Plasma Membrane Ca-ATPases in Mouse Chemosensory Cells

小鼠化学感应细胞中的质膜 Ca-ATP 酶

• 批准号: 6760443
• 负责人: JUDITH L VAN HOUTEN
• 金额: $ 22.73万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6760443
• 关键词: biological models; calcium flux; calcium indicator; calcium transporting ATPase; genetically modified animals; immunocytochemistry; immunofluorescence technique; laboratory mouse; long term potentiation; olfactory nerve; olfactory stimulus; protein isoforms; protein localization; respiratory epithelium; sensory mechanism; sensory signal detection; vomeronasal systems; western blottings

DESCRIPTION (provided by applicant): Calcium dynamics are important in the physiology of olfactory and vomeronasal sensory neurons (OSNs and VSNs). The focus of attention is primarily on Ca2+ influx, because Ca2+ carries the current of the cyclic nucleotide gated channel (CNGC) that opens upon odorant stimulation of the OSN and both internal stores and extracellular Ca 2+ contribute to the rise in intracellular Ca 2+ in VSNs upon stimulation with pheromone. Once Ca 2+ enters, the kinetics of its removal become important because, for example in OSNs, Ca 2+ can amplify the odor evoked currents by activating a CI conductance, amplify second messenger production, inhibit the CNGC, activate feed back regulation, inhibit key enzymes in signal transduction, among other effects. Therefore, removal of Ca2vand the kinetics of this removal can modulate the excitability and function of OSNs and VSNs, but less attention has been paid to Ca 2+ clearance than to increases. The Na/Ca exchanger plays a role in this Ca 2+ removal, but there is evidence from the past and our preliminary data that support a role for Plasma Membrane Calcium ATPases (Ca 2+ pumps, PMCAs) in this removal. We provide additional preliminary data that demonstrate the presence of PMCA isoforms in mouse OSNs and VNO. In particular, PMCAs 1 and 2 are found in OSNs and VNO, and PMCA2 may be localized to the OSN dendritic knob. These observations have implications for the functions of the PMCAs in OSNs and VSNs because each of the 4 PMCA isoforms has very different regulatory properties and affinities for Ca 2+. We propose three specific aims to partially test the hypotheses 1) PMCAs play a role in restoring calcium to basal levels after stimulation of OSNs and VSNs and 2) deletion of PMCA2 (in knock-out mice) leads to impairment in response to repetitive odor stimulation due to failure to return Ca 2v levels to basal in OSNs and VSNs. Specific Aim 1. Identify the PMCA isoforms in the olfactory and VNO epithelia and their cellular Iocalizations on the OSNs and VSNS using Western blotting and immunohistochemistry of epithelia and immuno-fluorescence of dissociated cells. Specific Aim 2. Test whether PMCAs among other Ca 2+ clearance mechanisms contribute to restoration of Ca2+ to basal levels after stimulation using calcium imaging of dissociated cells. Specific Aim 3. Examine the OSNs and VSNs from PMCA2 knock-out mice using the techniques of Specific Aims 1 and 2. This exploratory work will improve our understanding of the contributions of PMCAs to OSN and VSN Ca 2+ clearance, give new insight into VSN and OSN physiology and transduction regulation, and open up new avenues for chemosensory research. The exploratory research will take advantage of the mouse genetic model and also allows the PI to apply her expertise in PMCAs from other chemosensory systems to the mouse. The understanding of OSN and VSN function is important for understanding normal olfactory and VNO function in health and disease.

描述（由申请人提供）：钙动力学在嗅觉和呕吐性感觉神经元（OSN和VSN）的生理学中很重要。注意力的重点主要放在Ca2+涌入上，因为Ca2+带有环环核苷酸门控通道（CNGC）的电流，该通道（CNGC）在对OSN的气味刺激以及内部存储和细胞外Ca 2+方面打开，并在刺激刺激的VSN中有助于细胞内Ca 2+的上升。一旦Ca 2+进入，其去除的动力学就变得很重要，例如，在OSN中，Ca 2+可以通过激活CI电导，扩增第二信使产生，抑制CNGC，激活饲料背面调节，抑制其他效果中的信号转移中的关键酶。因此，去除CA2VAND的动力学可以调节OSN和VSN的兴奋性和功能，但是对Ca 2+清除的关注少于增加。 NA/CA交换器在此Ca 2+的去除中起作用，但是过去的证据和我们的初步数据支持质膜钙ATPases（Ca 2+泵，PMCAS）在此删除中的作用。我们提供其他初步数据，这些数据证明了小鼠OSN和VNO中PMCA同工型的存在。特别是，在OSN和VNO中发现了PMCAS 1和2，并且PMCA2可以定位于OSN树突旋钮。这些观察结果对PMCA在OSN和VSN中的功能具有影响，因为4种PMCA同工型中的每一种都具有Ca 2+的调节性质和亲和力非常不同。我们提出了三个特定的目的，以部分测试假设1）PMCA在刺激OSN和VSN后恢复钙至基础水平方面发挥作用，以及2）删除PMCA2（在敲除小鼠中）导致损害因响应CA 2V水平而导致的重复刺激，从而导致损害，而OSNS和VSNS和VSNS中的底层均导致了损害。具体目的1。使用蛋白质印迹和分离细胞的免疫荧光，识别嗅觉和VNO上皮及其在OSN和VSN上的细胞IOCALISATIS中的PMCA同工型。具体目标2。测试PMCA在其他Ca 2+清除机制中是否有助于使用解离细胞的钙成像刺激后Ca2+恢复到基础水平。特定目标3。使用特定目的1和2的技术检查PMCA2敲除小鼠的OSN和VSN。这项探索性工作将提高我们对PMCAS和VSN CA 2+批准的贡献的理解，对VSN和OSN生理学和转导调节的新洞察力，并开设了新的Aspulation，以及开设新的化学意义研究。探索性研究将利用小鼠遗传模型，还允许PI将其专业知识应用于其他化学感应系统的PMCAS。对OSN和VSN功能的理解对于理解健康和疾病中正常的嗅觉和VNO功能很重要。