RESUMO
Downregulation of G-protein-coupled receptors (GPCRs) provides an important mechanism for reducing neurotransmitter signaling during sustained stimulation. Chronic stimulation of M(2) muscarinic receptors (M(2)Rs) causes internalization of M(2)R and G-protein-activated inwardly rectifying potassium (GIRK) channels in neuronal PC12 cells, resulting in loss of function. Here, we show that coexpression of GABA(B) R2 receptors (GBR2s) rescues both surface expression and function of M(2)R, including M(2)R-induced activation of GIRKs and inhibition of cAMP production. GBR2 showed significant association with M(2)R at the plasma membrane but not other GPCRs (M(1)R, mu-opioid receptor), as detected by fluorescence resonance energy transfer measured with total internal reflection fluorescence microscopy. Unique regions of the proximal C-terminal domains of GBR2 and M(2)R mediate specific binding between M(2)R and GBR2. In the brain, GBR2, but not GBR1, biochemically coprecipitates with M(2)R and overlaps with M(2)R expression in cortical neurons. This novel heteromeric association between M(2)R and GBR2 provides a possible mechanism for altering muscarinic signaling in the brain and represents a previously unrecognized role for GBR2.
Assuntos
Receptor Muscarínico M2/metabolismo , Receptores de GABA-B/metabolismo , Transdução de Sinais/fisiologia , Animais , Membrana Celular/metabolismo , Humanos , Células PC12 , Ligação Proteica/fisiologia , Ratos , Receptor Muscarínico M2/química , Receptores de GABA-B/químicaRESUMO
Many inhibitory neurotransmitters in the brain activate Kir3 channels by stimulating pertussis toxin (PTX)-sensitive G-protein-coupled receptors. Here, we investigated the regulation of native muscarinic receptors and Kir3 channels expressed in NGF-differentiated PC12 cells, which are similar to sympathetic neurons. Quantitative reverse transcription-PCR and immunocytochemistry revealed that NGF treatment significantly upregulated mRNA and protein for m2 muscarinic receptors, PTX-sensitive G alpha(o) G-proteins, and Kir3.2c channels. Surprisingly, these upregulated muscarinic receptor/Kir3 signaling complexes were functionally silent. Ectopic expression of m2 muscarinic receptors or Kir3.2c channels was unable to produce muscarinic receptor-activated Kir3 currents with oxotremorine. Remarkably, pretreatment with muscarinic (m2/m4) receptor antagonists resulted in robust oxotremorine-activated Kir3 currents. Thus, sustained cholinergic stimulation of natively expressed m2/m4 muscarinic receptors controlled cell surface expression and functional coupling of both receptors and Kir3 channels. This new pathway for controlling Kir3 signaling could help limit the potential harmful effects of excessive Kir3 activity in the brain.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Receptor Muscarínico M2/metabolismo , Receptores de Superfície Celular/metabolismo , Regulação para Cima/fisiologia , Análise de Variância , Animais , Diferenciação Celular/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Agonistas Muscarínicos/farmacologia , Antagonistas Muscarínicos/farmacologia , Fator de Crescimento Neural/farmacologia , Oxotremorina/análogos & derivados , Oxotremorina/farmacologia , Células PC12/efeitos dos fármacos , Técnicas de Patch-Clamp/métodos , RNA Mensageiro/biossíntese , Ratos , Receptor Muscarínico M2/genética , Receptores de Superfície Celular/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Regulação para Cima/efeitos dos fármacos , Ácido gama-Aminobutírico/farmacologiaRESUMO
We found the voltage-gated K+ channel Kv12.2 to be a potent regulator of excitability in hippocampal pyramidal neurons. Genetic deletion and pharmacologic block of Kv12.2 substantially reduced the firing threshold of these neurons. Kv12.2-/- (also known as Kcnh3-/-) mice showed signs of persistent neuronal hyperexcitability including frequent interictal spiking, spontaneous seizures and increased sensitivity to the chemoconvulsant pentylenetetrazol.
Assuntos
Epilepsia/fisiopatologia , Canais de Potássio Éter-A-Go-Go/metabolismo , Hipocampo/fisiopatologia , Neurônios/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Linhagem Celular , Células Cultivadas , Convulsivantes/toxicidade , Epilepsia/induzido quimicamente , Epilepsia/genética , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Canais de Potássio Éter-A-Go-Go/genética , Feminino , Hipocampo/efeitos dos fármacos , Humanos , Técnicas In Vitro , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/efeitos dos fármacos , Pentilenotetrazol/toxicidade , Células Piramidais/efeitos dos fármacos , Células Piramidais/fisiopatologia , Convulsões/induzido quimicamente , Convulsões/genética , Convulsões/fisiopatologia , Gravação em Vídeo , XenopusRESUMO
Voltage-gated potassium channels that activate near the neuronal resting membrane potential are important regulators of excitation in the nervous system, but their functional diversity is still not well understood. For instance, Kv12.2 (ELK2, KCNH3) channels are highly expressed in the cerebral cortex and hippocampus, and although they are most likely to contribute to resting potassium conductance, surprisingly little is known about their function or regulation. Here we demonstrate that the auxiliary MinK (KCNE1) and MiRP2 (KCNE3) proteins are important regulators of Kv12.2 channel function. Reduction of endogenous KCNE1 or KCNE3 expression by siRNA silencing, significantly increased macroscopic Kv12.2 currents in Xenopus oocytes by around 4-fold. Interestingly, an almost 9-fold increase in Kv12.2 currents was observed with the dual injection of KCNE1 and KCNE3 siRNA, suggesting an additive effect. Consistent with these findings, over-expression of KCNE1 and/or KCNE3 suppressed Kv12.2 currents. Membrane surface biotinylation assays showed that surface expression of Kv12.2 was significantly increased by KCNE1 and KCNE3 siRNA, whereas total protein expression of Kv12.2 was not affected. KCNE1 and KCNE3 siRNA shifted the voltages for half-maximal activation to more hyperpolarized voltages, indicating that KCNE1 and KCNE3 may also inhibit activation gating of Kv12.2. Native co-immunoprecipitation assays from mouse brain membranes imply that KCNE1 and KCNE3 interact with Kv12.2 simultaneously in vivo, suggesting the existence of novel KCNE1-KCNE3-Kv12.2 channel tripartite complexes. Together these data indicate that KCNE1 and KCNE3 interact directly with Kv12.2 channels to regulate channel membrane trafficking.
Assuntos
Canais de Potássio Éter-A-Go-Go/metabolismo , Proteínas de Membrana/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA , Canais de Potássio Éter-A-Go-Go/química , Imunoprecipitação , Técnicas In Vitro , Camundongos , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , RNA Interferente Pequeno , Homologia de Sequência de Aminoácidos , XenopusRESUMO
Neuronal G-protein-gated inwardly rectifying potassium (Kir3; GIRK) channels are activated by G-protein-coupled receptors that selectively interact with PTX-sensitive (Galphai/o) G proteins. Although the Gbetagamma dimer is known to activate GIRK channels, the role of the Galphai/o subunit remains unclear. Here, we established that Galphao subunits co-immunoprecipitate with neuronal GIRK channels. In vitro binding studies led to the identification of six amino acids in the GIRK2 C-terminal domain essential for Galphao binding. Further studies suggested that the Galphai/obetagamma heterotrimer binds to the GIRK2 C-terminal domain via Galpha and not Gbetagamma. Galphai/o binding-impaired GIRK2 channels exhibited reduced receptor-activated currents, but retained normal ethanol- and Gbetagamma-activated currents. Finally, PTX-insensitive Galphaq or Galphas subunits did not bind to the GIRK2 C-terminus. Together, these results suggest that the interaction of PTX-sensitive Galphai/o subunit with the GIRK2 C-terminal domain regulates G-protein receptor coupling, and may be important for establishing specific Galphai/o signaling pathways.
Assuntos
Membrana Celular/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Neurônios/fisiologia , Toxina Pertussis/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Sequência de Aminoácidos/fisiologia , Animais , Sítios de Ligação/fisiologia , Encéfalo/fisiologia , Linhagem Celular , Etanol/farmacologia , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Subunidades alfa de Proteínas de Ligação ao GTP/efeitos dos fármacos , Subunidades beta da Proteína de Ligação ao GTP/efeitos dos fármacos , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas Heterotriméricas de Ligação ao GTP/efeitos dos fármacos , Humanos , Neurônios/metabolismo , Oócitos , Canais de Potássio Corretores do Fluxo de Internalização/química , Canais de Potássio Corretores do Fluxo de Internalização/efeitos dos fármacos , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologia , Ratos , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , XenopusRESUMO
Mucus hypersecretion is a feature of several respiratory diseases and frequently leads to obstruction of small airways where the principal source of mucous glycoproteins (mucins), the major macromolecular constituents of mucus, are goblet cells. Hence, inhibition of mucin secretion from these cells may be clinically beneficial. In this study, we have developed a lectin-based assay for mucin secretion from ovine airway goblet cells and used this assay to investigate the regulation of these cells by endothelin (ET)-1. ET-1 inhibited baseline mucin secretion (maximum inhibition: 60.3 +/- 4.2%, 50% inhibitory concentration: 0.8 +/- 0.17 nM). This response was abolished by the ET(A) antagonist, BQ-123 (1 muM), but not by the ET(B) antagonist, BQ-788 (1 muM). ET-1 (1 muM) did not affect mucin secretion stimulated by ATP (100 muM) but secretion in response to ATP (10 muM) was inhibited by 63.3 +/- 11.8%. This response could be eliminated by BQ-123, but not by BQ-788. Radioligand binding and immunohistochemistry indicated the expression of both ET(A)- and ET(B)-receptors on the epithelium. In summary, ET-1, acting via ET(A)-receptors, inhibits baseline and ATP-stimulated mucin secretion from ovine airway goblet cells. This represents the first report of a physiologic mechanism for inhibiting airway goblet cell mucin secretion; an understanding of this mechanism may provide opportunities for the treatment of obstructive airways disease.