Study on multimolecular modulations of ATP-sensitive K^+ channels in pancreatic β-cells and their disharmony in diabetes.

本文研究了由磺脲受体单元异聚体和内向整流K^+通道孔单元组成的atp敏感K^+ (KATP)通道对细胞内Ca^<2+>活性的影响。研究发现，细胞内Ca^<2+>的升高可抑制磺脲类药物与受体结合和通道关闭之间的功能转导。暴露于PIP_2的膜片增加了KATP通道的活性，并降低了通道的ATP敏感性。用绿色荧光蛋白（GFP）标记ph结构域探针测定膜上PIP_2浓度，发现乙酰胆碱作用下β-细胞膜上GFP强度降低。与对照移液管溶液相比，在移液管中有乙酰胆碱存在时，通道的ATP灵敏度在内向外模式下有所增加。在细胞贴附贴片实验中，乙酰胆碱作用于β细胞，可诱导作用电流。在通过制氨抑素穿孔模式记录的全细胞电流中，乙酰胆碱在葡萄糖浓度为2.8 mM时降低了KATP通道电流。从这些结果可以看出，受体刺激胰腺β细胞会降低与膜去极化相关的KATP通道的活性，并在葡萄糖阈值浓度附近增加胰岛素分泌的输出。糖尿病动物β-细胞在副交感神经刺激过程中是否存在乙酰胆碱不能使膜去极化从而导致胰岛素分泌紊乱的现象，有待进一步探讨。

ATP-sensitive K^+ (KATP) channels, composed of heteromultimers of sulfonylurea-receptor unit and a pore unit of inwardly rectifying K^+ channels, were studied with respect to effects of intracellular Ca^<2+> activity of the channels. It was found that elevation of intracellular Ca^<2+> conferred inhibition of functional transduction between binding of sulfonylureas to their receptors and closure of channels. Exposure of membrane patches to PIP_2 increased activity of KATP channels in association with reduction of ATP sensitivity of the channels. When measured membrane PIP_2 concentration by means of GFP (green fluorescent protein) labeled PH-domain contained probe, membrane fraction of GFP intensity decreased on exposure of β-cells to acetylcholine. The ATP sensitivity of the channel recorded in the presence of acetylcholine in the pipette in inside-out mode was increased as compared to that in control pipette solution. Acetylcholine, when it was applied to β-cell during cell-attached patch experiments, induced action currents. In whole-cell current recorded by nystatin-perforated mode, acetylcholine decreased the KATP channel currents at the glucose concentration of 2.8 mM. From these results, it is suggested that receptor-stimulation of pancreatic β-cells produces reduction of activity of the KATP channels associated with depolarization of membrane and an increase in output of insulin secretion around threshold concentrations of glucose. It is needed to further explore whether β-cells from diabetic animals have the inability of acetylcholine to depolarize the membrane and resultant perturbation of insulin secretion during parasympathetic neural stimulation.