Transduction of MIN6 β Cells with TAT-Syntaxin SNARE Motif Inhibits Insulin Exocytosis in Biphasic Insulin Release in a Distinct Mechanism Analyzed by Evanescent Wave Microscopy

Abstract

To investigate the in vivo interaction of syntaxin-mediated soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) assembly and insulin exocytosis in biphasic release, we examined the dynamics of insulin granule motion such as docking and fusion with the plasma membrane when the syntaxin SNARE motif (H3 domain) was transduced into living MIN6 ␤ cells. TAT-H3, produced by fusion of the protein transduction domain of human immunodeficiency virus-1 TAT to the syntaxin-H3 domain, was rapidly transduced into the subplasmalemmal region in living MIN6 cells. Immunoblotting analysis followed by immunoprecipitation on TAT-H3-treated MIN6 cells showed that TAT-H3 binds SNAP-25 and VAMP-2 in vivo. Transduction of MIN6 cells with TAT-H3 caused a decrease in both the first and second phase of insulin release. We therefore quantitatively analyzed approaching, docking, and fusing of green fluorescent proteinlabeled single insulin granules in TAT-H3-transduced MIN6 cells by evanescent wave microscopy. Under high glucose stimulation, TAT-H3 treatment not only reduced the fusion events from previously docked granules for the first 120 s (first phase of release) but also strongly inhibited the docking and fusion from newly recruited insulin granules after this point (second phase of release). During the second phase of release we observed a marked reduction in the accumulation of newly docked insulin granules; subsequently, fusion events were significantly decreased. TAT-H3 treatment by itself, however, did not alter the number of previously docked granules without stimulation. We conclude that introduction of the H3 domain into MIN6 cells inhibits biphasic insulin release by two mechanisms. 1) In the first phase of insulin release, the H3 domain interferes with previously docked granules to be fused, and 2) in the second phase of insulin release reduced fusion events result from a marked decline of newly docked granules. Thus, syntaxin-mediated SNARE assembly modulates insulin exocytosis in biphasic insulin release in a distinct way.In pancreatic ␤ cells, the hormone insulin is stored in large dense-core vesicles and is released by exocytosis when glucose levels rise (1). Soluble N-ethylmaleimide-sensitive fusion protein (NSF) 1 attachment protein receptors (SNAREs) are believed to be required for vesicular trafficking such as endoplasmic reticulum to Golgi, Golgi to the plasma membrane, and exocytosis in general (2, 3). Syntaxin is a key component of the SNARE complex involved in synaptic vesicle docking and fusion, and it functions as an organizing center for exocytosis (4,5). Syntaxin 1 consists of four ␣-helical domains (Habc domain and SNARE motif (H3 domain)) and a short C-terminal transmembrane domain (6, 7). The syntaxin-H3 domain contributes to one of the four ␣-helical bundles in the SNARE core complex; that is, one helix each from syntaxin-H3 and VAMP and two helices from SNAP-25 (8), suggesting the H3 domain is the most critical domain in the molecular function of syntaxin.SNAREs are exp...

Rab27 Effector Granuphilin Promotes the Plasma Membrane Targeting of Insulin Granules via Interaction with Syntaxin 1a

“…The importance of syntaxin 1a in the tethering or docking process of insulin granules also is supported by recent findings from evanescent wave microscopy. Ohara-Imaizumi et al (14) have shown that introduction of the H3 domain of syntaxin 1a into MIN6 cells inhibits new docking events of insulin granules during the glucose stimulation. We have found that the H3 fragment of syntaxin 1a directly binds to granuphilin in vitro and that its introduction into MIN6 cells induces a concomitant decrease in newly docked granules and the amount of endogenous granuphilin-syntaxin 1a complex.…”

“…Transduction of MIN6 Cells with TAT Fusion Proteins-Isolation of TAT-GFP and TAT-H3 was performed as described previously (14). MIN6 cells were transduced for 50 min with 70 g/ml of TAT fusion proteins, and the cell extracts were prepared as described for the immunoprecipitation experiments.…”

“…Human immunodeficiency virus-1 TAT protein can cross a biological membrane efficiently and promote delivery of fused peptides into cells. Ohara-Imaizumi et al (14) showed that the introduction of TAT-H3 inhibits the plasma membrane docking of newly arrived insulin granules as well as the membrane fusion of predocked granules. These findings suggest that syntaxin 1a is required for replenishment of insulin granules into the docked pool.…”

“…Complex-Ohara-Imaizumi et al (14) recently examined the effect of a TAT-fused H3 fragment of syntaxin 1a on the exocytosis of insulin granules in MIN6 cells using an evanescent wave microscopic technique. Human immunodeficiency virus-1 TAT protein can cross a biological membrane efficiently and promote delivery of fused peptides into cells.…”

Polymorphisms in the gene encoding the voltage-dependent Ca2+ channel CaV2.3 (CACNA1E) are associated with type 2 diabetes and impaired insulin secretion

“…Impaired, or even completely absent first-phase insulin secretion is an early feature of type 2 diabetes, whereas second-phase insulin secretion deteriorates with progression of the disease. Data from rodent models suggest that firstphase insulin secretion results from exocytosis of insulin granules already present at the plasma membrane, whereas second-phase secretion involves recruitment of insulin granules from the beta cell interior [12,13]. Triggering of biphasic insulin secretion requires generation of beta cell electrical activity, which results from a functional interplay between the beta cell ATP-sensitive K + channel (which, in part, is encoded by the KCNJ11 gene) and voltagedependent Ca 2+ channels (VDCC) [14].…”