Rupnik M, Kreft M, Sikdar S K, Grilc S, Romih R, Zupancic G, Martin T F, Zorec R
Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Medical School, Ljubljana, Slovenia SI-1001.
Proc Natl Acad Sci U S A. 2000 May 9;97(10):5627-32. doi: 10.1073/pnas.090359097.
Although many proteins essential for regulated neurotransmitter and peptide hormone secretion have been identified, little is understood about their precise roles at specific stages of the multistep pathway of exocytosis. To study the function of CAPS (Ca(2+)-dependent activator protein for secretion), a protein required for Ca(2+)-dependent exocytosis of dense-core vesicles, secretory responses in single rat melanotrophs were monitored by patch-clamp membrane capacitance measurements. Flash photolysis of caged Ca(2+) elicited biphasic capacitance increases consisting of rapid and slow components with distinct Ca(2+) dependencies. A threshold of approximately 10 microM Ca(2+) was required to trigger the slow component, while the rapid capacitance increase was recorded already at a intracellular Ca(2+) activity < 10 microM. Both kinetic membrane capacitance components were abolished by botulinum neurotoxin B or E treatment, suggesting involvement of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-dependent vesicle fusion. The rapid but not the slow component was inhibited by CAPS antibody. These results were further clarified by immunocytochemical studies that revealed that CAPS was present on only a subset of dense-core vesicles. Overall, the results indicate that dense-core vesicle exocytosis in melanotrophs occurs by two parallel pathways. The faster pathway exhibits high sensitivity to Ca(2+) and requires the presence of CAPS, which appears to act at a late stage in the secretory pathway.
尽管已经鉴定出许多对神经递质和肽激素分泌调节至关重要的蛋白质，但对于它们在胞吐作用多步骤途径的特定阶段的确切作用却知之甚少。为了研究CAPS（Ca(2+)依赖性分泌激活蛋白）的功能，一种致密核心囊泡Ca(2+)依赖性胞吐作用所需的蛋白质，通过膜片钳膜电容测量监测了单个大鼠黑素细胞的分泌反应。笼锁Ca(2+)的闪光光解引发了双相电容增加，包括具有不同Ca(2+)依赖性的快速和慢速成分。触发慢速成分需要约10 microM Ca(2+)的阈值，而在细胞内Ca(2+)活性<10 microM时就记录到了快速电容增加。肉毒杆菌神经毒素B或E处理消除了两个动力学膜电容成分，表明涉及SNARE（可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体）依赖性囊泡融合。CAPS抗体抑制了快速成分而非慢速成分。免疫细胞化学研究进一步阐明了这些结果，该研究表明CAPS仅存在于致密核心囊泡的一个子集中。总体而言，结果表明黑素细胞中致密核心囊泡的胞吐作用通过两条平行途径发生。较快的途径对Ca(2+)表现出高敏感性，并且需要CAPS的存在，CAPS似乎在分泌途径的后期起作用。
