RESUMO
Dendritic spines compartmentalize information in the brain, and their morphological characteristics are thought to underly synaptic plasticity. Here we identify copine-6 as a novel modulator of dendritic spine morphology. We found that brain-derived neurotrophic factor (BDNF) - a molecule essential for long-term potentiation of synaptic strength - upregulated and recruited copine-6 to dendritic spines in hippocampal neurons. Overexpression of copine-6 increased mushroom spine number and decreased filopodia number, while copine-6 knockdown had the opposite effect and dramatically increased the number of filopodia, which lacked PSD95. Functionally, manipulation of post-synaptic copine-6 levels affected miniature excitatory post-synaptic current (mEPSC) kinetics and evoked synaptic vesicle recycling in contacting boutons, and post-synaptic knockdown of copine-6 reduced hippocampal LTP and increased LTD. Mechanistically, copine-6 promotes BDNF-TrkB signaling and recycling of activated TrkB receptors back to the plasma membrane surface, and is necessary for BDNF-induced increases in mushroom spines in hippocampal neurons. Thus copine-6 regulates BDNF-dependent changes in dendritic spine morphology to promote synaptic plasticity.
Assuntos
Proteínas de Transporte/metabolismo , Espinhas Dendríticas/fisiologia , Hipocampo/citologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Neurônios/ultraestrutura , Vesículas Sinápticas/fisiologia , Animais , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Proteínas de Transporte/genética , Células Cultivadas , Espinhas Dendríticas/ultraestrutura , Proteína 4 Homóloga a Disks-Large/metabolismo , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/genética , Técnicas de Cultura de Órgãos , Ratos , Receptor trkB/genética , Receptor trkB/metabolismo , Sinapses/efeitos dos fármacos , Sinapses/fisiologia , Sinapses/ultraestrutura , Potenciais Sinápticos/efeitos dos fármacos , Potenciais Sinápticos/genética , Vesículas Sinápticas/efeitos dos fármacos , Sinaptossomos/metabolismo , Sinaptossomos/ultraestrutura , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismoRESUMO
The copine family of proteins contains nine members with a similar domain structure, namely two N-terminal C2-domains (C2A and C2B) and a C-terminal A-domain. The former are thought to be responsible for binding to the inner face of the plasma membrane following increases in intracellular calcium levels, whereas the A-domain has been suggested to be a protein-binding structure. In this study, we examined the effects of mutagenesis of selected residues in the linker area between the C2-domains and the A-domain, and mutagenesis of the aspartates of the C2-domains, which are predicted to bind calcium and promote membrane association of the copines. We found that Lys282-Lys284 of the linker area are important for the folding of the intact protein. We showed that substitution with asparagine, single or multiple, of the aspartates in the C2A-domain had no effect on the calcium-mediated membrane association of copine-2, copine-6, or copine-7. Similar mutagenesis of a single residue in the C2B-domain of copine-6 (but not copine-2 and copine-7) was sufficient to eliminate its calcium-mediated membrane binding, and simultaneous substitution of all four of the asparagines in the C2B-domain resulted in constitutive membrane association of copine-2, copine-6 and copine-7 with the plasma membrane. These data show that the C2B-domains of copine-2, copine-6 and copine-7 are the domains responsible for the protein calcium-dependent membrane association.
Assuntos
Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Carbacol/farmacologia , Proteínas de Transporte/genética , Membrana Celular/química , Células HEK293 , Humanos , Proteínas de Membrana/genética , Dados de Sequência Molecular , Mutação , Dobramento de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de AminoácidosRESUMO
The obligatory heterodimerization of the GABAB receptor (GBR) raises fundamental questions about molecular mechanisms controlling its signaling efficacy. Here, we show that NEM sensitive fusion (NSF) protein interacts directly with the GBR heterodimer both in rat brain synaptosomes and in CHO cells, forming a ternary complex that can be regulated by agonist stimulation. Inhibition of NSF binding with a peptide derived from GBR2 (TAT-Pep-27) did not affect basal signaling activity but almost completely abolished agonist-promoted GBR desensitization in both CHO cells and hippocampal slices. Taken with the role of PKC in the desensitization process, our observation that TAT-Pep-27 prevented both agonist-promoted recruitment of PKC and receptor phosphorylation suggests that NSF is a priming factor required for GBR desensitization. Given that GBR desensitization does not involve receptor internalization, the NSF/PKC coordinated action revealed herein suggests that NSF can regulate GPCR signalling efficacy independently of its role in membrane trafficking. The functional interaction between three bona fide regulators of neurotransmitter release, such as GBR, NSF and PKC, could shed new light on the modulation of presynaptic GBR action.