RESUMEN
The intracellular domains of connexins are essential for the assembly of gap junctions. For connexin 36 (Cx36), the major neuronal connexin, it has been shown that a dysfunctional PDZ-binding motif interferes with electrical synapse formation. However, it is still unknown how this motif coordinates the transport of Cx36. In the present study, we characterize a phenotype of Cx36 mutants that lack a functional PDZ-binding motif using HEK293T cells as an expression system. We provide evidence that an intact PDZ-binding motif is critical for proper endoplasmic reticulum (ER) export of Cx36. Removing the PDZ-binding motif of Cx36 results in ER retention and the formation of multimembrane vesicles containing gap junction-like connexin aggregates. Using a combination of site-directed mutagenesis and electron micrographs, we reveal that these vesicles consist of Cx36 channels that docked prematurely in the ER. Our data suggest a model in which ER-retained Cx36 channels reshape the ER membrane into concentric whorls that are released into the cytoplasm.
Asunto(s)
Conexinas , Retículo Endoplásmico , Uniones Comunicantes , Humanos , Conexinas/genética , Conexinas/metabolismo , Retículo Endoplásmico/metabolismo , Uniones Comunicantes/metabolismo , Células HEK293 , Dominios Proteicos , Secuencias de Aminoácidos , Sinapsis Eléctricas/fisiología , Mutación , Transporte de Proteínas/genética , Vesículas Sinápticas/patología , Vesículas Sinápticas/ultraestructura , Microscopía Electrónica de Rastreo , Proteína delta-6 de Union ComunicanteRESUMEN
Ca2+/calmodulin-dependent protein kinase II (CaMKII) binding and phosphorylation of mammalian connexin-36 (Cx36) potentiate electrical coupling. To explain the molecular mechanism of how Cx36 modifies plasticity at gap junctions, we investigated the roles of ionotropic N-methyl-D-aspartate receptors and pannexin1 (Panx1) channels in regulating Cx36 binding to CaMKII. Pharmacological interference and site-directed mutagenesis of protein interaction sites shows that NMDA receptor activation opens Cx36 channels, causing the Cx36- CaMKII binding complex to adopt a compact conformation. Ectopic Panx1 expression in a Panx1 knock-down cell line is required to restore CaMKII mediated opening of Cx36. Furthermore, blocking of Src-family kinase activation of Panx1 is sufficient to prevent the opening of Cx36 channels. Our research demonstrates that the efficacy of Cx36 channels requires convergent calcium-dependent signaling processes in which activation of ionotropic N-methyl-D-aspartate receptor, Src-family kinase, and Pannexin1 open Cx36. Our results add to the best of our knowledge a new twist to mounting evidence for molecular communication between these core components of electrical and chemical synapses.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Conexinas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transducción de Señal , Animales , Calcio/metabolismo , Señalización del Calcio , Línea Celular , Uniones Comunicantes/metabolismo , Ratones , Neuronas/metabolismo , Mapas de Interacción de Proteínas , Proteína delta-6 de Union ComunicanteRESUMEN
The gap junctional protein connexin 36 (Cx36) has been co-purified with the lipid raft protein caveolin-1 (Cav-1). The relevance of an interaction between the two proteins is unknown. In this study, we explored the significance of Cav-1 interaction in the context of intracellular and membrane transport of Cx36. Coimmunoprecipitation assays and Förster resonance energy transfer analysis (FRET) were used to confirm the interaction between the two proteins in the Neuro 2a cell line. We found that the Cx36 and Cav-1 interaction was dependent on the intracellular calcium levels. By employing different microscopy techniques, we demonstrated that Cav-1 enhances the vesicular transport of Cx36. Pharmacological interventions coupled with cell surface biotinylation assays and FRET analysis revealed that Cav-1 regulates membrane localization of Cx36. Our data indicate that the interaction between Cx36 and Cav-1 plays a role in the internalization of Cx36 by a caveolin-dependent pathway.
Asunto(s)
Calcio/metabolismo , Caveolas/metabolismo , Caveolina 1/genética , Conexinas/genética , Endocitosis/genética , Microdominios de Membrana/metabolismo , Animales , Cationes Bivalentes , Caveolas/ultraestructura , Caveolina 1/metabolismo , Línea Celular Tumoral , Conexinas/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Regulación de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Transporte Iónico , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microdominios de Membrana/ultraestructura , Ratones , Microscopía Fluorescente , Neuronas/metabolismo , Neuronas/ultraestructura , Unión Proteica , Transducción de Señal , Proteína delta-6 de Union ComunicanteRESUMEN
Pannexin 1 (Panx1) is a ubiquitously expressed hexameric integral membrane protein known to function as an adenosine triphosphate (ATP) release channel. Panx1 proteins exist in unglycosylated core form (Gly0). They undergo critical post-translational modifications forming the high mannose glycosylation state (Gly1) in the endoplasmic reticulum (ER) and the complex glycosylation state (Gly2) in the Golgi apparatus. The regulation of transition from the ER to the cell membrane is not fully understood. Using site-specific mutagenesis, dye uptake assays, and interaction testing, we identified two conserved aromatic residues, Trp123 and Tyr205, in the transmembrane domains 2 and 3 of the zebrafish panx1a protein. Results suggest that both residues primarily govern the assembly of panx1a subunits into channels, with mutant proteins failing to interact. The results provide insight into a mechanism enabling regulation of Panx1 oligomerization, glycosylation, and trafficking.