A Role Of Snapin In Neurosecretion

Neurotransmitters and hormones are released through exocytosis of vesicles. The vesicular exocytosis consists of complex serial events of trafficking, tethering, docking, priming, fusing with cell membrane. SNARE protein complex is essential molecular machinery in most, if not all, exocytosis events studied so far, and supply the energy for vesicle fusion with their target membrane. Synaptotagmins are Ca2+ sensors in rgulative exocytosis molecular machinery. Its association with SNARE complex is essential for membrane fusion, and the complex of synaptotagmin with SNARE proteins is basic molecular machinery for vesicle fusion. Exocytosis is controlled and regulated strickly by regulative proteins, such as α-SNAP, NSF, Munc18/nSec1, Munc13, Rab, Rab effectors (including rabphilin, Rim, and Noc2), and calcium-binding proteins. Priming of viscle is a biochemical process which prepares membranes for fusion. It is a speed-limited step in exocytosis, and a low level calcium-, ATP-, and phosphatidyl inositol metabolites-depended process. The primed vesicles constitute releasable pool (RP). The size of releasable pool is depended on the size of un-primed pool, and rate of pimimg, un-priming, and exocytosis. The priming step maybe corresponds to the assembly of the SNARE complex, in which the vesicle-associated v-SNARE protein synaptobrevin (VAMP) interacts with two plasma membrane-associated t-SNARE proteins, SNAP-25 and syntaxin, to form a stable SNARE complex. The priming process is most likely aided by the action of Munc13. Furthermore, maturation into a release-ready vesicle requires synaptotagmin. Synaptotagmins, integral Ca²⁺-binding proteins and Ca2+ sensors of vesicle membrane, provide Ca2+-dependent regulation of the fusion machinery. However, the molecular mechanisms underlying regulation of the structural and functional coupling of the calcium sensor with the SNARE-based fusion machinery during priming and maturation remains unclear. Snapin was first identified as a SNAP-25 binding protein that associates with the SNARE complex and enhances the association of synaptotagmin with the SNARE complex. Snapin is a ubiquitously expressed soluble protein that is present in both cytosol and peripheral membrane-associated fractions. In addition to binding to SNAP-25, Snapin can form a complex with SNAP-23, the nonneuronal isoform of SNAP-25. Both Snapin and synaptotagmin IX interact in vitro and in vivo with the vanilloid receptor-1 (TRPV1) and consequently modulate PKA-mediated recruitment of the functional TRPV1 channel to the plasma membrane in dorsal root ganglion neurons via SNARE-dependent exocytosis. Snapin was also identified as a subunit of biogenesis of lysosome-related organelles Complex-1 (BLOC-1), suggesting its potential role in the endocytic pathway. Snapin is an important target of PKA, and phosphorylation modulates the efficacy of Snapin action on release. In the present study, we generated null mutations in the mouse snapin gene via homologous recombination to abolished snapin expression in mice, and functionally evaluated Snapin's role in neuroexocytosis by state-of-the-art biochemical and biophysical techniques. We found that the absence of Snapin expression did not affect the expression level of a large variety of proteins involved in synaptic vesicle exocytosis, indicates that snapin deletion does not result in any apparent compensatory changes in the expression of known proteins that are required for presynaptic function. Furthermore, our biochemical data demonstrate that the formation of the SNARE complex is not affected by deletion of snapin. We detected a marked reduction (34 ±3%, from 11 littermates, p<0.01) in the association of SNAP-25 and synaptotagmin in brain homogenates of snapin KO mice when compared to wild-type littermates. Using flash photolysis of caged calcium as a fast stimulus and high-temporal-resolution measurement of membrane capacitance and catecholamine release, we found that deletion of snapin in chromaffin cells led to a 45 % reduction of calcium-dependent burst exocytosis, while the sustained release phase remained unaffected. Furthermore, reintroduction of Snapin into the mutant cells could fully rescue this inhibitory effect within hours, indicating the specificity of the observed burst reduction upon snapin deletion. Our electron microscopy analysis demonstrated no significant differences between these groups either in the distribution of the vesicles relative to the plasma membrane or in the total number of vesicles per cell section, suggesting that Snapin is not directly involved in morphological docking of LDCVs in chromaffin cells. Furthermore, our calcium ramp experiments showed that neither the Ca2+ sensitivity nor the Ca2+ cooperativity of secretion is changed in the absence of Snapin, excluding the possibility that Snapinis directly involved in the final Ca2+-triggered fusion reaction. Thus, our biochemical and electrophysiological studies using snapin knockout mice indicate that Snapin plays a critical role in modulating neurosecretion, likely by stabilizing synaptotagmin-SNARE complex, decreasing the rate of de-priming, and thus stabilizing the release-ready vesicles. We presented a short introduction of biophysics techniques frequently used for study of exocytosis. We investigated the docking, undocking, and fusing with plasma membrane process of vesicle in PC12 cells by total internal reflection fluorescence microscopy, our data strongly support the original hypothesis that vesicle docking was a reversible process.