Activation and inactivation of neuronal nitric oxide synthase: characterization of Ca2+-dependent [125I]Calmodulin binding

Weissman, B.A., Jones, C.L., Liu, Q., Gross, S.S. and Smith, C.L. 2002. Activation and inactivation of neuronal nitric oxide synthase: characterization of Ca2+-dependent [125I]Calmodulin binding. European Journal of Pharmacology. 435 (1), pp. 9-18. https://doi.org/10.1016/S0014-2999(01)01560-6

TitleActivation and inactivation of neuronal nitric oxide synthase: characterization of Ca2+-dependent [125I]Calmodulin binding
AuthorsWeissman, B.A., Jones, C.L., Liu, Q., Gross, S.S. and Smith, C.L.
Abstract

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.

JournalEuropean Journal of Pharmacology
Journal citation435 (1), pp. 9-18
ISSN0014-2999
YearJan 2002
Digital Object Identifier (DOI)https://doi.org/10.1016/S0014-2999(01)01560-6
Publication dates
PublishedJan 2002
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