| Research Abstract |
Purpose Calcium ion acts as a ubiquitous second messenger, especially in neural tissues where many of its physiological responses are mediated by the Ca^2-binding protein calmodulin (CaM). The actions of CaM are mediated by its association with specific target proteins, some of which are known as CaM-binding proteins, including the nitric oxide synthases (NOSs) which catalyze the formation of nitric oxide (NO) and L-citrulline from L-arginine. In order to elucidate the activation mechanisms of neuronal NOS (nNOS) at the molecular level, the following projects were undertaken : 1. Structure-activity relationship of nNOS.2. Discovery of a small molecule inhibitor for nNOS.3. Molecular cloning and characterization of novel Ca^<2+>/CaM-dependent proteins.
Results and discussions 1. A specific hydrophobic/basic amino acid cluster in the rat nNOS sequence, Lys^<732>LysLeu, critical for its CaM and membrane binding is identified. We also show intracellular nNOS-iNOS dimerization within engineered cells and would suggest that nNOS-iNOS dimerization might result in generating Ca^<2+>/CaM stimulated activity of the heteromeric enzyme. 2. A newly synthesized isoquinolinesulfonamide, HMN-1180 (1-(5-isoquinolinylsulfonyl)-7-methylhomopiperazine), was shown to have selective inhibitory action against nNOS with a Ki value of 5.4 muM.HMN-1180 was also found to inhibit glutamate stimulated NO production generated by nNOS in the human neuroblastoma cell line SK-N-MC.3. We report the molecular cloning and expression of a cDNA encoding bovine brain NCalpha, a neuron specific Ca^<2+>-binding protein with three EF-hand motifs. We also demonstrate the existence of an isoform-specific activation mechanism of CaM-kinase I.Future prospect Based on our present studies, the analyses for an important component of the "cross-talk" between NO and kinases or Ca^<2+>-binding proteins in calcium signal will need to be determined.
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