Activation and inactivation of neuronal nitric oxide synthase: characterization of Ca(2+)-dependent [125I]Calmodulin binding.
Constitutive isoforms of nitric oxide synthase (NOS) are activated by transient binding of Ca(2+)/Calmodulin. Here, we characterize the binding of Calmodulin to purified neuronal NOS (nNOS). [125I]Calmodulin bound to a single class of non-interacting and high affinity sites on nNOS. [125I]Calmodulin binding achieved rapid saturation, was linear with nNOS concentration, and exhibited a strict dependence on [Ca(2+)]. Neither affinity nor extent of [125I]Calmodulin binding was affected by L-arginine, NADPH or Tetrahydrobiopterin. Native Calmodulin and engineered Calmodulin homologs [i.e., duplicated N-terminal (CaMNN)] potently displaced [125I]Calmodulin. CaMNN supported nNOS catalysis, but required approximately five-fold more Ca(2+) for comparable activity with native Calmodulin. Taken with results from kinetic analyses of [125I]Calmodulin association and dissociation, our findings suggest four sequential steps in activation of nNOS by Calmodulin: (1) Ca(2+) binds to Calmodulin's C-lobe, (2) the C-lobe of Calmodulin binds NOS, (3) Ca(2+) binds to the N-lobe of Calmodulin, and (4) the N-lobe binds to nNOS. Activation of nNOS only occurs after completion of step (4), with the displacement of nNOS's autoinhibitory insert. Upon intracellular Ca(2+) sequestration, deactivation of nNOS would proceed in reverse order.
Weissman
Ben A
BA
Department of Pharmacology, Cornell University Medical College, New York, NY 10021, USA. baw@iibr.gov.il
Jones
Caroline L
CL
Liu
Qing
Q
Gross
Steven S
SS
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D004789:Enzyme Activation; D007457:Iodine Radioisotopes; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D011869:Radioligand Assay; D051381:Rats; D013379:Substrate Specificity
D002147:Calmodulin; D007457:Iodine Radioisotopes; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; D002118:Calcium