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Aims/hypothesis. Syntaxin-1A (Syn-1A) is known to play a negative regulatory role in insulin secretion but the precise mechanisms for its action are not clear. Syn-2, ±3 and ±4 are also present in islet beta cells but their functions are not known. Here, we investigated the role of these syntaxins in the insulin secretory process. Methods. We examined the following effects of Syn-1, ±2, ±3 and ±4 expression in insulinoma beta-cell lines. Endogenous insulin secretion was measured by batch radioimmunoassay (RIA) and single cell patch clamp capacitance measurements. The l-type Ca 2+ channel activity was studied by patch clamp electrophysiology. Insulin gene transcription was examined by Northern blotting and measurement of insulin gene promoter activity by the co-expression of cyan fluorescent protein-labelled rat insulin promoter. Results. Syn-1A or ±3, but not Syn-2 or ±4 overexpression, inhibited K + -induced insulin release as determined by RIA (49.7 5.5 % and 49.1 6.2 %, respectively) and electrophysiologic membrane capacitance measurements (68.0 21.0 % and 58.0 13.2 %, respectively). Overexpressed Syn-1A and ±3, but not Syn-2, inhibited Ca 2+ channel current amplitude by 39.5 11.6 % and 52.7 6.0 %, respectively. Of note, overexpression of Syn-1A and ±3 also reduced single cell (by confocal microscopy) and total cellular endogenous insulin content (by RIA) by 24.8 4.2 % and 31.8 3.9 %, respectively. This correlated to a reduction in endogenous insulin mRNA by 24.5 4.2 % and 25.7 4.2 %, respectively. This inhibition of insulin biosynthesis is mainly at the level of insulin gene transcription as demonstrated by an inhibition of insulin gene promoter activity (53.3 9.15 % and 39.0 6.8 %, respectively). Conclusions/interpretation. These results demonstrate that Syn-1A and ±3 possess strong inhibitory actions on both insulin exocytosis and insulin biosynthesis whereas Syn-2 and ±4 do not inhibit the insulin secretory process. [Diabetologia (2002)

The ATP-sensitive potassium (K ATP ) channel in pancreatic islet beta cells consists of four pore-forming (Kir6.2) subunits and four regulatory sulfonylurea receptor (SUR1) subunits. In beta cells, the K ATP channel links intracellular metabolism to the dynamic regulation of the cell membrane potential that triggers insulin secretion. Syntaxin 1A (Syn-1A) is a SNARE protein that not only plays a direct role in exocytosis, but also binds and modulates voltage-gated K ؉ and Ca 2؉ channels to fine tune exocytosis. We recently reported that wild type Syn-1A inhibits rat islet beta cell K ATP channels and binds both nucleotide-binding folds (NBF-1 and NBF-2) of SUR1. However, wild type Syn-1A inhibition of rat islet beta cell K ATP channels seems to be mediated primarily via NBF-1. During exocytosis, Syn-1A undergoes a conformational change from a closed form to an open form, which would fully expose its active domain, the C-terminal H3 domain. Here, we show that the constitutively open form Syn-1A

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Previous reports showed that cleavage of vesicle-associated membrane protein-2 (VAMP-2) and synaptosomal-associated protein of 25 kDa (SNAP-25) by clostridial neurotoxins in permeabilized insulin-secreting beta-cells inhibited Ca(2+)-evoked insulin secretion. In these reports, the soluble N-ethylmaleimide-sensitive factor attachment protein target receptor proteins might have formed complexes, which preclude full accessibility of the putative sites for neurotoxin cleavage. In this work, VAMP-2 and SNAP-25 were effectively cleaved before they formed toxin-insensitive complexes by transient transfection of insulinoma HIT or INS-1 cells with tetanus toxin (TeTx) or botulinum neurotoxin A (BoNT/A), as shown by immunoblotting and immunofluorescence microscopy. This resulted in an inhibition of Ca(2+) (glucose or KCl)-evoked insulin release proportionate to the transfection efficiency (40-50%) and an accumulation of insulin granules. With the use of patch-clamp capacitance measurements, Ca(2+)-evoked exocytosis by membrane depolarization to -10 mV was abolished by TeTx (6% of control) but only moderately inhibited by BoNT/A (30% of control). Depolarization to 0 mV to maximize Ca(2+) influx partially overcame BoNT/A (50% of control) but not TeTx inhibition. Of note, cAMP activation potentiated Ca(2+)-evoked secretion by 129% in control cells but only 55% in BoNT/A-transfected cells and had negligible effects in TeTx-transfected cells. These results indicate that, whereas VAMP-2 is absolutely necessary for insulin exocytosis, the effects of SNAP-25 depletion on exocytosis, perhaps on insulin granule pool priming or mobilization steps, could be partially reversed by higher levels of Ca(2+) or cAMP potentiation.