CALMODULIN-DEPENDENT REGULATION OF INDUCIBLE AND NEURONAL NITRIC-OXIDE SYNTHASE

Neuronal and endothelial nitric-oxide synthases depend upon Ca2+/calmo dulin for activation, whereas the activity of the inducible nitric-oxi de synthase is Ca2+-independent, presumably due to tightly bound calmo dulin, To study these different mechanisms, a series of chimeras deriv ed from neuronal and inducible nitric-oxide synthases were analyzed. C himeras containing only the oxygenase domain, calmodulin-binding regio n, or reductase domain of inducible nitric-oxide synthase did not conf er significant Ca2+-independent activity. However, each chimera was mo re sensitive to Ca2+ than the neuronal isoform, The calmodulin-binding region of inducible nitric-oxide synthase with either its oxygenase o r reductase domains resulted in significant, but not total, Ca2+-indep endent activity. Co-immunoprecipitation experiments showed no calmodul in associated with the former chimera in the absence of Ca2+. Trifluop erazine also inhibited this chimera in the absence of Ca2+. The combin ed interactions of calmodulin bound to inducible nitric-oxide synthase calmodulin-binding region with the oxygenase domain may be weaker tha n with the reductase domain. Thus, Ca2+-independent activity of induci ble nitric-oxide synthase appears to result from the concerted interac tions of calmodulin with both the oxygenase and reductase domains in a ddition to the canonical calmodulin-binding region. The neuronal isofo rm is not regulated by a unique autoinhibitory element in its reductas e domain.