OBJECTIVE-To characterize the voltage-gated ion channels in human -cells from nondiabetic donors and their role in glucosestimulated insulin release.RESEARCH DESIGN AND METHODS-Insulin release was measured from intact islets. Whole-cell patch-clamp experiments and measurements of cell capacitance were performed on isolated -cells. The ion channel complement was determined by quantitative PCR.RESULTS-Human -cells express two types of voltage-gated K ϩ currents that flow through delayed rectifying (K V 2.1/2.2) and large-conductance Ca 2ϩ -activated K ϩ (BK) channels. Blockade of BK channels (using iberiotoxin) increased action potential amplitude and enhanced insulin secretion by 70%, whereas inhibition of K V 2.1/2.2 (with stromatoxin) was without stimulatory effect on electrical activity and secretion. Voltage-gated tetrodotoxin (TTX)-sensitive Na ϩ currents (Na V 1.6/1.7) contribute to the upstroke of action potentials. Inhibition of Na ϩ currents with TTX reduced glucose-stimulated (6 -20 mmol/l) insulin secretion by 55-70%. Human -cells are equipped with L-(Ca V 1.3), P/Q-(Ca V 2.1), and T-(Ca V 3.2), but not N-or R-type Ca 2ϩ channels. Blockade of L-type channels abolished glucosestimulated insulin release, while inhibition of T-and P/Q-type Ca 2ϩ channels reduced glucose-induced (6 mmol/l) secretion by 60 -70%. Membrane potential recordings suggest that L-and T-type Ca 2ϩ channels participate in action potential generation. Blockade of P/Q-type Ca 2ϩ channels suppressed exocytosis (measured as an increase in cell capacitance) by Ͼ80%, whereas inhibition of L-type Ca 2ϩ channels only had a minor effect.CONCLUSIONS-Voltage-gated T-type and L-type Ca 2ϩ channels as well as Na ϩ channels participate in glucose-stimulated electrical activity and insulin secretion. Ca 2ϩ -activated BK channels are required for rapid membrane repolarization. Exocytosis of insulin-containing granules is principally triggered by Ca 2ϩ influx through P/Q-type Ca 2ϩ channels. Diabetes 57:1618-1628, 2008
SummaryGlucagon, secreted by pancreatic islet α cells, is the principal hyperglycemic hormone. In diabetes, glucagon secretion is not suppressed at high glucose, exacerbating the consequences of insufficient insulin secretion, and is inadequate at low glucose, potentially leading to fatal hypoglycemia. The causal mechanisms remain unknown. Here we show that α cell KATP-channel activity is very low under hypoglycemic conditions and that hyperglycemia, via elevated intracellular ATP/ADP, leads to complete inhibition. This produces membrane depolarization and voltage-dependent inactivation of the Na+ channels involved in action potential firing that, via reduced action potential height and Ca2+ entry, suppresses glucagon secretion. Maneuvers that increase KATP channel activity, such as metabolic inhibition, mimic the glucagon secretory defects associated with diabetes. Low concentrations of the KATP channel blocker tolbutamide partially restore glucose-regulated glucagon secretion in islets from type 2 diabetic organ donors. These data suggest that impaired metabolic control of the KATP channels underlies the defective glucose regulation of glucagon secretion in type 2 diabetes.
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