Neuronal Ca2+ sensor 1 - Characterization of the myristoylated protein, its cellular effects in permeabilized adrenal chromaffin cells, Ca2+-independent membrane association, and interaction with binding proteins, suggestinga role in rapid Ca2+ signal transduction

Overexpression of frequenin and its orthologue neuronal Ca2+ sensor 1 (NCS- 1) has been shown to increase evoked exocytosis in neurons and neuroendocri ne cells. The site of action of NCS-1 and its biochemical targets that affe ct exocytosis are unknown. To allow further investigation of NCS-1 function , we have demonstrated that NCS-1 is a substrate for N-myristoyltransferase and generated recombinant myristoylated NCS-1, The bacterially expressed N CS-1 shows Ca2+-induced conformational changes. The possibility that NCS-1 directly interacts with the exocytotic machinery to enhance exocytosis was tested using digitonin-permeabilized chromaffin cells. Exogenous NCS-1 was retained in permeabilized cells but had no effect on Ca2+-dependent release of catecholamine. In addition, exogenous NCS-1 did not regulate cyclic nuc leotide levels in this system. These data suggest that the effects of NCS-1 seen in intact cells are likely to be due to an action on the early steps of stimulus-secretion coupling or on Ca2+ homeostasis. Myristoylated NCS-1 bound to membranes in the absence of Ca2+ and endogenous NCS-1 was tightly membrane-associated. Using biotinylated NCS-1, a series of specific binding proteins were detected in cytosol, chromaffin granule membrane, and micros ome fractions of adrenal medulla. These included proteins distinct from tho se detected by biotinylated calmodulin, demonstrating the presence of multi ple specific Ca2+-independent and Ca2+-dependent binding proteins as putati ve targets for NCS-1 action. A model for NCS-1 function, from these data, i ndicates a constitutive membrane association independent of Ca2+. This diff ers from the Ca2+ myristoyl switch model for the closely related recoverin and suggests a possible action in rapid Ca2+ signal transduction in respons e to local Ca2+ signals.