Pouli A E, Karagenc N, Wasmeier C, Hutton J C, Bright N, Arden S, Schofield J G, Rutter G A
Department of Biochemistry, School of Medical Sciences, University Walk, University of Bristol, Bristol BS8 1TD, U.K.
Biochem J. 1998 Mar 15;330 ( Pt 3)(Pt 3):1399-404. doi: 10.1042/bj3301399.
Microdomains of high Ca2+ concentration ([Ca2+]) may be critical to the control of intracellular processes such as secretion and metabolism without compromising other cell functions. To explore changes in [Ca2+] in the outer mantle (< 30 nm deep) that surrounds the surface of dense-core secretory granules, we have designed a recombinant chimaera between the granule protein phogrin and aequorin. When expressed in populations of insulin-secreting MIN6 or phaeochromocytoma PC12 cells, the chimaera was targeted to secretory granules as expected. The recombinant protein reported a similar [Ca2+] at the granule surface to that in the bulk cytosol, measured with untargeted aequorin. This was the case both at rest (-Ca2+- = 80-120 nM) and after stimulation with agents that provoke Ca2+ entry or Ca2+ mobilization from intracellular pools, and during activated secretion. Thus depolarization of MIN6 cell populations with high K+ increased [Ca2+] both in the bulk cytosol and close to the granules to approx. 4 microM, with near-identical kinetics of increase and recovery. Similarly, stimulation of PC12 cells with ATP provoked an increase in -Ca2+- in either domain to 1.3 microM. These data argue that, in MIN6 and PC12 neuroendocrine cells (i) significant mobilization of Ca2+ from most secretory granules probably does not occur during activated Ca2+ influx or mobilization of internal Ca2+ stores, and (ii) agonist-stimulated Ca2+-dependent secretion can occur without development of a large gradient of [Ca2+] between the surface of most secretory vesicles and the rest of the cytosol.
高钙离子浓度（[Ca2+]）的微区对于控制细胞内过程（如分泌和代谢）可能至关重要，同时又不会损害其他细胞功能。为了探究围绕致密核心分泌颗粒表面的外被膜（深度<30 nm）中[Ca2+]的变化，我们设计了一种颗粒蛋白phogrin与水母发光蛋白之间的重组嵌合体。当在分泌胰岛素的MIN6细胞群或嗜铬细胞瘤PC12细胞中表达时，该嵌合体如预期那样靶向分泌颗粒。用非靶向水母发光蛋白测量发现，重组蛋白报告的颗粒表面[Ca2+]与胞质溶胶中的相似。在静息状态（-Ca2+- = 80 - 120 nM）下、在用引发Ca2+内流或从细胞内池动员Ca2+的试剂刺激后以及在激活分泌期间均是如此。因此，用高钾使MIN6细胞群去极化会使胞质溶胶和颗粒附近的[Ca2+]都增加到约4 microM，增加和恢复的动力学几乎相同。同样，用ATP刺激PC12细胞会使任一区域的-Ca2+-增加到1.3 microM。这些数据表明，在MIN6和PC12神经内分泌细胞中：（i）在激活的Ca2+内流或内部Ca2+储存动员期间，大多数分泌颗粒可能不会发生显著的Ca2+动员；（ii）激动剂刺激的Ca2+依赖性分泌可以在大多数分泌小泡表面与胞质溶胶其余部分之间不形成大的[Ca2+]梯度的情况下发生。
