OMP:ROLE IN OLFACTORY SIGNAL DETECTION AND TRANSDUCTION

描述（由申请人提供）：钙调素（CaM）是一种细胞内钙传感器，是许多信号通路（包括嗅觉信号转导的几乎所有方面）的关键参与者。然而，CaM太重要了，不能让它自己发挥作用。存在调节CaM和Ca/CaM在细胞内信号级联的每个步骤的可及性的机制。我们建议，嗅觉标记蛋白（OMP）是一个关键成员，这样的钙调蛋白调节过程中的嗅觉转导。我们假设OMP是一种新机制的关键参与者，该机制与Bex蛋白一起调节Ca/CaM对嗅觉感觉神经元（OSN）信号转导级联中多个效应器的参与和靶向。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检验的框架。此外，该提议将OMP定位为从Ca 2+进入到消除的整个嗅觉转导级联中的多个步骤中的关键、不可或缺的组分。OMP/Bex/Ca/CaM假说得到了实验室实验观察的支持：1-Ca ~（2+）通过Na ~+/Ca ~（2+）交换器（NCX）从OSNs流出，在OMP-KO小鼠中受到损害，2-OMP形成短寿命的二聚体，与Bex 1和2结合，3-Bex 1和2是OMP的胞内蛋白伴侣，与Ca/CaM相互作用; 4-Na+/Ca 2+交换剂NCX 1、2和3的细胞内环各自具有Ca/CaM结合位点; 5-Bex 1-KO小鼠具有与OMP-KO小鼠相似的EOG和行为缺陷; 6-NCX 1与CaM-琼脂糖结合; 7-NCX 1活性被药理学CaM拮抗剂抑制。我们现在建议应用一个协调的，多学科的方法来表征这个OMP/Bex/CaM假说在分子，细胞和整个动物水平，以增加我们的理解的基本机制调节嗅觉转导级联。为了实现这一点，我们将讨论以下假设：1-Na+/Ca ~（2+）交换器（NCX）活性通过其与Ca/CaM、Bex 1和2以及OMP的细胞内相互作用来调节，该活性使OSN中升高的细胞内Ca ~（2+）恢复到刺激前水平; 2-在嗅觉转导中，Ca/CaM对Ca ~（2+）激活的氯离子通道（Ca/ClC）的功效通过CaM、OMP和Bex 1和2之间的相互作用来调节; 3-OMP和Bex 1和2蛋白之间的相互作用，调节细胞内Ca/Ca 2+的功效。CaM和影响NCX活性导致OSNs在体内的行为和电生理改变。这些协调的分析将为OSNs的分子机制提供新的见解。嗅觉信号转导级联反应的调控，将有广泛的影响，了解钙调素调控的信号转导机制。

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.