Synaptotagmins: mediators of Ca -regulated vesicle fusion. Focus on “Stable gene silencing of synaptotagmin I in rat PC12 cells inhibits Ca -evoked release of catecholamine”

EXOCYTOSIS IS A GENERAL PROCESS by which intracellular vesicles containing cargo are transported to and then fuse with the plasma membrane. Exocytic vesicles either fuse constitutively or they accumulate under the plasma membrane, where they await fusion until an appropriate physiological signal is detected in a process called regulated exocytosis (4, 21). Examples of exocytic vesicles that undergo regulated exocytosis include discoidal vesicles that fuse with the apical plasma membrane of umbrella cells in response to stretch, synaptic vesicles that fuse with the presynaptic membrane in response to action potentials, and dense-core/secretory vesicles that fuse with the plasma membrane of neuroendocrine/endocrine cells downstream of hormonal stimuli (6, 21, 24). Dense-core granules are formed in the trans-Golgi network and then accumulate in the cell cytoplasm forming a reserve pool of vesicles. A fraction of this pool is delivered to the plasma membrane where vesicles are docked. The vesicles then undergo a Ca and ATP-dependent priming step(s) that is incompletely understood, but is thought to produce an asynchronous, slowly releasable pool of vesicles (21). Vesicle priming is likely to depend in part on the assembly of soluble N-ethylmaleimidesensitive factor attachment protein receptors (SNAREs) that reside on the vesicle SNARE (v-SNARE) and target membrane SNARE (t-SNARE), which drive the final steps in vesicle fusion and cargo release (6, 21). Upon priming, a synchronous rapidly releasable pool of vesicles is generated. Fusion of both pools of vesicles occurs in response to micromolar concentrations of intracellular Ca , and as their names imply, the rapidly releasable pool is quickly exocytosed (in the range of 0.1–1 s), whereas the slowly releasable pool is exocytosed over a longer time scale (several seconds). The molecular basis of these distinct rates of vesicle fusion is only poorly understood. Ca -regulated fusion is thought to depend on a family of proteins called the synaptotagmins (syts), the putative Ca sensors that trigger exocytosis (6, 14, 23). The syt family in humans and mice is comprised of 16 members, but 90 syt members are present in the published plant and animal genomes (7). Some isoforms of this family, syt VII in particular, have multiple splice variants, but the functional consequence of these different forms is unknown (22). Syts are localized to secretory vesicles and the plasma membrane, and have a conserved domain structure that includes a short extracellular domain, a transmembrane domain, and a cytoplasmic domain that is folded into a membrane proximal C2A domain and a membrane distal C2B region (6). The C2 domains of most syt isoforms bind Ca , however, syts VI, VIII, XII, and XIII lack consensus Ca -binding sites (22). Both biochemical and genetic data point to an important role for syts in Ca -dependent fusion. The C2A and C2B domains of syt I (a major component of synaptic vesicles) penetrate into the plasma membrane lipids in a Ca -dependent manner and the C2B domain specifically binds to phosphatidylinositol 4,5-bisphosphate, a lipid primarily associated with the plasma membrane (2, 6). Significantly, syt I associates with the membrane-proximal region of the t-SNAREs syntaxin 1 and SNAP25, and may regulate Ca -dependent fusion by promoting SNARE assembly and fusion (5, 6, 21, 26). Synaptotagmins undergo Ca -dependent homoand heterooligomerization, which may potentially aid SNARE fusion by bringing opposing membrane and SNARE complexes in close proximity (3, 10, 22). Ca -dependent fusion can be reconstituted in vitro by the addition of the cytoplasmic domain of syt I to an assay that measures SNARE-dependent fusion of liposomes (26). Strikingly, not only does the syt I cytoplasmic domain make the reaction Ca dependent, it also stimulates the rate of vesicle fusion, consistent with a role for synaptotagmins in promoting the fusion reaction. Genetic experiments show that expression of mutant syt I in mice, Caenorhabditis elegans, or Drosophila melanogaster disrupts exocytosis of the rapidly releasable pool of synaptic vesicles (12, 15, 17, 29). Similarly, exocytosis of a release-ready pool of dense core vesicles is perturbed in chromaffin cells isolated from syt I-deficient mice (27). In all cases, significant Ca -dependent exocytosis remains, but the rate of residual exocytosis is slower, and may reflect the utilization of other syt isoforms. A widely used model system to study syt function is the rat pheochromocytoma PC12 cell line. These cells have a population of dense core granules that contain catecholamines, and express multiple syt isoforms including I, III, IV, VII, and IX (9, 16, 28). Syt I and IX are thought to be expressed at relatively high levels, whereas expression of the other isoforms is low and somewhat variable depending on the PC12 cell line. Evidence from mechanically perforated PC12 cells show that both syt III and syt VII C2A domains inhibit exocytosis (25). Syt IX may also play a significant role in dense-core vesicle exocytosis as antibodies to the syt IX C2A domain inhibits exocytosis in permeabilized PC12 cells (11), and downregulation of syt IX by interfering RNA (RNAi) impairs dense-core granule exocytosis (9). The function of syt I in PC12 cells is controversial because earlier studies indicate that it may be dispensable. Naturally occurring variant PC12 cell lines, which have low levels of syt I expression, still undergo significant Ca -dependent exocytosis (9, 20). Furthermore, knockdown of syt I by transient RNAi expression has no impact on Address for reprint requests and other correspondence: G. Apodaca, Univ. of Pittsburgh, Renal Division, 982 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15261 (e-mail: [email protected]). Am J Physiol Cell Physiol 291: C234–C236, 2006; doi:10.1152/ajpcell.00129.2006.