Dynamic regulation of neuronal NO synthase transcription by calcium influxthrough a CREB family transcription factor-dependent mechanism

Neuronal nitric oxide (NO) synthase (nNOS) is dynamically regulated in resp onse to a variety of physiologic and pathologic stimuli. Although the dynam ic regulation of nNOS is well established, the molecular mechanisms by whic h such diverse stimuli regulate nNOS expression have not yet been identifie d. We describe experiments demonstrating that Ca2+ entry through voltage-se nsitive Ca2+ channels regulates nNOS expression through alternate promoter usage in cortical neurons and that nNOS exon 2 contains the regulatory sequ ences that respond to Ca2+. Deletion and mutational analysis of the nNOS ex on 2 promoter reveals two critical cAMP/Ca2+ response elements (CREs) that are immediately upstream of the transcription start site. CREB binds to the CREs within the nNOS gene. Mutation of the nNOS CREs as well as blockade o f CREB function results in a dramatic loss of nNOS transcription. These fin dings suggest that nNOS is a Ca2+-regulated gene through the interactions o f CREB on the CREs within the nNOS exon 2 promoter and that these interacti ons are likely to be centrally involved in the regulation of nNOS in respon se to neuronal injury and activity-dependent plasticity.