Nitric oxide (NO) production by nitric oxide synthases (NOSs) plays an important role in physiology and the aberration from normal production leads to disease development. The activity of endothelial NOS (eNOS) is regulated by Ca<sup>2+</sup> flux through the binding of calmodulin (CaM) which allows electron transfer from the NOS reductase domain to oxygenase domain to occur. Recent studies also indicate that Caveolin 1 (Cav-1) associated eNOS in cell caveolae structures is inactive and its activity is restored by CaM binding, suggesting the interplay between Cav-1 and CaM. Here we attempted to provide thermodynamic and kinetic parameters in order to understand the interplay among eNOS, CaM and Cav-1 peptide (Cav-1p) in relation to NO production. Our preliminary data has shown that NO production is inhibited by the addition of Cav-1p and the eNOS/Cav-1p complex was observed by using surface plasmon resonance (SPR). We had recently constructed the CaM and Troponin C (TnC) chimeras with one of each of the CaM EF-hands replaced by TnC EF-hand motifs, and investigated the interactions with the CaM-binding domain (CMBD) of Orai. Our results indicate these chimera proteins serve as a good model for studying the interaction between CaM and target enzymes. Thus we are analyzing the interaction between the chimeras and eNOS using isothermal titration calorimetry to rationalize their impacts on NO production.  We have also constructed several fused fluorescent proteins or fluorescent-labeled CaM and Cav-1, which will allow us to test the hypothesis that CaM plays a role in dissociating eNOS from Caveoin-1 and activating NO production using fluorescence resonance energy transfer (FRET) and a NO assay.<br/>

Regulatory mechanism of nitric oxide synthase by cav-1 and calmodulin

255th National Meeting (2018)

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