Animales , Encéfalo/metabolismo , Canales de Calcio Tipo L , Cartilla de ADN/genética , Regulación de la Expresión Génica , Miocardio/metabolismo , Oocitos/metabolismo , Técnicas de Placa-Clamp , Ratas , Proteínas Recombinantes/genética , Eliminación de Secuencia , XenopusRESUMEN
During sustained depolarization, voltage-gated Ca2+ channels progressively undergo a transition to a nonconducting, inactivated state, preventing Ca2+ overload of the cell. This transition can be triggered either by the membrane potential (voltage-dependent inactivation) or by the consecutive entry of Ca2+ (Ca2+-dependent inactivation), depending on the type of Ca2+ channel. These two types of inactivation are suspected to arise from distinct underlying mechanisms, relying on specific molecular sequences of the different pore-forming Ca2+ channel subunits. Here we report that the voltage-dependent inactivation (of the alpha1A Ca2+ channel) and the Ca2+-dependent inactivation (of the alpha1C Ca2+ channel) are similarly influenced by Ca2+ channel beta subunits. The same molecular determinants of the beta subunit, and therefore the same subunit interactions, influence both types of inactivation. These results strongly suggest that the voltage and the Ca2+-dependent transitions leading to channel inactivation use homologous structures of the different alpha1 subunits and occur through the same molecular process. A model of inactivation taking into account these new data is presented.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Activación del Canal Iónico , Secuencia de Bases , Canales de Calcio/genética , Cartilla de ADN , Cinética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Ca2+ current potentiation by conditioning depolarization is a general mechanism by which excitable cells can control the level of Ca2+ entry during repetitive depolarizations. Several types of Ca2+ channels are sensitive to conditioning depolarization, however, using clearly distinguishable mechanisms. In the case of L-type Ca2+ channels, prepulse-induced current facilitation can only be recorded when the pore-forming alpha1C subunit is coexpressed with the auxiliary beta1, beta3, or beta4, but not beta2, subunit. These four beta subunits are composed of two conserved domains surrounded by central, N-terminal, and C-terminal variable regions. Using different deleted and chimeric forms of the beta1 and beta2 subunits, we have mapped essential sequences for L-type Ca2+ channel facilitation. A first sequence, located in the second conserved domain of all beta subunits, is responsible for the promotion of current facilitation by the beta subunit. A second sequence of 16 amino acids, located on the N-terminal tail of the beta2 subunit, induces a transferable block of L-type current facilitation. Site-specific mutations reveal the essential inhibitory role played by three positive charges on this segment. The lack of prepulse-induced current facilitation recorded with some truncated forms of the beta2 subunit suggests the existence of an additional inhibitory sequence in the beta2 subunit.
Asunto(s)
Canales de Calcio/fisiología , Proteínas del Tejido Nervioso/fisiología , Plasticidad Neuronal , Secuencia de Aminoácidos , Animales , Calcio/metabolismo , Canales de Calcio/química , Canales de Calcio/genética , Canales de Calcio Tipo L , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Oocitos/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Conformación Proteica , Proteínas Recombinantes de Fusión/metabolismo , Relación Estructura-Actividad , XenopusRESUMEN
This article describes a study designed to assess the feasibility of using recombinant adenovirus for delivering therapeutic peptides in vivo in the guinea pig middle ear cleft. A recombinant adenoviral vector AdCMVsp1 LacZ containing the Escherichia coli beta-galactosidase was injected into the middle ear space. Qualitative assessment of cell middle ear transfection was performed on day 2 by light microscopy study, after injecting a multiplicity of infection (MOI) ranging from 0 to 1000. At an MOI of 30, 30% of the promontory area epithelial cells were stained. An MOI of 50 stained 60% of the cells and an MOI of 100 or more stained more than 90% of the cells. The duration of cell transfection was studied after injecting an MOI of 50. The percentage of stained cells was 60% on day 2, 10% on day 7, and 0% on day 14. Middle ear mucosal inflammation, consisting of a granulocytic infiltrate, was observed when an MOI above 50 was used. Even at a high MOI (500), no staining could be found in the cochlea, in the facial nerve, in the brain, or in visceral organs. These data suggest that recombinant adenovirus vectors can be used to transfer genes in the middle ear. This method appears to be safe, and may be envisaged as a short-duration treatment to transfer genes in vivo in the treatment of middle ear diseases.
Asunto(s)
Adenoviridae/genética , ADN Recombinante/genética , Oído Medio , Técnicas de Transferencia de Gen , Terapia Genética , Animales , Difusión , Estudios de Factibilidad , Femenino , Cobayas , Semivida , Operón Lac , Membrana MucosaRESUMEN
Metabotropic glutamate (mGlu), Ca2+-sensing, gamma-aminobutyric acidB, and a large number of pheromone receptors constitute a peculiar family of G protein-coupled receptors. They possess a large extracellular domain that has been proposed to constitute their ligand binding domain. The aim of the current study was to examine whether this large ligand binding domain had any influence on the G protein-coupling selectivity of the receptor, and vice versa. We chose mGlu receptors, which are classified into three groups according to their sequence homology and pharmacology, as representatives of this receptor family. To define a G protein-coupling profile for these receptors, we used a set of exogenous phospholipase C-activating G proteins in the same way that synthetic ligands are used to define agonist and antagonist pharmacological profiles. This set includes Galpha15, Galpha16, Galphaq, and chimeric Galphaq proteins with the last few amino acids of either Galphai2 (Galphaqi), Galphao (Galphaqo), or Galphaz (Galphaqz). Cotransfection of mGlu receptors with these G proteins and examination of their coupling to phospholipase C revealed that group I, II, and III receptors have distinct G protein-coupling profiles. By swapping the extracellular domains of the most distantly related mGlu receptors (the rat group I mGlu1a and the Drosophila melanogaster group II DmGluA receptors), we show that the extracellular domain determines the agonist pharmacological profile and that this domain does not modify the G protein-coupling profile determined by the seven-transmembrane-domain region of mGlu receptors.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/fisiología , Receptores de Glutamato Metabotrópico/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Drosophila melanogaster/genética , Embrión de Mamíferos , Embrión no Mamífero , Espacio Extracelular/metabolismo , Proteínas de Unión al GTP/agonistas , Ácido Glutámico/farmacología , Humanos , Riñón , Ligandos , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Ratas , Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/genética , Proteínas Recombinantes de Fusión/agonistas , Proteínas Recombinantes de Fusión/síntesis química , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Novel mRNA isoforms for two members of the group III metabotropic glutamate receptors (mGluRs), called mGluR7b and mGluR8b, were identified from rat brain cerebral cortex and hippocampus. In both cases, the alternative splicing is generated by a similar out-of-frame insertion in the carboxyl-terminus that results in the replacement of the last 16 amino acids of mGluR7 and mGluR8 by 23 and 16 different amino acids, respectively. Distribution analysis for mGluR7 and mGluR8 isoforms revealed that the two splice variants are generally coexpressed in the same brain areas. The few exceptions were the olfactory bulb, in which only the mGluR7a form could be detected by reverse transcription-polymerase chain reaction, and the lateral reticular and ambiguous nuclei, which showed only mGluR8a labelling. Despite expression in the same regions, different mRNA abundance for the two variants of each receptor were observed. When transiently coexpressed in HEK 293 cells with the phospholipase C-activating chimeric G alpha qi9-G-protein, the a and b forms for both receptor subtypes showed a similar pharmacological profile. The rank order of potencies for both was: DL-amino-4-phosphonobutyrate > L-serine-O-phosphate > glutamate. However, the agonist potencies were significantly higher for mGluR8a, b compared with mGluR7a,b. In Xenopus oocytes, glutamate evoked currents only with mGluR8 when coexpressed with Kir 3.1 and 3.4. Glutamate-induced currents were antagonized by the group II/III antagonist (RS)-alpha-cyclopropyl-4-phosphonophenylglycine. In conclusion, the two isoforms of each receptor have identical pharmacological profiles when expressed in heterologous systems, despite structural differences in the carboxyl-terminal domains.
Asunto(s)
Empalme Alternativo/fisiología , Canales de Potasio de Rectificación Interna , ARN Mensajero/análisis , Receptores de Glutamato Metabotrópico/genética , Animales , Elementos sin Sentido (Genética) , Secuencia de Bases , Células Cultivadas , Corteza Cerebral/química , Clonación Molecular , Cartilla de ADN , Electrofisiología , Antagonistas de Aminoácidos Excitadores/farmacología , Canales de Potasio Rectificados Internamente Asociados a la Proteína G , Ácido Glutámico/farmacología , Glicina/análogos & derivados , Glicina/farmacología , Hipocampo/química , Humanos , Fosfatos de Inositol/análisis , Riñón/citología , Potenciales de la Membrana/efectos de los fármacos , Datos de Secuencia Molecular , Oocitos/fisiología , Canales de Potasio/fisiología , Propionatos/farmacología , Ratas , Homología de Secuencia de Aminoácido , XenopusRESUMEN
Together with the calcium-sensing receptor, the metabotropic glutamate receptors (mGluRs) share no sequence homology with the other G protein-coupled receptors (GPCRs) and therefore constitute a new family of receptors. Recently, it was reported that G alpha 15 and G alpha 16 subunits allow many GPCRs to activate phospholipase C (PLC). Furthermore, the exchange of a few carboxyl-terminal residues of G alpha q by those of G alpha 12 or G alpha o allows the resulting chimeric G alpha subunits (G alpha ql and G alpha qol respectively) to couple Gi-coupled receptors to PLC. We report that mGluR2 and mGluR4, two receptors negatively coupled to adenylyl cyclase, activate PLC when coexpressed with G alpha 15, G alpha ql or G alpha qo. This indicates that the carboxyl-terminal end of the G alpha subunit also plays an important role in the specific interaction between mGluRs and the G proteins. In addition, the measurement of PLC activation by Gi-coupled mGluRs coexpressed with these G alpha subunits constitutes an easy functional assay for the pharmacological characterization of these receptors. The rank order of potency of antagonists was found to be (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine approximately (R,S)- alpha-methyl-4-phosphonophenylglycine > (R,S)-alpha-methyl-4-sulfonophenylglycine > (R,S)-alpha-methyl-4-tetrazolylphenylglycine = (S)-2-amino-2-methyl-4-phosphonobutyrate for mGluR2 and to be (R,S)-alpha-methyl-4-phosphonophenylglycine > or = (S)-2-amino-2-methyl-4-phosphonobutyrate > > (R,S)-alpha-methyl-4-sulfonophenylglycine [(R,S)-alpha-methyl-4-tetrazolylphenylglycine and (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine being inactive at 1 mM] for mGluR4. Using this functional assay, (R,S)-alpha-methyl-4-phosphonophenylglycine was found to have a similar KB value for mGluR2 and mGluR4.