ATP Modulates Interaction of Syntaxin-1A with Sulfonylurea Receptor 1 to Regulate Pancreatic β-Cell KATP Channels

Abstract

ATP-sensitive potassium (K ATP ) channels are regulated by a variety of cytosolic factors (adenine nucleotides, Mg2؉ , phospholipids, and pH). We previously reported that K ATP channels are also regulated by endogenous membrane-bound SNARE protein syntaxin-1A (Syn-1A), which binds both nucleotide-binding folds of sulfonylurea receptor (SUR)1 and 2A, causing inhibition of K ATP channel activity in pancreatic islet ␤-cells and cardiac myocytes, respectively. In this study, we show that ATP dose-dependently inhibits Syn-1A binding to SUR1 at physiological concentrations, with the addition of Mg 2؉ causing a decrease in the ATP-induced inhibitory effect. This ATP disruption of Syn-1A binding to SUR1 was confirmed by FRET analysis in living HEK293 cells. Electrophysiological studies in pancreatic ␤-cells demonstrated that reduced ATP concentrations increased K ATP channel sensitivity to Syn-1A inhibition. Depletion of endogenous Syn-1A in insulinoma cells by botulinum neurotoxin C1 proteolysis followed by rescue with exogenous Syn-1A showed that Syn-1A modulates K ATP channel sensitivity to ATP. Thus, our data indicate that although both ATP and Syn-1A independently inhibit ␤-cell K ATP channel gating, they could also influence the sensitivity of K ATP channels to each other. These findings provide new insight into an alternate mechanism by which ATP regulates pancreatic ␤-cell K ATP channel activity, not only by its direct actions on Kir6.2 pore subunit, but also via ATP modulation of Syn-1A binding to SUR1.The ATP-sensitive potassium (K ATP ) 3 channel couples intracellular metabolic changes to plasma membrane electrical activity in many cell types (1-3). In pancreatic ␤-cells, increase in ATP/ADP ratio from glucose metabolism closes plasma membrane K ATP channels, causing membrane depolarization that leads to opening of L-type voltage-dependent calcium channels. Ensuing calcium influx triggers exocytosis of docked and primed insulin granules in part by acting on the exocytic SNARE (soluble NSF attachment protein receptor) complex (4). The pancreatic ␤-cell K ATP channel is a heterooctamer of pore-forming Kir 6.2 and regulatory sulfonylurea receptor 1 (SUR1) subunits, with a 4:4 stoichiometry (5-7). SURs are members of the ATP-binding cassette protein superfamily (8), including cystic fibrosis transmembrane conductance regulator and P-glycoprotein/MDR1 (multidrug resistance 1). These ATP-binding cassette proteins uniformly contain two nucleotide-binding folds (NBF1 and NBF2), with each NBF containing Walker A and B motifs, which are sites of the action by adenine nucleotides. Regulation of K ATP channels by adenine nucleotides is complex, with ATP and ADP having both stimulatory and inhibitory effects (9 -11). Structure-function studies have demonstrated that ATP inhibits channel activity by interactions with Kir6.2 (12), whereas ADP acts on the SUR subunit to stimulate channel activity in an Mg 2ϩ -dependent manner (13). The importance of SUR1 as a regulator of K ATP channel activity is demonstrated by the fact th...

The skeleton in the closet: actin cytoskeletal remodeling in β-cell function

“…MLCK (myosin light chain kinase) phosphorylates myosin IIA (event 8), which induces its relocalization and association with the actin ring surrounding granules [important for maintenance of the fusion pore in an open conformation and for providing energy for discharge of granule contents (event 9) as well as cortical actin remodeling (event 10)] to allow insulin granules to approach the basal membrane. reversibly with K ATP channels (61), and both syntaxin and SNAP-25 also bind to L-type voltage-gated Ca 2ϩ channels (60,145). This is reminiscent of vascular smooth muscle cells, in which integrin engagement can stimulate L-type Ca 2ϩ channels via FAK (151).…”

Stromal Interaction Molecule 1 (STIM1) Regulates ATP-sensitive Potassium (KATP) and Store-operated Ca2+ Channels in MIN6 β-Cells

“…NBF1 has previously been shown to play a role in binding EPAC2 (37) and syntaxin-1A (38,39). IP studies revealed that STIM1 and FLAG-NBF1 co-immunoprecipitated, suggesting that NBF1 forms at least one interaction site between STIM1 and SUR1 proteins (Fig.…”

“…An increase in [Ca 2ϩ ] ER will induce translocation of STIM1 away from the plasma membrane to inactivate SOCE. A second possibility is that the rise in intracellular ATP will cause displacement of STIM1 from binding to SUR1, analogous to the effects of ATP on syntaxin binding (39). Displacement of STIM1 from SUR1 by ATP would contribute to the inhibition of K ATP channels after a rise in blood glucose.…”

Current understanding of KATP channels in neonatal diseases: focus on insulin secretion disorders

“…First, syntaxin 1A interacts with the NBD1 of SUR subunits via its C-terminal H3 domain to decrease the activity of existing plasma membrane K ATP channels when ATP levels are lowered [58][59][60] . This effect is subject to ATP regulation, such that ATP dose-dependently inhibits syntaxin 1A binding to SUR1 subunits at physiological concentrations [61] . Second, it causes downregulation of K ATP channel expression, either by accelerating endocytosis of existing surface channels, or by decreasing the biogenesis of K ATP channels in the early secretory pathway [62] .…”

“…Since diazoxide is not very effective for CHI patients with K ATP channel mutations, alternative approaches for treating these patients may be to examine novel regulators of K ATP channels. One such molecule is syntaxin 1A, which inhibits K ATP channels [61] . Therefore, decreases in syntaxin 1A levels can increase K ATP channel activity.…”