RESUMO
Synaptic vesicle fusion is catalyzed by assembly of synaptic SNARE complexes, and is regulated by the synaptic vesicle GTP-binding protein Rab3 that binds to RIM and to rabphilin. RIM is a known physiological regulator of fusion, but the role of rabphilin remains obscure. We now show that rabphilin regulates recovery of synaptic vesicles from use-dependent depression, probably by a direct interaction with the SNARE protein SNAP-25. Deletion of rabphilin dramatically accelerates recovery of depressed synaptic responses; this phenotype is rescued by viral expression of wild-type rabphilin, but not of mutant rabphilin lacking the second rabphilin C2 domain that binds to SNAP-25. Moreover, deletion of rabphilin also increases the size of synaptic responses in synapses lacking the vesicular SNARE protein synaptobrevin in which synaptic responses are severely depressed. Our data suggest that binding of rabphilin to SNAP-25 regulates exocytosis of synaptic vesicles after the readily releasable pool has either been physiologically exhausted by use-dependent depression, or has been artificially depleted by deletion of synaptobrevin.
Assuntos
Vesículas Sinápticas/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Deleção de Genes , Camundongos , Camundongos Knockout , Modelos Moleculares , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas R-SNARE/metabolismo , Ratos , Sacarose/farmacologia , Vesículas Sinápticas/efeitos dos fármacos , Proteína rab3A de Ligação ao GTP/metabolismoRESUMO
Synaptotagmin acts as a Ca(2+) sensor in neurotransmitter release through its two C(2) domains. Ca(2+)-dependent phospholipid binding is key for synaptotagmin function, but it is unclear how this activity cooperates with the SNARE complex involved in release or why Ca(2+) binding to the C(2)B domain is more crucial for release than Ca(2+) binding to the C(2)A domain. Here we show that Ca(2+) induces high-affinity simultaneous binding of synaptotagmin to two membranes, bringing them into close proximity. The synaptotagmin C(2)B domain is sufficient for this ability, which arises from the abundance of basic residues around its surface. We propose a model wherein synaptotagmin cooperates with the SNAREs in bringing the synaptic vesicle and plasma membranes together and accelerates membrane fusion through the highly positive electrostatic potential of its C(2)B domain.