Varadi A, Grant A, McCormack M, Nicolson T, Magistri M, Mitchell K J, Halestrap A P, Yuan H, Schwappach B, Rutter G A
Henry Wellcome Laboratories for Integrated Cell Signalling and Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol, BS8 1TD, UK.
Diabetologia. 2006 Jul;49(7):1567-77. doi: 10.1007/s00125-006-0257-9. Epub 2006 May 12.
AIMS/HYPOTHESIS: ATP-sensitive K(+) (K(ATP)) channels located on the beta cell plasma membrane play a critical role in regulating insulin secretion and are targets for the sulfonylurea class of antihyperglycaemic drugs. Recent reports suggest that these channels may also reside on insulin-containing dense-core vesicles and mitochondria. The aim of this study was to explore these possibilities and to test the hypothesis that vesicle-resident channels play a role in the control of organellar Ca(2+) concentration or pH.
To quantify the subcellular distribution of the pore-forming subunit Kir6.2 and the sulfonylurea binding subunit SUR1 in isolated mouse islets and clonal pancreatic MIN6 beta cells, we used four complementary techniques: immunoelectron microscopy, density gradient fractionation, vesicle immunopurification and fluorescence-activated vesicle isolation. Intravesicular and mitochondrial concentrations of free Ca(2+) were measured in intact or digitonin-permeabilised MIN6 cells using recombinant, targeted aequorins, and intravesicular pH was measured with the recombinant fluorescent probe pHluorin.
SUR1 and Kir6.2 immunoreactivity were concentrated on dense-core vesicles and on vesicles plus the endoplasmic reticulum/Golgi network, respectively, in both islets and MIN6 cells. Reactivity to neither subunit was detected on mitochondria. Glibenclamide, tolbutamide and diazoxide all failed to affect Ca(2+) uptake into mitochondria, and K(ATP) channel regulators had no significant effect on intravesicular free Ca(2+) concentrations or vesicular pH.
CONCLUSIONS/INTERPRETATION: A significant proportion of Kir6.2 and SUR1 subunits reside on insulin-secretory vesicles and the distal secretory pathway in mouse beta cells but do not influence intravesicular ion homeostasis. We propose that dense-core vesicles may serve instead as sorting stations for the delivery of channels to the plasma membrane.
目的/假设：位于β细胞质膜上的ATP敏感性钾（K(ATP)）通道在调节胰岛素分泌中起关键作用，并且是磺脲类抗高血糖药物的作用靶点。最近的报道表明，这些通道也可能存在于含胰岛素的致密核心囊泡和线粒体上。本研究的目的是探索这些可能性，并检验囊泡驻留通道在控制细胞器钙（Ca(2+)）浓度或pH值中起作用的假设。
为了量化分离的小鼠胰岛和克隆的胰腺MIN6β细胞中形成孔道的亚基Kir6.2和磺脲类结合亚基SUR1的亚细胞分布，我们使用了四种互补技术：免疫电子显微镜、密度梯度分级分离、囊泡免疫纯化和荧光激活囊泡分离。使用重组靶向水母发光蛋白在完整的或洋地黄皂苷通透的MIN6细胞中测量囊泡内和线粒体内游离钙（Ca(2+)）的浓度，并用重组荧光探针pHluorin测量囊泡内pH值。
在胰岛和MIN6细胞中，SUR1和Kir6.2免疫反应性分别集中在致密核心囊泡以及囊泡加上内质网/高尔基体网络上。在线粒体上未检测到对任何一个亚基的反应性。格列本脲、甲苯磺丁脲和二氮嗪均未能影响钙（Ca(2+)）摄入线粒体，并且K(ATP)通道调节剂对囊泡内游离钙（Ca(2+)）浓度或囊泡pH值没有显著影响。
结论/解读：相当一部分Kir6.2和SUR1亚基存在于小鼠β细胞的胰岛素分泌囊泡和远端分泌途径上，但不影响囊泡内离子稳态。我们提出致密核心囊泡可能反而作为将通道递送至质膜的分选站。
