OMP: Role in Olfactory Signal Detection and Transduction

钙离子在嗅觉感受器神经元的信号转导通路中发挥着重要作用。Ca2+ 通过cAMP门控通道进入气味配体受体激活的G蛋白介导的增加 腺酰环化酶活力。Ca~(2+)内流是哺乳动物嗅觉化学感觉转导的关键步骤 刺激钙激活的氯离子通道以放大转导电流。Ca~(2+)也是一种 适应、脱敏、恢复和转导-翻译耦合。已经做出了很大的努力来 了解ORN中的这些事件。对下游事件的关注要少得多，因为这些事件 Orns细胞内稳定的钙离子水平。我们对嗅觉标记蛋白(OMP)缺失小鼠的鉴定 在之前的资助周期中，使用电生理、生化和行为指标的表型使我们 假设OMP缺失表型是由于调节细胞内钙离子水平的缺陷所致。具体地说。 我们推测OMP参与调控ONS中钙分泌过程的活性。这个 OMP在成熟脊椎动物中表达的特异性、其序列在所有脊椎动物中的保守性以及OMP缺失 小鼠的表型均表明OMP对嗅觉功能有重要作用。我们最近观察到一个重要的 OMP缺失小鼠刺激后ORN树突状小结中钙离子动力学的变化。几个基因 参与Ca~(2+)外排，包括Na~+/Ca~(2+)交换器(NCX)和Na~+/Ca~(2+)-K~+交换器(NCKX)。 其中每一个都是由三个基因和大量的mna剪接变异体在细胞和组织中表达的。 模式。免疫细胞化学和电生理报告表明，这些交换器在 维持细胞内ORN钙离子水平。这些报告，再加上我们的新数据，即OMP调节钙离子 挤压进一步支持了我们的假设以及表征分子和生化基础的重要性 在奥恩斯的钙离子挤出过程。这是我们在这项提议中所解决的知识上的一个重大差距。 我们将(L)-表征NCX和NCKX基因在嗅觉组织中的表达，特别是在嗅觉组织中 发展和对损伤的反应；(2)-识别特定的NCX或NCKX蛋白的部分， 参与OMP介导的钙离子水平的调节；(3)-决定其他 这一过程的参与者，例如最近发现的与OMP结合的Bex蛋白。这些研究将提供 对Orns细胞内钙离子水平调节机制的新见解 治疗嗅觉障碍或嗅觉障碍的化疗机会。 BFORFORMANCE SITE(S)(组织、城市、州) 关键人员，请参阅第11页的说明。根据需要使用续页，以如下所示的格式提供所需信息。 命名项目中的组织角色 马里兰州马格利斯大学弗兰克·L 马里兰大学乔伊斯·W·马戈利斯研究专家 马里兰大学载亨古博士学位 马蒂娜·M·皮尔斯基马里兰大学博士后 基里尔·乌哈诺夫马里兰大学博士后 丹·舒尔茨马里兰大学合作者 马里兰大学唐娜·M·巴尔梅西合作者 阿卜杜勒·M·鲁克努丁马里兰大学合作者 特蕾西·莱因德斯--马里兰祖福尔大学顾问 小灵通398(4/98版)第2页BB 在整个应用程序的底部连续编号页码。不要使用3a、3b等后缀。 Rnncipai n^ptigaior/rrogram uiReceior(最后，nrsi，miaa/ey.)^Md^Miib，Rimus. 在每张打印页和每张续页的顶部键入首席调查员/项目主任的姓名。(有关类型规格，请参阅说明 (见第6页。) 研究补助金 目录 页码 脸页？1 描述，

Calcium plays many roles in the signal-transduction cascade in olfactory receptor neurons (ORNs). Ca2+ enters through cAMP-gated channels in response to odor-ligand receptor activation of G-protein-mediated increases in adenylyl cyclase activity. Ca2+ entry is a key step in mammalian olfactory chemosensory transduction and stimulates a Ca2+-activated chloride channel to amplify the transduction current. Ca2+ is also a regulator of adaptation, desensitization, recovery and transduction-translation coupling. Much effort has been directed towards understanding these events in the ORN. Much less attention has been paid to downstream events that maintain stable intracellular Ca2+ levels in ORNs. Our characterization of the olfactory marker protein (OMP)-null mouse phenotype using electrophysiological, biochemical and behavioral measures in the previous funding cycle have led us to hypothesize that the OMP-null phenotvpe is due to a defect in regulating intracellular Ca2+ levels. Specifically. we hypothesize that OMP participates in regulating the activity of Ca extrusion processes in ORNs. The specificity of OMP expression in mature ORNs, its sequence conservation across all vertebrates, and the OMP-null mouse phenotype all indicate OMP is important to olfactory function. We recently observed a significant alteration in Ca2+ kinetics in ORN dendritic knobs in OMP-null mice following stimulation. Several genes participate in Ca2+ extrusion including the Na+/Ca2+ exchangers (NCX), and the Na+/Ca2+-K+ exchangers (NCKX). Each of these is represented by three genes and numerous mRNA splice variants expressed in cell- and tissue-specific patterns. Immunocytochemical and electrophysiological reports suggest that these exchangers are important in maintaining intracellular ORN Ca2+ levels. These reports, coupled with our new data that OMP modulates Ca2+ extrusion further supports our hypothesis and the importance of characterizing the molecular and biochemical bases of Ca2+ extrusion processes in ORNs. This is a significant gap in our knowledge that this proposal addresses. We will (l)-characterize NCX and NCKX gene expression in olfactory tissue, and especially in ORNs, during development and in response to lesions; (2)-identify that part of the specific NCX or NCKX protein that participates in the OMP-mediated regulation of Ca + levels; (3)-determine the nature and function of other participants in this process e.g. the recently discovered Bex proteins that bind to OMP. These studies will provide novel insights to the mechanisms by which intracellular Ca2+ levels are regulated in ORNs and may generate chemotherapeutic opportunities for treatment of anosmias or dysosmias. bERFORMANCE SITE(S) (organization, city, State) KEY PERSONNEL, see instructions on Page 11. Use continuation pages as needed to provide the required information in the format shown below. Name Organization Role on Project Frank L Margolis University of Maryland PI Joyce W. Margolis University of Maryland Research Specialist Jae Hyung Koo University of Maryland Post Doc Martina M. Pyrski University of Maryland Post Doc Kyrill Ukhanov University of Maryland Post Doc Dan Schulze University of Maryland Collaborator Donna M. Baldisseri University of Maryland Collaborator Abdul M. Ruknudin University of Maryland Collaborator Trese Leinders-Zufall University of Maryland Consultant PHS 398 (Rev. 4/98) Page 2 BB Number pages consecutively at the bottom throughout the application. Do not use suffixes such as 3a, 3b. rnncipai n^ptigaior/rrogram uirecior (Last, nrsi, miaa/ey. ^md^Miib, rimus. Type the name of the principal investigator/programdirector at the top of each printed page and each continuation page. (For type specifications, see instructions on page 6.) RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page ¿. 1 Description,