Deletion of Fas in the pancreatic beta-cells leads to enhanced insulin secretion.

Fas/Fas ligand belongs to the tumor necrosis factor superfamily of receptors/ligands and is best known for its role in apoptosis. However, recent evidence supports its role in other cellular responses, including proliferation and survival. Although Fas has been implicated as an essential mediator of beta-cell death in the pathogenesis of type 1 diabetes, the essential role of Fas specifically in pancreatic beta-cells has been found to be controversial. Moreover, the role of Fas on beta-cell homeostasis and function is not clear. The objective of this study is to determine the role of Fas specifically in beta-cells under both physiological and diabetes models. Mice with Fas deletion specifically in the beta-cells were generated using the Cre-loxP system. Cre-mediated Fas deletion was under the control of the rat insulin promoter. Absence of Fas in beta-cells leads to complete protection against FasL-induced cell death. However, Fas is not essential in determining beta-cell mass or susceptibility to streptozotocin- or HFD-induced diabetes. Importantly, Fas deletion in beta-cells leads to increased p65 expression, enhanced glucose tolerance, and glucose-stimulated insulin secretion, with increased exocytosis as manifested by increased changes in membrane capacitance and increased expression of Syntaxin1A, VAMP2, and munc18a. Together, our study shows that Fas in the beta-cells indeed plays an essential role in the canonical death receptor-mediated apoptosis but is not essential in regulating beta-cell mass or diabetes development. However, beta-cell Fas is critical in the regulation of glucose homeostasis through regulation of the exocytosis machinery.

High beta-cell mass prevents streptozotocin-induced diabetes in thioredoxin-interacting protein-deficient mice.

Thioredoxin-interacting protein (TxNIP) is an endogenous inhibitor of thioredoxin, a ubiquitous thiol oxidoreductase, that regulates cellular redox status. Diabetic mice exhibit increased expression of TxNIP in pancreatic islets, and recent studies suggest that TxNIP is a proapoptotic factor in beta-cells that may contribute to the development of diabetes. Here, we examined the role of TxNIP deficiency in vivo in the development of insulin-deficient diabetes and whether it impacted on pancreatic beta-cell mass and/or insulin secretion. TxNIP-deficient (Hcb-19/TxNIP(-/-)) mice had lower baseline glycemia, higher circulating insulin concentrations, and higher total pancreatic insulin content and beta-cell mass than control mice (C3H). Hcb-19/TxNIP(-/-) did not develop hyperglycemia when injected with standard multiple low doses of streptozotocin (STZ), in contrast to C3H controls. Surprisingly, although beta-cell mass remained higher in Hcb-19/TxNIP(-/-) mice compared with C3H after STZ exposure, the relative decrease induced by STZ was as great or even greater in the TxNIP-deficient animals. Consistently, cultured pancreatic INS-1 cells transfected with small-interfering RNA against TxNIP were more sensitive to cell death induced by direct exposure to STZ or to the combination of inflammatory cytokines interleukin-1beta, interferon-gamma, and tumor necrosis factor-alpha. Furthermore, when corrected for insulin content, isolated pancreatic islets from TxNIP(-/-) mice exhibited reduced glucose-induced insulin secretion. These data indicate that TxNIP functions as a regulator of beta-cell mass and influences insulin secretion. In conclusion, the relative resistance of TxNIP-deficient mice to STZ-induced diabetes appears to be because of an increase in beta-cell mass. However, TxNIP deficiency is associated with sensitization to STZ- and cytokine-induced beta-cell death, indicating complex regulatory roles of TxNIP under different physiological and pathological conditions.

Interaction between Munc13-1 and RIM is critical for glucagon-like peptide-1 mediated rescue of exocytotic defects in Munc13-1 deficient pancreatic beta-cells.

OBJECTIVE: Glucagon-like peptide-1 (GLP-1) rescues insulin secretory deficiency in type 2 diabetes partly via cAMP actions on exchange protein directly activated by cAMP (Epac2) and protein kinase A (PKA)-activated Rab3A-interacting molecule 2 (Rim2). We had reported that haplodeficient Munc13-1(+/-) mouse islet beta-cells exhibited reduced insulin secretion, causing glucose intolerance. Munc13-1 binds Epac2 and Rim2, but their functional interactions remain unclear. RESEARCH DESIGN AND METHODS: We used Munc13-1(+/-) islet beta-cells to examine the functional interactions between Munc13-1 and Epac2 and PKA. GLP-1 stimulation of Munc13-1(+/-) islets normalized the reduced biphasic insulin secretion by its actions on intact islet cAMP production and normal Epac2 and Rim2 levels. RESULTS: To determine which exocytotic steps caused by Munc13-1 deficiency are rescued by Epac2 and PKA, we used patch-clamp capacitance measurements, showing that 1) cAMP restored the reduced readily releasable pool (RRP) and partially restored refilling of a releasable pool of vesicles in Munc13-1(+/-) beta-cells, 2) Epac-selective agonist [8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate] partially restored the reduced RRP and refilling of a releasable pool of vesicles, and 3) PKA blockade by H89 (leaving Epac intact) impaired cAMP ability to restore the RRP and refilling of a releasable pool of vesicles. Conversely, PKA-selective agonist (N(6)-benzoyladenosine-cAMP) completely restored RRP and partially restored refilling of a releasable pool of vesicles. To determine specific contributions within Epac-Rim2-Munc13-1 interaction sites accounting for cAMP rescue of exocytosis caused by Munc13-1 deficiency, we found that blockade of Rim2-Munc13-1 interaction with Rim-Munc13-1-binding domain peptide abolished cAMP rescue, whereas blockade of Epac-Rim2 interaction with Rim2-PDZ peptide only moderately reduced refilling with little effect on RRP. CONCLUSIONS: cAMP rescue of priming defects caused by Munc13-1 deficiency via Epac and PKA signaling pathways requires downstream Munc13-1-Rim2 interaction.

Distinct in vivo roles of caspase-8 in beta-cells in physiological and diabetes models.

Inadequate pancreatic beta-cell mass resulting from excessive beta-cell apoptosis is a key defect in type 1 and type 2 diabetes. Caspases are the major molecules involved in apoptosis; however, in vivo roles of specific caspases in diabetes are unclear. The purpose of this study is to examine the role of Caspase (Casp)8 in beta-cells in vivo. Using the Cre-loxP system, mice lacking Casp8 in beta-cells (RIPcre(+)Casp8(fl/fl) mice) were generated to address the role of Casp8 in beta-cells in physiological and diabetes models. We show that islets isolated from RIPcre(+)Casp8(fl/fl) mice were protected from Fas ligand (FasL)-and ceramide-induced cell death. Furthermore, RIPcre(+)Casp8(fl/fl) mice were protected from in vivo models of type 1 and type 2 diabetes. In addition to being the central mediator of apoptosis in diabetes models, we show that Casp8 is critical for maintenance of beta-cell mass under physiological conditions. With aging, RIPcre(+)Casp8(fl/fl) mice gradually develop hyperglycemia and a concomitant decline in beta-cell mass. Their islets display decreased expression of molecules involved in insulin/IGF-I signaling and show decreased pancreatic duodenal homeobox-1 and cAMP response element binding protein expression. At the level of individual islets, we observed increased insulin secretory capacity associated with increased expression of exocytotic proteins. Our results show distinct context-specific roles of Casp8 in physiological and disease states; Casp8 is essential for beta-cell apoptosis in type 1 and type 2 diabetes models and in regulating beta-cell mass and insulin secretion under physiological conditions.

Effects of palmitate on insulin secretion and exocytotic proteins in islets of diabetic Goto-Kakizaki rats.

OBJECTIVES: We examined how lipotoxicity contributes to pancreatic beta-cell secretory dysfunction. METHODS: Effects of palmitate (0.2 mmol/L) were assessed on insulin secretion and soluble N-ethylmaleimide-sensitive factor attachment protein receptor exocytotic machinery in isolated pancreatic islets of type 2 diabetic Goto-Kakizaki (GK) rats and control Wistar (W) rats. RESULTS: One-day palmitate treatment enhanced basal glucose (3.3 mmol/L)-mediated insulin release 5-fold in W and 3.3-fold in GK islets, but had no effect at high glucose (16.7 mmol/L) on W islets while enhancing GK islet insulin release 2-fold. After 3-day palmitate treatment, high-glucose-induced insulin release in W islets was reduced (by 69%), whereas in GK islets, it increased 2-fold. Insulin response to arginine was reduced in both islet types, but more so in GK islets. Exocytotic proteins (syntaxin 1A, VAMP-2, SNAP-25, nSec1) were reduced in GK islets by 56% to 69% compared with W islets. In W islets, palmitate treatment caused no changes in the levels of these proteins but increased actin levels. In GK islets, whereas 1-day palmitate treatment had no effect, 3-day treatment further reduced SNAP-25 and nSec1 levels. CONCLUSIONS: Lipotoxic-induced secretory insufficiency in normal islets may be attributed to lack of compensatory increase in levels of exocytotic proteins and/or excess actin. However, in GK islets, palmitate treatment moderately enhanced insulin secretion, likely by acting on proximal metabolic pathways capable of compensating for the defective soluble N-ethylmaleimide-sensitive factor attachment protein receptor exocytotic machinery. These results were different from prolonged glucose treatment we previously reported, indicating differences between glucotoxic and lipotoxic actions on the insulin secretory machinery.

Target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (t-SNAREs) differently regulate activation and inactivation gating of Kv2.2 and Kv2.1: Implications on pancreatic islet cell Kv channels.

We have hypothesized that the plasma membrane protein components of the exocytotic soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) complex, syntaxin 1A and SNAP-25, distinctly regulate different voltage-gated K+ (Kv) channels that are differentially distributed. Neuroendocrine islet cells (alpha, beta, delta) uniformly contain both syntaxin 1A and SNAP-25. However, using immunohistochemistry, we show that the different pancreatic islet cells contain distinct dominant Kv channels, including Kv2.1 in beta cells and Kv2.2 in alpha and delta cells, whose interactions with the SNARE proteins would, respectively regulate insulin, glucagon and somatostatin secretion. We therefore examined the regulation by syntaxin 1A and SNAP-25 of these two channels. We have shown that Kv2.1 interacts with syntaxin 1A and SNAP-25 and, based on studies in oocytes, suggested a model of two distinct modes of interaction of syntaxin 1A and the complex syntaxin 1A/SNAP-25 with the C terminus of the channel. Here, we characterized the interactions of syntaxin 1A and SNAP-25 with Kv2.2 which is highly homologous to Kv2.1, except for the C-terminus. Comparative two-electrode voltage clamp analysis in oocytes between Kv2.2 and Kv2.1 shows that Kv2.2 interacts only with syntaxin 1A and, in contrast to Kv2.1, it does not interact with the syntaxin 1A/SNAP-25 complex and hence is not sensitive to the assembly/disassembly state of the complex. The distinct regulation of these closely related channels by SNAREs may be attributed to differences in their C termini. Together with the differential distribution of these channels among islet cells, their distinct regulation suggests that the documented profound down-regulation of islet SNARE levels in diabetes could distort islet cell ion channels and secretory responses in different ways, ultimately contributing to the abnormal glucose homeostasis.

Two populations of pancreatic islet alpha-cells displaying distinct Ca2+ channel properties.

In low or absence of glucose, alpha-cells generate rhythmic action potentials and secrete glucagon. alpha-Cell T-type Ca(2+) channels are believed to be pacemaker channels, which are expected to open near the resting membrane potential (around -60 mV) to initiate a small depolarization. A previous publication, however, showed that alpha-cell T-type Ca(2+) channels have an activation threshold of -40 mV, which does not appear to fulfill their role as pacemakers. In this work, we investigated the Ca(2+) channel characteristics in alpha-cells of mouse-insulin-promoter green-fluorescent-protein (MIP-GFP) mouse. The beta-cells of MIP-GFP were conveniently distinguished as green cells, while immunostaining indicated that the majority of non-green cells were alpha-cells. We found that majority of alpha-cells possessed T-type Ca(2+) channels having an activation threshold of -40 mV; these cells also had high-voltage-activated (HVA) Ca(2+) channels (activation threshold of -20 mV). A novel finding here is that a minority of alpha-cells had T-type Ca(2+) channels with an activation threshold of -60 mV. This minor population of alpha-cells was, surprisingly, devoid of HVA Ca(2+) channels. We suggest that this alpha-cell subpopulation may act as pacemaker cells in low or absence of glucose.

Munc13-1 deficiency reduces insulin secretion and causes abnormal glucose tolerance.

Munc13-1 is a diacylglycerol (DAG) receptor that is essential for synaptic vesicle priming. We recently showed that Munc13-1 is expressed in rodent and human islet beta-cells and that its levels are reduced in islets of type 2 diabetic humans and rat models, suggesting that Munc13-1 deficiency contributes to the abnormal insulin secretion in diabetes. To unequivocally demonstrate the role of Munc13-1 in insulin secretion, we studied heterozygous Munc13-1 knockout mice (+/-), which exhibited elevated glucose levels during intraperitoneal glucose tolerance tests with corresponding lower serum insulin levels. Munc13-1(+/-) mice exhibited normal insulin tolerance, indicating that a primary islet beta-cell secretory defect is the major cause of their hyperglycemia. Consistently, glucose-stimulated insulin secretion was reduced 50% in isolated Munc13-1(+/-) islets and was only partially rescued by phorbol ester potentiation. The corresponding alterations were minor in mice expressing one allele of a Munc13-1 mutant variant, which does not bind DAG (H567K/+). Capacitance measurements of Munc13-1(+/-) and Munc13-1(H567k/+) islet beta-cells revealed defects in granule priming, including the initial size and refilling of the releasable pools, which become accentuated by phorbol ester potentiation. We conclude that Munc13-1 plays an important role in glucose-stimulated insulin secretion and that Munc13-1 deficiency in the pancreatic islets as occurs in diabetes can reduce insulin secretion sufficient to cause abnormal glucose homeostasis.

Insulin regulates islet alpha-cell function by reducing KATP channel sensitivity to adenosine 5'-triphosphate inhibition.

Glucose regulates pancreatic islet alpha-cell glucagon secretion directly by its metabolism to generate ATP in alpha-cells, and indirectly via stimulation of paracrine release of beta-cell secretory products, particularly insulin. How the cellular substrates of these pathways converge in the alpha-cell is not well known. We recently reported the use of the MIP-GFP (mouse insulin promoter-green fluorescent protein) mouse to reliably identify islet alpha- (non-green cells) and beta-cells (green cells), and characterized their ATP-sensitive K(+) (K(ATP)) channel properties, showing that alpha-cell K(ATP) channels exhibited a 5-fold higher sensitivity to ATP inhibition than beta-cell K(ATP) channels. Here, we show that insulin exerted paracrine regulation of alpha-cells by markedly reducing the sensitivity of alpha-cell K(ATP) channels to ATP (IC(50) = 0.18 and 0.50 mM in absence and presence of insulin, respectively). Insulin also desensitized beta-cell K(ATP) channels to ATP inhibition (IC(50) = 0.84 and 1.23 mM in absence and presence of insulin, respectively). Insulin effects on both islet cell K(ATP) channels were blocked by wortmannin, indicating that insulin acted on the insulin receptor-phosphatidylinositol 3-kinase signaling pathway. Insulin did not affect alpha-cell A-type K(+) currents. Glutamate, known to also inhibit alpha-cell glucagon secretion, did not activate alpha-cell K(ATP) channel opening. We conclude that a major mechanism by which insulin exerts paracrine control on alpha-cells is by modulating its K(ATP) channel sensitivity to ATP block. This may be an underlying basis for the proposed sequential glucose-insulin regulation of alpha-cell glucagon secretion, which becomes distorted in diabetes, leading to dysregulated glucagon secretion.

Impaired gene and protein expression of exocytotic soluble N-ethylmaleimide attachment protein receptor complex proteins in pancreatic islets of type 2 diabetic patients.

Exocytosis of insulin is dependent on the soluble N-ethylmaleimide attachment protein receptor (SNARE) complex proteins in the B-cells. We assessed insulin release as well as gene and protein expression of SNARE complex protein in isolated pancreatic islets of type 2 diabetic patients (n = 4) and nondiabetic control subjects (n = 4). In islets from the diabetic patients, insulin responses to 8.3 and 16.7 mmol/l glucose were markedly reduced compared with control islets (4.7 +/- 0.3 and 8.4 +/- 1.8 vs. 17.5 +/- 0.1 and 24.3 +/- 1.2 microU . islet(-1) . h(-1), respectively; P < 0.001). Western blot analysis revealed decreased amounts of islet SNARE complex and SNARE-modulating proteins in diabetes: syntaxin-1A (21 +/- 5% of control levels), SNAP-25 (12 +/- 4%), VAMP-2 (7 +/- 4%), nSec1 (Munc 18; 34 +/- 13%), Munc 13-1 (27 +/- 4%), and synaptophysin (64 +/- 7%). Microarray gene chip analysis, confirmed by quantitative PCR, showed that gene expression was decreased in diabetes islets: syntaxin-1A (27 +/- 2% of control levels), SNAP-25 (31 +/- 7%), VAMP-2 (18 +/- 3%), nSec1 (27 +/- 5%), synaptotagmin V (24 +/- 2%), and synaptophysin (12 +/- 2%). In conclusion, these data support the view that decreased islet RNA and protein expression of SNARE and SNARE-modulating proteins plays a role in impaired insulin secretion in type 2 diabetic patients. It remains unclear, however, to which extent this defect is primary or secondary to, e.g., glucotoxicity.

Electrophysiological characterization of pancreatic islet cells in the mouse insulin promoter-green fluorescent protein mouse.

We recently reported a transgenic [mouse insulin promoter (MIP)-green fluorescent protein (GFP)] mouse in which GFP expression is targeted to the pancreatic islet beta-cells to enable convenient identification of beta-cells as green cells. The GFP-expressing beta-cells of the MIP-GFP mouse were functionally indistinguishable from beta-cells of normal mice. Here we characterized the ionic channel properties and exocytosis of MIP-GFP mouse islet beta- and alpha-cells. Beta-cells displayed delayed rectifying K+ and high-voltage-activated Ca2+ channels and exhibited Na+ currents only at hyperpolarized holding potential. Alpha-cells were nongreen and had both A-type and delayed rectifier K+ channels, both low-voltage-activated and high-voltage-activated Ca2+ channels, and displayed Na+ currents readily at -70 mV holding potential. Alpha-cells had ATP-sensitive K+ channel (KATP) channel density as high as that in beta-cells, and, surprisingly, alpha-cell KATP channels were more sensitive to ATP inhibition (IC50=0.16+/-0.03 mM) than beta-cell KATP channels (IC50=0.86+/-0.10 mM). Whereas alpha-cells were rather uniform in size [2-4.5 picofarad (pF)], beta-cells varied vastly in size (2-12 pF). Of note, small beta-cells (<4.5 pF) showed little exocytosis, whereas medium beta-cells (5-8 pF) exhibited vigorous exocytosis, but large beta-cells (>8 pF) had weaker exocytosis. We found no correlation between beta-cell size and their Ca2+ channel density, suggesting that Ca2+ influx may not be the cause of the heterogeneity in exocytotic responses. The MIP-GFP mouse therefore offers potential to further explore the functional heterogeneity in beta-cells of different sizes. The MIP-GFP mouse islet is therefore a reliable model to efficiently examine alpha-cell and beta-cell physiology and should greatly facilitate examination of their pathophysiology when the MIP-GFP mice are crossed with diabetic models.

Transgenic mouse overexpressing syntaxin-1A as a diabetes model.

Soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) protein syntaxin-1A (STX-1A) plays a role not only in exocytosis, but also binds and regulates Ca(2+) and K(+) (voltage-gated K(+) and ATP-sensitive K(+) channels) to influence the sequence of events leading to secretion. Islet levels of STX-1A and cognate SNARE proteins are reduced in type 2 diabetic rodents, suggesting their role in dysregulated insulin secretion contributing to the abnormal glucose homeostasis. We investigated the specific role of STX-1A in pancreatic beta-cells by generating transgenic mice, which express a moderately increased level ( approximately 30% higher) of STX-1A in pancreatic islets (hereafter called STX-1A mice). The STX-1A mice displayed fasting hyperglycemia and a more sustained elevation of plasma glucose levels after an intraperitoneal glucose tolerance test, with correspondingly reduced plasma insulin levels. Surprisingly, beta-cells from the STX-1A male mice also exhibited abnormal insulin tolerance. To unequivocally determine the beta-cell secretory defects, we used single-cell analyses of exocytosis by patch clamp membrane capacitance measurements and ion channel recordings. Depolarization-evoked membrane capacitance increases were reduced in the STX-1A mouse islet beta-cells. The STX-1A mouse also exhibited reduced currents through the Ca(2+) channels but little change in the voltage-gated K(+) channel or ATP-sensitive K(+) channel. These results suggest that fluctuation of islet STX-1A levels in diabetes could influence the pathological and differential regulation of beta-cell ion channels and the exocytotic machinery, collectively contributing to the impaired insulin secretion.

Caspase-3-dependent beta-cell apoptosis in the initiation of autoimmune diabetes mellitus.

beta-Cell apoptosis is a key event contributing to the pathogenesis of type 1 diabetes mellitus. In addition to apoptosis being the main mechanism by which beta cells are destroyed, beta-cell apoptosis has been implicated in the initiation of type 1 diabetes mellitus through antigen cross-presentation mechanisms that lead to beta-cell-specific T-cell activation. Caspase-3 is the major effector caspase involved in apoptotic pathways. Despite evidence supporting the importance of beta-cell apoptosis in the pathogenesis of type 1 diabetes, the specific role of caspase-3 in this process is unknown. Here, we show that Caspase-3 knockout (Casp3(-/-) mice were protected from developing diabetes in a multiple-low-dose streptozotocin autoimmune diabetes model. Lymphocyte infiltration of the pancreatic islets was completely absent in Casp3(-/-) mice. To determine the role of caspase-3-dependent apoptosis in disease initiation, a defined antigen-T-cell receptor transgenic system, RIP-GP/P14 double-transgenic mice with Casp3 null mutation, was examined. beta-cell antigen-specific T-cell activation and proliferation were observed only in the pancreatic draining lymph node of RIP-GP/P14/Casp3(+/-) mice, but not in mice lacking caspase-3. Together, our findings demonstrate that caspase-3-mediated beta-cell apoptosis is a requisite step for T-cell priming, a key initiating event in type 1 diabetes.

Regional differences in cholinergic regulation of potassium current in feline esophageal circular smooth muscle.

Potassium channels are important contributors to membrane excitability in smooth muscles. There are regional differences in resting membrane potential and K(+)-channel density along the length of the feline circular smooth muscle esophagus. The aim of this study was to assess responses of K(+)-channel currents to cholinergic (ACh) stimulation along the length of the feline circular smooth muscle esophageal body. Perforated patch-clamp technique assessed K(+)-channel responses to ACh stimulation in isolated smooth muscle cells from the circular muscle layer of the esophageal body at 2 (distal)- and 4-cm (proximal) sites above the lower esophageal sphincter. Western immunoblots assessed ion channel and receptor expression. ACh stimulation produced a transient increase in outward current followed by inhibition of spontaneous transient outward currents. These ACh-induced currents were abolished by blockers of large-conductance Ca(2+)-dependent K(+) channels (BK(Ca)). Distal cells demonstrated a greater peak current density in outward current than cells from the proximal region and a longer-lasting outward current increase. These responses were abolished by atropine and the specific M(3) receptor antagonist 4-DAMP but not the M(1) receptor antagonist pirenzipine or the M(2) receptor antagonist methoctramine. BK(Ca) expression along the smooth muscle esophagus was similar, but M(3) receptor expression was greater in the distal region. Therefore, ACh can differentially activate a potassium channel (BK(Ca)) current along the smooth muscle esophagus. This activation probably occurs through release of intracellular calcium via an M(3) pathway and has the potential to modulate the timing and amplitude of peristaltic contraction along the esophagus.

Open form of syntaxin-1A is a more potent inhibitor than wild-type syntaxin-1A of Kv2.1 channels.

We have shown that SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) proteins not only participate directly in exocytosis, but also regulate the dominant membrane-repolarizing Kv channels (voltage-gated K+ channels), such as Kv2.1, in pancreatic beta-cells. In a recent report, we demonstrated that WT (wild-type) Syn-1A (syntaxin-1A) inhibits Kv2.1 channel trafficking and gating through binding to the cytoplasmic C-terminus of Kv2.1. During beta-cell exocytosis, Syn-1A converts from a closed form into an open form which reveals its active H3 domain to bind its SNARE partners SNAP-25 (synaptosome-associated protein of 25 kDa) and synaptobrevin. In the present study, we compared the effects of the WT Syn-1A and a mutant open form Syn-1A (L165A, E166A) on Kv2.1 channel trafficking and gating. When co-expressed in HEK-293 cells (human embryonic kidney-293 cells), the open form Syn-1A decreased Kv2.1 current density more than (P<0.05) the WT Syn-1A (166+/-35 and 371+/-93 pA/pF respectively; control=911+/-91 pA/pF). Confocal microscopy and biotinylation experiments showed that both the WT and open form Syn-1A inhibited Kv2.1 expression at the plasma membrane to a similar extent, suggesting that the stronger reduction of Kv2.1 current density by the open form compared with the WT Syn-1A is probably due to a stronger direct inhibition of channel activity. Consistently, dialysis of the recombinant open form Syn-1A protein into Kv2.1-expressing HEK-293 cells caused stronger inhibition of Kv2.1 current amplitude (P<0.05) than the WT Syn-1A protein (73+/-2 and 82+/-3% of the control respectively). We found that the H3 but not H(ABC) domain is the putative active domain of Syn-1A, which bound to and inhibited the Kv2.1 channel. When co-expressed in HEK-293 cells, the open-form Syn-1A slowed down Kv2.1 channel activation (tau=12.3+/-0.8 ms) much more than (P<0.05) WT Syn-1A (tau=7.9+/-0.8 ms; control tau=5.5+/-0.6 ms). In addition, only the open form Syn-1A, but not the WT Syn-1A, caused a significant (P<0.05) left-shift in the steady-state inactivation curve (V(1/2)=33.1+/-1.3 and -29.4+/-1.1 mV respectively; control V(1/2)=-24.8+/-2 mV). The present study therefore indicates that the open form of Syn-1A is more potent than the WT Syn-1A in inhibiting the Kv2.1 channel. Such stronger inhibition by the open form of Syn-1A may limit K+ efflux and thus decelerate membrane repolarization during exocytosis, leading to optimization of insulin release.

H3 domain of syntaxin 1A inhibits KATP channels by its actions on the sulfonylurea receptor 1 nucleotide-binding folds-1 and -2.

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 mutant (L165A/E166A) has a similar affinity to NBF-1 and NBF-2 as wild type Syn-1A and was equally effective in inhibiting the K(ATP) channels of rat pancreatic beta cells and a cell line (BA8) stably expressing SUR1/Kir6.2. Although dialysis of NBF-1 into BA8 and islet beta cells effectively blocked wild type and open form Syn-1A inhibition of the K(ATP) current, NBF-2 was also effective in blocking the open form Syn-1A inhibition. This prompted us to examine the specific domains within Syn-1A that would mediate its action on the K(ATP) channels. The C-terminal H3 domain of Syn-1A (Syn-1A-H3), but not the N-terminal H(ABC) domain (Syn-1A-H(ABC)), binds the SUR1 protein of BA8 cells, causing an inhibition of K(ATP) currents, and this inhibition was mediated via both NBF-1 and NBF-2. It therefore appears that the H3 domain of Syn-1A is the putative domain, which binds SUR1, but its distinct actions on the NBFs may depend on the conformation of Syn-1A occurring during exocytosis.

Syntaxin-1A inhibits cardiac KATP channels by its actions on nucleotide binding folds 1 and 2 of sulfonylurea receptor 2A.

ATP-sensitive potassium (KATP) channels couple the metabolic status of the cell to its membrane potential to regulate a number of cell actions, including secretion (neurons and neuroendocrine cells) and muscle contractility (skeletal, cardiac, and vascular smooth muscle). KATP channels consist of regulatory sulfonylurea receptors (SUR) and pore-forming (Kir6.X) subunits. We recently reported (Pasyk, E. A., Kang, Y., Huang, X., Cui, N., Sheu, L., and Gaisano, H. Y. (2004) J. Biol. Chem. 279, 4234-4240) that syntaxin-1A (Syn-1A), known to mediate exocytotic fusion, was capable of binding the nucleotide binding folds (NBF1 and C-terminal NBF2) of SUR1 to inhibit the KATP channels in insulin-secreting pancreatic islet beta cells. This prompted us to examine whether Syn-1A might modulate cardiac SUR2A/KATP channels. Here, we show that Syn-1A is present in the plasma membrane of rat cardiac myocytes and binds the SUR2A protein (of rat brain, heart, and human embryonic kidney 293 cells expressing SUR2A/Kir6. 2) at its NBF1 and NBF2 domains to decrease KATP channel activation. Unlike islet beta cells, in which Syn-1A inhibition of the channel activity was apparently mediated only via NBF1 and not NBF2 of SUR1, both exogenous recombinant NBF1 and NBF2 of SUR2A were found to abolish the inhibitory actions of Syn-1A on K(ATP) channels in rat cardiac myocytes and HEK293 cells expressing SUR2A/Kir6.2. Together with our recent report, this study suggests that Syn-1A binds both NBFs of SUR1 and SUR2A but appears to exhibit distinct interactions with NBF2 of these SUR proteins in modulating the KATP channels in islet beta cells and cardiac myocytes.

Disruption of pancreatic beta-cell lipid rafts modifies Kv2.1 channel gating and insulin exocytosis.

In pancreatic beta-cells, the predominant voltage-gated Ca(2+) channel (Ca(V)1.2) and K(+) channel (K(V)2.1) are directly coupled to SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor) proteins. These SNARE proteins modulate channel expression and gating and closely associate these channels with the insulin secretory vesicles. We show that K(V)2.1 and Ca(V)1.2, but not K(V)1.4, SUR1, or Kir6.2, target to specialized cholesterol-rich lipid raft domains on beta-cell plasma membranes. Similarly, the SNARE proteins syntaxin 1A, SNAP-25, and VAMP-2, but not Munc-13-1 or n-Sec1, are associated with lipid rafts. Disruption of the lipid rafts by depleting membrane cholesterol with methyl-beta-cyclodextrin shunts K(V)2.1, Ca(V)1.2, and SNARE proteins out of lipid rafts. Furthermore, methyl-beta-cyclodextrin inhibits K(V)2.1 but not Ca(V)1.2 channel activity and enhances single-cell exocytic events and insulin secretion. Membrane compartmentalization of ion channels and SNARE proteins in lipid rafts may be critical for the temporal and spatial coordination of insulin release, forming what has been described as the excitosome complex.

Alcoholic chronic pancreatitis involves displacement of Munc18c from the pancreatic acinar basal membrane surface.

The minimal machinery for fusion of secretory vesicles with the cell membrane is a cognate set of v- and t-SNAREs on opposing membranes. Spontaneous SNARE complex assembly leading to unregulated membrane fusion is prevented by Munc18 proteins that bind membrane SNAREs syntaxins. Munc18 blocks syntaxin interactions with cognate SNARE proteins and thereby act as an inhibitor of exocytosis. The pancreatic acinar cell contains several sets of cognate SNAREs and Munc18 proteins that mediate the distinct exocytic events. We had reported that in the rat pancreas, Munc18c co-localizes with t-SNAREs syntaxin4 and SNAP23 on the acinar cell basolateral plasma membrane. Under conditions that induce pancreatitis in vivo, displacement of Munc18c from the basolateral plasma membrane relieved its blockade of SNARE-mediated membrane fusion in this region and thereby redirected apical exocytosis to the basal membrane surface. Here we show in a case of human mild alcoholic chronic pancreatitis that Munc18c is also displaced from the plasma membrane of intact acinar cells, which would render these cells receptive to pathologic basolateral exocytosis and further episodes of pancreatitis.

Syntaxin-1A binds the nucleotide-binding folds of sulphonylurea receptor 1 to regulate the KATP channel.

ATP-sensitive potassium (KATP) channels in neuron and neuroendocrine cells consist of a pore-forming Kir6.2 and regulatory sulfonylurea receptor (SUR1) subunits, which are regulated by ATP and ADP. SNARE protein syntaxin 1A (Syn-1A) is known to mediate exocytic fusion, and more recently, to also bind and modulate membrane-repolarizing voltage-gated K+ channels. Here we show that Syn-1A acts as an endogenous regulator of KATP channels capable of closing these channels when cytosolic ATP concentrations were lowered. Botulinum neurotoxin C1 cleavage of endogenous Syn-1A in insulinoma HIT-T15 cells resulted in the increase in KATP currents, which could be subsequently inhibited by recombinant Syn-1A. Whereas Syn-1A binds both nucleotide-binding folds (NBF-1 and NBF-2) of SUR1, the functional inhibition of KATP channels in rat islet beta-cells by Syn-1A seems to be mediated primarily by its interactions with NBF-1. These inhibitory actions of Syn-1A can be reversed by physiologic concentrations of ADP and by diazoxide. Syn-1A therefore acts to fine-tune the regulation of KATP channels during dynamic changes in cytosolic ATP and ADP concentrations. These actions of Syn-1A on KATP channels contribute to the role of Syn-1A in coordinating the sequence of ionic and exocytic events leading to secretion.