Xiaohang Huang

The 25-kDa Synaptosome-associated Protein (SNAP-25) Binds and Inhibits Delayed Rectifier Potassium Channels in Secretory Cells

Delayed-rectifier K+ channels (KDR) are important regulators of membrane excitability in neurons and neuroendocrine cells. Opening of these voltage-dependent K+ channels results in membrane repolarization, leading to the closure of the Ca2+channels and cessation of insulin secretion in neuroendocrine islet β cells. Using patch clamp techniques, we have demonstrated that the activity of the KDR channel subtype, KV1.1, identified by its specific blocker dendrodotoxin-K, is inhibited by SNAP-25 in insulinoma HIT-T15 β cells. A co-precipitation study of rat brain confirmed that SNAP-25 interacts with the KV1.1 protein. Cleavage of SNAP-25 by expression of botulinum neurotoxin A in HIT-T15 cells relieved this SNAP-25-mediated inhibition of KDR. This inhibitory effect of SNAP-25 is mediated by the N terminus of KV1.1, likely by direct interactions with KVα1.1 and/or KVβ subunits, as revealed by co-immunoprecipitation performed in the Xenopus oocyte expression system and in vitro binding. Taken together we have concluded that SNAP-25 mediates secretion not only through its participation in the exocytotic SNARE complex but also by regulating membrane potential and calcium entry through its interaction with KDR channels.

Cholecystokinin-regulated exocytosis in rat pancreatic acinar cells is inhibited by a C-terminus truncated mutant of SNAP-23.

Introduction Exocytosis is thought to result from the fusion of vesicle and plasma membranes caused by the formation of a trans-complex between proteins of the vesicle-associated membrane protein (VAMP) family on the vesicle with members of the syntaxin and synaptosomal-associated protein of 25 kd (SNAP-25) families on the plasma membrane. In the pancreatic acinar cell, synaptosomal-associated protein of 23 kd (SNAP-23) is the major SNAP-25 isoform expressed in pancreatic acinar cells, but its role in acinar cell exocytosis has not been determined. Aims To examine the role of SNAP-23 in regulated exocytosis in acinar cells, we subcloned into adenoviral vectors SNAP-23, SNAP-25, and dominant negative mutants in which the C-terminal domains corresponding to the botulinum neurotoxin A cleavage sites are deleted. Methodology and Results High-efficiency infection of rat pancreatic acini in culture with these adenoviruses by subcellular fractionation showed that the overexpressed SNAP-23, SNAP-25, and their truncated mutant proteins were uniformly targeted to the zymogen granules and plasma membrane. To maximally stimulate apical exocytosis from these infected acini, we used the cholecystokinin–phenylethyl ester analog (CCK-OPE), which does not show inhibition of secretion from maximal levels at high doses. CCK-OPE–stimulated amylase release from adenovirus–cytomegalovirus (AdCMV)–SNAP-23 or AdCMV–SNAP-25–infected acini to the same extent as from acini infected with the empty vector. In contrast, CCK-OPE–evoked enzyme secretion from AdCMV–SNAP-23&Dgr;C8– and AdCMV–SNAP-25 1−197 –infected acini were inhibited by 60% and 40%, respectively. The identical targeting of the mutant SNAP-23 and SNAP-25 proteins to the same membrane compartments as SNAP-23 suggests that the inhibition of secretion was a result of their competition against endogenous SNAP-23. This is supported by the fact that this inhibition by the mutant proteins was partially reversed or rescued when the AdCMV–SNAP-25&Dgr;C8– or AdCMV–SNAP-25 1−197 –infected acini were co-infected with wild-type SNAP-23 or SNAP-25. Conclusion From these results, we conclude that SNAP-23 plays a role in CCK-evoked regulated exocytosis in the acinar cells.

Effects of selective endocrine or exocrine induction of AR42J on SNARE and Munc18 protein expression.

We used the amphicrine AR42J as an excellent model to study the differentiation of the secretory machinery of pancreatic endocrine and exocrine cells. Dexamethasone treatment induced the AR42J to differentiate towards the exocrine phenotype capable of secreting amylase in response to cholecystokinin. In contrast, activin A plus hepatocyte growth factor treatment of a subclone of AR42J, AR42J-B13, induced this cell to differentiate morphologically and functionally toward an insulin-containing and insulin-secreting endocrine phenotype. We took advantage of these unique properties of selective exocrine and endocrine induction of the AR42J to reveal which distinct combinations of exocytic SNARE complex proteins (syntaxin, SNAP-25 and VAMP) and associated Munc18 proteins were preferentially expressed to play a role in enzyme and insulin secretion. Results and Conclusion: To our surprise, both endocrine and exocrine induction of AR42J and AR42J-B13 caused very similar upregulation in the expression of the exocytic member isoforms of the syntaxin, SNAP-25, VAMP, and Munc18 families. We conclude that whereas the differentiation of the proximal components of the secretory machinery of the exocrine acinar and endocrine islet &bgr;-cells is distinct, the differentiation of the distal components of exocytosis between these two cell types is very similar.