(Verbatim from the application): Calmodulin (CaM) is the primary mediator of calcium (Ca) signal transduction processes, regulating more than 30 enzymes and ion channels. Nature produces a variety of CaM isoforms and we found that two isoforms, ScaM-1 and ScaM-4, exhibit dramatically different activation or inhibition of 9 target enzymes. ScaM-1 activates 8 enzymes (including myosin light chain kinase (MLCK), Calcineurin (CaN), NAD-kinase and Ca-ATPase), but it is a selective competitive antagonist of nitric oxide synthase (NOS). The opposite is true for ScaM-4 that activates NOS and 4 of the enzymes activated by ScaM-1, but are a competitive antagonist of MLCK, CaN, NAD-kinase and Ca-ATPase. Each of these CaM isoforms allows the selective activation and inhibition of particular target enzymes, allowing a bifurcation of the Ca-CaM signal transduction pathway and possibly alterations in cell function. The applicant determined that a point mutation (M144V) is responsible for ScaM-1 selective inhibition of NOS. The applicant will use site-directed mutagenesis to map the specific regions of ScaM-4, of human CaM isoform (hCLP) and of mutant paramecium CaM's to determine which are responsible for their differential enzyme activation and altered Ca binding. The applicant will determine the effect of EF-hand factors (acidic chelating residues, non-chelating residues in the Ca binding loop and helical hydrophobicity) on Ca affinity and Ca exchange rates in CaM. This will allow the applicant to alter CaM's N and C-terminal Ca affinity and produce CaM mutants which exhibit very different Ca-dependent activation of target enzymes and altered rates of activation and inactivation in response to Ca transients. The applicant will produce a constitutively active CaM mutant which remains active even in the absence of Ca. These studies should provide new insight into how CaM binds and activates its various target enzymes during Ca transients and perhaps provide new therapeutic tools for altering Ca signal transduction.
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