In B cells from dispersed rat islet of Langerhans we have identified an inward rectifying voltage-independent K+ channel whose behavior parallels the metabolically regulated potassium permeability (PK) found in tracer flux and microelectrode recording studies. cell-attached patches membrane, is closed when any one several substrates (glucose, mannose, leucine, or glyceraldehyde) added to cell's bathing solution but reopened on addition appropriate metabolic inhibitor, which prevents...

A fluorescent sensor for catecholamines, NS510, is presented. The based on a quinolone fluorophore incorporating boronic acid recognition element that gives it high affinity catecholamines and turn-on response to norepinephrine. results in punctate staining of norepinephrine-enriched chromaffin cells visualized using confocal microscopy indicating stains the norepinephrine secretory vesicles. Amperometry conjunction with total internal reflection fluorescence (TIRF) demonstrates can be used...

Intracellular ATP (ATPi)-sensitive K+ [K+(ATP)] channels are now a recognized site of action clinically useful hypoglycemic and hyperglycemic sulfonamides. We have further examined the these agents on single in rat pancreatic B-cells 1) Tolbutamide glyburide, two sulfonylureas which decrease K+(ATP) channel activity cell-attached patch, affect kinetics manner similar to glucose. They shorten duration “burst,” or cluster open events, while lengthening intervals between bursts. 2) The...

High-throughput electrode arrays are required for advancing devices testing the effect of drugs on cellular function. In this paper, we present design criteria a potentiostat circuit that is capable measuring transient amperometric oxidation currents at surface an with submillisecond time resolution and picoampere current resolution. The regulated cascode stage in which high-gain amplifier maintains voltage through negative feedback loop. uses new shared structure all amplifiers given row...

Fusion of a single vesicle induces quantal response, which is critical in determining synaptic strength. Quantal size varies at most synapses. Its underlying mechanisms are not well understood. Here, we examined five sources variation: vesicular glutamate concentration ([Glu] v ), volume, ultrafast fusion pore closure, the postsynaptic receptor, and location between release receptor cluster glutamatergic, calyx Held By averaging 2.66 million events from 459 synapses, resolved capacitance...

A method for the selective labeling and imaging of catecholamines in live fixed secretory cells is reported. The integrates a tailored approach using novel fluorescence-based turn-on molecular sensor (NeuroSensor 521) that can exploit high concentration neurotransmitters acidic environment within vesicles recognition norepinephrine dopamine. utility was demonstrated by selectively such discrimination between norepinephrine- epinephrine-enriched populations chromaffin observed. This validated...

We have used membrane capacitance measurements and carbon-fiber amperometry to assay exocytosis triggered by photorelease of caged Ca2+ directly measure the sensitivity from INS-1 insulin-secreting cell line. find heterogeneity release in that a small proportion granules makes up highly Ca2+-sensitive pool (HCSP), whereas bulk lower Ca2+. A substantial HCSP remains after brief depolarization, suggesting majority with high are not located close channels. The is enhanced size glucose, cAMP,...

Gating of the cystic fibrosis transmembrane conductance regulator (CFTR) involves a coordinated action ATP on two nucleotide binding domains (NBD1 and NBD2). Previous studies using nonhydrolyzable analogues NBD mutant CFTR have suggested that hydrolysis at NBD1 is required for opening channel, while nucleotides NBD2 controls channel closing. We studied ATP-dependent gating in excised inside-out patches from stably transfected NIH3T3 cells. Single kinetics different [ATP] were analyzed. The...

Carbon-fiber amperometry has been extensively used to monitor the time course of catecholamine release from cells as individual secretory granules discharge their contents during process quantal exocytosis, but microfabricated devices offer promise higher throughput. Here we report development a microchip device that uses transparent indium tin oxide (ITO) electrodes measure exocytosis in microfluidic channels. ITO films on glass substrate were patterned 20-μm-wide stripes using...

Electrochemical microelectrodes are commonly used to detect spikes of amperometric current that correspond exocytosis oxidizable transmitter from individual vesicles, i.e., quantal exocytosis. We developing transparent multielectrochemical electrode arrays on microchips in order automate measurement Here, we report development an improved device target cells each microelectrode array. Efficient targeting (∼75%) is achieved using cell-sized microwell traps fabricated SU-8 photoresist together...

Nucleic acids can undergo conformational changes upon binding small molecules. These be exploited to develop new therapeutic strategies through control of gene expression or triggering cellular responses and also used sensors for molecules such as neurotransmitters. Many analytical approaches detect dynamic change nucleic acids, but they need labeling, are expensive, have limited time resolution. The nanopore approach provide a snapshot each acid molecule detected, has not been reported in...

Maintaining homeostasis of metabolites such as amino acids is critical for cell survival. Dysfunction nutrient balance can result in human diseases diabetes. Much remains to be discovered about how cells transport, store, and utilize due limited research tools. Here we developed a novel, pan-amino acid fluorescent turn-on sensor, NS560. It detects 18 the 20 proteogenic visualized mammalian cells. Using NS560, identified pools lysosomes, late endosomes, surrounding rough endoplasmic...

We have used flash photolysis of caged Ca 2+ and membrane capacitance measurements to probe exocytosis in chromaffin cells at low concentrations intracellular ([Ca ] i ) (<10 μM). observed a small pool granules that is more sensitive [Ca than the previously described “readily releasable pool.” Upon activation PKC, this “highly -sensitive pool” enhanced size greater extent readily but eliminated upon expression C-terminal deletion mutant (Δ9) synaptosome-associated protein 25 kDa...

We have fabricated electrochemical electrodes in picolitersized wells for measuring catecholamine release from individual cells with millisecond resolution. Each well-electrode roughly conforms to the shape of cell order capture a large fraction released high time Using this device, we can resolve spikes amperometric current corresponding quantal via exocytosis. In addition, combined recording on chip patch-clamp recordings membrane capacitance as an assay A quantitative comparison two...

We describe the application of ‘perforated patch recording’ using pore‐forming antibiotic nystatin, to monitor electrical activity and underlying ionic currents rat human pancreatic islet B cells. demonstrate that glucose‐induced is seen even in single cells during current‐clamp recordings lasting hours ‘ L ‐type’ Ca 2+ ‐channel can also be monitored over this period time. This technique may prove useful examining hormone neurotransmitter modulation cells, while minimizing effects...

The phosphorylation targets that mediate the enhancement of exocytosis by PKC are unknown. phosporylates SNARE protein SNAP-25 at Ser-187. We expressed mutants using Semliki Forest Virus system in bovine adrenal chromaffin cells and then directly measured Ca2+ dependence photorelease caged together with patch-clamp capacitance measurements. A flash UV light used to elevate [Ca2+]i several μM release highly Ca2+-sensitive pool (HCSP) vesicles was followed a train depolarizing pulses elicit...

Activation of diacylglycerol (DAG) signaling pathways with phorbol esters dramatically enhances Ca2+-triggered exocytosis from both endocrine cells and neurons, however the relevant targets DAG are controversial. A possible effector mechanism for this pathway is phosphorylation SNAP-25 (25 kDa synaptosome-associated protein) at Ser187 by PKC. Here, we investigated role in enhancement ester PMA (phorbol 12-myristate 13-acetate). We used patch-clamp measurements membrane capacitance together...

With human islets isolated for transplantation, we examined the applicability to humans of a metabolic fuel hypothesis glucose transduction and Ca2+ depolarization-secretion coupling, both previously proposed rodent islet beta-cells. We report that several features beta-cell physiology are well accounted by these hypotheses. whole-islet perifusion, demonstrated insulin secretion induced glucose, tolbutamide, or elevated K+ is dependent on extracellular Ca2+. Insulin release secretagogues...

In patch-clamped surface cells of human islets, we identified an inwardly rectifying, voltage-independent K+ channel that may be a crucial link between substrate metabolism and depolarization-induced insulin secretion. It is the major open at rest. closes on exposure cell to secretagogue concentrations glucose or other metabolic fuels oral hypoglycemic sulfonylureas but reopens addition either inhibitor prevents utilization hyperglycemic sulfonamide diazoxide. Onset electrical activity...