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. ? ? ?
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