Compressive Force Activation of the Neuronal Nitric Oxide Synthase Enzyme

Calmodulin (CaM), a calcium-sensing protein, regulates the production of nitric oxide (NO) by the nitric oxide synthase (NOS) enzyme. It is interesting to decipher the control mechanism of CaM-dependent NO biosynthesis by the NOS isoforms, as NO is a ubiquitous intercellular signaling molecule in numerous physiological processes. It is generally accepted that 4Ca²⁺-bound CaM associates with the NOS enzyme and activates NO production, while lower-level calcium has also been reported to allow for NOS enzymatic function under certain circumstances (e.g., shear stress). However, NO production by apo-CaM-activated NOS under an external force has not been directly demonstrated. Herein, we have utilized an atomic force microscopy (AFM)-correlated confocal microscopy technique to probe NO production by neuronal NOS (nNOS) enzyme, where compressive force-manipulated apo-CaM acts as a mechanosensing protein. DAR-4M was used as an NO-sensing fluorescent probe. Our results indicate that compressive force may induce necessary conformation changes of apo-CaM in binding and activating the nNOS enzyme.