RESUMO
Investigating subunit assembly of ionotropic glutamate receptor complexes and their trafficking to the plasma membrane under physiological conditions in live cells has been challenging. By confocal imaging of fluorescently labeled kainate receptor (KAR) subunits combined with digital co-localization and fluorescence resonance energy (FRET) transfer analyses, we investigated the assembly of homomeric and heteromeric receptor complexes and identified the subcellular location of subunit interactions. Our data provide direct evidence for oligomerization of KAR subunits as early as following their biosynthesis in the endoplasmic reticulum (ER). These oligomeric assemblies pass through the Golgi apparatus en route to the plasma membrane. We show that the amino acid at the Q/R editing site of the KAR subunit GluR6 neither determines subunit oligomerization in the ER nor ER exit or plasma membrane expression, and that it does not alter GluR6 interaction with KA2. This finding sets KARs apart from alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors, where in the absence of auxiliary proteins Q isoforms exit the ER much more efficiently than R isoforms. Furthermore, although KA2 subunits do not form functional homotetrameric complexes, we visualized their oligomerization (at least dimerization) in the ER. Finally, we demonstrate that plasma membrane expression of GluR6/KA2 heteromeric complexes is modulated not only by GluR6 but also KA2.
Assuntos
Retículo Endoplasmático/metabolismo , Subunidades Proteicas/metabolismo , Receptores de Ácido Caínico/química , Receptores de Ácido Caínico/metabolismo , Proteínas de Bactérias/genética , Linhagem Celular Transformada , Membrana Celular/genética , Membrana Celular/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Regulação da Expressão Gênica/genética , Humanos , Proteínas Luminescentes/genética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Microscopia Confocal/métodos , Mutagênese Sítio-Dirigida , Técnicas de Patch-Clamp/métodos , Multimerização Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/genética , Transporte Proteico/fisiologia , Edição de RNA/fisiologia , Receptores de Ácido Caínico/classificação , Receptores de Ácido Caínico/genética , Transfecção/métodosRESUMO
While many health and human service innovations are sustained and replicated, it has been a puzzle how to sustain and replicate the performance of the better ones. What knowledge, skills, and conditions are required to reproduce across space and time the effectiveness of those innovations that are the most worthwhile? An extensive body of literature and experience is reviewed to suggest a comprehensive conceptual framework of programmatic, organizational, and environmental factors that may shape the circumstances for sustaining and replicating effectiveness.
