OMP:ROLE IN OLFACTORY SIGNAL DETECTION AND TRANSDUCTION

描述（由申请人提供）：钙调素（Calmodulin, CaM）是一种细胞内钙传感器，是许多信号通路的关键参与者，包括嗅觉信号转导的几乎所有方面。然而，CaM太重要了，不能让它自己发挥作用。在细胞内信号级联的每个步骤中，存在调节CaM和Ca/CaM的可及性的机制。我们认为嗅觉标记蛋白（OMP）是嗅觉转导过程中CaM调控过程的关键成员。我们假设OMP是一个新机制的关键参与者，该机制与Bex蛋白一致，调节Ca/CaM在嗅觉感觉神经元（OSN）信号转导级联中的参与和靶向多个效应器。这种新的OMP/Bex/CaM假说将OMP的功能与CaM联系起来，为解释OMP- ko小鼠的所有行为和电生理表型提供了一个可测试的框架。此外，本研究将OMP定位为从Ca2+进入到消除整个嗅觉转导级联过程中多个步骤的关键组成部分。这种新颖的OMP/Bex/Ca/CaM假说得到了我实验室几个经验观察的支持：在OMP- ko小鼠中，通过Na+/Ca2+交换器（NCX）活性从OSNs流出的1-Ca2+受到损害；2- omp形成一个短寿命二聚体，与Bex1和2结合；OMP的胞内蛋白伴侣3-Bex1和2与Ca/CaM相互作用；4 . Na+/Ca2+交换器NCX1、2和3的胞内环各有一个Ca/CaM结合位点；5 . Bex1-KO小鼠的EOG和行为缺陷与OMP-KO小鼠相似；6-NCX1与cam -琼脂糖结合；7-NCX1活性被CaM拮抗剂抑制。我们现在建议采用协调的多学科方法在分子，细胞和整个动物水平上表征OMP/Bex/CaM假说，以增加我们对调节嗅觉转导级联的基本机制的理解。为了实现这一目标，我们将提出以下假设：1- Na+/Ca2+交换器（NCX）活性通过其与Ca/CaM、Bex1和2以及OMP的细胞内相互作用来调节，NCX活性使osn细胞内升高的Ca2+恢复到刺激前水平；2 . Ca/CaM对Ca2+激活氯离子通道（Ca/ClC）嗅觉转导的影响是由CaM、OMP和Bex1和bex2相互作用调节的；OMP和Bex1和2蛋白之间的相互作用调节细胞内Ca/CaM的功效并影响NCX的活性，导致OSNs的行为和电生理在体内发生改变。这些协同分析将为嗅觉转导级联调控的分子机制提供新的见解，并将对理解CaM调控信号转导机制产生广泛的影响。

DESCRIPTION (provided by applicant): Calmodulin (CaM) is an intracellular calcium sensor that is a critical participant in many signaling pathways including almost every aspect of olfactory signal transduction. However, CaM is too important to be allowed to function by itself. Mechanisms exist to regulate the accessibility of CaM and Ca/CaM at each step in intracellular signaling cascades. We propose that the olfactory marker protein (OMP) is a key member of such a CaM regulatory process in olfactory transduction. We hypothesize that OMP is a pivotal participant in a novel mechanism that, in concert with the Bex proteins, regulates the participation and targeting of Ca/CaM to multiple effectors in the olfactory sensory neuron (OSN) signal transduction cascade. This novel OMP/Bex/CaM hypothesis linking the function of OMP to CaM provides a testable framework to explain all the behavioral and electrophysiological phenotypes of the OMP-KO mouse. Furthermore, this proposal positions OMP as a key, integral component at multiple steps throughout the entire olfactory transduction cascade from Ca2+ entry to elimination. This novel OMP/Bex/Ca/CaM hypothesis is supported by several empirical observations from my lab: 1-Ca2+ efflux from OSNs via Na+/Ca2+ exchanger (NCX) activity is compromised in the OMP-KO mouse; 2-OMP forms a short lived dimer that binds to Bex1 and 2; 3-Bex1 and 2, the intracellular protein partners of OMP, interact with Ca/CaM; 4-the intracellular loops of Na+/Ca2+ exchangers NCX1, 2 and 3 each have a Ca/CaM binding site; 5-the Bex1-KO mouse has EOG and behavioral deficits similar to those of the OMP-KO mouse; 6-NCX1 binds to CaM-agarose; 7-NCX1 activity is inhibited by pharmacological CaM antagonists. We now propose to apply a coordinated, multidisciplinary approach to characterize this OMP/Bex/CaM hypothesis at the molecular, cellular and whole animal levels to increase our understanding of the basic mechanisms regulating the olfactory transduction cascade. To achieve this we will address the following hypotheses:1-The Na+/Ca2+ exchanger (NCX) activity that returns elevated intracellular Ca2+ to prestimulus levels in OSNs is modulated by its intracellular interactions with Ca/CaM, Bex1 and 2 and OMP; 2-The efficacy of Ca/CaM on the Ca2+-activated chloride channel (Ca/ClC) in olfactory transduction is modulated by the interactions among CaM, OMP and Bex1 and 2; 3-The interactions among OMP and Bex1 and 2 proteins that modulate the efficacy of intracellular Ca/CaM and influence NCX activity cause in vivo alterations in behavior and electrophysiology of OSNs.These coordinated analyses will provide new insights to the molecular mechanisms by which the olfactory transduction cascade is regulated and will have broad impact on understanding CaM regulation of signal transduction mechanisms.