An ATP-dependent activity of NSF (N-ethylmaleimidesensitive factor) that rearranges soluble NSF attachment protein (SNAP) receptor (SNARE) protein complexes was proposed to be the driving force for membrane fusion. The Ca 2؉-activated fusion of secretory vesicles with the plasma membrane in permeable PC12 cells requires ATP; however, the ATP requirement is for a priming step that precedes the Ca 2؉ -triggered fusion reaction. While phosphoinositide phosphorylation is a key reaction required for priming, additional ATP-dependent reactions are also necessary. Here we report that the NSF-catalyzed rearrangement of SNARE protein complexes occurs during ATP-dependent priming. NSF with ␣-SNAP (soluble NSF attachment protein) were required for ATP-dependent priming but not Ca 2؉ -triggered fusion, indicating that NSF acts at an ATP-dependent prefusion step rather than at fusion itself. NSFcatalyzed activation of SNARE proteins may reorganize membranes to generate a vesicle-plasma membrane prefusion intermediate that is poised for conversion to full fusion by Ca 2؉ -dependent mechanisms.The regulated fusion of vesicles with the plasma membrane in neural and endocrine cells requires a core complex of proteins (synaptobrevin, syntaxin, and SNAP-25) that are specific substrates for clostridial neurotoxin proteases (1-4). This complex is proposed to function in vesicle targeting, docking or fusion. Identification of these neuronal synaptic proteins (termed SNAREs) 1 as receptors for SNAP proteins that mediate the membrane association of NSF, a protein required for constitutive membrane fusion (1), suggested that NSF may be required for Ca 2ϩ -regulated neurosecretion (5). Genetic studies in Drosophila have established an essential role for NSF in neural function (6). Stimulatory effects of ␣-SNAP on neurotransmitter secretion from chromaffin cells and squid neurons have been reported (7,8). However, the precise stage in the regulated secretory pathway at which NSF acts has not been directly established. In vitro biochemical studies demonstrated that a 20 S complex of SNAREs, NSF, and ␣/␤-SNAP was disassembled by the ATP-dependent activity of NSF, and it was suggested that NSF-catalyzed SNARE protein rearrangements drive membrane fusion (9). However, previous studies with permeable PC12 and adrenal cells had shown that MgATP was required for a priming step that precedes the final fusion steps triggered by Ca 2ϩ (10,11). In the present studies, the execution point of NSF and ␣-SNAP was established as the ATP-dependent priming step that precedes Ca 2ϩ -activated fusion. EXPERIMENTAL PROCEDURES Preparation of Permeable PC12 Cells and Secretion Assays-PC12 cells were labeled with [3 H]norepinephrine (NE; Amersham Corp.) and permeabilized with a ball homogenizer (10, 12). Two stage secretion assays were in KGlu buffer (20 mM HEPES, pH 7.2, 120 mM potassium glutamate, 20 mM potassium acetate, 2 mM EGTA) with 0.1% bovine serum albumin. Thirty-min priming incubations at 30°C contained 2 mM MgATP and 1.0 mg/ml rat brain cytosol, whic...

We have characterized some properties of the caffeine-sensitive Ca2+ store in bovine chromaffin cells. Addition of 10 mM-caffeine to permeabilized cells that were allowed to sequester Ca2+ in the presence of the precipitating anion pyrophosphate induced a transient rise in free Ca2+ concentration that was blocked by 10 microM-Ruthenium Red. Caffeine was able to release Ca2+ after the InsP3-sensitive Ca2+ pool had been completely emptied, and 10 microM-InsP3 still released Ca2+ in the presence of a high dose (50 mM) of caffeine, indicating that there are selectively sensitive Ca2+ pools in these cells. The progressive hydrolysis of pyrophosphate by a cytosolic pyrophosphatase induced a spontaneous Ca2+ release after a latency. Caffeine prevented this spontaneous Ca2+ release, indicating that the pyrophosphate-sensitive Ca2+ pool was caffeine-sensitive. On varying the free Ca2+ concentration within the caffeine-sensitive pool (by using methylenediphosphonic acid, pyrophosphate or no precipitating anion), we observed that the Ca(2+)-releasing effect of caffeine was dependent on an elevated intraluminal free Ca2+ concentration. In conclusion, the caffeine-sensitive Ca2+ store in bovine chromaffin cells is largely distinct from the InsP3-sensitive Ca2+ store, and its release mechanism shares characteristics with the ryanodine receptor of muscle cells.

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We have investigated the modulation of stimulus-induced changes in intracellular Ca2+ concentration ([Ca2+]i) by a caffeine-and ryanodine-sensitive Ca2+ store in PC12 cells. In populations of fura-2-loaded cells, caffeine cause a concentration-dependent increase in [Ca2+]i that was saturable, reversible and inhibited in a use-dependent fashion by ryanodine. Maximal Ca2+ release occurred with 40 mM caffeine, with an EC50 of 13 mM caffeine and a Hill coefficient (h) of 2.7, indicating that the release mechanism was co-operative. Pretreatment of intact cell populations with increasing concentrations of caffeine in nominally Ca(2+)-free medium inhibited the subsequent Ca2+ response to a maximal concentration of ATP, in a dose-dependent manner. In permeabilized cells, a maximal concentration (40 microM) of InsP3 still released Ca2+ in the presence of a supramaximal concentration (50 mM) of caffeine, whereas caffeine was unable to release Ca2+ after the InsP3-sensitive store had been completely emptied. These data suggest that PC12 cells contain a uniquely InsP3-sensitive Ca2+ store, and a store that is sensitive to both InsP3 and caffeine. Depletion of the caffeine-sensitive Ca2+ store by caffeine and ryanodine pretreatment in intact cells attenuated the Ca2+ response to ATP, but not to 55 mM K+, suggesting that the caffeine-sensitive Ca2+ store acts as a Ca2+ source after ATP stimulation, but not after depolarization with 55 mM K+. Pretreatment of intact cells with ATP and ryanodine resulted in a use-dependent block of both caffeine- and ATP-mediated Ca2+ release, confirming that ATP stimulation of PC12 cells brings about activation of ryanodine receptors. The rate of recovery, but not the magnitude or rate of onset, of the depolarization-induced [Ca2+]i transient was modulated by the state of filling of the caffeine-sensitive Ca2+ store such that recovery was prolonged if the store was either full, or empty and unable to refill. We conclude that the caffeine- and ryanodine-sensitive Ca2+ store can act as a Ca2+ source and a Ca2+ sink in PC12 cells, and that its role may in part be governed by the nature of the stimulating agent.

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