Calmodulin-dependent Regulation of Inducible and Neuronal Nitric-oxide Synthase

Shiow-Ju Lee,James T Stull

doi:10.1074/jbc.273.42.27430

Abstract

Neuronal and endothelial nitric-oxide synthases depend upon Ca2+/calmodulin for activation, whereas the activity of the inducible nitric-oxide synthase is Ca2+-independent, presumably due to tightly bound calmodulin. To study these different mechanisms, a series of chimeras derived from neuronal and inducible nitric- oxide synthases were analyzed. Chimeras containing only the oxygenase domain, calmodulin-binding region, or reductase domain of inducible nitric-oxide synthase did not confer significant Ca2+-independent activity. However, each chimera was more sensitive to Ca2+ than the neuronal isoform. The calmodulin-binding region of inducible nitric-oxide synthase with either its oxygenase or reductase domains resulted in significant, but not total, Ca2+-independent activity. Co-immunoprecipitation experiments showed no calmodulin associated with the former chimera in the absence of Ca2+. Trifluoperazine also inhibited this chimera in the absence of Ca2+. The combined interactions of calmodulin bound to inducible nitric-oxide synthase calmodulin-binding region with the oxygenase domain may be weaker than with the reductase domain. Thus, Ca2+-independent activity of inducible nitric-oxide synthase appears to result from the concerted interactions of calmodulin with both the oxygenase and reductase domains in addition to the canonical calmodulin-binding region. The neuronal isoform is not regulated by a unique autoinhibitory element in its reductase domain.

Highlights

Three NOS isoforms were originally identified based on the tissue source: nNOS, eNOS, and iNOS [5]
As described under “Materials and Methods,” all expressed chimeric enzymes were catalytically active with activities ranging from 100 to 30% relative to iNOS. nNOSL696V and iNOSL475V containing a conserved point mutation designed to make some of the chimeric NOSs were catalytically active, and their dependence on different [Ca2ϩ] were the same as their respective wild-type enzymes (Fig. 2)
Previous results obtained on chimeras with the respective calmodulin-binding sequences from iNOS substituted in eNOS or nNOS showed the sequence was necessary but not sufficient for Ca2ϩ-independent activity or calmodulin binding [11, 12]

Summary

Introduction

Three NOS isoforms were originally identified based on the tissue source: nNOS, eNOS, and iNOS [5]. All of the putative calmodulin-binding regions from nNOS, eNOS, and iNOS satisfy the criteria for properties of a canonical calmodulin-binding region, a basic amphipathic ␣-helical sequence containing 12 basic and hydrophobic residues [8]. In addition to the canonical calmodulin-binding region, sites in the reductase domain confer Ca2ϩ-independent binding of calmodulin, which is presumably responsible for the iNOS Ca2ϩindependent activity. Salerno et al [13] proposed an autoinhibitory segment in the FMN-binding domains of nNOS and eNOS, but not iNOS. Calmodulin binding may displace this unique autoinhibitory segment, resulting in catalysis These two models for Ca2ϩ/calmodulin regulation of NOS activities were investigated by the characterization of chimeric enzymes made from nNOS and iNOS

Methods

Results

Conclusion