ATP-sensitive potassium (''K ATP '') channels are rapidly inhibited by intracellular ATP. This inhibition plays a crucial role in the coupling of electrical activity to energy metabolism in a variety of cells. The K ATP channel is formed from four each of a sulfonylurea receptor (SUR) regulatory subunit and an inwardly rectifying potassium (K ir 6.2) pore-forming subunit. We used systematic chimeric and point mutagenesis, combined with patch-clamp recording, to investigate the molecular basis of ATP-dependent inhibition gating of mouse pancreatic ␤ cell K ATP channels expressed in Xenopus oocytes. We identified distinct functional domains of the presumed cytoplasmic C-terminal segment of the K ir 6.2 subunit that play an important role in this inhibition. Our results suggest that one domain is associated with inhibitory ATP binding and another with gate closure.The ATP-sensitive potassium (K ATP ) channel couples membrane electrical activity to energy metabolism in a variety of cells and is important in several physiological systems. In pancreatic ␤ cells, for example, it is essential for coupling the rate of insulin release to blood glucose levels. Although the channel's name reflects its characteristic inhibition by intracellular ATP, little is known about the molecular nature of this property.To understand the molecular mechanisms underlying ATPdependent inhibition gating in the K ATP channel, one must identify those parts of the channel complex that form the ATP-binding site, the inhibition gate, and the ''linkage'' domains handling signal flow between them. The K ATP channel is assembled from four each of two subunit types, a regulatory sulfonylurea receptor (SUR) or SUR1 and a potassium poreforming subunit or K ir 6.2 (1-4). SUR1 is a member of the ATP-binding cassette (ABC) family of proteins featuring two cytoplasmic nucleotide-binding folds, viewed initially as likely mediators of inhibition by ATP (5, 6). However, a mutant K ir 6.2 in which the C-terminal 26 residues are deleted remarkably gives rise to potassium channels that retain much ATP sensitivity in the absence of SUR1 (7). The truncation does, however, diminish ATP sensitivity of the K ATP channel 10-fold, as does SUR2 when coexpressed with wild-type K ir 6.2 (6). These results have led to opposing models in which ATP acts either on K ir 6.2 or on SUR to inhibit the K ATP channel.We tested whether major components of the ATPdependent inhibition gating mechanism reside in the poreforming subunit. By systematically mutating K ir 6.2, we localized molecular components of ATP-dependent inhibition gating to distinct regions of its cytoplasmic C-terminal segment. Our results confirm and extend the findings of Tucker et al. (7) that the primary site of action of inhibitory ATP lies on K ir 6.2. We go on to show that one of the regions we identified is likely associated with inhibitory ATP binding, whereas a second region appears to be associated with inhibition gate closure. MATERIALS AND METHODSMolecular Biology. Cloning of mouse SUR1 and K ...

Improving the long-term performance of neural electrode interfaces requires overcoming severe biological reactions such as neuronal cell death, glial cell activation, and vascular damage in the presence of implanted intracortical devices. Past studies traditionally observe neurons, microglia, astrocytes, and blood-brain barrier (BBB) disruption around inserted microelectrode arrays. However, analysis of these factors alone yields poor correlation between tissue inflammation and device performance. Additionally, these studies often overlook significant biological responses that can occur during acute implantation injury. The current study employs additional histological markers that provide novel information about neglected tissue components—oligodendrocytes and their myelin structures, oligodendrocyte precursor cells, and BBB -associated pericytes—during the foreign body response to inserted devices at 1, 3, 7, and 28 days post-insertion. Our results reveal unique temporal and spatial patterns of neuronal and oligodendrocyte cell loss, axonal and myelin reorganization, glial cell reactivity, and pericyte deficiency both acutely and chronically around implanted devices. Furthermore, probing for immunohistochemical markers that highlight mechanisms of cell death or patterns of proliferation and differentiation have provided new insight into inflammatory tissue dynamics around implanted intracortical electrode arrays.

With ATP sites on K ir 6.2 that inhibit activity and ADP sites on SUR1 that antagonize the inhibition, ATPsensitive potassium channels (K ATP channels) are designed as exquisite sensors of adenine nucleotide levels that signal changes in glucose metabolism. If pancreatic K ATP channels localize to the insulin secretory granule, they would be well positioned to transduce changes in glucose metabolism into changes in granule transport and exocytosis. Tests for pancreatic K ATP channels localized to insulin secretory granules led to the following observations: fluorescent sulfonylureas that bind the pancreatic K ATP channel specifically label intracellular punctate structures in cells of the endocrine pancreas. The fluorescent glibenclamides colocalize with Ins-C-GFP, a live-cell fluorescent reporter of insulin granules. Expression of either SUR1-GFP or K ir 6.2-GFP fusion proteins, but not expression of GFP alone, directs GFP fluorescence to insulin secretory granules. An SUR1 antibody specifically labels insulin granules identified by anti-insulin. Two different K ir 6.2 antibodies specifically label insulin secretory granules identified by antiinsulin. Immunoelectron microscopy showed K ir 6.2 antibodies specifically label perimeter membrane regions of the secretory granule. Relatively little or no labeling of other structures, including the plasma membrane, was found. Our results demonstrate that the insulin secretory granule is the major site of K ATP channels of the endocrine pancreas. Diabetes 52: 767-776, 2003 A major question in insulin secretion is the cellular site of action of sulfonylureas, which are taken daily by millions of diabetic subjects to correct hyperglycemia. One site of sulfonylurea action is at the cytoplasmic face of the plasma membrane sulfonylurea receptor (1-5) of the ATP-sensitive potassium channel (K ATP channel) (6). Hundreds of K ATP channels are localized to the plasma membrane of insulin-secreting ␤-cells (7). The pancreatic K ATP channel comprises four regulatory sulfonylurea receptor (SUR1) subunits and four potassium pore-forming (K ir 6.2) subunits (8 -10). The plasma membrane K ATP channel acts like an on/off switch. When on, potassium ions flow out through the channel electrically hyperpolarizing the ␤-cell, putting a brake on the signal flow controlling insulin secretion (11). When off, the K ATP channel, which is inhibited by sulfonylureas or by the increased ATP/ADP ratio from glucose metabolism (12,13), removes this brake, allowing initiation of insulin release by elevating intracellular calcium (14 -17), which triggers the exocytotic fusion of insulin granule and ␤-cell membrane.The calcium signal, however, is insufficient for regulating insulin release in response to glucose under typical conditions (11,18). While pharmacologically either opening or inhibiting the plasma membrane K ATP channel, maneuvers that elevate not only intracellular calcium but also glucose metabolism are necessary to confer glucose dose dependency to stimulated insulin release. By an unknown m...

α-Synuclein has been studied in numerous cell types often associated with secretory processes. In pancreatic β-cells, α-synuclein might therefore play a similar role by interacting with organelles involved in insulin secretion. We tested for α-synuclein localizing to insulin-secretory granules and characterized its role in glucose-stimulated insulin secretion. Immunohistochemistry and fluorescent sulfonylureas were used to test for α-synuclein localization to insulin granules in β-cells, immunoprecipitation with Western blot analysis for interaction between α-synuclein and K(ATP) channels, and ELISA assays for the effect of altering α-synuclein expression up or down on insulin secretion in INS1 cells or mouse islets, respectively. Differences in cellular phenotype between α-synuclein knockout and wild-type β-cells were found by using confocal microscopy to image the fluorescent insulin biosensor Ins-C-emGFP and by using transmission electron microscopy. The results show that anti-α-synuclein antibodies labeled secretory organelles within β-cells. Anti-α-synuclein antibodies colocalized with K(ATP) channel, anti-insulin, and anti-C-peptide antibodies. α-Synuclein coimmunoprecipitated in complexes with K(ATP) channels. Expression of α-synuclein downregulated insulin secretion at 2.8 mM glucose with little effect following 16.7 mM glucose stimulation. α-Synuclein knockout islets upregulated insulin secretion at 2.8 and 8.4 mM but not 16.7 mM glucose, consistent with the depleted insulin granule density at the β-cell surface membranes observed in these islets. These findings demonstrate that α-synuclein interacts with K(ATP) channels and insulin-secretory granules and functionally acts as a brake on secretion that glucose stimulation can override. α-Synuclein might play similar roles in diabetes as it does in other degenerative diseases, including Alzheimer's and Parkinson's diseases.

We combined confocal and live-cell imaging with a novel molecular strategy aimed at revealing mechanisms underlying glucose-regulated insulin vesicle secretion. The 'Ins-C-GFP' reporter monitors secretory peptide targeting, trafficking, and exocytosis without directly tagging the mature secreted peptide. We trapped a green fluorescent protein (GFP) reporter in equimolar quantity within the secretory vesicle by fusing it within the C peptide of proinsulin which only after nascent vesicle sealing and acidification is cleaved from the mature secreted A and B chains of insulin. Ins-C-GFP expression in mouse islets without fail exhibited punctate distribution of green fluorescence by confocal microscopy. Ins-C-GFP colocalized GFP with insulin at vesicle dense cores by immuno-electron microscopy. Glucose stimulation decreased vesicle fluorescence coordinately with enhanced secretion from islets of C-GFP detected by anti-GFP Western blots, and of insulin detected by antiinsulin radioimmunoassay. An insulin secretagogue with a red fluorescent label, glibenclamide BODIPY A TR, was applied to islets expressing Ins-C-GFP. The stimulus response was imaged as a rise in red secretagogue leading to marked loss in green granules. Since neuropeptides as well as peptide hormones are processed from propeptides after sealing of secretory granules, vesicle trapping likely is widely applicable for studies on targeting, trafficking, and regulated release of secretory peptides. Glucose-stimulated insulin secretion is regulated over a wide range of time scales. It exhibits day to hour regulation of nuclear transcription (5), hour to minute regulation of cytoplasmic translation (6-8), hour to minute regulation of vesicular trafficking and recycling (9-13), and minute to millisecond regulation of exocytosis at the plasma membrane (14-23). Live-cell monitoring of the spatial and temporal features of the underlying molecules and mechanisms would be facilitated by a method that avoids modification of the mature bioactive secretory peptide for not only glucose-regulated insulin secretion but also regulated peptide secretion in general.Regulated secretory peptide granule trafficking and exocytosis have been typically measured by using insulin antibodies labeled by radioactivity or enzymes (24), by using capacitance changes (16), or by using amperometry (25). By far the most popular measure of insulin vesicle trafficking and secretion in the basic research lab or clinic is by immunoassays. The assays depend on the use of a relatively largesized biopsy or perfusate tissue and costly I 125 radioisotopes or enzymes conjugated to a secondary antibody. Though in widespread use, these methods reveal little about the underlying molecular and cellular mechanisms at work within the cell, in a live-cell, real-time format with dynamic spatial (subcellular, one-cell, multi-cell, islet) and temporal (millisecond, second, min, to hour) monitoring.The fluorescent labeling approach developed here provides the following advantages for studying complex molecular...