RESUMEN
GABA(B) receptors are the G-protein-coupled receptors for gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the brain. They are expressed in almost all neurons of the brain, where they regulate synaptic transmission and signal propagation by controlling the activity of voltage-gated calcium (Ca(v)) and inward-rectifier potassium (K(ir)) channels. Molecular cloning revealed that functional GABA(B) receptors are formed by the heteromeric assembly of GABA(B1) with GABA(B2) subunits. However, cloned GABA(B(1,2)) receptors failed to reproduce the functional diversity observed with native GABA(B) receptors. Here we show by functional proteomics that GABA(B) receptors in the brain are high-molecular-mass complexes of GABA(B1), GABA(B2) and members of a subfamily of the KCTD (potassium channel tetramerization domain-containing) proteins. KCTD proteins 8, 12, 12b and 16 show distinct expression profiles in the brain and associate tightly with the carboxy terminus of GABA(B2) as tetramers. This co-assembly changes the properties of the GABA(B(1,2)) core receptor: the KCTD proteins increase agonist potency and markedly alter the G-protein signalling of the receptors by accelerating onset and promoting desensitization in a KCTD-subtype-specific manner. Taken together, our results establish the KCTD proteins as auxiliary subunits of GABA(B) receptors that determine the pharmacology and kinetics of the receptor response.
Asunto(s)
Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Multimerización de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Receptores de GABA-B/química , Receptores de GABA-B/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Conductividad Eléctrica , Agonistas de Receptores GABA-B , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Cinética , Ratones , Neuronas/metabolismo , Oocitos/metabolismo , Potasio/metabolismo , Canales de Potasio/metabolismo , Estructura Terciaria de Proteína , Ratas , Ratas Wistar , Transducción de Señal , XenopusRESUMEN
The properties of glycine receptors (GlyRs) depend upon their subunit composition. While the prevalent adult forms of GlyRs are heteromers, previous reports suggested functional α homomeric receptors in mature nervous tissues. Here we show two functionally different GlyRs populations in the rat medial nucleus of trapezoid body (MNTB). Postsynaptic receptors formed α1/ß-containing clusters on somatodendritic domains of MNTB principal neurons, colocalizing with glycinergic nerve endings to mediate fast, phasic IPSCs. In contrast, presynaptic receptors on glutamatergic calyx of Held terminals were composed of dispersed, homomeric α1 receptors. Interestingly, the parent cell bodies of the calyces of Held, the globular bushy cells of the cochlear nucleus, expressed somatodendritic receptors (α1/ß heteromers) and showed similar clustering and pharmacological profile as GlyRs on MNTB principal cells. These results suggest that specific targeting of GlyR ß-subunit produces segregation of GlyR subtypes involved in two different mechanisms of modulation of synaptic strength.
Asunto(s)
Vías Auditivas/metabolismo , Receptores de Glicina/metabolismo , Sinapsis/metabolismo , Animales , Espinas Dendríticas/fisiología , Estimulación Eléctrica , Fenómenos Electrofisiológicos , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Glicina/fisiología , Glicinérgicos/farmacología , Inmunohistoquímica , Cinética , Microscopía Inmunoelectrónica , Terminaciones Nerviosas/metabolismo , Técnicas de Placa-Clamp , Ratas , Ratas Wistar , Receptores de Glicina/efectos de los fármacos , Receptores Presinapticos/metabolismoRESUMEN
Inhibitory circuits in the auditory brainstem undergo multiple postnatal changes that are both activity-dependent and activity-independent. We tested to see if the shift from GABA- to glycinergic transmission, which occurs in the rat medial nucleus of the trapezoid body (MNTB) around the onset of hearing, depends on sound-evoked neuronal activity. We prevented the activity by bilateral cochlear ablations in early postnatal rats and studied ionotropic GABA and glycine receptors in MNTB neurons after hearing onset. The removal of the cochlea decreased responses of GABAA and glycine receptors to exogenous agonists as well as the amplitudes of inhibitory postsynaptic currents. The reduction was accompanied by a decrease in the number of glycine receptor- or vesicular GABA transporter-immunopositive puncta. Furthermore, the ablations markedly affected the switch in presynaptic GABAA to glycine receptors. The increase in the expression of postsynaptic glycine receptors and the shift in inhibitory transmitters were not prevented. The results suggest that inhibitory transmission in the MNTB is subject to multiple developmental signals and support the idea that auditory experience plays a role in the maturation of the brainstem glycinergic circuits.
Asunto(s)
Técnicas de Ablación , Cóclea/fisiopatología , Cóclea/cirugía , Inhibición Neural/fisiología , Transmisión Sináptica , Cuerpo Trapezoide/fisiología , Animales , Animales Recién Nacidos , Potenciales Evocados Auditivos del Tronco Encefálico/fisiología , Agonistas de Receptores de GABA-A/farmacología , Potenciales Postsinápticos Inhibidores/fisiología , Masculino , Inhibición Neural/efectos de los fármacos , Ratas , Receptores de GABA-A/fisiología , Receptores de Glicina/agonistas , Receptores de Glicina/metabolismo , Receptores de Glicina/fisiología , Proteínas del Transporte Vesicular de Aminoácidos Inhibidores/metabolismoRESUMEN
The physiological functions of glycine receptors (GlyRs) depend on their subcellular locations. In axonal terminals of the central neurons, GlyRs trigger a slow facilitation of presynaptic transmitter release; however, their spatial relationship to the release sites is not known. In this study, we examined the distribution of GlyRs in the rat glutamatergic calyx of Held nerve terminal using high-resolution pre-embedding immunoelectron microscopy. We performed a quantitative analysis of GlyR-associated immunogold (IG) labeling in 3D reconstructed calyceal segments. A variable density of IG particles and their putative accumulations, inferred from the frequency distribution of inter-IG distances, indicated a non-uniform distribution of the receptors in the calyx. Subsequently, increased densities of IG particles were found in calyceal swellings, structures characterized by extensive exocytosis of glutamate. In swellings as well as in larger calyceal stalks, IG particles did not tend to accumulate near the glutamate releasing zones. On the other hand, GlyRs in swellings (but not in stalks) preferentially occupied membrane regions, unconnected to postsynaptic cells and presumably accessible by ambient glycine. Furthermore, the sites with increased GlyR concentrations were found in swellings tightly juxtaposed with GABA/glycinergic nerve endings. Thus, the results support the concept of an indirect mechanism underlying the modulatory effects of calyceal GlyRs, activated by glycine spillover. We also suggest the existence of an activity-dependent mechanism regulating the surface distribution of α homomeric GlyRs in axonal terminals of central neurons.
Asunto(s)
Tronco Encefálico/citología , Neuronas/citología , Terminales Presinápticos/metabolismo , Receptores de Glicina/metabolismo , Sinapsis/metabolismo , Animales , Glicina/metabolismo , Técnicas In Vitro , Masculino , Terminales Presinápticos/ultraestructura , Ratas , Ratas Wistar , Receptores de Glicina/ultraestructura , Estadísticas no Paramétricas , Sinapsis/ultraestructura , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
G-protein-coupled receptors (GPCRs) have been shown to form dimers, but the relevance of this phenomenon in G-protein activation is not known. Among the large GPCR family, metabotropic glutamate (mGlu) receptors are constitutive dimers. Here we examined whether both heptahelical domains (HDs) are turned on upon full receptor activation. To that aim, we measured G-protein coupling efficacy of dimeric mGlu receptors in which one subunit bears specific mutations. We show that a mutation in the third intracellular loop (i3 loop) known to prevent G-protein activation in a single subunit decreases coupling efficacy. However, when a single HD is blocked in its inactive state using an inverse agonist, 2-methyl-6-(phenylethynyl)pyridine (MPEP), no decrease in receptor activity is observed. Interestingly, in a receptor dimer in which the subunit that binds MPEP is mutated in its i3 loop, MPEP enhances agonist-induced activity, reflecting a 'better' activation of the adjacent HD. These data are consistent with a model in which a single HD is turned on upon activation of such homodimeric receptors and raise important issues in deciphering the functional role of GPCR dimer formation for G-protein activation.