| Rapid neurotransmitter release upon nerve stimulation is critical for neuronal signaling. This is achieved by fast, Ca2+-dependent fusion of the neurotransmitter-filled vesicles with the plasma membrane in the presynaptic terminal. Elucidating the molecular mechanisms of Ca 2+-triggered exocytosis is therefore crucial for understanding the basic principles of neuronal communication. Furthermore, as the molecular components mediating different membrane trafficking pathways are conserved, our study also provides valuable insights into the general principles underlying intracellular membrane fusion.;To study Ca2+-stimulated exocytosis, we utilized a cracked cell secretion assay, in which PC12 cells are mechanically permeabilized and reconstituted for Ca2+-triggered release of [3H]-norepinephrine from dense core vesicles.;SNARE proteins (syntaxin, SNAP-25, and VAMP) are known to be essential for synaptic and dense core vesicle exocytosis. However, it has been controversial whether SNAREs are involved mainly in the tethering of these vesicles to the plasma membrane or in the final membrane fusion process. Using a novel rescue approach, we were able to manipulate SNARE complexes inside PC12 cells while monitoring their function. Our results strongly suggest that SNARE complex formation is triggered by a Ca2+ signal and catalyzes the membrane fusion reaction.;Specific pairing of SNARE proteins has been proposed to mediate the fidelity of intracellular vesicle trafficking. However, this remains to be tested in a physiological membrane fusion system. We assayed a large number of SNAREs for their stimulatory or inhibitory effects in our cracked cell system. Our results showed that the known cognate SNAREs inhibited or rescued more effectively than non-cognate SNAREs, suggesting that SNAREs do contribute to the specificity of membrane fusion in a cellular context.;The molecular mechanisms by which Ca2+ regulates exocytosis are not well understood. We found that Hrs-2, an ATPase that binds to SNAP-25 in a Ca2+-sensitive manner, inhibited PC12 cell secretion, implicating Hrs-2 in Ca2+-regulated exocytosis. In addition, using a biochemical fractionation approach, we identified calmodulin and protein kinase C as two Ca2+-sensitive positive regulators of the membrane-attached exocytic machinery. While the exact mechanisms through which Ca 2+ regulates the basic membrane trafficking apparatus still remains a mystery, our work has provided important leads for future studies. |
