Generation and analysis of dominant negative mutants of neuronal nitric oxide synthase. • 231

Abstract

Since the functional form of neuronal nitric oxide synthase (nNOS) exists as a homodimer, nNOS protein mutations disrupting homodimer formation may serve as dominant negative mutations. To test this hypothesis two sets of expression plasmids were designed and constructed. The first set expressed the heme and reductase domains of rat nNOS either singly or as a fusion protein separated by a short hydrophilic linker peptide. The second set expressed the rat nNOS heme domain and the reductase domains of adrenodoxin reductase, oxido reductase, and the reductase domain from cytochrome P450 BM-3. Again the two domains were linked via a short hydrophilic linker peptide. All the constructs were tested for their ability to metabolize L-arginine (the substrate of NOS) after their transient transfection into COS-7 cells. All the constructs had activities of <8% of the wild-type nNOS protein similarly expressed in COS-7 cells. The dominant negative effect of the constructs was then determined by their ability to reduce wild-type nNOS activity when transfected into a COS-7 stable cell line expressing rat nNOS. Results obtained demonstrated that all the constructs significantly reduced the activity of the wild type nNOS. The nNOS heme-reductase fusion was then used to transiently transfect rat PC12 cells to determine if this construct could exhibit a dominant negative effect on nerve growth factor (NGF)-induced PC12 cell differentiation. Transfection with this construct reduced NGF-induced neurite outgrowth (a measure of PC12 cell differentiation) from 63.0% ± 9.6% to 28.7%±6.5%. These constructs demonstrate the utility of producing dominant negative mutants of NOS isoforms and should prove to be valuable in analyzing the role of nNOS in biological systems.