RESUMEN
In synaptic transmission, vesicles are proposed to dock at presynaptic active zones by the association of synaptobrevin (v-SNARE) with syntaxin (t-SNARE). We test this hypothesis in Drosophila strains lacking neural synaptobrevin (n-synaptobrevin) or syntaxin. We showed previously that loss of either protein completely blocks synaptic transmission. Here, we attempt to establish the level of this blockade. Ultrastructurally, vesicles are still targeted to the presynaptic membrane and dock normally at specialized release sites. These vesicles are mature and functional since spontaneous vesicle fusion persists in the absence of n-synaptobrevin and since vesicle fusion is triggered by hyperosmotic saline in the absence of syntaxin. We conclude that the SNARE hypothesis cannot fully explain the role of these proteins in synaptic transmission. Instead, both proteins play distinct roles downstream of docking.
Asunto(s)
Drosophila/fisiología , Proteínas de la Membrana/fisiología , Proteínas del Tejido Nervioso/fisiología , Vesículas Sinápticas/metabolismo , Animales , Animales Modificados Genéticamente , Sitios de Unión , Araña Viuda Negra , Calcio/farmacología , Drosophila/embriología , Drosophila/genética , Fusión de Membrana/fisiología , Proteínas de la Membrana/genética , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas Qa-SNARE , Proteínas R-SNARE , Venenos de Araña/farmacología , Sinapsis/fisiología , Transmisión Sináptica/fisiología , Vesículas Sinápticas/ultraestructuraRESUMEN
Synaptotagmin (syt), a synaptic vesicle-specific protein known to bind Ca2+ in the presence of phospholipids, has been proposed to mediate Ca(2+)-dependent neurotransmitter release. We have addressed the role of syt in neurotransmitter release in vivo by generating mutations in synaptotagmin (syt) in the fruitfly and assaying the subsequent effects on neurotransmission. Most embryos that lack syt fail to hatch and exhibit very reduced, uncoordinated muscle contractions. Larvae with partial lack-of-function mutations show almost no evoked excitatory junctional potentials (EJPs) in 0.4 mM Ca2+ and a 15-fold reduction in EJP amplitude in 1.0 mM Ca2+ when compared with heterozygous controls. In contrast, we observe an increase in the frequency of spontaneous miniature EJPs in the mutants. These results provide in vivo evidence that syt plays a key role in Ca2+ activation of neurotransmitter release and indicate the existence of separate pathways for evoked and spontaneous neurotransmitter release.