MgATP-dependent and MgATP-independent [3H]noradrenaline release from perforated synaptosomes both use N-ethylmaleimide-sensitive fusion protein.
In streptolysin-O (SLO)-perforated rat brain cortical synaptosomes, Ca2+-induced [3H]noradrenaline (3H-NA) release began with a phase lasting about 1 min that did not depend on MgATP. Subsequent release became increasingly MgATP-dependent. The first phase involved release from previously "primed" synaptic vesicles. MgATP-dependent release, on the other hand, was release from unprimed vesicles that needed to be primed by ATP hydrolysis before they could be fused with the presynaptic membrane. Vesicle depriming was detected by observing that the initial release decreased when the synaptosomes were perforated and incubated for 2 min in the absence of MgATP before increasing Ca2+ to promote release. One millimolar N-ethylmaleimide (NEM) inhibited both MgATP-dependent and MgATP-independent release at all times of incubation (0.5-5 min), and inhibition by NEM was partially reversed at short (0.5 min) and longer (5 min) times by adding intact N-ethylmaleimide sensitive fusion protein (NSF) to the perforated synaptosomes. Polyclonal antibodies against the N-terminal domain of NSF produced dose-dependent inhibition of Ca2+-induced 3H-NA release. This inhibition occurred in both early and late release phases and was highly significant at early times if the perforated synaptosomes were preincubated for 2 min with anti-NSF. These results indicate participation of NSF both after vesicular fusion, probably for separation of SNARE proteins in v/t-SNARE complexes before endocytosis, and, surprisingly, after docking, possibly to maintain vesicles in a primed state and reverse depriming during regulated secretion.
Zheng
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Department of Chemistry, Texas Christian University, Fort Worth 76129, USA.
Bobich
J A
JA
D000255:Adenosine Triphosphate / Q000037:antagonists & inhibitors / Q000502:physiology*; D000818:Animals; D002118:Calcium / Q000037:antagonists & inhibitors / Q000502:physiology; D002352:Carrier Proteins / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology*; D007106:Immune Sera / Q000494:pharmacology; D008297:Male; D008561:Membrane Fusion / Q000187:drug effects; D052066:N-Ethylmaleimide-Sensitive Proteins; D009638:Norepinephrine / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013572:Synaptic Vesicles / Q000187:drug effects / Q000378:metabolism; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism*; D014316:Tritium; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D007106:Immune Sera; D033921:Vesicular Transport Proteins; D014316:Tritium; D000255:Adenosine Triphosphate; D052066:N-Ethylmaleimide-Sensitive Proteins; C498737:Nsf protein, rat; D005033:Ethylmaleimide; D002118:Calcium; D009638:Norepinephrine