RESUMEN
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.
Asunto(s)
Calcio/farmacología , Fusión de Membrana/efectos de los fármacos , Fosfolípidos/metabolismo , Sinaptotagminas/metabolismo , Animales , Sitios de Unión , Calcio/metabolismo , Liposomas/química , Liposomas/metabolismo , Modelos Biológicos , Modelos Moleculares , Docilidad , Unión Proteica/efectos de los fármacos , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ratas , Espectrometría de Fluorescencia , Vesículas Sinápticas/efectos de los fármacos , Vesículas Sinápticas/metabolismo , Sinaptotagminas/químicaAsunto(s)
Proteínas de Unión al Calcio , Calcio/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Animales , Sitios de Unión , Señalización del Calcio , Técnicas In Vitro , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Neuronas/metabolismo , Estructura Terciaria de Proteína , SinaptotagminasRESUMEN
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.