We have uncovered a family of calcium binding proteins that are homologous to Drosophila frequenin and are highly conserved between species. These proteins, which we have termed neuronal calcium sensors (NCS) are present in post-mitotic neurons and have been shown in in vitro assays to inhibit rhodopsin phosphorylation. One member of this family, NCS-1, was also shown in in vitro assays to substitute for calmodulin in the activation of nitric oxide synthase (NOS) and calcineurin. We wished to know their distribution within the adult nervous system and whether they colocalize with NOS or calcineurin. Immunolocalization experiments were carried out for one member of this family, NCS-1, in collaboration with the National Center for Microscopy and Imaging Research. Confocai imaging experiments indicated that NCS-1 was widely distributed throughout the central nervous system and at the neuromusclularjunction. Double labeling experiments indicate that both NCS-1 and NOS are highly expressed in the basket cell axons in the cerebellum. Light level analysis and immunoperoxidase labeling at the EM level indicated that it was possibly associated with cytoskeletal elements and the post-synaptic density. However, higher resolution localization is necessary to determine the subcellular localization. Unfortunately, immunolocalization using colloidal gold and cryosections was unsuccessful: the degree of labeling was too low to determine localization conclusively. Experiments are currently underway using photo oxidation of eosin-labeled immunoreagents for high resolution electron microscopic localization. Preliminary experiments using this technique have indicated that NCS-1 may be associated with neurofilaments in myelinated axons. The manuscript detailing this work is in preparation.
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