An autoinhibitory control element defines calcium-regulated isoforms of nitric oxide synthase

Nitric oxide synthases (NOSs) are classified functionally, based on whether calmodulin binding is Ca2+-dependent (cNOS) or Ca2+-independent (iNOS). This key dichotomy has not been defined at the molecular level. Here we show that cNOS isoforms contain a unique polypeptide insert in their FMN binding domains which is not shared with iNOS or other related flavoproteins. Previously identified autoinhibitory domains in calmodulin-regulated enzymes raise the possibility that the polypeptide insert is the autoinhibitory domain of cNOSs. Consistent with this possibility, three-dimensional molecular modeling suggested that the insert originates from a site immediately adjacent to the calmodulin binding sequence. Synthetic peptides derived from the 45-amino acid insert of endothelial NOS were found to potently inhibit binding of calmodulin and activation of cNOS isoforms. This inhibition was associated with peptide binding to NOS, rather than free calmodulin, and inhibition could be reversed by increasing calmodulin concentration. In contrast, insert-derived peptides did not interfere with the arginine site of cNOS, as assessed from [3H]NG-nitro-L-arginine binding, nor did they potently effect iNOS activity. Limited proteolysis studies showed that calmodulin's ability to gate electron flow through cNOSs is associated with displacement of the insert polypeptide; this is the first specific calmodulin-induced change in NOS conformation to be identified. Together, our findings strongly suggest that the insert is an autoinhibitory control element, docking with a site on cNOSs which impedes calmodulin binding and enzymatic activation. The autoinhibitory control element molecularly defines cNOSs and offers a unique target for developing novel NOS activators and inhibitors

Shared and selective neural correlates of inhibition, facilitation, and shifting processes during executive control

A network of prefrontal and parietal regions has been implicated in executive control processes. However, the extent to which individual regions within this network are engaged in component control processes, such as inhibition of task-irrelevant stimulus attributes or shifting (switching) between attentional foci, remains controversial. Participants (N = 17) underwent functional magnetic resonance imaging while performing a global–local task in which the global and local levels could facilitate or interfere with one another. Stimuli were presented in blocks in which participants either constantly shifted between the global and local levels, or consistently responded to one level only. Activations related to inhibition and shifting processes were observed in a large network of bilateral prefrontal, parietal, and basal ganglia regions. Region of interest analyses were used to classify each region within this network as being common to inhibition and shifting, or preferential to one component process. Several regions were classified as being preferential to inhibition, including regions within the dorsolateral and ventrolateral prefrontal cortex, the parietal lobes, and the temporal–parietal junction. A limited set of regions in the parietal lobes and left dorsolateral prefrontal cortex were classified as preferential to shifting. There was a very large set of regions displaying activation common to both inhibition and shifting processes, including regions within the dorsolateral prefrontal cortex, anterior cingulate, and basal ganglia. Several of these common regions were also involved during facilitation, suggesting that they are responsive to the number of task-salient channels of information, rather than purely to demands on control processes.National Institute of Mental Health (U.S.) (MH061426)National Institute on Aging (AG021847