We have continued our development of an analytical description for the final steps of calcium-triggered exocytosis that relies, on average, only upon the calcium-dependence of fusion complexes at vesicle docking sites. The fusion complex has been defined as a functional entity representing the minimal complex that catalyzes vesicle fusion. Our hypothesis is that an increase in calcium increases the average number of participating fusion complexes, and that the number of activated fusion complexes is distributed among vesicles as a Poisson random variable. At sub-optimal calcium concentrations, some vesicles will not have an activated complex and do not fuse while at maximally saturating calcium concentrations essentially every complex is activated and all vesicles fuse. N-ethyl maleimide (NEM) inhibition of fusion was used to estimate the maximum number of fusion complexes per vesicle at docking sites. This number indicates that the system operates with a reserve capacity. Under conditions of reserve capacity it is inappropriate to directly compare changes in potential protein components of the fusion complex with the extent of fusion because the loss of fusion response occurs in a regime where reserve capacity is minimal. With reserve capacity, changes in the rate rather than the extent of fusion are a much better measure for the involvement of selectively modified proteins in the fusion process. The prediction of this model is that random removal of fusion complexes using targeted protein modification will diminish the reserve capacity under maximally saturating calcium concentrations with minimal change in the extent of fusion but a progressive decrease in the maximum rate of fusion. Data obtained from NEM and protease treatment of both calcium-triggered vesicle-vesicle fusion and exocytosis supports this interpretation. Predicted variations in kinetics are an important tool in identifying those proteins essential to the final steps of calcium-triggered membrane fusion.While the relationship between exocytosis and calcium is fundamental both to synaptic and non-neuronal secretory function, analysis is problematic because of the temporal and spatial properties of calcium, and coupled vesicle transport, priming, retrieval and recycling. Synaptic vesicles are profoundly important in neuronal communication. However, their small size limits the use of fluorescence techniques in studying their calcium dynamics. The relatively large secretory vesicles of the sea urchin egg share many similarities with their neuronal counterparts. Remarkably, vesicles and other organelles are labeled using the calcium fluorophore, Fluo-4 penta-potassium salt in essentially calcium-free solutions. Using confocal microscopy, we have detected temporal fluctuations in fluorescence, suggesting that the intra-vesicular calcium concentration is dynamic in nature. Dynamic changes in calcium concentration at vesicle docking sites may influence the distribution of calcium thresholds that defines the activity in this system. Our kinetic analysis requires a conversion of the fusion complex from an inactive to an active state; this conversion is dependent upon the calcium concentration. In addition to calcium dynamics in mitochondria and endoplasmic reticulum, intravesicular calcium may be a modulator of calcium-triggered release at this key kinetic step.
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