In B cells from dispersed rat islet of Langerhans we have identified an inward rectifying voltageindependent K+ channel whose behavior parallels the metabolically regulated potassium permeability (PK) found in tracer flux and microelectrode recording studies. In cell-attached patches of membrane, the channel is closed when any one of several substrates (glucose, mannose, leucine, or glyceraldehyde) is added to the cell's bathing solution but is reopened on addition of an appropriate metabolic inhibitor, which prevents utilization of that substrate. In inside-out excised patches, a K+ channel with nearly identical kinetic features is closed by addition of micromolar concentrations of ATP to the "cytoplasmic" solution. The ATP sensitivity of channel activity is modified by addition of ADP, suggesting competition at a nucleotide binding site. These results suggest the presence of a metabolically regulated K+ channel gated by intracellular concentrations of ATP or the ratio of ATP/ADP concentrations.A popular hypothesis for stimulus-secretion coupling in pancreatic islet B cells is: nutrient metabolism by B cells decreased K+ permeability (PK) -* membrane depolarization --voltage-dependent Ca2 entry --insulin granule exocytosis (1, 2). Using single-channel recording techniques, several groups have identified, in cell-attached patches of pancreatic islet cells, a potassium-selective channel whose activity is reduced by raising the glucose concentration of the medium bathing the rest of the cell (3-6). Others have reported the existence in inside-out excised patches of a potassium channel of similar conductance whose opening frequency is reduced by micromolar concentrations of ATP (4-8). Here we demonstrate that both channels represent the same "metabolite-regulated" K+ channel in two different recording configurations and describe some regulatory effects of cytoplasmic nucleotides on channel gating. METHODS AND MATERIALSRat pancreatic islets, isolated by collagenase digestion of chopped pancreases of adult male Sprague-Dawley rats were dispersed into small clumps of cells by incubation with the enzyme "dispase" (9). Cells prepared in this manner increase their insulin secretion nearly 3-fold above baseline in the presence of 10 mM glucose media (10). Clumps of cells were added to 35-mm tissue culture dishes "seeded" with appropriately sized glass coverslips and were cultured for 1-10 days at 37°C in modified CMRL medium (GIBCO) containing 10% heat-inactivated fetal bovine serum, 0.5% penicillin, and 0.5% streptomycin in 5% C02/95% air. Individual coverslip chips were then transferred to the recording chamber containing modified physiological saline solutions at 20-23°C. The basic zero-glucose extracellular saline solution (zeroglucose ES solution) consisted of 138 mM NaCl, 5.5 mM KCl, 0.5 mM CaCl2, 2 mM MgCl2, and 20 mM Hepes titrated with NaOH to pH 7.35. The solution used to fill patch pipettes as well as bathe inside-out excised patches (IS solution) consisted of 138 mM KCl, 2 mM Mg2+, 5-20 AM CaCl2, and 2...

Intracellular ATP (ATPi)-sensitive K+ [K+(ATP)] channels are now a recognized site of action of clinically useful hypoglycemic and hyperglycemic sulfonamides. We have further examined the action of these agents on single K+ channels in rat pancreatic B-cells 1) Tolbutamide and glyburide, two hypoglycemic sulfonylureas which decrease K+(ATP) channel activity in the cell-attached patch, affect the kinetics of K+(ATP) channel in a manner similar to glucose. They shorten the duration of the "burst," or cluster of open channel events, while lengthening the intervals between bursts. 2) The hyperglycemic vasodilator diazoxide increases mean K+(ATP) channel activity in the cell-attached patch as well as in the inside-out excised patch exposed to ATPi. It appears to lengthen channel bursts and shorten the intervals between them. Two structurally similar diuretics, hydrochlorothiazide and furosemide, which have mild hyperglycemic effects, do not increase K+(ATP) channel activity even at clinically toxic concentrations. 3) Neither the sulfonylureas nor diazoxide directly affect the activity of single delayed rectifier K+ channels or single calcium and voltage-activated K+ channels in normal B-cells.

When exposed to a hypotonic bathing solution, clonal N1E115 neuroblastoma cells initially swell and then undergo a regulatory volume decrease (RVD). Using cellattached patch-clamp recording, we have found that the activity of a stretch-sensitive, nonselective cation [C+(SA)J channel increases shortly after the onset of osmotically induced cell swelling; this depolarizes the cells as much as 30 mV. Shortly thereafter, and roughly coincident with the onset of RVD, two types of voltage-dependent channels open at the new resting potential; these are (i) a delayed-rectifier type K+ [K+(DR)J channel and (ii) a large-conductance anion channel. We suggest that opening of the C+(SA) channel may contribute to the volume "sensor" mechanism, while the depolarizationinduced opening of the K+(DR) and anion channels may constitute a significant K+ salt exit pathway, operating in RVD.Volume regulation is a feature common to many vertebrate cells (1). When placed in a hypotonic bathing solution, these cells initially swell but then, over several minutes, shrink back to near their resting volumes. The regulatory volume decrease (RVD) is usually accompanied by the loss of intracellular K+ and anions, including Cl-. Recently, attention has turned to stretch-activated ion channels, which appear to be ubiquitous (2-8), as possible candidates for "sensor" mechanisms and K+ and anion exit pathways for cell-volume regulation (6-8). Here, we report that neuroblastoma cells of the clone N1E115, which display RVD, express numerous stretch-activated nonselective cation [C+(SA)] channels. The activity of the C+(SA) channel increases shortly after the onset of osmotically induced cell swelling and then decreases during RVD. Cell depolarization caused by the opening of C+(SA) channels opens voltage-dependent, delayed-rectifier type K+ [K+(DR)] channels and seems to be responsible for the activation of multiple-conductance-state anion channels. These three types of channels may serve as a volume "sensor" and volume "effector" pathways for RVD. Some of this work has been presented in abstract form (9). METHODS AND MATERIALSExperiments were performed on 20-to 50-,um-diameter NiE115 cells grown for 3-15 days on glass coverslips at 37°C in Dulbecco's modified Eagle's medium supplemented with 10% (vol/vol) fetal calf serum, 1-2% dimethyl sulfoxide, 0.5% penicillin, and 0.5% streptomycin in the presence of 5% C02/95% air. Though the cell population was quite pleomorphic, with some cells resembling hemiellipsoids and others having complex neuritic processes, all cells examined displayed nearly identical ionic channel currents as well as action potential activity in response to depolarization or anodal break. The standard extracellular-like bathing solution, ES, whose osmolarity was 285 mosM and whose pH was 7.3, contained 130 mM NaCI, 5.5 mM KCI, 2.5 mM CaCI2, 1.25 mM MgCl2, 20 mM NaOH-adjusted Hepes buffer, and 10 mM glucose. Bath osmolarity was reduced to 225 milliosmolar (mosM) or 185 mosM by diluting ES with distilled water, hence reducing...

We describe the application of 'perforated patch recording' using the pore-forming antibiotic nystatin, to monitor the electrical activity and underlying ionic currents of rat and human pancreatic islet B cells. We demonstrate that glucoseinduced electrical activity is seen even in single B cells during current-clamp recordings lasting hours 'L-type' Ca2+-channel currents can also be monitored over this period of time. This technique may prove useful in examining hormone and neurotransmitter modulation of electrical activity in B cells, while minimizing the effects of cytoplasmic 'wash-out'.

Stretch-activated ion channels have been described in animal cells, where they might serve as mechanoreceptors, baroreceptors or osmoreceptors, as well as in yeast and bacteria, where osmoregulatory functions have been suggested. Here we report a large conductance, stretch-activated, anion-selective channel in protoplasts of a higher plant, tobacco, and discuss its possible role in osmoregulation.