Localization, Interactions, And Functions of Synaptotagmins in the Pituitary

描述（由申请人提供）：Synaptotagmin 1，一种在突触囊泡上发现的蛋白质，在神经递质释放中作为Ca2+传感器。这种蛋白质有16个同源物，尽管人们普遍认为这些其他蛋白质具有相关功能，但人们对它们的位置和作用却一无所知。本研究中提出的初步数据表明，在垂体后叶和/或垂体前叶中存在多种突触塔蛋白同工型，这些腺体释放肽激素，控制哺乳动物生理的所有重要方面。在这些新的初步结果之前，垂体已被确定为一种适合定量生物物理研究的制剂，用于研究含有肽的囊泡如何与质膜融合并将其内容物释放到循环中。因此，在垂体中发现新的synaptotagmin亚型提供了一个独特的机会来详细测试它们的功能，并阐明它们在兴奋-分泌耦合中的确切作用。本项目将采用互补的方法研究垂体synaptotagmin亚型，研究它们在体外的结合特性，以及它们在垂体中的定位和生理功能。功能研究采用了一种策略，使用转基因小鼠对特定的synaptotagmin亚型进行基因消融，并通过基因靶向来识别表达这些亚型的特定细胞群以进行电记录。通过在鉴定的细胞和神经末梢进行电容记录，我们将确定系统同种异构体如何在Ca2+敏感性和融合孔方面调节胞外分泌。本文提出的实验将测试synaptotagmins 4、7、9、10、11和12在控制生长、代谢、应激、体液平衡和繁殖的激素释放中的功能。

DESCRIPTION (provided by applicant): Synaptotagmin 1, a protein found on synaptic vesicles, serves as a Ca2+ sensor in neurotransmitter release. This protein has 16 homologues, and while these other proteins are widely thought to serve related functions essentially nothing is known about where they are or what they do. Preliminary data presented in this application shows that several synaptotagmin isoforms are abundant in the posterior and/or anterior pituitary, glands that release peptide hormones that control essentially all vital aspects of mammalian physiology. Prior to these new preliminary results, the pituitary had been established as a preparation amenable to quantitative biophysical study of how peptide-containing vesicles fuse with the plasma membrane to release their content into the circulation. Thus, the finding of novel synaptotagmin isoforms in the pituitary offers a unique opportunity to test their function in detail and elucidate their precise roles in excitation-secretion coupling. This project will study the pituitary synaptotagmin isoforms with complementary approaches, investigating their binding properties in vitro, and their localization and physiological function in the pituitary. The functional studies employ a strategy of using transgenic mice for gene ablation of specific synaptotagmin isoforms, and gene targeting to identify specific cell populations that express those isoforms for electrical recording. By performing capacitance recording in identified cells and nerve terminals we will determine how syt isoforms regulate exocytosis in terms of Ca2+ sensitivity and fusion pores. The experiments proposed here will test the functions of synaptotagmins 4, 7, 9, 10, 11, and 12 in the release of hormones that control growth, metabolism, stress, fluid balance, and reproduction. PUBLIC HEALTH RELEVANCE: These experiments will shed light on the function of a class of proteins with broad roles in neurological, mental, and endocrine function and thus contribute to improving treatment of neurological disorders and mental illness. By illuminating the mechanisms of control of the pituitary hormones oxytocin, vasopressin, growth hormone, adrenocorticotropin, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, and prolactin, these studies will improve our understanding of growth defects, abnormal stress responses, metabolic disorders, and reproductive health.