A metabolite-regulated potassium channel in rat pancreatic B cells.

Abstract

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...

Block of pancreatic ATP‐sensitive K+ channels and insulinotrophic action by the antiarrhythmic agent, cibenzoline

“…Singlechannel conductance was calculated by a least-square fit to the negative limb of the relation and was 62.1 + 0.3 pS. Taken together, the channels so far recorded are compatible with rat pancreatic KATP channels reported previously (Cook & Hales, 1984;Ashcroft et al, 1984;Misler et al, 1986;Tsuura et al, 1992).…”

“…Closure of them depolarizes /3 cells and promotes Ca2" influx through the voltage-dependent Ca2" channels, thereby triggering hormone secretion (Cook & Hales, 1984;Ashcroft et al, 1984;Rorsman & Trube, 1985;Misler et al, 1986; for review see Ashcroft, 1988). Sulphonylureas, potent insulin secretagogues, were found to act through a block of pancreatic and cardiac KATP channels via an intramembrane pathway (Zunkler et al, 1989;Findlay, 1992).…”

Clonidine inhibits ATP‐sensitive K+channels in mouse pancreatic β‐cells

“…In contrast, adrenaline (100pM) was without effect on 86Rb efflux under the same conditions. In 6mm glucose, most ATP-sensitive K+ channels are closed (Rorsman & Trube, 1985;Misler et al, 1986;Ashcroft et al, 1988) and 86Rb effiux is low and stable. Diazoxide (100pM), an opener of ATP-sensitive K+ channels in fl-cells (Trube et al, 1986), caused a marked acceleration of 86Rb effiux.…”

Amantadine and sparteine inhibit ATP‐regulated K‐currents in the insulin‐secreting β‐cell line, HIT‐T15

“…This slow depolarization is mediated by a decrease in membrane K + permeability which results from the inhibition of ATP-regulated K-channels (K-ATP channels) (Ashcroft et al, 1984;Rorsman & Trube, 1985;Misler et al, 1986). It is believed that an increase in the cytosolic ATP/ADP ratio links glucose metabolism to channel inhibition (Cook & Hales, 1984;Ashcroft & Rorsman, 1989).…”